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Nappo S, Toriello M, Maisto G, Mirabelli P, Topo F, Gallo L, Castellano G, Esposito M, Auriemma L, Madalese D, Cacace F, Picardi A, Tambaro FP, Penta de Vera d'Aragona R. Gelofusine as alternative to Dextran40-based solution for washing cryopreserved hematopoietic stem cell products prior to infusion: Validation and application to clinical practice. Transfusion 2025. [PMID: 40366301 DOI: 10.1111/trf.18272] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2025] [Revised: 04/15/2025] [Accepted: 04/16/2025] [Indexed: 05/15/2025]
Abstract
BACKGROUND Cryopreservation is an essential step for autologous hematopoietic stem cell (HSC) transplantation and umbilical cord blood units (CBUs), and for allogeneic peripheral blood stem cells (PBSCs) or bone marrow (BM) when immediate infusion is not possible. However, the cryoprotectant dimethyl sulfoxide (DMSO) used for HSC cryopreservation can be toxic to cells post-thaw and to patients during infusion. The Rubinstein solution is validated to wash HSCs, but the unavailability of Dextran40 in Italy prompted a search for alternatives. This report discusses the use of Gelofusine, a 4% modified gelatin solution, as a substitute for Dextran40-based solutions in washing cryopreserved stem cell products. STUDY DESIGN AND METHODS The study includes: (1) validation of Gelofusine in 10 CBUs unsuitable for transplantation; (2) outcomes of the first 93 transplanted units washed with Gelofusine; (3) comparisons of recovery and viability in five paired autologous PBSC products washed with Gelofusine and Rubinstein-solution; and (4) comparisons of engraftment times in patients receiving units washed with Gelofusine and Rubinstein-solution. RESULTS AND DISCUSSION For 10 CBUs washed with Gelofusine, CD34+ and TNC viability and recovery were 96%, 87%, 71%, and 75% respectively, higher than our reference values. In transplanted products, CD34+ and TNC viability and recovery were 96%, 89%, 82%, and 91% respectively. Comparisons with Rubinstein solution revealed similar TNC and CD34+ recovery but significantly higher TNC (89% vs. 68%) and CD34+ (97% vs. 89%) viability with Gelofusine. Engraftment times for both solutions were similar. These findings support Gelofusine as an effective and valid alternative to Rubinstein-solution for washing cryopreserved HSCs.
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Affiliation(s)
- Stefania Nappo
- Ba.S.C.O., Cell Processing and Immunogenetics Unit, Oncology, Hematology and Cellular Therapy Department, AORN Santobono-Pausilipon, Naples, Italy
| | - Mario Toriello
- Ba.S.C.O., Cell Processing and Immunogenetics Unit, Oncology, Hematology and Cellular Therapy Department, AORN Santobono-Pausilipon, Naples, Italy
| | - Giovanna Maisto
- Transfusion Medicine Unit, Oncology, Hematology and Cellular Therapy Department, AORN Santobono-Pausilipon, Naples, Italy
| | - Peppino Mirabelli
- Research Laboratories and Biobanking Unit, AORN Santobono-Pausilipon, Naples, Italy
| | - Francesco Topo
- Ba.S.C.O., Cell Processing and Immunogenetics Unit, Oncology, Hematology and Cellular Therapy Department, AORN Santobono-Pausilipon, Naples, Italy
| | - Lucia Gallo
- Ba.S.C.O., Cell Processing and Immunogenetics Unit, Oncology, Hematology and Cellular Therapy Department, AORN Santobono-Pausilipon, Naples, Italy
| | - Giovanni Castellano
- Ba.S.C.O., Cell Processing and Immunogenetics Unit, Oncology, Hematology and Cellular Therapy Department, AORN Santobono-Pausilipon, Naples, Italy
| | - Martina Esposito
- Ba.S.C.O., Cell Processing and Immunogenetics Unit, Oncology, Hematology and Cellular Therapy Department, AORN Santobono-Pausilipon, Naples, Italy
| | - Laura Auriemma
- Ba.S.C.O., Cell Processing and Immunogenetics Unit, Oncology, Hematology and Cellular Therapy Department, AORN Santobono-Pausilipon, Naples, Italy
| | - Donato Madalese
- Ba.S.C.O., Cell Processing and Immunogenetics Unit, Oncology, Hematology and Cellular Therapy Department, AORN Santobono-Pausilipon, Naples, Italy
| | - Fabiana Cacace
- Stem Cell Transplantation and Cell Therapy Unit, AORN Santobono-Pausilipon, Naples, Italy
| | | | | | - Roberta Penta de Vera d'Aragona
- Ba.S.C.O., Cell Processing and Immunogenetics Unit, Oncology, Hematology and Cellular Therapy Department, AORN Santobono-Pausilipon, Naples, Italy
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Modaresi S, Pacelli S, Chakraborty A, Coyle A, Luo W, Singh I, Paul A. Engineering a Microfluidic Platform to Cryopreserve Stem Cells: A DMSO-Free Sustainable Approach. Adv Healthc Mater 2024; 13:e2401264. [PMID: 39152923 PMCID: PMC11582517 DOI: 10.1002/adhm.202401264] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2024] [Revised: 06/24/2024] [Indexed: 08/19/2024]
Abstract
Human adipose-derived stem cells (hASCs) are cryopreserved traditionally using dimethyl sulfoxide (DMSO) as the cryoprotectant agent. DMSO penetrates cell membranes and prevents cellular damage during cryopreservation. However, DMSO is not inert to cells, inducing cytotoxic effects by causing mitochondrial dysfunction, reduced cell proliferation, and impaired hASCs transplantation. Additionally, large-scale production of DMSO and contamination can adversely impact the environment. A sustainable, green alternative to DMSO is trehalose, a natural disaccharide cryoprotectant agent that does not pose any risk of cytotoxicity. However, the cellular permeability of trehalose is less compared to DMSO. Here, a microfluidic chip is developed for the intracellular delivery of trehalose in hASCs. The chip is designed for mechanoporation, which creates transient pores in cell membranes by mechanical deformation. Mechanoporation allows the sparingly permeable trehalose to be internalized within the cell cytosol. The amount of trehalose delivered intracellularly is quantified and optimized based on cellular compatibility and functionality. Furthermore, whole-transcriptome sequencing confirms that less than 1% of all target genes display at least a twofold change in expression when cells are passed through the chip compared to untreated cells. Overall, the results confirm the feasibility and effectiveness of using this microfluidic chip for DMSO-free cryopreservation of hASCs.
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Affiliation(s)
- Saman Modaresi
- Department of Chemical and Petroleum Engineering, Bioengineering Graduate Program, School of Engineering, The University of Kansas, Lawrence, KS, 66045, USA
| | - Settimio Pacelli
- Department of Biomedical Engineering, Illinois Institute of Technology, Chicago, IL, 60616, USA
| | - Aishik Chakraborty
- Department of Chemical and Biochemical Engineering, The University of Western Ontario, London, ON, N6A 5B9, Canada
- Collaborative Specialization in Musculoskeletal Health Research and Bone and Joint Institute, The University of Western Ontario, London, ON, N6A 5B9, Canada
| | - Ali Coyle
- School of Biomedical Engineering, The University of Western Ontario, London, ON, N6A 5B9, Canada
| | - Wei Luo
- Department of Chemical and Biochemical Engineering, The University of Western Ontario, London, ON, N6A 5B9, Canada
| | - Irtisha Singh
- Department of Cell Biology and Genetics, College of Medicine, Texas A&M University, Bryan, TX, 77807, USA
- Department of Biomedical Engineering, College of Engineering, Texas A&M University, College Station, TX, 77843, USA
- Interdisciplinary Program in Genetics and Genomics, Texas A&M University, College Station, TX, 77840, USA
| | - Arghya Paul
- Department of Chemical and Biochemical Engineering, The University of Western Ontario, London, ON, N6A 5B9, Canada
- Collaborative Specialization in Musculoskeletal Health Research and Bone and Joint Institute, The University of Western Ontario, London, ON, N6A 5B9, Canada
- School of Biomedical Engineering, The University of Western Ontario, London, ON, N6A 5B9, Canada
- Department of Chemistry, The Center for Advanced Materials and Biomaterials Research, The University of Western Ontario, London, ON, N6A 5B9, Canada
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Fang WH, Vangsness CT. Orthobiologic Products: Preservation Options for Orthopedic Research and Clinical Applications. J Clin Med 2024; 13:6577. [PMID: 39518716 PMCID: PMC11546119 DOI: 10.3390/jcm13216577] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2024] [Revised: 10/14/2024] [Accepted: 10/28/2024] [Indexed: 11/16/2024] Open
Abstract
The biological products used in orthopedics include musculoskeletal allografts-such as bones, tendons, ligaments, and cartilage-as well as biological therapies. Musculoskeletal allografts support the body's healing process by utilizing preserved and sterilized donor tissue. These allografts are becoming increasingly common in surgical practice, allowing patients to avoid more invasive procedures and the risks associated with donor site morbidity. Bone grafting is one of the most frequently used procedures in orthopedics and traumatology. Biologic approaches aim to improve clinical outcomes by enhancing the body's natural healing capacity and reducing inflammation. They serve as an alternative to surgical interventions. While preliminary results from animal studies and small-scale clinical trials have been promising, the field of biologics still lacks robust clinical evidence supporting their efficacy. Biological therapies include PRP (platelet-rich plasma), mesenchymal stem cells (MSCs)/stromal cells/progenitor cells, bone marrow stem/stromal cells (BMSCs), adipose stem/stromal cells/progenitor cells (ASCs), cord blood (CB), and extracellular vesicles (EVs), including exosomes. The proper preservation and storage of these cellular therapies are essential for future use. Preservation techniques include cryopreservation, vitrification, lyophilization, and the use of cryoprotective agents (CPAs). The most commonly used CPA is DMSO (dimethyl sulfoxide). The highest success rates and post-thaw viability have been achieved by preserving PRP with a rate-controlled freezer using 6% DMSO and storing other cellular treatments using a rate-controlled freezer with 5% or 10% DMSO as the CPA. Extracellular vesicles (EVs) have shown the best results when lyophilized with 50 mM or 4% trehalose to prevent aggregation and stored at room temperature.
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Affiliation(s)
- William H. Fang
- Department of Orthopedic Surgery, Valley Health Systems, 620 Shadow Lane, Las Vegas, NV 89106, USA
| | - C. Thomas Vangsness
- Department of Orthopedic Surgery, Keck School of Medicine of USC, Los Angeles, CA 90033, USA
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Murray A, Kilbride P, Gibson MI. Trehalose in cryopreservation. Applications, mechanisms and intracellular delivery opportunities. RSC Med Chem 2024; 15:2980-2995. [PMID: 39309363 PMCID: PMC11411628 DOI: 10.1039/d4md00174e] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2024] [Accepted: 07/08/2024] [Indexed: 09/25/2024] Open
Abstract
Cryopreservation is crucial to fields including immune and stem cell therapies, reproductive technology, blood banking, regenerative medicine and across all biotechnology. During cryopreservation, cryoprotectants are essential to protect cells from the damage caused by exposure to freezing temperatures. The most common penetrating cryoprotectants, such as DMSO and glycerol do not give full recovery and have a cytotoxicity limit on the concentration which can be applied. The non-reducing disaccharide trehalose has been widely explored and used to supplement these, inspired by its use in nature to aid survival at extreme temperatures and/or desiccation. However, trehalose has challenges to its use, particular its low membrane permeability, and how its protective role compares to other sugars. Here we review the application of trehalose and its reported benefit and seek to show where chemical tools can improve its function. In particular, we highlight emerging chemical methods to deliver (as cargo, or via selective permeation) into the intracellular space. This includes encapsulation, cell penetrating peptides or (selective) modification of hydroxyls on trehalose.
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Affiliation(s)
- Alex Murray
- Department of Chemistry, University of Warwick CV4 7AL UK
- Division of Biomedical Sciences, Warwick Medical School, University of Warwick CV4 7AL UK
| | | | - Matthew I Gibson
- Department of Chemistry, University of Warwick CV4 7AL UK
- Division of Biomedical Sciences, Warwick Medical School, University of Warwick CV4 7AL UK
- Asymptote, Cytiva Chivers Way Cambridge CB24 9BZ USA
- Department of Chemistry, University of Manchester Oxford Road Manchester M13 9PL UK
- Manchester Institute of Biotechnology, University of Manchester 131 Princess Street Manchester M1 7DN UK
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Traversari G, Locci AM, Concas A, Lai N, Cincotti A. Osmotic injury and cytotoxicity for hMSCs in contact with Me2SO: The effect of cell size distribution. Cryobiology 2024; 116:104943. [PMID: 39033954 DOI: 10.1016/j.cryobiol.2024.104943] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2023] [Revised: 07/17/2024] [Accepted: 07/17/2024] [Indexed: 07/23/2024]
Abstract
The paper discusses the impact of cell size on cytotoxicity and expansion lysis during the osmotic excursions resulting from the contact of hMSCs from UCB with Me2SO. It builds upon the mathematical model recently presented by the authors, which pertains to a population of cells with uniform size. The objective is to enhance the model's relevance by incorporating the more realistic scenario of cell size distribution, utilizing a Population Balance Equations approach. The study compares the capability of the multiple-sized model to the single-sized one to describe system behavior experimentally measured through cytofluorimetry and Coulter counter when, first, suspending hMSCs in hypertonic solutions of Me2SO (at varying osmolality, system temperature, and contact times), and then (at room temperature) pelleting by centrifugation before suspending the cells back to isotonic conditions. Simulations demonstrate that expansion lysis and cytotoxic effect are not affected by cell size for the specific system hMSCs/Me2SO, thus confirming what was found so far by the authors through a single-size model. On the other hand, simulations show that, when varying the adjustable parameters of the model that are expected to change from cell to cell lineages, expansion lysis is sensitive to cell size, while cytotoxicity is not, being mainly influenced by external CPA concentration and contact duration. More specifically, it is found that smaller cells suffer expansion lysis more than larger ones. The findings suggest that different cells from hMSCs may require a multiple-sized model to assess cell damage during osmotic excursions in cryopreservation.
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Affiliation(s)
- Gabriele Traversari
- Dipartimento di Ingegneria Meccanica, Chimica e Dei Materiali, Facoltà di Ingegneria e Architettura, Università Degli Studi di Cagliari, Via Marengo 2, 09123, Cagliari, Italy
| | - Antonio Mario Locci
- Dipartimento di Ingegneria Meccanica, Chimica e Dei Materiali, Facoltà di Ingegneria e Architettura, Università Degli Studi di Cagliari, Via Marengo 2, 09123, Cagliari, Italy
| | - Alessandro Concas
- Dipartimento di Ingegneria Meccanica, Chimica e Dei Materiali, Facoltà di Ingegneria e Architettura, Università Degli Studi di Cagliari, Via Marengo 2, 09123, Cagliari, Italy
| | - Nicola Lai
- Dipartimento di Ingegneria Meccanica, Chimica e Dei Materiali, Facoltà di Ingegneria e Architettura, Università Degli Studi di Cagliari, Via Marengo 2, 09123, Cagliari, Italy
| | - Alberto Cincotti
- Dipartimento di Ingegneria Meccanica, Chimica e Dei Materiali, Facoltà di Ingegneria e Architettura, Università Degli Studi di Cagliari, Via Marengo 2, 09123, Cagliari, Italy.
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Kumar N, Bidkhori HR, Yawno T, Lim R, Inocencio IM. Therapeutic potential of extracellular vesicles derived from human amniotic epithelial cells for perinatal cerebral and pulmonary injury. Stem Cells Transl Med 2024; 13:711-723. [PMID: 38895873 PMCID: PMC11328935 DOI: 10.1093/stcltm/szae037] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2023] [Accepted: 04/19/2024] [Indexed: 06/21/2024] Open
Abstract
Lung and brain injury that occurs during the perinatal period leads to lifelong disability and is often driven and/or exacerbated by inflammation. Human amniotic epithelial cells (hAEC), which demonstrate immunomodulatory, anti-fibrotic, and regenerative capabilities, are being explored as a therapeutic candidate for perinatal injury. However, limitations regarding scalable manufacturing, storage, transport, and dose-related toxicity have impeded clinical translation. Isolated therapeutic extracellular vesicles (EVs) from stem and stem-like cells are thought to be key paracrine mediators of therapeutic efficacy. The unique characteristics of EVs suggest that they potentially circumvent the limitations of traditional cell-based therapies. However, given the novelty of EVs as a therapeutic, recommendations around ideal methods of production, isolation, storage, and delivery have not yet been created by regulatory agencies. In this concise review, we discuss the pertinence and limitations of cell-based therapeutics in perinatal medicine. We also review the preclinical evidence supporting the use of therapeutic EVs for perinatal therapy. Further, we summarize the arising considerations regarding adequate cell source, biodistribution, isolation and storage methods, and regulatory roadblocks for the development of therapeutic EVs.
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Affiliation(s)
- Naveen Kumar
- The Ritchie Centre, The Hudson Institute of Medical Research, Clayton 3168, Victoria, Australia
- Department of Obstetrics and Gynaecology, Monash University, Clayton 3168, Victoria, Australia
| | - Hamid Reza Bidkhori
- The Ritchie Centre, The Hudson Institute of Medical Research, Clayton 3168, Victoria, Australia
- Department of Obstetrics and Gynaecology, Monash University, Clayton 3168, Victoria, Australia
| | - Tamara Yawno
- The Ritchie Centre, The Hudson Institute of Medical Research, Clayton 3168, Victoria, Australia
- Department of Obstetrics and Gynaecology, Monash University, Clayton 3168, Victoria, Australia
- Department of Paediatrics, Monash University, Clayton 3168, Victoria, Australia
| | - Rebecca Lim
- The Ritchie Centre, The Hudson Institute of Medical Research, Clayton 3168, Victoria, Australia
- Department of Obstetrics and Gynaecology, Monash University, Clayton 3168, Victoria, Australia
| | - Ishmael Miguel Inocencio
- The Ritchie Centre, The Hudson Institute of Medical Research, Clayton 3168, Victoria, Australia
- Department of Obstetrics and Gynaecology, Monash University, Clayton 3168, Victoria, Australia
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Eltelbanei MA, El-Bassiouny NA, Abdalla MS, Khalaf M, Werida RH. Clinical and safety outcomes of BeEAM (Bendamustine, Etoposide, Cytarabine, Melphalan) versus CEM (Carboplatin, Etoposide, Melphalan) in lymphoma patients as a conditioning regimen before autologous hematopoietic cell transplantation. BMC Cancer 2024; 24:1002. [PMID: 39134959 PMCID: PMC11320894 DOI: 10.1186/s12885-024-12694-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2024] [Accepted: 07/24/2024] [Indexed: 08/15/2024] Open
Abstract
BACKGROUND Autologous stem cell transplantation (ASCT) is a pivotal treatment for lymphoma patients. The BeEAM regimen (Bendamustine, Etoposide, Cytarabine, Melphalan) traditionally relies on cryopreservation, whereas the CEM regimen (Carboplatin, Etoposide, Melphalan) has been optimized for short-duration administration without the need for cryopreservation. This study rigorously compares the clinical and safety profiles of the BeEAM and CEM regimens. METHODS A controlled, randomized clinical trial was conducted with 58 lymphoma patients undergoing ASCT at the International Medical Center (IMC) in Cairo, Egypt. Patients were randomly assigned to either the BeEAM (n = 29) or CEM (n = 29) regimen, with an 18-month follow-up period. Clinical and safety outcomes were meticulously compared, focusing on time to engraftment for neutrophils and platelets, side effects, length of hospitalization, transplant-related mortality (TRM), and survival rates. RESULTS The findings demonstrate a significant advantage for the CEM regimen. Neutrophil recovery was markedly faster in the CEM group, averaging 8.5 days compared to 14.5 days in the BeEAM group (p < 0.0001). Platelet recovery was similarly expedited, with 11 days in the CEM group versus 23 days in the BeEAM group (p < 0.0001). Hospitalization duration was substantially shorter for CEM patients, averaging 18.5 days compared to 30 days for those on BeEAM (p < 0.0001). Furthermore, overall survival (OS) was significantly higher in the CEM group at 96.55% (95% CI: 84.91-99.44%) compared to 79.31% (95% CI: 63.11-89.75%) in the BeEAM group (p = 0.049). Progression-free survival (PFS) was also notably superior in the CEM group, at 86.21% (95% CI: 86.14-86.28%) versus 62.07% (95% CI: 61.94-62.20%) in the BeEAM group (p = 0.036). CONCLUSION The CEM regimen might demonstrate superiority over the BeEAM regimen, with faster neutrophil and platelet recovery, reduced hospitalization time, and significantly improved overall and progression-free survival rates. Future studies with longer duration and larger sample sizes are warranted. TRIAL REGISTRATION This study is registered on ClinicalTrials.gov under the registration number NCT05813132 ( https://clinicaltrials.gov/ct2/show/NCT05813132 ). (The first submitted registration date: is March 16, 2023).
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Affiliation(s)
- Mohamed A Eltelbanei
- Senior Clinical Pharmacist in Hematology, Oncology, and Stem Cell Transplantation at International Medical Center (IMC) Hospital, Clinical pharmacy & pharmacy practice master candidate at Faculty of pharmacy, Damanhour University, Cairo, Egypt.
| | - Noha A El-Bassiouny
- Clinical Pharmacy & Pharmacy Practice Department, Faculty of Pharmacy, Damanhour University, Damanhour, 22514, Egypt
| | - Mahmoud Salah Abdalla
- Hematology& BMT Department at International Medical Center (IMC) Hospital, Cairo, Egypt
| | - Mohamed Khalaf
- Consultant of Hematology & BMT Department at Maadi Military Hospital, Cairo, Egypt.
| | - Rehab H Werida
- Clinical Pharmacy & Pharmacy Practice Department, Faculty of Pharmacy, Damanhour University, Damanhour, 22514, Egypt.
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Tan Y, Salkhordeh M, Murray ABP, Souza-Moreira L, Stewart DJ, Mei SHJ. Key quality parameter comparison of mesenchymal stem cell product cryopreserved in different cryopreservation solutions for clinical applications. Front Bioeng Biotechnol 2024; 12:1412811. [PMID: 39148941 PMCID: PMC11324487 DOI: 10.3389/fbioe.2024.1412811] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2024] [Accepted: 07/12/2024] [Indexed: 08/17/2024] Open
Abstract
Introduction Cryopreservation is a critical process of cell products for achieving a commercial viability through wide scale adoption. By preserving cells in a lower temperature, cryopreservation enables a product to be off-the-shelf and ready for infusion. An optimized cryopreservation strategy can maintain the viability, phenotype, and potency of thawed mesenchymal stromal/stem cells (MSCs) while being regulatory compliant. We compared three clinical-ready formulations with one research cryopreservation solutions and evaluated key quality parameters of post thawed MSCs. Method and result MSCs were cryopreserved at 3, 6, and 9 million cells/mL (M/mL) in four different cryopreservation solutions: NutriFreez (10% dimethyl sulfoxide [DMSO]), Plasmalyte A (PLA)/5% human albumin (HA)/10% DMSO (PHD10), CryoStor CS5 (5% DMSO), and CryoStor CS10 (10% DMSO). To establish post thaw viability, cells were evaluated with no dilution of DMSO (from 3 M/mL), 1:1 dilution (from 6 M/mL), or 1:2 dilution (from 9 M/mL) with PLA/5% HA, to achieve uniform concentration at 3 M/mL. Cell viability was measured at 0-, 2-, 4-, and 6-h post thaw with Trypan blue exclusion and Annexin V/PI staining. Dilution (1:2) of final cell products from 9M/mL resulted in an improvement of cell viability over 6 h but showed a trend of decreased recovery. MSCs cryopreserved in solutions with 10% DMSO displayed comparable viabilities and recoveries up to 6 h after thawing, whereas a decreasing trend was noted in cell viability and recovery with CS5. Cells from all groups exhibited surface marker characteristics of MSCs. We further evaluated cell proliferation after 6-day recovery in culture. While cells cryopreserved in NutriFreez and PHD10 presented similar cell growth post thaw, MSCs cryopreserved in CS5 and CS10 at 3 M/mL and 6M/mL showed 10-fold less proliferative capacity. No significant differences were observed between MSCs cryopreserved in NutriFreez and PHD10 in their potency to inhibit T cell proliferation and improve monocytic phagocytosis. Conclusion MSCs can be cryopreserved up to 9 M/mL without losing notable viability and recovery, while exhibiting comparable post thaw potency with NutriFreez and PHD10. These results highlight the importance of key parameter testing for selecting the optimal cryopreservation solution for MSC-based therapy.
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Affiliation(s)
- Yuan Tan
- Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada
- Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada
| | - Mahmoud Salkhordeh
- Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Aidan B P Murray
- Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Luciana Souza-Moreira
- Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Duncan J Stewart
- Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada
- Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada
| | - Shirley H J Mei
- Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada
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Valentini CG, Pellegrino C, Teofili L. Pros and Cons of Cryopreserving Allogeneic Stem Cell Products. Cells 2024; 13:552. [PMID: 38534396 PMCID: PMC10968795 DOI: 10.3390/cells13060552] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2024] [Revised: 03/16/2024] [Accepted: 03/18/2024] [Indexed: 03/28/2024] Open
Abstract
The COVID-19 pandemic has precipitously changed the practice of transplanting fresh allografts. The safety measures adopted during the pandemic prompted the near-universal graft cryopreservation. However, the influence of cryopreserving allogeneic grafts on long-term transplant outcomes has emerged only in the most recent literature. In this review, the basic principles of cell cryopreservation are revised and the effects of cryopreservation on the different graft components are carefully reexamined. Finally, a literature revision on studies comparing transplant outcomes in patients receiving cryopreserved and fresh grafts is illustrated.
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Affiliation(s)
- Caterina Giovanna Valentini
- Dipartimento di Diagnostica per Immagini, Radioterapia Oncologica ed Ematologia, Fondazione Policlinico Universitario “A. Gemelli” IRCCS, 00168 Rome, Italy; (C.G.V.); (C.P.)
| | - Claudio Pellegrino
- Dipartimento di Diagnostica per Immagini, Radioterapia Oncologica ed Ematologia, Fondazione Policlinico Universitario “A. Gemelli” IRCCS, 00168 Rome, Italy; (C.G.V.); (C.P.)
- Sezione di Ematologia, Dipartimento di Scienze Radiologiche ed Ematologiche, Università Cattolica del Sacro Cuore, 00168 Rome, Italy
| | - Luciana Teofili
- Dipartimento di Diagnostica per Immagini, Radioterapia Oncologica ed Ematologia, Fondazione Policlinico Universitario “A. Gemelli” IRCCS, 00168 Rome, Italy; (C.G.V.); (C.P.)
- Sezione di Ematologia, Dipartimento di Scienze Radiologiche ed Ematologiche, Università Cattolica del Sacro Cuore, 00168 Rome, Italy
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Madaci L, Gard C, Nin S, Sarrabay A, Baier C, Venton G, Rihet P, Puthier D, Loriod B, Costello R. Single-Cell Transcriptome Analysis of Acute Myeloid Leukemia Cells Using Methanol Fixation and Cryopreservation. Diseases 2023; 12:1. [PMID: 38275564 PMCID: PMC10814800 DOI: 10.3390/diseases12010001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2023] [Revised: 12/08/2023] [Accepted: 12/18/2023] [Indexed: 01/27/2024] Open
Abstract
INTRODUCTION The application of single-cell RNA sequencing has greatly improved our understanding of various cellular and molecular mechanisms involved in physiological and pathophysiological processes. However, obtaining living cells for this technique can be difficult under certain conditions. To solve this problem, the methanol fixation method appeared as a promising alternative for routine clinical use. MATERIALS AND METHODS In this study, we selected two AML samples that had been fixed in methanol for 12-18 months. Once the cells were rehydrated, these samples were subjected to single-cell RNA sequencing. We then compared the results obtained from these samples with those obtained from the same samples cryopreserved in DMSO. RESULTS We used a previously validated methanol fixation protocol to perform scRNA-seq on DMSO cryopreserved cells and cells fixed in methanol for more than one year. Preliminary results show that methanol fixation induces some genetic and transcriptional modification compared with DMSO cryopreservation but remains a valuable method for single-cell analysis of primary human leukemia cells. CONCLUSIONS The initial findings from this study highlight certain resemblances in methanol fixation over a 12-month period and cryopreservation with DMSO, along with associated transcriptional level modifications. However, we observed genetic degradation in the fixation condition when extending beyond one year. Despite certain study limitations, it is evident that short-term methanol fixation can be effectively used for leukemia blast samples. Its ease of implementation holds the potential to simplify the integration of this technique into routine clinical practice.
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Affiliation(s)
- Lamia Madaci
- TAGC, TGML, INSERM, UMR1090, Aix-Marseille University, Parc Scientifique de Luminy, 13009 Marseille, France; (L.M.); (C.G.); (S.N.); (G.V.); (P.R.); (D.P.); (B.L.)
| | - Charlyne Gard
- TAGC, TGML, INSERM, UMR1090, Aix-Marseille University, Parc Scientifique de Luminy, 13009 Marseille, France; (L.M.); (C.G.); (S.N.); (G.V.); (P.R.); (D.P.); (B.L.)
| | - Sébastien Nin
- TAGC, TGML, INSERM, UMR1090, Aix-Marseille University, Parc Scientifique de Luminy, 13009 Marseille, France; (L.M.); (C.G.); (S.N.); (G.V.); (P.R.); (D.P.); (B.L.)
| | - Alexandre Sarrabay
- TAGC, TGML, INSERM, UMR1090, Aix-Marseille University, Parc Scientifique de Luminy, 13009 Marseille, France; (L.M.); (C.G.); (S.N.); (G.V.); (P.R.); (D.P.); (B.L.)
| | - Céline Baier
- Advanced BioDesign, Parc Technologique de Lyon, 655 Allée des Parcs, 69800 Saint Priest, France
| | - Geoffroy Venton
- TAGC, TGML, INSERM, UMR1090, Aix-Marseille University, Parc Scientifique de Luminy, 13009 Marseille, France; (L.M.); (C.G.); (S.N.); (G.V.); (P.R.); (D.P.); (B.L.)
- Hematology and Cellular Therapy Department, Conception University Hospital, 13005 Marseille, France
| | - Pascal Rihet
- TAGC, TGML, INSERM, UMR1090, Aix-Marseille University, Parc Scientifique de Luminy, 13009 Marseille, France; (L.M.); (C.G.); (S.N.); (G.V.); (P.R.); (D.P.); (B.L.)
| | - Denis Puthier
- TAGC, TGML, INSERM, UMR1090, Aix-Marseille University, Parc Scientifique de Luminy, 13009 Marseille, France; (L.M.); (C.G.); (S.N.); (G.V.); (P.R.); (D.P.); (B.L.)
| | - Béatrice Loriod
- TAGC, TGML, INSERM, UMR1090, Aix-Marseille University, Parc Scientifique de Luminy, 13009 Marseille, France; (L.M.); (C.G.); (S.N.); (G.V.); (P.R.); (D.P.); (B.L.)
| | - Régis Costello
- TAGC, TGML, INSERM, UMR1090, Aix-Marseille University, Parc Scientifique de Luminy, 13009 Marseille, France; (L.M.); (C.G.); (S.N.); (G.V.); (P.R.); (D.P.); (B.L.)
- Hematology and Cellular Therapy Department, Conception University Hospital, 13005 Marseille, France
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11
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Gokarn A, Tembhare PR, Syed H, Sanyal I, Kumar R, Parab S, Khanka T, Punatar S, Kedia S, Ghogale SG, Deshpande N, Nikam Y, Girase K, Mirgh S, Jindal N, Bagal B, Chichra A, Nayak L, Bonda A, Rath S, Hiregoudar S, Poojary M, Saha S, Ojha S, Subramanian PG, Khattry N. Long-Term Cryopreservation of Peripheral Blood Stem Cell Harvest Using Low Concentration (4.35%) Dimethyl Sulfoxide with Methyl Cellulose and Uncontrolled Rate Freezing at -80 °C: An Effective Option in Resource-Limited Settings. Transplant Cell Ther 2023; 29:777.e1-777.e8. [PMID: 37678607 DOI: 10.1016/j.jtct.2023.08.032] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2023] [Revised: 08/30/2023] [Accepted: 08/31/2023] [Indexed: 09/09/2023]
Abstract
Long-term cryopreservation of peripheral blood stem cells (PBSCs) is highly useful in the setting of tandem/multiple transplantations or treatment of relapse in the autologous hematopoietic stem cell transplantation (HSCT) setting. Even in allogeneic HSCT, donor lymphocyte infusions may be stored for months to years if excess stem cells are collected from donors. Cryopreservation is a delicate, complex, and costly procedure, and higher concentrations of dimethyl sulfoxide (DMSO), a commonly used cryoprotectant, can be toxic to cells and cause adverse effects in the recipient during infusions. In this study, we examined the effect of long-term cryopreservation using 4.35% DMSO (as final concentration) with methyl cellulose and uncontrolled rate freezing in a mechanical freezer (-80 °C) on the viability and colony-forming ability of CD34+ human PBSCs. For patients undergoing autologous HSCT, PBSCs were cryopreserved using DMSO (final concentration of 4.35%) with methyl cellulose. The post-thaw viability of PBSCs was determined using Trypan blue exclusion and flow cytometry-based 7-amino-actinomycin-D (FC-7AAD) methods. Concentrations of CD34+ stem cells and immune cell subsets in post-thaw PBSC harvest samples were assessed using multicolor flow cytometry, and the clonogenic potential of post-thaw stem cells was studied using a colony-forming unit (CFU) assay. CD34+ stem cell levels were correlated with the prestorage CD34 levels using the Pearson correlation test. The viability results in the Trypan blue dye exclusion method and the flow cytometry-based method were compared using Bland-Altman plots. We studied 26 PBSC harvest samples with a median cryopreservation duration of 6.6 years (range, 3.8 to 11.5 years). The median viability of post-thaw PBSCs was >80% using both methods, with a weak agreement between them (r = .03; P = .5). The median CD34+ stem cell count in the post-thaw samples was 9.13 × 106/kg (range, .44 to 26.27 × 106/kg). The CFU assay yielded a good proliferation and differentiation potential in post-thaw PBSCs, with a weak correlation between granulocyte macrophage CFU and CD34+ stem cell levels (r = .4; P = .05). Two samples that had been cryopreserved for >8 years showed low viability. Cryopreservation of PBSCs using 4.35% DMSO with methyl cellulose and uncontrolled freezing in a mechanical freezer at -80 °C allows the maintenance of long-term viability of PBSC for up to 8 years.
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Affiliation(s)
- Anant Gokarn
- Department of Medical Oncology, Advanced Centre for Treatment, Research, and Education in Cancer, Tata Memorial Center, Navi Mumbai, India; Homi Bhabha National Institute, Mumbai, India
| | - Prashant R Tembhare
- Homi Bhabha National Institute, Mumbai, India; Hematopathology Laboratory, Advanced Centre for Treatment, Research, and Education in Cancer, Tata Memorial Center, Navi Mumbai, India
| | - Hasan Syed
- Homi Bhabha National Institute, Mumbai, India; Hasan Laboratory, Advanced Centre for Treatment, Research, and Education in Cancer, Tata Memorial Center, Navi Mumbai, India
| | - Isha Sanyal
- Hematopathology Laboratory, Advanced Centre for Treatment, Research, and Education in Cancer, Tata Memorial Center, Navi Mumbai, India
| | - Rohit Kumar
- Hasan Laboratory, Advanced Centre for Treatment, Research, and Education in Cancer, Tata Memorial Center, Navi Mumbai, India
| | - Sarika Parab
- Department of Transfusion Medicine, Advanced Centre for Treatment, Research, and Education in Cancer, Tata Memorial Center, Navi Mumbai, India
| | - Twinkle Khanka
- Hematopathology Laboratory, Advanced Centre for Treatment, Research, and Education in Cancer, Tata Memorial Center, Navi Mumbai, India
| | - Sachin Punatar
- Department of Medical Oncology, Advanced Centre for Treatment, Research, and Education in Cancer, Tata Memorial Center, Navi Mumbai, India; Homi Bhabha National Institute, Mumbai, India
| | - Shweta Kedia
- Hematopathology Laboratory, Advanced Centre for Treatment, Research, and Education in Cancer, Tata Memorial Center, Navi Mumbai, India
| | - Sitaram G Ghogale
- Hematopathology Laboratory, Advanced Centre for Treatment, Research, and Education in Cancer, Tata Memorial Center, Navi Mumbai, India
| | - Nilesh Deshpande
- Hematopathology Laboratory, Advanced Centre for Treatment, Research, and Education in Cancer, Tata Memorial Center, Navi Mumbai, India
| | - Yuvraj Nikam
- Hasan Laboratory, Advanced Centre for Treatment, Research, and Education in Cancer, Tata Memorial Center, Navi Mumbai, India
| | - Karishma Girase
- Hematopathology Laboratory, Advanced Centre for Treatment, Research, and Education in Cancer, Tata Memorial Center, Navi Mumbai, India
| | - Sumeet Mirgh
- Department of Medical Oncology, Advanced Centre for Treatment, Research, and Education in Cancer, Tata Memorial Center, Navi Mumbai, India; Homi Bhabha National Institute, Mumbai, India
| | - Nishant Jindal
- Department of Medical Oncology, Advanced Centre for Treatment, Research, and Education in Cancer, Tata Memorial Center, Navi Mumbai, India; Homi Bhabha National Institute, Mumbai, India
| | - Bhausaheb Bagal
- Department of Medical Oncology, Advanced Centre for Treatment, Research, and Education in Cancer, Tata Memorial Center, Navi Mumbai, India; Homi Bhabha National Institute, Mumbai, India
| | - Akanksha Chichra
- Department of Medical Oncology, Advanced Centre for Treatment, Research, and Education in Cancer, Tata Memorial Center, Navi Mumbai, India; Homi Bhabha National Institute, Mumbai, India
| | - Lingaraj Nayak
- Department of Medical Oncology, Advanced Centre for Treatment, Research, and Education in Cancer, Tata Memorial Center, Navi Mumbai, India; Homi Bhabha National Institute, Mumbai, India
| | - Avinash Bonda
- Department of Medical Oncology, Advanced Centre for Treatment, Research, and Education in Cancer, Tata Memorial Center, Navi Mumbai, India; Homi Bhabha National Institute, Mumbai, India
| | - Sushmita Rath
- Department of Medical Oncology, Advanced Centre for Treatment, Research, and Education in Cancer, Tata Memorial Center, Navi Mumbai, India; Homi Bhabha National Institute, Mumbai, India
| | - Sumathi Hiregoudar
- Homi Bhabha National Institute, Mumbai, India; Department of Transfusion Medicine, Advanced Centre for Treatment, Research, and Education in Cancer, Tata Memorial Center, Navi Mumbai, India
| | - Minal Poojary
- Homi Bhabha National Institute, Mumbai, India; Department of Transfusion Medicine, Advanced Centre for Treatment, Research, and Education in Cancer, Tata Memorial Center, Navi Mumbai, India
| | - Suryatapa Saha
- Homi Bhabha National Institute, Mumbai, India; Department of Transfusion Medicine, Advanced Centre for Treatment, Research, and Education in Cancer, Tata Memorial Center, Navi Mumbai, India
| | - Shashank Ojha
- Homi Bhabha National Institute, Mumbai, India; Department of Transfusion Medicine, Advanced Centre for Treatment, Research, and Education in Cancer, Tata Memorial Center, Navi Mumbai, India
| | - Papagudi G Subramanian
- Homi Bhabha National Institute, Mumbai, India; Hematopathology Laboratory, Advanced Centre for Treatment, Research, and Education in Cancer, Tata Memorial Center, Navi Mumbai, India
| | - Navin Khattry
- Department of Medical Oncology, Advanced Centre for Treatment, Research, and Education in Cancer, Tata Memorial Center, Navi Mumbai, India; Homi Bhabha National Institute, Mumbai, India
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12
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Jia Rong TL, Basker G, Yong Hoe C, Hein T, Poon LMM, Yeow Tee G. Impact of the COVID-19 pandemic on hematopoietic stem cell transplant programmes in Singapore. BLOOD CELL THERAPY 2023; 6:139-144. [PMID: 38149024 PMCID: PMC10749730 DOI: 10.31547/bct-2023-019] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/07/2023] [Accepted: 08/29/2023] [Indexed: 12/28/2023]
Abstract
Background Hematopoietic stem cell transplantation (HSCT) has been performed in Singapore since 1985. Currently, more than 100 transplants are performed annually across the public and private sectors. In 2020, the COVID-19 pandemic resulted in unprecedented disruptions to global healthcare systems, and Singapore was no exception. In particular, the field of HSCT faced additional, unique challenges aside from those borne by the healthcare system at large, and appropriate measures were necessary to ensure that HSCT remained available to patients who needed it. Methods The expert opinions of six hematologists from various institutions across Singapore were gathered through individual interviews and summarized. This was supplemented by a literature review on bone marrow donation and HSCT in Singapore. Main Findings and Conclusion In Singapore, the COVID-19 pandemic has had significant implications for HSCT, ranging from the implementation of additional infection control measures in hospitals to an accelerated rise in haploidentical transplants. Further research is required to better understand and quantify these impacts, improve existing processes, and investigate the effects of COVID-19 and its treatment modalities on patients with HSCT.
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Affiliation(s)
- Timothy Lam Jia Rong
- Asian Medical Student Association Singapore SGMarrow Research Committee, Singapore
- Yong Loo Lin School of Medicine, National University of Singapore, Singapore
| | - Gayathri Basker
- Asian Medical Student Association Singapore SGMarrow Research Committee, Singapore
- Yong Loo Lin School of Medicine, National University of Singapore, Singapore
| | - Chan Yong Hoe
- Asian Medical Student Association Singapore SGMarrow Research Committee, Singapore
- Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore
| | - Than Hein
- Department of Haematology, Singapore General Hospital, Singapore
| | - Li Mei Michelle Poon
- Department of Haematology-Oncology, National University Hospital Singapore, Singapore
| | - Goh Yeow Tee
- Department of Haematology, Singapore General Hospital, Singapore
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13
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Joshi P, Rabin Y. Thermomechanical stress analyses of nanowarming-assisted recovery from cryopreservation by vitrification in human heart and rat heart models. PLoS One 2023; 18:e0290063. [PMID: 37585446 PMCID: PMC10431620 DOI: 10.1371/journal.pone.0290063] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2023] [Accepted: 08/01/2023] [Indexed: 08/18/2023] Open
Abstract
This study investigates thermomechanical stress in cryopreservation by vitrification of the heart, while exploring the effects of nanowarming-assisted recovery from cryogenic storage. This study expands upon a recently published study, combining experimental investigation and thermal analysis of cryopreservation on a rat heart model. Specifically, this study focuses on scenarios with variable concentrations of silica-coated iron-oxide nanoparticles (sIONPs), while accounting for loading limitations associated with the heart physiology, as well as the properties of cryoprotective agent (CPA) solution and the geometry of the container. Results of this study suggest that variable sIONP concentration based on the heart physiology will elevate mechanical stresses when compared with the mathematically simplified, uniform distribution case. The most dangerous part of rewarming is below glass transition and at the onset of nanowarming past the glass transition temperature on the way for organ recovery from cryogenic storage. Throughout rewarming, regions that rewarm faster, such as the chambers of the heart (higher sIONP concentration), undergo compressive stresses, while the slower rewarming regions, such as the heart myocardium (low sIONP concentration), undergo tension. Being a brittle material, the vitrified organ is expected to fail under tension in lower stresses than in compression. Unfortunately, the location and magnitude of the maximum stress in the investigated cases varied, while general rules were not identified. This investigation demonstrates the need to tailor the thermal protocol of heart cryopreservation on a case-by-case basis, since the location, orientation, magnitude, and instant at which the maximum mechanical stress is found cannot be predicted a priori. While thermomechanical stress poses a significant risk to organ integrity, careful design of the thermal protocol can be instrumental in reducing the likelihood of structural damage, while taking full advantage of the benefits of nanowarming.
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Affiliation(s)
- Purva Joshi
- Department of Mechanical Engineering, Biothermal Technology Laboratory, Carnegie Mellon University, Pittsburgh, PA, United States of America
| | - Yoed Rabin
- Department of Mechanical Engineering, Biothermal Technology Laboratory, Carnegie Mellon University, Pittsburgh, PA, United States of America
- Department of Mechanical Engineering, Forbes Avenue, Pittsburgh, PA, United States of America
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14
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Parihar A, Kumar A, Panda U, Khan R, Parihar DS, Khan R. Cryopreservation: A Comprehensive Overview, Challenges, and Future Perspectives. Adv Biol (Weinh) 2023; 7:e2200285. [PMID: 36755194 DOI: 10.1002/adbi.202200285] [Citation(s) in RCA: 19] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2022] [Revised: 01/05/2023] [Indexed: 02/10/2023]
Abstract
Cryopreservation is the most prevalent method of long-term cell preservation. Effective cell cryopreservation depends on freezing, adequate storage, and correct thawing techniques. Recent advances in cryopreservation techniques minimize the cellular damage which occurs while processing samples. This article focuses on the fundamentals of cryopreservation techniques and how they can be implemented in a variety of clinical settings. The article presents a brief description of each of the standard cryopreservation procedures, such as slow freezing and vitrification. Alongside that, the membrane permeating and nonpermeating cryoprotectants are briefly discussed, along with current advancements in the field of cryopreservation and variables influencing the cryopreservation process. The diminution of cryoinjury incurred by the cell via the resuscitation process will also be highlighted. In the end application of cryopreservation techniques in many fields, with a special emphasis on stem cell preservation techniques and current advancements presented. Furthermore, the challenges while implementing cryopreservation and the futuristic scope of the fields are illustrated herein. The content of this review sheds light on various ways to enhance the output of the cell preservation process and minimize cryoinjury while improving cell revival.
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Affiliation(s)
- Arpana Parihar
- Industrial Waste Utilization, Nano and Biomaterials, CSIR-Advanced Materials and Processes Research Institute (AMPRI), Hoshangabad Road, Bhopal, Madhya Pradesh, 462026, India
| | - Avinash Kumar
- Department of Mechanical Engineering, Indian Institute of Information Technology, Design & Manufacturing (IIITD&M), Kancheepuram, 600127, India
| | - Udwesh Panda
- Department of Mechanical Engineering, Indian Institute of Information Technology, Design & Manufacturing (IIITD&M), Kancheepuram, 600127, India
| | - Rukhsar Khan
- Department of Biosciences, Barkatullah University, Bhopal, Madhya Pradesh, 462026, India
| | | | - Raju Khan
- Industrial Waste Utilization, Nano and Biomaterials, CSIR-Advanced Materials and Processes Research Institute (AMPRI), Hoshangabad Road, Bhopal, Madhya Pradesh, 462026, India
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15
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Meric N, Parmaksız A, Gulbas Z. Patients experienced serious adverse reactions within one hour of hematopoietic stem-cell infusion. Transfus Clin Biol 2023; 30:82-86. [PMID: 35987476 DOI: 10.1016/j.tracli.2022.08.141] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2022] [Revised: 08/11/2022] [Accepted: 08/16/2022] [Indexed: 02/07/2023]
Abstract
METHODS SARs were examined occurred within 1 hour after initiating HSC product infusions in all HSCT done in Turkey's Anadolu Medical Center Hospital accredited for HSCTs between 2013 and 2015, targeting 315 patients. RESULTS SARs were carefully evaluated in this study based on a comparison of the amount of stem cells infused, age, frozen sample (FS) / non-frozen samples (NFS) between HSCs sources. Rate of SARs is significantly higher in FS infusions supports the hypothesis that DMSO plays an important role in the development of SAR. CONCLUSION The rate of SARs is significantly higher in infusions given using FSs confirms the hypothesis that the preservative agent DMSO plays an important role in the development of SAR. Our study provides guidance for future studies on the necessity of reducing the amount of DMSO in the HSCT product and using other alternative freezing agents instead of DMSO.
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Affiliation(s)
- Neslihan Meric
- Faculty of Engineering and Natural Sciences, Department of Molecular Biology and Genetics, Kütahya University of Health Sciences, Kütahya, Turkey.
| | - Ayhan Parmaksız
- Faculty of Medicine, Istanbul Health and Technology University, Istanbul, Turkey
| | - Zafer Gulbas
- Anadolu Health Center, Bone Marrow Transplantation Center, Kocaeli, Turkey
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16
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Kuang G, Zhang Q, Jia J, Yu Y. Freezing biological organisms for biomedical applications. SMART MEDICINE 2022; 1:e20220034. [PMID: 39188743 PMCID: PMC11235656 DOI: 10.1002/smmd.20220034] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 11/29/2022] [Accepted: 12/07/2022] [Indexed: 08/28/2024]
Abstract
Biological organisms play important roles in human health, either in a commensal or pathogenic manner. Harnessing inactivated organisms or living organisms is a promising way to treat diseases. As two types of freezing, cryoablation makes it simple to inactivate organisms that must be in a non-pathogenic state when needed, while cryopreservation is a facile way to address the problem of long-term storage challenged by living organism-based therapy. In this review, we present the latest studies of freezing biological organisms for biomedical applications. To begin with, the freezing strategies of cryoablation and cryopreservation, as well as their corresponding technical essentials, are illustrated. Besides, biomedical applications of freezing biological organisms are presented, including transplantation, tissue regeneration, anti-infection therapy, and anti-tumor therapy. The challenges and prospects of freezing living organisms for biomedical applications are well discussed. We believe that the freezing method will provide a potential direction for the standardization and commercialization of inactivated or living organism-based therapeutic systems, and promote the clinical application of organism-based therapy.
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Affiliation(s)
- Gaizhen Kuang
- Pharmaceutical Sciences LaboratoryÅbo Akademi UniversityTurkuFinland
- Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health)Wenzhou InstituteUniversity of Chinese Academy of SciencesWenzhouChina
| | - Qingfei Zhang
- Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health)Wenzhou InstituteUniversity of Chinese Academy of SciencesWenzhouChina
| | - Jinxuan Jia
- Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health)Wenzhou InstituteUniversity of Chinese Academy of SciencesWenzhouChina
| | - Yunru Yu
- Pharmaceutical Sciences LaboratoryÅbo Akademi UniversityTurkuFinland
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17
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Yoshikawa K. Quantitative evaluation of DNA double-strand breaks (DSBs) through single-molecule observation. Enzymes 2022; 51:7-27. [PMID: 36336410 DOI: 10.1016/bs.enz.2022.08.002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/16/2023]
Abstract
By adapting the method of single molecular observation for individual DNAs, it will be shown that reliable analysis of double-strand breaks, DSBs, becomes possible for various kinds of damage sources. Single DNA above the size of several-tens kilo base-pairs exhibits the length scale above several μm, indicating that their whole conformation is visible with fluorescence microscopy by adding suitable fluoresce dye to the solution. Various examples of the quantitative evaluation on DSBs are described, together with the evaluation of the protective effects of anti-oxidants.
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Affiliation(s)
- Kenichi Yoshikawa
- Faculty of Life and Medical Sciences, Doshisha University, Kyoto, Japan; Center for Integrative Medicine and Physics, Institute for Advanced Study, Kyoto University, Kyoto, Japan.
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18
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Li M, Morse B, Kassim S. Development and clinical translation considerations for the next wave of gene modified hematopoietic stem and progenitor cells therapies. Expert Opin Biol Ther 2022; 22:1177-1191. [PMID: 35833356 DOI: 10.1080/14712598.2022.2101361] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/04/2022]
Abstract
INTRODUCTION Consistent and reliable manufacture of gene modified hematopoietic stem and progenitor cell (HPSC) therapies will be of the utmost importance as they become more mainstream and address larger populations. Robust development campaigns will be needed to ensure that these products will be delivered to patients with the highest quality standards. AREAS COVERED Through publicly available manuscripts, press releases, and news articles - this review touches on aspects related to HSPC therapy, development, and manufacturing. EXPERT OPINION Recent advances in genome modification technology coupled with the longstanding clinical success of HSPCs warrants great optimism for the next generation of engineered HSPC-based therapies. Treatments for some diseases that have thus far been intractable now appear within reach. Reproducible manufacturing will be of critical importance in delivering these therapies but will be challenging due to the need for bespoke materials and methods in combination with the lack of off-the-shelf solutions. Continued progress in the field will manifest in the form of industrialization which currently requires attention and resources directed toward the custom reagents, a focus on closed and automated processes, and safer and more precise genome modification technologies that will enable broader, faster, and safer access to these life-changing therapies.
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Affiliation(s)
| | - Brent Morse
- Dark Horse Consulting Group, Walnut Creek, CA, USA
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19
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Franke GN, Pfannes R, Heyn S, Brückner M, Rieprecht S, Bach E, Remane Y, Leiblein S, Pönisch W, Niederwieser D, Schwind S, Platzbecker U, Jentzsch M, Vucinic V. Analysis of stem cell collections in adult patients with Ewing sarcoma. Transfusion 2022; 62:1612-1618. [PMID: 35801531 DOI: 10.1111/trf.17013] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2022] [Revised: 05/14/2022] [Accepted: 05/30/2022] [Indexed: 10/17/2022]
Abstract
BACKGROUND Ewing sarcoma is one of the most frequent soft-tissue tumors in pediatric patients. The current treatment protocols recommend stem cell apheresis (SCA) after completion of the second course of induction therapy with vincristine, ifosfamide, doxorubicine, and etoposide (VIDE). The feasibility of SCA and graft compositions in adult patients with Ewing sarcoma have not been previously analyzed. METHODS AND MATERIALS The authors analyzed 29 stem cell collections of 19 adult patients (9 male, 10 female) at a median age of 27 (range 19-53) years mobilized after VIDE (n = 17), cyclophosphamide/topotecan (n = 1) or vincristine, dactinomycin and ifosfamide (n = 1) chemotherapy. All patients were mobilized with filgrastim 5 μg/kg twice daily from day +7 of chemotherapy. The collections were performed if CD34+ cell count in peripheral blood was >10/μL. The target yields were ≥4×106 CD34+ cells/kg body weight. RESULTS Median CD34+ cells/μL in peripheral blood before SCA were 45.8 (range 6.7-614.4)/μL. The median cumulative yields were 10.6 (range 1.5-38.8) CD34+ cells/kg body weight and ≥2×106 in all but two patients (89%). CD34, CD3, and CD56 yields in collections after the third VIDE and after later courses did not differ. Four patients underwent high-dose therapy with autologous transplantation, and all were engrafted. DISCUSSION Stem cell mobilization is feasible in most Ewing sarcoma patients. Additionally, the present study's data suggest that it is safe to postpone stem cell collection to a later VIDE chemotherapy cycle if medically indicated.
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Affiliation(s)
- Georg-Nikolaus Franke
- Medical Clinic for Hematology, Celltherapy and Hemostaseology, Leipzig University Medical Center, Leipzig, Germany
| | - Roald Pfannes
- Medical Clinic for Hematology, Celltherapy and Hemostaseology, Leipzig University Medical Center, Leipzig, Germany
| | - Simone Heyn
- Medical Clinic for Hematology, Celltherapy and Hemostaseology, Leipzig University Medical Center, Leipzig, Germany
| | - Mandy Brückner
- Medical Clinic for Hematology, Celltherapy and Hemostaseology, Leipzig University Medical Center, Leipzig, Germany
| | | | - Enrica Bach
- Medical Clinic for Hematology, Celltherapy and Hemostaseology, Leipzig University Medical Center, Leipzig, Germany
| | - Yvonne Remane
- Pharmacy, Leipzig University Medical Center, Leipzig, Germany
| | - Sabine Leiblein
- Medical Clinic for Hematology, Celltherapy and Hemostaseology, Leipzig University Medical Center, Leipzig, Germany
| | - Wolfram Pönisch
- Medical Clinic for Hematology, Celltherapy and Hemostaseology, Leipzig University Medical Center, Leipzig, Germany
| | - Dietger Niederwieser
- Medical Clinic for Hematology, Celltherapy and Hemostaseology, Leipzig University Medical Center, Leipzig, Germany
| | - Sebastian Schwind
- Medical Clinic for Hematology, Celltherapy and Hemostaseology, Leipzig University Medical Center, Leipzig, Germany
| | - Uwe Platzbecker
- Medical Clinic for Hematology, Celltherapy and Hemostaseology, Leipzig University Medical Center, Leipzig, Germany
| | - Madlen Jentzsch
- Medical Clinic for Hematology, Celltherapy and Hemostaseology, Leipzig University Medical Center, Leipzig, Germany
| | - Vladan Vucinic
- Medical Clinic for Hematology, Celltherapy and Hemostaseology, Leipzig University Medical Center, Leipzig, Germany
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20
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Impact of Cryopreservation and Freeze-Thawing on Therapeutic Properties of Mesenchymal Stromal/Stem Cells and Other Common Cellular Therapeutics. CURRENT STEM CELL REPORTS 2022; 8:72-92. [PMID: 35502223 PMCID: PMC9045030 DOI: 10.1007/s40778-022-00212-1] [Citation(s) in RCA: 71] [Impact Index Per Article: 23.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/28/2022] [Indexed: 12/19/2022]
Abstract
Purpose of Review Cryopreservation and its associated freezing and thawing procedures–short “freeze-thawing”–are among the final steps in economically viable manufacturing and clinical application of diverse cellular therapeutics. Translation from preclinical proof-of-concept studies to larger clinical trials has indicated that these processes may potentially present an Achilles heel to optimal cell product safety and particularly efficacy in clinical trials and routine use. Recent Findings We review the current state of the literature on how cryopreservation of cellular therapies has evolved and how the application of this technique to different cell types is interlinked with their ability to engraft and function upon transfer in vivo, in particular for hematopoietic stem and progenitor cells (HSPCs), their progeny, and therapeutic cell products derived thereof. We also discuss pros and cons how this may differ for non-hematopoietic mesenchymal stromal/stem cell (MSC) therapeutics. We present different avenues that may be crucial for cell therapy optimization, both, for hematopoietic (e.g., effector, regulatory, and chimeric antigen receptor (CAR)-modified T and NK cell based products) and for non-hematopoietic products, such as MSCs and induced pluripotent stem cells (iPSCs), to achieve optimal viability, recovery, effective cell dose, and functionality of the cryorecovered cells. Summary Targeted research into optimizing the cryopreservation and freeze-thawing routines and the adjunct manufacturing process design may provide crucial advantages to increase both the safety and efficacy of cellular therapeutics in clinical use and to enable effective market deployment strategies to become economically viable and sustainable medicines.
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21
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Pilbauerova N, Schmidt J, Soukup T, Prat T, Nesporova K, Velebny V, Suchanek J. Innovative Approach in the Cryogenic Freezing Medium for Mesenchymal Stem Cells. Biomolecules 2022; 12:610. [PMID: 35625538 PMCID: PMC9138570 DOI: 10.3390/biom12050610] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2022] [Accepted: 04/14/2022] [Indexed: 02/07/2023] Open
Abstract
The physical stresses during cryopreservation affect stem cell survival and further proliferation. To minimize or prevent cryoinjury, cryoprotective agents (CPAs) are indispensable. Despite the widespread use of 10% dimethyl sulfoxide (DMSO), there are concerns about its potential adverse effects. To bypass those effects, combinations of CPAs have been investigated. This study aimed to verify whether high-molecular-hyaluronic acid (HMW-HA) serves as a cryoprotectant when preserving human mesenchymal stem cells (hMSCs) to reduce the DMSO concentration in the cryopreservation medium. We studied how 0.1% or 0.2% HMW-HA combined with reduced DMSO concentrations (from 10% to 5%, and 3%) affected total cell count, viability, immunophenotype, and differentiation potential post-cryopreservation. Immediately after cell revival, the highest total cell count was observed in 10% DMSO-stored hMSC. However, two weeks after cell cultivation an increased cell count was seen in the HMW-HA-stored groups along with a continued increase in hMSCs stored using 3% DMSO and 0.1% HMW-HA. The increased total cell count corresponded to elevated expression of stemness marker CD49f. The HA-supplemented cryomedium did not affect the differential potential of hMSC. Our results will participate in producing a ready-to-use product for cryopreservation of mesenchymal stem cells.
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Affiliation(s)
- Nela Pilbauerova
- Department of Dentistry, Charles University, Faculty of Medicine in Hradec Kralove and University Hospital Hradec Kralove, Sokolska 581, 50005 Hradec Kralove, Czech Republic; (N.P.); (J.S.); (J.S.)
| | - Jan Schmidt
- Department of Dentistry, Charles University, Faculty of Medicine in Hradec Kralove and University Hospital Hradec Kralove, Sokolska 581, 50005 Hradec Kralove, Czech Republic; (N.P.); (J.S.); (J.S.)
| | - Tomas Soukup
- Department of Histology and Embryology, Faculty of Medicine in Hradec Kralove, Charles University, Simkova 870, 50003 Hradec Kralove, Czech Republic;
| | - Tomas Prat
- Contipro a.s., Dolni Dobrouc 401, 56102 Dolni Dobrouc, Czech Republic; (K.N.); (V.V.)
| | - Kristina Nesporova
- Contipro a.s., Dolni Dobrouc 401, 56102 Dolni Dobrouc, Czech Republic; (K.N.); (V.V.)
| | - Vladimir Velebny
- Contipro a.s., Dolni Dobrouc 401, 56102 Dolni Dobrouc, Czech Republic; (K.N.); (V.V.)
| | - Jakub Suchanek
- Department of Dentistry, Charles University, Faculty of Medicine in Hradec Kralove and University Hospital Hradec Kralove, Sokolska 581, 50005 Hradec Kralove, Czech Republic; (N.P.); (J.S.); (J.S.)
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22
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Reich‐Slotky R, Vasovic LV, Land KJ, Halpenny M, Woeltz J, Mathew AJ, Fournier D, Alder B, Stasko K, Mahmud N. Cryopreserved hematopoietic stem/progenitor cells stability program‐development, current status and recommendations: A brief report from the AABB‐ISCT joint working group cellular therapy product stability project team. Transfusion 2022; 62:651-662. [DOI: 10.1111/trf.16820] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2021] [Revised: 01/24/2022] [Accepted: 01/25/2022] [Indexed: 11/28/2022]
Affiliation(s)
- Ronit Reich‐Slotky
- John Theurer Cancer Center Hackensack University Medical Center Hackensack New Jersey USA
| | | | - Kevin J. Land
- Vitalant Biotherapies Phoenix Arizona USA
- University of Texas Health Science Center San Antonio, Department of Pathology Transfusion Medicine San Antonio Texas USA
| | | | - Joan Woeltz
- Blood & Marrow Transplantation and Cellular Therapy Stanford Health Care Stanford California USA
| | | | | | - Brenda Alder
- Northside Hospital, Blood and Marrow Transplant Program, Cell Therapy Lab Atlanta Georgia USA
| | - Karl Stasko
- Dana‐Farber Cancer Institute Cell Manipulation Core Facility Boston Massachusetts USA
| | - Nadim Mahmud
- Division of Hematology/Oncology, Department of Medicine University of Illinois College of Medicine Chicago Illinois USA
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23
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Makhani SS, Oza SP, Reich-Slotky R, Munshi PN, Biran N, Donato ML, Siegel DS, Vesole DH, Naam S, Rowley SD. Sustained Hematopoietic Engraftment Potential after Prolonged Storage of Cryopreserved Hematopoietic Stem Cells (HSC) Used in Salvage Autologous Stem Cell Transplantation: Prolonged Storage of HSCs Sustains Hematological Reconstitution Potential. Transplant Cell Ther 2022; 28:306.e1-306.e7. [PMID: 35248777 DOI: 10.1016/j.jtct.2022.02.023] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2022] [Revised: 02/17/2022] [Accepted: 02/27/2022] [Indexed: 10/18/2022]
Abstract
BACKGROUND Salvage autologous hematopoietic stem cell transplantation (HSCT) is an effective treatment for patients with relapsed multiple myeloma (MM). Peripheral blood stem cells (PBSCs), a source of hematopoietic stem cells (HSCs), are collected prior to first transplant and adequate quantities of PBSCs can be collected and stored potentially for years to support at least two transplants for eligible patients. To ensure the safety of salvage HSCT used in the treatment of patients in subsequent relapse, PBSCs must retain the potential to engraft even after several years of cryopreservation. Although PBSC viability has been extensively studied using in vitro techniques, few publications describe the most rigorous functional potency measure: of patients receiving a myeloablative conditioning regimen. This study describes a large single-institution experience evaluating the engraftment kinetics of PBSCs used in salvage transplantation after multiple years of storage in comparison to first transplantation of the same patients in the treatment of MM. STUDY DESIGN AND METHODS A retrospective chart review of patients with MM undergoing HSCT from 2000 to 2021, identified 89 patients who received salvage autologous PBSC stored > 1 year after first HSCT. PBSC were cryopreserved and stored in vapor-phase liquid nitrogen refrigerators at a temperature of ≤ -150°C. All patients received a PBSC product for both transplants from the same collection cycle. Differences in CD34+ cell doses and days to engraftment between the first and salvage transplant were tested using a paired 2-tailed t-test and Wilcoxon signed-rank test. Univariate and multivariable linear regressions were used to determine association between storage time and days to engraftment, adjusting for CD34+ cell dose and conditioning regimen in the multivariable model. RESULTS The median storage time between day of initial collection and salvage transplant was 5.4 years (range, 1.0 - 19.7). Engraftment kinetics demonstrated a sustained neutrophil engraftment (absolute neutrophil count (ANC) > 0.5 × 109 cells/L) at a median of 11 days after both the first and salvage transplant (ranges, 8 - 15 and 8 - 19 respectively, p<0.05). The median time to sustained platelet engraftment (> 20 × 109 cells/L without transfusion support) was 13.5 days after first HSCT and 14 days after salvage HSCT (ranges, 9 - 27 and 10 - 56 respectively, p = 0.616). After adjusting for CD34+ cell doses and conditioning regimens, there was no association between the duration of cryopreservation and days to neutrophil (r = 0.178, p = 0.130) or platelet (r = 0.244, p = 0.100) engraftments. CONCLUSION Engraftment kinetics of the salvage HSCT are comparable to the first HSCT even when products are stored in vapor-phase nitrogen refrigerators for a median time of 5.4 years. There is no association between storage duration and time to engraftment when controlling for CD34+ dose and conditioning regimens. Prolonged storage of cryopreserved HSC products is a safe practice for MM patients undergoing salvage autologous HSCT.
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Affiliation(s)
- Sarah S Makhani
- Herbert Wertheim College of Medicine, Florida International University, Miami, FL
| | - Samir P Oza
- John Theurer Cancer Center, Hackensack University Medical Center, Hackensack, NJ
| | - Ronit Reich-Slotky
- John Theurer Cancer Center, Hackensack University Medical Center, Hackensack, NJ.
| | - Pashna N Munshi
- Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC
| | - Noa Biran
- John Theurer Cancer Center, Hackensack University Medical Center, Hackensack, NJ
| | - Michele L Donato
- John Theurer Cancer Center, Hackensack University Medical Center, Hackensack, NJ
| | - David S Siegel
- John Theurer Cancer Center, Hackensack University Medical Center, Hackensack, NJ
| | - David H Vesole
- John Theurer Cancer Center, Hackensack University Medical Center, Hackensack, NJ
| | - Suzan Naam
- John Theurer Cancer Center, Hackensack University Medical Center, Hackensack, NJ
| | - Scott D Rowley
- John Theurer Cancer Center, Hackensack University Medical Center, Hackensack, NJ; Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC
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24
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Joshi P, Ehrlich LE, Gao Z, Bischof JC, Rabin Y. Thermal Analyses of Nanowarming-Assisted Recovery of the Heart From Cryopreservation by Vitrification. JOURNAL OF HEAT TRANSFER 2022; 144:031202. [PMID: 35833152 PMCID: PMC8823202 DOI: 10.1115/1.4053105] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/15/2021] [Revised: 11/19/2021] [Indexed: 05/09/2023]
Abstract
This study explores thermal design aspects of nanowarming-assisted recovery of the heart from indefinite cryogenic storage, where nanowarming is the volumetric heating effect of ferromagnetic nanoparticles excited by a radio frequency electromagnet field. This study uses computational means while focusing on the human heart and the rat heart models. The underlying nanoparticle loading characteristics are adapted from a recent, proof-of-concept experimental study. While uniformly distributed nanoparticles can lead to uniform rewarming, and thereby minimize adverse effects associated with ice crystallization and thermomechanical stress, the combined effects of heart anatomy and nanoparticle loading limitations present practical challenges which this study comes to address. Results of this study demonstrate that under such combined effects, nonuniform nanoparticles warming may lead to a subcritical rewarming rate in some parts of the domain, excessive heating in others, and increased exposure potential to cryoprotective agents (CPAs) toxicity. Nonetheless, the results of this study also demonstrate that computerized planning of the cryopreservation protocol and container design can help mitigate the associated adverse effects, with examples relating to adjusting the CPA and/or nanoparticle concentration, and selecting heart container geometry, and size. In conclusion, nanowarming may provide superior conditions for organ recovery from cryogenic storage under carefully selected conditions, which comes with an elevated complexity of protocol planning and optimization.
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Affiliation(s)
- Purva Joshi
- Biothermal Technology Laboratory, Department of Mechanical Engineering, Carnegie Mellon University, Pittsburgh, PA 15237
| | - Lili E. Ehrlich
- Biothermal Technology Laboratory, Department of Mechanical Engineering, Carnegie Mellon University, Pittsburgh, PA 15237
| | - Zhe Gao
- Department of Mechanical Engineering, University of Minnesota, Minneapolis, MN 55455
| | - John C. Bischof
- Department of Mechanical Engineering, University of Minnesota, Minneapolis, MN 55455
| | - Yoed Rabin
- Biothermal Technology Laboratory, Department of Mechanical Engineering, Carnegie Mellon University, 5000 Forbes Avenue, Pittsburgh, PA 15213
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25
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Reich-Slotky R, Vasovic LV, Land KJ, Halpenny M, Woeltz J, Mathew AJ, Fournier D, Alder B, Stasko K, Mahmud N. Cryopreserved hematopoietic stem/progenitor cells stability program-development, current status and recommendations: A brief report from the AABB-ISCT joint working group cellular therapy product stability project team. Cytotherapy 2022; 24:473-481. [DOI: 10.1016/j.jcyt.2022.03.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022]
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26
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Joshi P, Rabin Y. Analysis of crystallization during rewarming in suboptimal vitrification conditions: a semi-empirical approach. Cryobiology 2021; 103:70-80. [PMID: 34543621 PMCID: PMC9248894 DOI: 10.1016/j.cryobiol.2021.09.007] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2021] [Revised: 09/13/2021] [Accepted: 09/14/2021] [Indexed: 10/30/2022]
Abstract
Circumventing ice formation is critical to successful cryopreservation by vitrification of large organs. While ice formation during the cooling part of the cryogenic protocol is dictated by the evolving thermal conditions, ice formation during the rewarming part of the cryogenic protocol is also dependent on the history of cooling and storage conditions. Furthermore, while the exothermic effect of ice crystallization during cooling tends to adversely slow down the desired high cooling rates to ensure ice-free preservation, the same effect under some conditions tends to assist acceleration of rewarming during recovery of the specimen from cryogenic storage when limited crystallization does occur. The current study proposes a computational framework to study the thermal effects of crystallization during recovery from cryogenic storage, using a semi-empirical approach to account for the relationship between latent heat effects and the rewarming rate. This study adds another layer of computational capabilities to a recent study investigating similar effects during cooling. Results of this study demonstrate that the thermal effects of crystallization on the local cooling and rewarming rates cannot be neglected. It further explains how crystallization during rewarming helps in increasing the rewarming rate and, thereby, affects rewarming-phase crystallization. Counterintuitively, this study suggests that the fastest possible rewarming rate at the outer surface of the domain in an inwards rewarming problem is not always advantageous, while the proposed computational tool is essential to find an intermediate optimal rate.
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Affiliation(s)
- Purva Joshi
- Department of Mechanical Engineering Carnegie Mellon University, 5000 Forbes Avenue, Pittsburgh, PA, 15213, United States
| | - Yoed Rabin
- Department of Mechanical Engineering Carnegie Mellon University, 5000 Forbes Avenue, Pittsburgh, PA, 15213, United States.
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27
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Xie A, Tsvetkova I, Liu Y, Ye X, Hewavitharanage P, Dragnea B, Cadena-Nava RD. Hydrophobic Cargo Encapsulation into Virus Protein Cages by Self-Assembly in an Aprotic Organic Solvent. Bioconjug Chem 2021; 32:2366-2376. [PMID: 34730939 DOI: 10.1021/acs.bioconjchem.1c00420] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
While extensive studies of virus capsid assembly in environments mimicking in vivo conditions have led to an understanding of the thermodynamic driving forces at work, applying this knowledge to virus assembly in other solvents than aqueous buffers has not been attempted yet. In this study, Brome mosaic virus (BMV) capsid proteins were shown to preserve their self-assembly abilities in an aprotic polar solvent, dimethyl sulfoxide (DMSO). This facilitated protein cage encapsulation of nanoparticles and dye molecules that favor organic solvents, such as β-NaYF4-based upconversion nanoparticles and BODIPY dye. Assembly was found to be robust relative to a surprisingly broad range of DMSO concentrations. Cargos with poor initial stability in aqueous solutions were readily encapsulated at high DMSO concentrations and then transferred to aqueous solvents, where they remained stable and preserved their function for months.
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Affiliation(s)
- Amberly Xie
- Department of Chemistry, Indiana University, 800 E Kirkwood Avenue, Bloomington, Indiana 47405, United States
| | - Irina Tsvetkova
- Department of Chemistry, Indiana University, 800 E Kirkwood Avenue, Bloomington, Indiana 47405, United States
| | - Yang Liu
- Department of Chemistry, Indiana University, 800 E Kirkwood Avenue, Bloomington, Indiana 47405, United States
| | - Xingchen Ye
- Department of Chemistry, Indiana University, 800 E Kirkwood Avenue, Bloomington, Indiana 47405, United States
| | - Priyadarshine Hewavitharanage
- Chemistry Department, University of Southern Indiana, 8600 University Boulevard, Evansville, Indiana 47712, United States
| | - Bogdan Dragnea
- Department of Chemistry, Indiana University, 800 E Kirkwood Avenue, Bloomington, Indiana 47405, United States
| | - Ruben D Cadena-Nava
- Department of Chemistry, Indiana University, 800 E Kirkwood Avenue, Bloomington, Indiana 47405, United States
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28
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Gilfanova R, Callegari A, Childs A, Yang G, Luarca M, Gutierrez AG, Medina KI, Mai J, Hui A, Kline M, Wei X, Norris PJ, Muench MO. A bioinspired and chemically defined alternative to dimethyl sulfoxide for the cryopreservation of human hematopoietic stem cells. Bone Marrow Transplant 2021; 56:2644-2650. [PMID: 34155359 PMCID: PMC8563414 DOI: 10.1038/s41409-021-01368-w] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2021] [Revised: 05/19/2021] [Accepted: 05/26/2021] [Indexed: 02/08/2023]
Abstract
The cryopreservation of hematopoietic cells using dimethyl sulfoxide (DMSO) and serum is a common procedure used in transplantation. However, DMSO has clinical and biological side effects due to its toxicity, and serum introduces variation and safety risks. Inspired by natural antifreeze proteins, a novel class of ice-interactive cryoprotectants was developed. The corresponding DMSO-, protein-, and serum-free cryopreservation media candidates were screened through a series of biological assays using human cell lines, peripheral blood cells, and bone marrow cells. XT-Thrive-A and XT-Thrive-B were identified as lead candidates to rival cryopreservation with 10% DMSO in serum based on post-thaw cell survival and short-term proliferation assays. The effectiveness of the novel cryopreservation media in freezing hematopoietic stem cells from human whole bone marrow was assessed by extreme limiting dilution analysis in immunodeficient mice. Stem cell frequencies were measured 12 weeks after transplant based on bone marrow engraftment of erythroid, myeloid, B-lymphoid, and CD34+ progenitors measured by flow cytometry. The recovered numbers of cryopreserved stem cells were similar among XT-Thrive A, XT-Thrive B, and DMSO with serum groups. These findings show that cryoprotectants developed through biomimicry of natural antifreeze proteins offers a substitute for DMSO-based media for the cryopreservation of hematopoietic stem cells.
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Affiliation(s)
| | | | | | | | | | | | | | - Justin Mai
- Vitalant Research Institute, San Francisco, CA, USA
| | - Alvin Hui
- Vitalant Research Institute, San Francisco, CA, USA
| | | | | | - Philip J Norris
- Vitalant Research Institute, San Francisco, CA, USA
- Department of Laboratory Medicine, University of California, San Francisco, CA, USA
| | - Marcus O Muench
- Vitalant Research Institute, San Francisco, CA, USA.
- Department of Laboratory Medicine, University of California, San Francisco, CA, USA.
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29
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Kaushal R, Jahan S, McGregor C, Pineault N. Dimethyl sulfoxide-free cryopreservation solutions for hematopoietic stem cell grafts. Cytotherapy 2021; 24:272-281. [PMID: 34654640 DOI: 10.1016/j.jcyt.2021.09.002] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2021] [Revised: 08/18/2021] [Accepted: 09/05/2021] [Indexed: 02/08/2023]
Abstract
BACKGROUND AIMS The use of effective methods for the cryopreservation of hematopoietic stem cells (HSCs) is vital to retain the maximum engraftment activity of cord blood units (CBUs). Current protocols entail the use of dimethyl sulfoxide (DMSO) as intracellular cryoprotective agent (CPA) and dextran and plasma proteins as extracellular CPAs, but DMSO is known to be cytotoxic, and its infusion in patients is associated with mild to moderate side effects. However, new, commercially available, DMSO-free cryopreservation solutions have been developed, but their capacity to protect HSCs remains poorly investigated. METHODS Herein the authors compared the capacity of four DMSO-free freezing media to cryopreserve cord blood (CB) HSCs: CryoProtectPureSTEM (CPP-STEM), CryoScarless (CSL), CryoNovo P24 (CN) and Pentaisomaltose (PIM). Clinical-grade DMSO/dextran solution was used as control. RESULTS Of the four cryopreservation solutions tested, the best post-thaw cell viability, recovery of viable CD45+ and CD34+ cells and potency were achieved with CPP-STEM, which was equal or superior to that seen with the control DMSO. CSL provided the second best post-thaw results followed by PIM, whereas CN was associated with modest viability and potency. Further work with CPP-STEM revealed that CB CD34-enriched HSCs and progenitors cryopreserved with CPP-STEM maintained high viability and growth expansion activity. In line with this, a pilot transplantation assay confirmed that CPP-STEM-protected CB grafts supported normal short- and long-term engraftment kinetics. CONCLUSIONS The authors' results suggest that new, valuable alternatives to DMSO are now available for the cryopreservation of HSCs and grafts, including CBUs.
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Affiliation(s)
- Richa Kaushal
- Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Canada; Centre for Innovation, Canadian Blood Services, Ottawa, Canada
| | - Suria Jahan
- Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Canada; Centre for Innovation, Canadian Blood Services, Ottawa, Canada
| | | | - Nicolas Pineault
- Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Canada; Centre for Innovation, Canadian Blood Services, Ottawa, Canada.
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30
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Novitzky-Basso I, Remberger M, Chen C, Pasić I, Lam W, Law A, Gerbitz A, Viswabandya A, Lipton JH, Kim DD, Kumar R, Mattsson J, Michelis FV. Anti-thymocyte globulin and post-transplant cyclophosphamide predisposes to inferior outcome when using cryopreserved stem cell grafts. Eur J Haematol 2021; 108:61-72. [PMID: 34606661 DOI: 10.1111/ejh.13714] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2021] [Revised: 09/26/2021] [Accepted: 09/28/2021] [Indexed: 12/14/2022]
Abstract
During 2020, the concurrent novel COVID-19 pandemic lead to widespread cryopreservation of allogeneic hematopoietic cell transplant grafts based on National Marrow Donor Program and European Society of Blood and Marrow Transplantation recommendations, in order to secure grafts before the start of conditioning chemotherapy. We sought to examine the impact of this change in practice on patient outcomes. We analyzed the outcomes of 483 patients who received hematopoietic stem cell transplantation (HSCT) between August 2017 and August 2020, at Princess Margaret Cancer Centre, Canada, in the retrospective study, comparing the outcomes between those who received cryopreserved or fresh peripheral blood stem cell grafts. Overall compared with those who received fresh grafts (n = 348), patients who received cryopreserved grafts (n = 135) had reduced survival and GRFS, reduced incidence of chronic graft-versus-host disease (GvHD), delay in neutrophil engraftment, and higher graft failure (GF), with no significant difference in relapse incidence or acute GvHD. However, recipients of cryopreserved matched-related donor HSCT showed significantly worse OS, NRM, GRFS compared with fresh grafts. Multivariable analysis of the entire cohort showed significant impact of cryopreservation on OS, relapse, cGvHD, GF, and GRFS. We conclude that cryopreservation was associated with inferior outcomes post-HSCT, possibly due to the combination of ATG and post-transplant cyclophosphamide impacting differential tolerance to cryopreservation on components of the stem cell graft; further studies are warranted to elucidate mechanisms for this observation.
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Affiliation(s)
- Igor Novitzky-Basso
- Hans Messner Allogeneic Transplant Program, Toronto, ON, Canada.,University of Toronto, Toronto, ON, Canada
| | - Mats Remberger
- Department of Medical Sciences, Uppsala University Hospital, Uppsala University and KFUE, Uppsala, Sweden
| | - Carol Chen
- Hans Messner Allogeneic Transplant Program, Toronto, ON, Canada
| | - Ivan Pasić
- Hans Messner Allogeneic Transplant Program, Toronto, ON, Canada.,University of Toronto, Toronto, ON, Canada
| | - Wilson Lam
- Hans Messner Allogeneic Transplant Program, Toronto, ON, Canada.,University of Toronto, Toronto, ON, Canada
| | - Arjun Law
- Hans Messner Allogeneic Transplant Program, Toronto, ON, Canada.,University of Toronto, Toronto, ON, Canada
| | - Armin Gerbitz
- Hans Messner Allogeneic Transplant Program, Toronto, ON, Canada.,University of Toronto, Toronto, ON, Canada
| | - Auro Viswabandya
- Hans Messner Allogeneic Transplant Program, Toronto, ON, Canada.,University of Toronto, Toronto, ON, Canada
| | - Jeffrey H Lipton
- Hans Messner Allogeneic Transplant Program, Toronto, ON, Canada.,University of Toronto, Toronto, ON, Canada
| | - Dennis D Kim
- Hans Messner Allogeneic Transplant Program, Toronto, ON, Canada.,University of Toronto, Toronto, ON, Canada
| | - Rajat Kumar
- Hans Messner Allogeneic Transplant Program, Toronto, ON, Canada.,University of Toronto, Toronto, ON, Canada
| | - Jonas Mattsson
- Hans Messner Allogeneic Transplant Program, Toronto, ON, Canada.,University of Toronto, Toronto, ON, Canada.,Gloria and Seymour Epstein Chair in Cell Therapy and Transplantation, University of Toronto, Toronto, Ontario, Canada
| | - Fotios V Michelis
- Hans Messner Allogeneic Transplant Program, Toronto, ON, Canada.,University of Toronto, Toronto, ON, Canada
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Měřička P, Janoušek L, Benda A, Lainková R, Sabó J, Dalecká M, Prokšová P, Salmay M, Špunda R, Pecha O, Jandová M, Gregor J, Štěrba L, Špaček M, Lindner J. Cell Viability Assessment Using Fluorescence Vital Dyes and Confocal Microscopy in Evaluating Freezing and Thawing Protocols Used in Cryopreservation of Allogeneic Venous Grafts. Int J Mol Sci 2021; 22:ijms221910653. [PMID: 34638994 PMCID: PMC8509073 DOI: 10.3390/ijms221910653] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2021] [Revised: 09/24/2021] [Accepted: 09/26/2021] [Indexed: 12/29/2022] Open
Abstract
The authors present their contribution to the improvement of methods suitable for the detection of the freezing and thawing damage of cells of cryopreserved venous grafts used for lower limb revascularization procedures. They studied the post-thaw viability of cells of the wall of cryopreserved venous grafts (CVG) immediately after thawing and after 24 and 48 h culture at +37 °C in two groups of six CVG selected randomly for slow thawing in the refrigerator and rapid thawing in a water bath at +37 °C. The grafts were collected from multi-organ and tissue brain-dead donors, cryopreserved, and stored in a liquid nitrogen vapor phase for five years. The viability was assessed from tissue slices obtained by perpendicular and longitudinal cuts of the thawed graft samples using in situ staining with fluorescence vital dyes. The mean and median immediate post-thaw viability values above 70% were found in using both thawing protocols and both types of cutting. The statistically significant decline in viability after the 48-h culture was observed only when using the slow thawing protocol and perpendicular cutting. The possible explanation might be the “solution effect damage” during slow thawing, which caused a gentle reduction in the graft cellularity. The possible influence of this phenomenon on the immunogenicity of CVG should be the subject of further investigations.
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Affiliation(s)
- Pavel Měřička
- Tissue Bank, University Hospital, 500 05 Hradec Králové, Czech Republic; (P.M.); (M.J.); (J.G.); (L.Š.)
| | - Libor Janoušek
- Department of Transplantation Surgery, Institute for Clinical and Experimental Medicine, 140 21 Prague, Czech Republic;
| | - Aleš Benda
- Imaging Methods Core Facility at BIOCEV, Faculty of Science, Charles University, 252 50 Prague, Czech Republic; (A.B.); (J.S.); (M.D.); (P.P.)
| | - Radka Lainková
- 2nd Department of Surgery–Department of Cardiovascular Surgery, 1st Medical Faculty, Charles University and General University Hospital, 128 08 Prague, Czech Republic; (R.L.); (M.S.); (R.Š.); (J.L.)
| | - Ján Sabó
- Imaging Methods Core Facility at BIOCEV, Faculty of Science, Charles University, 252 50 Prague, Czech Republic; (A.B.); (J.S.); (M.D.); (P.P.)
| | - Markéta Dalecká
- Imaging Methods Core Facility at BIOCEV, Faculty of Science, Charles University, 252 50 Prague, Czech Republic; (A.B.); (J.S.); (M.D.); (P.P.)
- Department of Cell Biology, Charles University, Viničná 7, 128 00 Prague, Czech Republic
| | - Petra Prokšová
- Imaging Methods Core Facility at BIOCEV, Faculty of Science, Charles University, 252 50 Prague, Czech Republic; (A.B.); (J.S.); (M.D.); (P.P.)
| | - Myroslav Salmay
- 2nd Department of Surgery–Department of Cardiovascular Surgery, 1st Medical Faculty, Charles University and General University Hospital, 128 08 Prague, Czech Republic; (R.L.); (M.S.); (R.Š.); (J.L.)
| | - Rudolf Špunda
- 2nd Department of Surgery–Department of Cardiovascular Surgery, 1st Medical Faculty, Charles University and General University Hospital, 128 08 Prague, Czech Republic; (R.L.); (M.S.); (R.Š.); (J.L.)
| | - Ondřej Pecha
- Technology Centre of the Czech Academy of Sciences, 160 00 Prague, Czech Republic;
| | - Miroslava Jandová
- Tissue Bank, University Hospital, 500 05 Hradec Králové, Czech Republic; (P.M.); (M.J.); (J.G.); (L.Š.)
- Department of Anatomy, Histology and Embryology Medical Faculty in Hradec Králové, Charles University, 500 03 Hradec Králové, Czech Republic
| | - Jiří Gregor
- Tissue Bank, University Hospital, 500 05 Hradec Králové, Czech Republic; (P.M.); (M.J.); (J.G.); (L.Š.)
| | - Lubomír Štěrba
- Tissue Bank, University Hospital, 500 05 Hradec Králové, Czech Republic; (P.M.); (M.J.); (J.G.); (L.Š.)
| | - Miroslav Špaček
- 2nd Department of Surgery–Department of Cardiovascular Surgery, 1st Medical Faculty, Charles University and General University Hospital, 128 08 Prague, Czech Republic; (R.L.); (M.S.); (R.Š.); (J.L.)
- Correspondence:
| | - Jaroslav Lindner
- 2nd Department of Surgery–Department of Cardiovascular Surgery, 1st Medical Faculty, Charles University and General University Hospital, 128 08 Prague, Czech Republic; (R.L.); (M.S.); (R.Š.); (J.L.)
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Erol OD, Pervin B, Seker ME, Aerts-Kaya F. Effects of storage media, supplements and cryopreservation methods on quality of stem cells. World J Stem Cells 2021; 13:1197-1214. [PMID: 34630858 PMCID: PMC8474714 DOI: 10.4252/wjsc.v13.i9.1197] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/27/2021] [Revised: 04/21/2021] [Accepted: 08/24/2021] [Indexed: 02/06/2023] Open
Abstract
Despite a vast amount of different methods, protocols and cryoprotective agents (CPA), stem cells are often frozen using standard protocols that have been optimized for use with cell lines, rather than with stem cells. Relatively few comparative studies have been performed to assess the effects of cryopreservation methods on these stem cells. Dimethyl sulfoxide (DMSO) has been a key agent for the development of cryobiology and has been used universally for cryopreservation. However, the use of DMSO has been associated with in vitro and in vivo toxicity and has been shown to affect many cellular processes due to changes in DNA methylation and dysregulation of gene expression. Despite studies showing that DMSO may affect cell characteristics, DMSO remains the CPA of choice, both in a research setting and in the clinics. However, numerous alternatives to DMSO have been shown to hold promise for use as a CPA and include albumin, trehalose, sucrose, ethylene glycol, polyethylene glycol and many more. Here, we will discuss the use, advantages and disadvantages of these CPAs for cryopreservation of different types of stem cells, including hematopoietic stem cells, mesenchymal stromal/stem cells and induced pluripotent stem cells.
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Affiliation(s)
- Ozgur Dogus Erol
- Department of Stem Cell Sciences, Hacettepe University Graduate School of Health Sciences, Ankara 06100, Turkey
- Center for Stem Cell Research and Development, Hacettepe University, Ankara 06100, Turkey
| | - Burcu Pervin
- Department of Stem Cell Sciences, Hacettepe University Graduate School of Health Sciences, Ankara 06100, Turkey
- Center for Stem Cell Research and Development, Hacettepe University, Ankara 06100, Turkey
| | - Mehmet Emin Seker
- Department of Stem Cell Sciences, Hacettepe University Graduate School of Health Sciences, Ankara 06100, Turkey
- Center for Stem Cell Research and Development, Hacettepe University, Ankara 06100, Turkey
| | - Fatima Aerts-Kaya
- Department of Stem Cell Sciences, Hacettepe University Graduate School of Health Sciences, Ankara 06100, Turkey
- Center for Stem Cell Research and Development, Hacettepe University, Ankara 06100, Turkey
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Allogeneic transplant procurement in the times of COVID-19: Quality report from the central European cryopreservation site. J Transl Med 2021; 19:145. [PMID: 33832504 PMCID: PMC8027980 DOI: 10.1186/s12967-021-02810-9] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2021] [Accepted: 03/28/2021] [Indexed: 01/14/2023] Open
Abstract
Background Because of limitations of transportation imposed by the COVID-19 pandemic, current recommendation calls for cryopreservation of allogeneic stem cell transplants before patient conditioning. A single cell therapy laboratory was selected to function as the central cryopreservation hub for all European registry donor transplants intended for the Australian-Pacific region. We examined properties of these transplants to ascertain how quality is maintained. Methods We analyzed 100 pandemic-related allogeneic mobilized blood-derived stem cell apheresis products generated at 30 collection sites throughout Europe, shipped to and cryopreserved at our center between April and November of 2020. Products were shipped in the cool, subsequently frozen with DMSO as cryoprotectant. Irrespective of origin, all products were frozen within the prescribed shelf-life of 72 h. Results Prior to cryopreservation, viable stem cell and leukocyte count according to the collection site and our reference laboratory were highly concordant (r2 = 0.96 and 0.93, respectively) and viability was > 90% in all instances. Median nominal post-thaw recovery of viable CD34+ cells was 42%. Weakly associated with poorer CD34+ cell recovery was higher leukocyte concentration, but not time lag between apheresis or addition of cryopreservant, respectively, and start of freezing. The correlation between pre- and post-thaw CD34+ cell dose was high (r2 = 0.85), hence predictable. Neutrophil and platelet engraftment were prompt with no evidence of dose dependency within the range of administered cell doses (1.31–15.56 × 106 CD34+ cells/kg). Conclusions General cryopreservation of allogeneic stem cell transplants is feasible. While more than half of the CD34+ cell content is lost, the remaining stem cells ensure timely engraftment. Supplementary Information The online version contains supplementary material available at 10.1186/s12967-021-02810-9.
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Cryoimmunology: Opportunities and challenges in biomedical science and practice. Cryobiology 2021; 100:1-11. [PMID: 33639110 DOI: 10.1016/j.cryobiol.2021.02.005] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2020] [Revised: 01/28/2021] [Accepted: 02/18/2021] [Indexed: 12/26/2022]
Abstract
Autologous and allogeneic cryoimmunological medicine is a brand new branch of biomedical science and clinical practice that examines the features and formation of the immune response to immunogenic properties of normal and malignant biological structures altered by ultralow temperature, as well as specific changes in the structural and functional characteristics of immune cells and tissues after cryopreservation. Cryogenic protein denaturation phenomenon provides important insights into the mechanisms underlying the damage to cryogenic lesions immediately after freeze-thawing sessions in bioscience and medicine applications. The newly formed cryocoagulated protein components (cryomodified protein components) are crucial in cryoimmunology from the perspective of the formation of immunological substances at ultralow temperatures. Dendritic cells and cryocell detritus (cryocell debris) formed in living biological tissue after exposure to ultralow temperature in vivo may be an indication of one of the essential mechanisms involved in the cryoimmunological response of living structures to the impact of ultralow temperature exposure. Hence, the formation of new autologous and allogeneic cryoinduced immunogenic substances is a novel concept in biomedical research globally. Accordingly, this review focuses on issues concerning the peculiarities of the interaction of the immune system with a dominant malignant neoplasm tissue after exposure to subzero temperatures, considering the original cryogenic technical approaches. We present an overview of the state-of-the-art methods of cryoimmunology, and their major developments, past and present. The need for the delineation of structural and functional characteristics of the biological substrates of the immune system after cryopreservation that can be used in adoptive cell therapy, especially in cancer patients, is emphasized.
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Belisário AR, da Costa Funes AP, Luz JR, de Almeida Costa L, Furtado MDSBS, Martins MC, Cruz NG, Pederzoli PRMP, de Andrade RK, Libânio MRIS, de Lima Prata K. Influence of laboratory procedures on postthawing cell viability and hematopoietic engraftment after autologous peripheral blood stem cell transplantation. Transfusion 2021; 61:1202-1214. [PMID: 33569783 DOI: 10.1111/trf.16289] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2020] [Revised: 12/17/2020] [Accepted: 12/23/2020] [Indexed: 12/31/2022]
Abstract
BACKGROUND The kinetics of hematopoietic recovery after autologous stem cell transplantation (ASCT) may be affected by laboratory procedures. The aim of this study was to evaluate the influence of characteristics of the cryopreserved units of peripheral blood stem cells (PBSC) on postthawing cell viability and engraftment outcomes after ASCT. STUDY DESIGN AND METHODS This was a retrospective cohort study including individuals referred for ASCT. Cryopreservation was conducted at a single processing facility between 2014 and 2019, and patients received clinical care at six transplant centers. Covariates and outcome data were retrieved from participants' records. RESULTS The study population comprised 619 patients (345 [55.7%] male). Median age was 53 years. Multiple myeloma was the most common diagnosis (62.7%). Higher preapheresis CD34+ cell count, lower nucleated cell (NC) concentration per cryobag, and composition of the cryoprotectant solution (5% dimethyl sulfoxide [DMSO] and 6% hydroxyethyl starch) were statistically significantly associated with higher postthawing cell viability. The linear regression model for time to neutrophil and platelet engraftment included the infused CD34+ cell dose and the composition of the cryoprotectant solution. Patients who had PBSC cryopreserved using 10% DMSO solution presented six times higher odds (odds ratio [OR] = 6.9; 95% confidence interval [CI]: 2.2-21.1; p = .001) of delayed neutrophil engraftment (>14 days) and two times higher odds (OR = 2.3, 95%CI: 1.4-3.7; p = .001) of prolonged hospitalization (>18 days). DISCUSSION The study showed that mobilization efficacy, NC concentration, and the composition of the cryoprotectant solution significantly affected postthawing cell viability. In addition, the composition of the cryoprotectant solution significantly impacted engraftment outcomes and time of hospitalization after ASCT.
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Affiliation(s)
- André Rolim Belisário
- Centro de Tecidos Biológicos de Minas Gerais, Fundação Hemominas, Lagoa Santa, Minas Gerais, Brazil
| | - Ana Paula da Costa Funes
- Centro de Tecidos Biológicos de Minas Gerais, Fundação Hemominas, Lagoa Santa, Minas Gerais, Brazil
| | - Junio Rocha Luz
- Centro de Tecidos Biológicos de Minas Gerais, Fundação Hemominas, Lagoa Santa, Minas Gerais, Brazil
| | - Luciana de Almeida Costa
- Centro de Tecidos Biológicos de Minas Gerais, Fundação Hemominas, Lagoa Santa, Minas Gerais, Brazil
| | | | | | - Nathália Gomide Cruz
- Centro de Tecidos Biológicos de Minas Gerais, Fundação Hemominas, Lagoa Santa, Minas Gerais, Brazil
| | | | - Roberta Kelly de Andrade
- Centro de Tecidos Biológicos de Minas Gerais, Fundação Hemominas, Lagoa Santa, Minas Gerais, Brazil
| | | | - Karen de Lima Prata
- Centro de Tecidos Biológicos de Minas Gerais, Fundação Hemominas, Lagoa Santa, Minas Gerais, Brazil
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van Velthoven AJ, Bertolin M, Barbaro V, Sthijns MM, Nuijts RM, LaPointe VL, Dickman MM, Ferrari S. Increased Cell Survival of Human Primary Conjunctival Stem Cells in Dimethyl Sulfoxide-Based Cryopreservation Media. Biopreserv Biobank 2021; 19:67-72. [PMID: 33185460 PMCID: PMC7892306 DOI: 10.1089/bio.2020.0091] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Glycerol and dimethyl sulfoxide (DMSO) are widely used cryoprotectants for freezing human cell cultures. During the manufacturing process of ocular stem cell-based autographs, ex vivo cultivated ocular cells are cryopreserved for quality control purposes in accordance with regulatory requirements. The efficiency of the cryopreservation methods is limited by their effect on cell survival and quality. We compared two cryopreservation reagents, glycerol and DMSO, for their influence on the survival and quality of human primary conjunctival cultures. We found increased cell viability after cryopreservation in DMSO compared to cryopreservation in glycerol. The clonogenic and proliferative capacity was unaffected by the cryopreservation reagents, as shown by the colony forming efficiency and cumulative cell doubling. Importantly, the percentage of p63α- and keratin 19 (K19)-positive cells following cryopreservation in DMSO or glycerol was comparable. Taken together, our results demonstrate that cryopreservation in DMSO improves cell survival compared to cryopreservation in glycerol, with no subsequent effect on cell proliferative-, clonogenic-, or differentiation capacity. Therefore, we advise the use of a 10% DMSO-based cryopreservation medium for the cryopreservation of human primary conjunctival cells, as it will improve the number of cells available for the manufacturing of conjunctival stem cell-based autografts for clinical use.
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Affiliation(s)
- Arianne J.H. van Velthoven
- University Eye Clinic Maastricht, Maastricht University Medical Center+, Maastricht, the Netherlands
- Department of Cell Biology-Inspired Tissue Engineering, MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht University, Maastricht, the Netherlands
| | | | | | - Mireille M.J.P.E. Sthijns
- Department of Cell Biology-Inspired Tissue Engineering, MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht University, Maastricht, the Netherlands
| | - Rudy M.M.A. Nuijts
- University Eye Clinic Maastricht, Maastricht University Medical Center+, Maastricht, the Netherlands
| | - Vanessa L.S. LaPointe
- Department of Cell Biology-Inspired Tissue Engineering, MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht University, Maastricht, the Netherlands
| | - Mor M. Dickman
- University Eye Clinic Maastricht, Maastricht University Medical Center+, Maastricht, the Netherlands
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Abstract
PURPOSE OF REVIEW One aim in bone tissue engineering is to develop human cell-based, 3D in vitro bone models to study bone physiology and pathology. Due to the heterogeneity of cells among patients, patient's own cells are needed to be obtained, ideally, from one single cell source. This review attempts to identify the appropriate cell sources for development of such models. RECENT FINDINGS Bone marrow and peripheral blood are considered as suitable sources for extraction of osteoblast/osteocyte and osteoclast progenitor cells. Recent studies on these cell sources have shown no significant differences between isolated progenitor cells. However, various parameters such as medium composition affect the cell's proliferation and differentiation potential which could make the peripheral blood-derived stem cells superior to the ones from bone marrow. Peripheral blood can be considered a suitable source for osteoblast/osteocyte and osteoclast progenitor cells, being less invasive for the patient. However, more investigations are needed focusing on extraction and differentiation of both cell types from the same donor sample of peripheral blood.
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Affiliation(s)
- Sana Ansari
- Orthopaedic Biomechanics, Department of Biomedical Engineering and Institute for Complex Molecular Systems, Eindhoven University of Technology, P.O. Box 513, 5600 MB, Eindhoven, the Netherlands
| | - Keita Ito
- Orthopaedic Biomechanics, Department of Biomedical Engineering and Institute for Complex Molecular Systems, Eindhoven University of Technology, P.O. Box 513, 5600 MB, Eindhoven, the Netherlands
| | - Sandra Hofmann
- Orthopaedic Biomechanics, Department of Biomedical Engineering and Institute for Complex Molecular Systems, Eindhoven University of Technology, P.O. Box 513, 5600 MB, Eindhoven, the Netherlands.
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Jahan S, Kaushal R, Pasha R, Pineault N. Current and Future Perspectives for the Cryopreservation of Cord Blood Stem Cells. Transfus Med Rev 2021; 35:95-102. [PMID: 33640254 DOI: 10.1016/j.tmrv.2021.01.003] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2020] [Revised: 01/22/2021] [Accepted: 01/24/2021] [Indexed: 12/29/2022]
Abstract
Hematopoietic stem cell (HSC) transplantation is a well-established procedure for the treatment of many blood related malignancies and disorders. Before transplantation, HSC are collected and cryopreserved until use. The method of cryopreservation should preserve both the number and function of HSC and downstream progenitors responsible for long- and short-term engraftment, respectively. This is especially critical for cord blood grafts, since the cell number associated with this stem cell source is often limiting. Loss of function in cryopreserved cells occurs following cryoinjuries due to osmotic shock, dehydration, solution effects and mechanical damage from ice recrystallization during freezing and thawing. However, cryoinjuries can be reduced by 2 mitigation strategies; the use of cryoprotectants (CPAs) and use of control rate cooling. Currently, slow cooling is the most common method used for the cryopreservation of HSC graft. Moreover, dimethyl-sulfoxide (DMSO) and dextran are popular intracellular and extracellular CPAs used for HSC grafts, respectively. Yet, DMSO is toxic to cells and can cause significant side effects in stem cells' recipients. However, new CPAs and strategies are emerging that may soon replace DMSO. The aim of this review is to summarise key concepts in cryobiology and recent advances in the field of HSC cryobiology. Other important issues that need to be considered are also discussed such as transient warming events and thawing of HSC grafts.
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Affiliation(s)
- Suria Jahan
- Canadian Blood Services, Centre for Innovation, Ottawa, Ontario, Canada; Biochemistry, Microbiology and Immunology department, University of Ottawa, Ottawa, Ontario, Canada
| | - Richa Kaushal
- Canadian Blood Services, Centre for Innovation, Ottawa, Ontario, Canada; Biochemistry, Microbiology and Immunology department, University of Ottawa, Ottawa, Ontario, Canada
| | - Roya Pasha
- Canadian Blood Services, Centre for Innovation, Ottawa, Ontario, Canada
| | - Nicolas Pineault
- Canadian Blood Services, Centre for Innovation, Ottawa, Ontario, Canada; Biochemistry, Microbiology and Immunology department, University of Ottawa, Ottawa, Ontario, Canada.
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Solanki PK, Rabin Y. Thermomechanical stress analysis of rabbit kidney and human kidney during cryopreservation by vitrification with the application of radiofrequency heating. Cryobiology 2021; 100:180-192. [PMID: 33412158 PMCID: PMC8972895 DOI: 10.1016/j.cryobiol.2021.01.002] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2020] [Revised: 12/31/2020] [Accepted: 01/01/2021] [Indexed: 12/19/2022]
Abstract
This study presents a computational framework for thermomechanical stress analysis in a specimen undergoing cryopreservation, with emphasis on radiofrequency (RF) heating for recovering from cryogenic storage. In particular, this study addresses cryopreservation by vitrification, where the specimen is stored in the amorphous phase (vitreous means glassy). In broad terms, the relatively high cooling and rewarming rates necessary for vitrification result in differential thermal expansion in the specimen, which is the driving force for thermo-mechanical stress. Thermomechanical stress can lead to structural damage, such as fractures or plastic deformation, rendering the specimen useless. Not without technical difficulties, those hazardous effects during the rewarming phase of the protocol can be mitigated by applying volumetric heating, with RF heating as an attractive means. The proposed computational framework in this study addresses the coupled electromagnetic, thermal and solid mechanics fields, using commercially available solvers. This study advances from a spherical-case benchmark to realistic models of the rabbit kidney and the human kidney. Results of this study suggest that structural damage to the brittle material can be prevented when stress relaxation is facilitated around the glass transition temperature. Furthermore, this study suggests that volumetric heating is necessary to surpass the critical rewarming rate, while benefiting from lowering the overall thermomechanical stress during recovery from cryogenic storage. More broadly, the computational framework presented here can be used for the optimization of the RF heating parameters, chamber specifics, specimen container shape, and the thermal protocol in order to preserve structural integrity in the specimen.
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Affiliation(s)
- Prem K Solanki
- Biothermal Technology Laboratory, Department of Mechanical Engineering, Carnegie Mellon University, Pittsburgh, PA, 15213, USA
| | - Yoed Rabin
- Biothermal Technology Laboratory, Department of Mechanical Engineering, Carnegie Mellon University, Pittsburgh, PA, 15213, USA.
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Pacheco-Herrero M, Soto-Rojas LO, Reyes-Sabater H, Garcés-Ramirez L, de la Cruz López F, Villanueva-Fierro I, Luna-Muñoz J. Current Status and Challenges of Stem Cell Treatment for Alzheimer's Disease. J Alzheimers Dis 2021; 84:917-935. [PMID: 34633316 PMCID: PMC8673502 DOI: 10.3233/jad-200863] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 09/07/2021] [Indexed: 12/23/2022]
Abstract
Neurodegenerative diseases called tauopathies, such as Alzheimer's disease (AD), frontotemporal dementia, progressive supranuclear palsy, and Parkinson's disease, among others, are characterized by the pathological processing and accumulation of tau protein. AD is the most prevalent neurodegenerative disease and is characterized by two lesions: neurofibrillary tangles (NFTs) and neuritic plaques. The presence of NFTs in the hippocampus and neocortex in early and advanced stages, respectively, correlates with the patient's cognitive deterioration. So far, no drugs can prevent, decrease, or limit neuronal death due to abnormal pathological tau accumulation. Among potential non-pharmacological treatments, physical exercise has been shown to stimulate the development of stem cells (SCs) and may be useful in early stages. However, this does not prevent neuronal death from the massive accumulation of NFTs. In recent years, SCs therapies have emerged as a promising tool to repopulate areas involved in cognition in neurodegenerative diseases. Unfortunately, protocols for SCs therapy are still being developed and the mechanism of action of such therapy remains unclear. In this review, we show the advances and limitations of SCs therapy. Finally, we provide a critical analysis of its clinical use for AD.
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Affiliation(s)
- Mar Pacheco-Herrero
- Neuroscience Research Laboratory, Faculty of Health Sciences, Pontificia Universidad Católica Madre y Maestra, Dominican Republic
| | - Luis O. Soto-Rojas
- Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, State of Mexico, Mexico
| | - Heidy Reyes-Sabater
- Neuroscience Research Laboratory, Faculty of Health Sciences, Pontificia Universidad Católica Madre y Maestra, Dominican Republic
| | - Linda Garcés-Ramirez
- Escuela Nacional de Ciencias Biológicas, Depto de Fisiología, Instituto Politécnico Nacional, Mexico City, Mexico
| | - Fidel de la Cruz López
- Escuela Nacional de Ciencias Biológicas, Depto de Fisiología, Instituto Politécnico Nacional, Mexico City, Mexico
| | | | - José Luna-Muñoz
- National Dementia BioBank, Ciencias Biológicas, Facultad de Estudios Superiores Cuautitlán, UNAM, State of Mexico, Mexico
- Banco Nacional de Cerebros-UNPHU, Universidad Nacional Pedro Henríquez Ureña, Dominican Republic
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Zheng X, Liu J, Liu Z, Wang J. Bio-inspired Ice-controlling Materials for Cryopreservation of Cells and Tissues. ACTA CHIMICA SINICA 2021. [DOI: 10.6023/a21020043] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
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Cryopreservation of Stem Cells. Stem Cells 2021. [DOI: 10.1007/978-981-16-1638-9_3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
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Whaley D, Damyar K, Witek RP, Mendoza A, Alexander M, Lakey JRT. Cryopreservation: An Overview of Principles and Cell-Specific Considerations. Cell Transplant 2021; 30:963689721999617. [PMID: 33757335 PMCID: PMC7995302 DOI: 10.1177/0963689721999617] [Citation(s) in RCA: 145] [Impact Index Per Article: 36.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2021] [Revised: 01/28/2021] [Accepted: 02/12/2021] [Indexed: 12/21/2022] Open
Abstract
The origins of low-temperature tissue storage research date back to the late 1800s. Over half a century later, osmotic stress was revealed to be a main contributor to cell death during cryopreservation. Consequently, the addition of cryoprotective agents (CPAs) such as dimethyl sulfoxide (DMSO), glycerol (GLY), ethylene glycol (EG), or propylene glycol (PG), although toxic to cells at high concentrations, was identified as a necessary step to protect against rampant cell death during cryopreservation. In addition to osmotic stress, cooling and thawing rates were also shown to have significant influence on cell survival during low temperature storage. In general, successful low-temperature cell preservation consists of the addition of a CPA (commonly 10% DMSO), alone or in combination with additional permeating or non-permeating agents, cooling rates of approximately 1ºC/min, and storage in either liquid or vapor phase nitrogen. In addition to general considerations, cell-specific recommendations for hepatocytes, pancreatic islets, sperm, oocytes, and stem cells should be observed to maximize yields. For example, rapid cooling is associated with better cryopreservation outcomes for oocytes, pancreatic islets, and embryonic stem cells while slow cooling is recommended for cryopreservation of hepatocytes, hematopoietic stem cells, and mesenchymal stem cells. Yields can be further maximized by implementing additional pre-cryo steps such as: pre-incubation with glucose and anti-oxidants, alginate encapsulation, and selecting cells within an optimal age range and functional ability. Finally, viability and functional assays are critical steps in determining the quality of the cells post-thaw and improving the efficiency of the current cryopreservation methods.
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Affiliation(s)
- David Whaley
- Department of Surgery, University of California Irvine, Orange, CA, USA
| | - Kimia Damyar
- Department of Surgery, University of California Irvine, Orange, CA, USA
| | | | | | - Michael Alexander
- Department of Surgery, University of California Irvine, Orange, CA, USA
| | - Jonathan RT Lakey
- Department of Surgery, University of California Irvine, Orange, CA, USA
- Department of Biomedical Engineering, University of California Irvine, Irvine, CA, USA
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Meneghel J, Kilbride P, Morris GJ. Cryopreservation as a Key Element in the Successful Delivery of Cell-Based Therapies-A Review. Front Med (Lausanne) 2020; 7:592242. [PMID: 33324662 PMCID: PMC7727450 DOI: 10.3389/fmed.2020.592242] [Citation(s) in RCA: 63] [Impact Index Per Article: 12.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2020] [Accepted: 10/23/2020] [Indexed: 12/24/2022] Open
Abstract
Cryopreservation is a key enabling technology in regenerative medicine that provides stable and secure extended cell storage for primary tissue isolates and constructs and prepared cell preparations. The essential detail of the process as it can be applied to cell-based therapies is set out in this review, covering tissue and cell isolation, cryoprotection, cooling and freezing, frozen storage and transport, thawing, and recovery. The aim is to provide clinical scientists with an overview of the benefits and difficulties associated with cryopreservation to assist them with problem resolution in their routine work, or to enable them to consider future involvement in cryopreservative procedures. It is also intended to facilitate networking between clinicians and cryo-researchers to review difficulties and problems to advance protocol optimization and innovative design.
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Affiliation(s)
- Julie Meneghel
- Asymptote, Cytiva, Danaher Corporation, Cambridge, United Kingdom
| | - Peter Kilbride
- Asymptote, Cytiva, Danaher Corporation, Cambridge, United Kingdom
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Zhang TY, Tan PC, Xie Y, Zhang XJ, Zhang PQ, Gao YM, Zhou SB, Li QF. The combination of trehalose and glycerol: an effective and non-toxic recipe for cryopreservation of human adipose-derived stem cells. Stem Cell Res Ther 2020; 11:460. [PMID: 33129347 PMCID: PMC7602354 DOI: 10.1186/s13287-020-01969-0] [Citation(s) in RCA: 25] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2020] [Accepted: 10/07/2020] [Indexed: 12/31/2022] Open
Abstract
Background Adipose-derived stem cells (ADSCs) promote tissue regeneration and repair. Cryoprotective agents (CPAs) protect cells from cryodamage during cryopreservation. Safe and efficient cryopreservation of ADSCs is critical for cell-based therapy in clinical applications. However, most CPAs are used at toxic concentrations, limiting their clinical application. Objective The aim of this study is to develop a non-toxic xeno-free novel CPA aiming at achieving high-efficiency and low-risk ADSC cryopreservation. Methods We explored different concentrations of trehalose (0.3 M, 0.6 M, 1.0 M, and 1.25 M) and glycerol (10%, 20%, and 30% v/v) for optimization and evaluated and compared the outcomes of ADSCs cryopreservation between a combination of trehalose and glycerol and the commonly used CPA DMSO (10%) + FBS (90%). All samples were slowly frozen and stored in liquid nitrogen for 30 days. The effectiveness was evaluated by the viability, proliferation, migration, and multi-potential differentiation of the ADSCs after thawing. Results Compared with the groups treated with individual reagents, the 1.0 M trehalose (Tre) + 20% glycerol (Gly) group showed significantly higher efficiency in preserving ADSC activities after thawing, with better outcomes in both cell viability and proliferation capacity. Compared with the 10% DMSO + 90% FBS treatment, the ADSCs preserved in 1.0 M Tre + 20% Gly showed similar cell viability, surface markers, and multi-potential differentiation but a significantly higher migration capability. The results indicated that cell function preservation can be improved by 1.0 M Tre + 20% Gly. Conclusions The 1.0 M Tre + 20% Gly treatment preserved ADSCs with a higher migration capability than 10% DMSO + 90% FBS and with viability higher than that with trehalose or glycerol alone but similar to that with 10% DMSO + 90% FBS and fresh cells. Moreover, the new CPA achieves stemness and multi-potential differentiation similar to those in fresh cells. Our results demonstrate that 1.0 M Tre + 20% Gly can more efficiently cryopreserve ADSCs and is a non-toxic CPA that may be suitable for clinical applications.
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Affiliation(s)
- Tian-Yu Zhang
- Department of Plastic & Reconstructive Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, 639 Zhizhaoju Road, Shanghai, 200011, People's Republic of China.,College of Life Sciences, Shanghai Normal University, Shanghai, People's Republic of China
| | - Poh-Ching Tan
- Department of Plastic & Reconstructive Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, 639 Zhizhaoju Road, Shanghai, 200011, People's Republic of China
| | - Yun Xie
- Department of Plastic & Reconstructive Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, 639 Zhizhaoju Road, Shanghai, 200011, People's Republic of China
| | - Xiao-Jie Zhang
- Department of Plastic & Reconstructive Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, 639 Zhizhaoju Road, Shanghai, 200011, People's Republic of China.,College of Life Sciences, Shanghai Normal University, Shanghai, People's Republic of China
| | - Pei-Qi Zhang
- Department of Plastic & Reconstructive Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, 639 Zhizhaoju Road, Shanghai, 200011, People's Republic of China
| | - Yi-Ming Gao
- Department of Plastic & Reconstructive Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, 639 Zhizhaoju Road, Shanghai, 200011, People's Republic of China
| | - Shuang-Bai Zhou
- Department of Plastic & Reconstructive Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, 639 Zhizhaoju Road, Shanghai, 200011, People's Republic of China.
| | - Qing-Feng Li
- Department of Plastic & Reconstructive Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, 639 Zhizhaoju Road, Shanghai, 200011, People's Republic of China.
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Solanki PK, Rabin Y. Scaling Effects on the Residual Thermomechanical Stress During Ice-Free Cooling to Storage Temperature. JOURNAL OF APPLIED MECHANICS 2020; 87:101003. [PMID: 34168384 PMCID: PMC8208319 DOI: 10.1115/1.4047420] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/20/2020] [Revised: 06/01/2020] [Accepted: 06/01/2020] [Indexed: 05/17/2023]
Abstract
Cryopreservation via vitrification (glass formation) is a promising approach for long-term preservation of large-size tissues and organs. Unfortunately, thermomechanical stress, which is driven by the tendency of materials to change size with temperature, may lead to structural failure. This study focuses on analysis of thermomechanical stress in a realistic, pillow-like shape cryobag as it is cooled to cryogenic storage, subject to sufficiently high cooling rates to facilitate vitrification. Contrary to common perception, it is demonstrated in this study that the maximum stress in the specimen does not necessarily increase with increasing size of the specimen. In fact, the maximum stress is affected by the combination of two competing effects, associated with the extent of the temperature gradients within the specimen and its overall volume. On one hand, the increase in specimen size gives rise to more prominent temperature gradients, which can intensify the thermomechanical stress. On the other hand, the temperature distribution at the core of larger specimens is more uniform, which leads to a larger portion of the specimen transitioning from fluid to a glassy material almost instantaneously, which carries a moderating effect on the overall mechanical stress at the glassy state (i.e., lower residual stress). In conclusion, this study demonstrates the role of container shape optimization in reducing the thermomechanical stress during cooling.
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Affiliation(s)
- Prem K. Solanki
- Biothermal Technology Laboratory, Department of Mechanical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213
| | - Yoed Rabin
- Biothermal Technology Laboratory, Department of Mechanical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213
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Engineering a Humanised Niche to Support Human Haematopoiesis in Mice: Novel Opportunities in Modelling Cancer. Cancers (Basel) 2020; 12:cancers12082205. [PMID: 32781703 PMCID: PMC7466161 DOI: 10.3390/cancers12082205] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2020] [Revised: 07/31/2020] [Accepted: 08/01/2020] [Indexed: 12/18/2022] Open
Abstract
Despite the bone marrow microenvironment being widely recognised as a key player in cancer research, the current animal models that represent a human haematopoietic system lack the contribution of the humanised marrow microenvironment. Here we describe a murine model that relies on the combination of an orthotopic humanised tissue-engineered bone construct (ohTEBC) with patient-specific bone marrow (BM) cells to create a humanised bone marrow (hBM) niche capable of supporting the engraftment of human haematopoietic cells. Results showed that this model supports the engraftment of human CD34+ cells from a healthy BM with human haematopoietic cells migrating into the mouse BM, human BM compartment, spleen and peripheral blood. We compared these results with the engraftment capacity of human CD34+ cells obtained from patients with multiple myeloma (MM). We demonstrated that CD34+ cells derived from a diseased BM had a reduced engraftment potential compared to healthy patients and that a higher cell dose is required to achieve engraftment of human haematopoietic cells in peripheral blood. Finally, we observed that hematopoietic cells obtained from the mobilised peripheral blood of patients yields a higher number of CD34+, overcoming this problem. In conclusion, this humanised mouse model has potential as a unique and patient-specific pre-clinical platform for the study of tumour–microenvironment interactions, including human bone and haematopoietic cells, and could, in the future, serve as a drug testing platform.
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Gironi B, Kahveci Z, McGill B, Lechner BD, Pagliara S, Metz J, Morresi A, Palombo F, Sassi P, Petrov PG. Effect of DMSO on the Mechanical and Structural Properties of Model and Biological Membranes. Biophys J 2020; 119:274-286. [PMID: 32610089 PMCID: PMC7376087 DOI: 10.1016/j.bpj.2020.05.037] [Citation(s) in RCA: 47] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2019] [Revised: 04/08/2020] [Accepted: 05/22/2020] [Indexed: 12/23/2022] Open
Abstract
Dimethyl sulfoxide (DMSO) is widely used in a number of biological and biotechnological applications, mainly because of its effects on the cell plasma membrane, but the molecular origins of this action are yet to be fully clarified. In this work, we used two- and three-component synthetic membranes (liposomes) and the plasma membrane of human erythrocytes to investigate the effect of DMSO when added to the membrane-solvating environment. Fourier transform infrared spectroscopy and thermal fluctuation spectroscopy revealed significant differences in the response of the two types of liposome systems to DMSO in terms of the bilayer thermotropic behavior, available free volume of the bilayer, its excess surface area, and bending elasticity. DMSO also alters the mechanical properties of the erythrocyte membrane in a concentration-dependent manner and is capable of increasing membrane permeability to ATP at even relatively low concentrations (3% v/v and above). Taken in its entirety, these results show that DMSO is likely to have a differential effect on heterogeneous biological membranes, depending on their local composition and structure, and could affect membrane-hosted biological functions.
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Affiliation(s)
- Beatrice Gironi
- Dipartimento di Chimica Biologia e Biotecnologie, Università di Perugia, Perugia, Italy
| | - Zehra Kahveci
- Living Systems Institute and School of Biosciences, University of Exeter, Exeter, United Kingdom
| | - Beth McGill
- Department of Physics and Astronomy, University of Exeter, Exeter, United Kingdom
| | - Bob-Dan Lechner
- Department of Physics and Astronomy, University of Exeter, Exeter, United Kingdom
| | - Stefano Pagliara
- Living Systems Institute and School of Biosciences, University of Exeter, Exeter, United Kingdom
| | - Jeremy Metz
- Living Systems Institute and School of Biosciences, University of Exeter, Exeter, United Kingdom
| | - Assunta Morresi
- Dipartimento di Chimica Biologia e Biotecnologie, Università di Perugia, Perugia, Italy
| | - Francesca Palombo
- Department of Physics and Astronomy, University of Exeter, Exeter, United Kingdom
| | - Paola Sassi
- Dipartimento di Chimica Biologia e Biotecnologie, Università di Perugia, Perugia, Italy.
| | - Peter G Petrov
- Department of Physics and Astronomy, University of Exeter, Exeter, United Kingdom.
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Trummer T, Fox R, Koç JR, de Lima M, Otegbeye F. Cryopreservation of hematopoietic cells using a pre-constituted, protein-free cryopreservative solution with 5% dimethyl sulfoxide. Cytotherapy 2020; 22:613-616. [PMID: 32600975 DOI: 10.1016/j.jcyt.2020.05.006] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2020] [Revised: 05/18/2020] [Accepted: 05/19/2020] [Indexed: 11/17/2022]
Abstract
BACKGROUND AIMS Adequate cryopreservation techniques are critical to ensure optimal recovery of functional progenitor cells in hematopoietic cell (HC) transplantation, minimize risk of contamination and prevent infusion-related adverse events (irAEs). In this article, we provide graft function and infusion safety results observed by decreasing the concentration of dimethyl sulfoxide (DMSO) in cryopreservative media and by minimizing processor-dependent formulation. METHODS Ten HC products, collected after standard mobilization of multiple myeloma patients, were cryopreserved with PRIME-XV FreezIS (FreezIS) and compared with products previously cryopreserved with media formulated in-house to achieve a final DMSO concentration of 10% (Std10) and 5% (Std5). At infusion, HCs were analyzed for recovery of CD34+ cells and viability; irAEs and time to engraftment of neutrophils and platelets were also monitored. RESULTS Median CD34+ cell recovery for HC cryopreserved with Std10, Std5 and FreezIS was 38%, 78% and 68%, respectively (P = 0.0002). There were less frequent irAEs with Std5 and FreezIS (10%) compared with Std10 (80%) (P ≤ 0.0001). Median time to neutrophil engraftment was comparable (11 days) for all three groups, while platelet engraftment occurred at a median of 20, 19 and 17 days, respectively (p-values not significant). CONCLUSIONS FreezIS, a Good Manufacturing Practice-grade, pre-constituted cryopreservative with low DMSO content, maintains functional viability of the HC product while reducing the incidence of irAEs compared with 10% DMSO solutions. The pre-constituted nature of this agent also decreases processor-dependent handling, hence decreasing the risk of variability and infection.
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Affiliation(s)
- Tabatha Trummer
- University Hospitals Seidman Cancer Center, Cleveland, Ohio, USA
| | - Robert Fox
- University Hospitals Seidman Cancer Center, Cleveland, Ohio, USA
| | - Jane Reese Koç
- University Hospitals Seidman Cancer Center, Cleveland, Ohio, USA
| | - Marcos de Lima
- University Hospitals Seidman Cancer Center, Cleveland, Ohio, USA
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Pasha R, Howell A, Turner TR, Halpenny M, Elmoazzen H, Acker JP, Pineault N. Transient warming affects potency of cryopreserved cord blood units. Cytotherapy 2020; 22:690-697. [PMID: 32591113 DOI: 10.1016/j.jcyt.2020.04.039] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2020] [Accepted: 04/04/2020] [Indexed: 10/24/2022]
Abstract
BACKGROUND AIMS Cryopreserved cord blood units (CBUs) can be exposed to transient warming events (TWEs) during routine banking operations, which may affect their potency. NetCord-FACT guidelines recommend removal of these CBUs from inventory. The objective of this work was to evaluate warming kinetics of frozen CBUs in different settings to determine the optimal working environment and define the impact of different TWE scenarios on CB post-thaw quality and potency. METHODS The warming kinetics of frozen CBUs was influenced by both working surfaces and ambient working temperature, with cold plates providing better protection than vinyl or metal surfaces. Measurement of time for required operational activities revealed that CBUs are probably exposed to core temperatures greater than -150°C even when cold plates are used to reduce warming rates. RESULTS On the basis of the warming kinetics and observed operational activities, three TWE causing scenarios (control, typical, worst case) were investigated using a pool-and-split design and cell viability, recovery and potency (colony-forming unit [CFU]) assays were performed. TWEs were found to have little impact on the recovery of total nucleated cells or on the viability of CD34+ cells. In contrast, the viability and recovery of CD45+ cells in the smaller CBU compartments were reduced by TWEs. Moreover, the worst-case TWE reduced CFU recovery from CBUs, whereas the typical-scenario TWE had little effect. CONCLUSIONS Our results demonstrate that the distal segment underestimates the viability and potency of CBUs and that TWEs can affect the post-thaw viability and potency of CBUs. Although TWEs are almost inevitable during cord-blood banking operations, their effects must be diminished by reducing exposure time, using cold plates and strict operational protocols, to prevent worst-case TWEs.
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Affiliation(s)
- Roya Pasha
- Canadian Blood Services, Centre for Innovation, Ottawa, Ontario, Canada
| | - Anita Howell
- Canadian Blood Services, Centre for Innovation, Edmonton, Alberta, Canada
| | - Tracey R Turner
- Canadian Blood Services, Centre for Innovation, Edmonton, Alberta, Canada
| | - Mike Halpenny
- Canadian Blood Services, Cord Blood Bank and Stem Cell Manufacturing, Ottawa, Canada
| | - Heidi Elmoazzen
- Canadian Blood Services, Cord Blood Bank and Stem Cell Manufacturing, Ottawa, Canada
| | - Jason P Acker
- University of Alberta, Department of Laboratory Medicine and Pathology, Edmonton, Alberta, Canada
| | - Nicolas Pineault
- University of Ottawa, Biochemistry, Microbiology and Immunology Department, Ottawa, Canada.
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