• Piezo1
• Yoda1
• calcium signaling
• cell migration
• cell spheroids
• mechanosensitive channels
• mesenchymal stem cells

• Humans
• Spheroids, Cellular/metabolism
• Spheroids, Cellular/cytology
• Spheroids, Cellular/drug effects
• Mesenchymal Stem Cells/metabolism
• Mesenchymal Stem Cells/cytology
• Mesenchymal Stem Cells/drug effects
• Female
• Ion Channels/metabolism
• Ion Channels/genetics
• Endometrium/cytology
• Endometrium/metabolism
• Cell Movement
• Cells, Cultured
• Pyrazines
• Thiadiazoles

• 22-74-10037 Russian Science Foundation

There is a growing interest in utilizing 3D culture models for stem cell and cancer cell research due to their closer resemblance to in vivo environments. In this study, human mesenchymal stem cells (MSCs) were cultured using adipocytes and osteocytes as differentiative mediums on varying concentrations of chitosan substrate. Light microscopy was employed to capture cell images from the first day to the 21st day of differentiation. Accurate image segmentation is crucial for analyzing the morphological features of the spheroids during the experimental period and for understanding MSC differentiation dynamics for therapeutic applications. Therefore, we developed an innovative, weakly supervised model, aided by convolutional neural networks, to perform label-free spheroid segmentation. Since obtaining pixel-level ground truth labels through manual annotation is labor-intensive, our approach improves the overall quality of the ground-truth map by incorporating a multi-stage process within a weakly supervised learning framework. Additionally, we developed a robust learning scheme for spheroid detection, providing a reliable foundation to study MSC differentiation dynamics. The proposed framework was systematically evaluated using low-resolution microscopic data and challenging, noisy backgrounds. The experimental results demonstrate the effectiveness of our segmentation approach in accurately separating the spheroid from the background. Furthermore, it achieves performance comparable to fully supervised state-of-the-art approaches. To quantitatively evaluate our algorithm, extensive experiments were conducted using available annotated data, confirming the reliability and robustness of our method. Our computationally extracted features can confirm the experimental results regarding alterations in MSC viability, attachment, and differentiation dynamics among the substrates with three concentrations of chitosan used. We observed the formation of more compact spheroids with higher solidity and convex area, resulting improved cell attachment and viability on the 2% chitosan substrate. Additionally, this substrate exhibited a higher propensity for differentiation into osteocytes, as evidenced by the formation of smaller and more ellipsoid spheroids.

"Chitosan biofilms mimic in vivo environments for stem cell culture, advancing therapeutic and fundamental applications.” "Innovative weakly supervised model enables label-free spheroid segmentation in stem cell differentiation studies.” "Robust learning scheme achieves accurate spheroid separation, comparable to state-of-the-art approaches.”

• Convolutional neural network (CNN)
• Deep learning
• MSC differentiation dynamics
• Segmentation
• Spheroid
• Stem cells
• Weakly supervised learning

• Aminopolysaccharide
• Cell culture support
• Cold plasma
• Dip-coating
• Porous surface

• Chitosan/chemistry
• Gelatin/chemistry
• Polyesters/chemistry
• Fibroblasts/drug effects
• Fibroblasts/cytology
• Humans
• Surface Properties
• Gels/chemistry
• Ultraviolet Rays
• Plasma Gases/chemistry
• Hydrophobic and Hydrophilic Interactions
• Coated Materials, Biocompatible/chemistry
• Cross-Linking Reagents/chemistry
• Wettability

• growth
• human iPSCs
• hydrogel
• porosity
• spheroids
• stem cells
• viscoelastic properties

• NF-κB
• human amniotic fluid stem cells (hAFSCs)
• neural differentiation
• three-dimensional (3D) spheroid cultures

• Female
• Humans
• Pregnancy
• Amniotic Fluid/metabolism
• Cell Differentiation
• Nerve Tissue Proteins/metabolism
• NF-kappa B/metabolism
• RNA-Binding Proteins/metabolism
• Signal Transduction
• Stem Cells/metabolism
• Spheroids, Cellular
• Cell Culture Techniques, Three Dimensional/methods

• Na/K pump
• apoptosis
• human endometrial mesenchymal stem/stromal cells
• mitochondrial membrane potential
• ouabain
• potassium
• three-dimensional spheroids

The therapeutic potential of mesenchymal stem cells (MSCs) in the form of three-dimensional spheroids has been extensively demonstrated. The underlying mechanisms for the altered cellular behavior of spheroids have also been investigated. Cell membrane fluidity is a critically important physical property for the regulation of cell behavior, but it has not been studied for the spheroid-forming cells to date.

To explore the association between cell membrane fluidity and the morphological changes of MSC spheroids on the surface of biomaterials to elucidate the role of membrane fluidity during the spheroid-forming process of MSCs.

We generated three-dimensional (3D) MSC spheroids on the surface of various culture substrates including chitosan (CS), CS-hyaluronan (CS-HA), and polyvinyl alcohol (PVA) substrates. The cell membrane fluidity and cell morphological change were examined by a time-lapse recording system as well as a high-resolution 3D cellular image explorer. MSCs and normal/cancer cells were pre-stained with fluorescent dyes and co-cultured on the biomaterials to investigate the exchange of cell membrane during the formation of heterogeneous cellular spheroids.

We discovered that vesicle-like bubbles randomly appeared on the outer layer of MSC spheroids cultured on different biomaterial surfaces. The average diameter of the vesicle-like bubbles of MSC spheroids on CS-HA at 37 °C was approximately 10 μm, smaller than that on PVA substrates (approximately 27 μm). Based on time-lapse images, these unique bubbles originated from the dynamic movement of the cell membrane during spheroid formation, which indicated an increment of membrane fluidity for MSCs cultured on these substrates. Moreover, the membrane interaction in two different types of cells with similar membrane fluidity may further induce a higher level of membrane translocation during the formation of heterogeneous spheroids.

Changes in cell membrane fluidity may be a novel path to elucidate the complicated physiological alterations in 3D spheroid-forming cells.

• Cell membrane fluidity
• Cell-cell interaction
• Cellular spheroids
• Chitosan
• Hyaluronan

In recent years, conditioned medium (CM) obtained from the culture of mesenchymal stromal/stem cells (MSCs) has been shown to effectively promote tissue repair and modulate the immune response in vitro and in different animal models, with potential for application in regenerative medicine. Using CM offers multiple advantages over the implantation of MSCs themselves: 1) simpler storage, transport, and preservation requirements, 2) avoidance of the inherent risks of cell transplantation, and 3) potential application as a ready-to-go biologic product. For these reasons, a large amount of MSCs research has focused on the characterization of the obtained CM, including soluble trophic factors and vesicles, preconditioning strategies for enhancing paracrine secretion, such as hypoxia, a three-dimensional (3D) environment, and biochemical stimuli, and potential clinical applications. In vitro preconditioning strategies can increase the viability, proliferation, and paracrine properties of MSCs and therefore improve the therapeutic potential of the cells and their derived products. Specifically, dynamic cultivation conditions, such as fluid flow and 3D aggregate culture, substantially impact cellular behaviour. Increased levels of growth factors and cytokines were observed in 3D cultures of MSC grown on orbital or rotatory shaking platforms, in stirred systems, such as spinner flasks or stirred tank reactors, and in microgravity bioreactors. However, only a few studies have established dynamic culture conditions and protocols for 3D aggregate cultivation of MSCs as a scalable and reproducible strategy for CM production. This review summarizes significant advances into the upstream processing, mainly the dynamic generation and cultivation of MSC aggregates, for de CM manufacture and focuses on the standardization of the soluble factor production.

• Corporación de Fomento de la Producción
• Comisión Nacional de Investigación Científica y Tecnológica

• microsphere
• poly(ethylene glycol)
• polysaccharide nanogel
• regenerative medicine
• spheroid
• stem cell

• 3D printing
• cartilage
• gelatin scaffolds
• mesenchymal stem cells
• temporomandibular joint

• 3DM-spheroid culture
• Cell differentiation
• Gene expression
• MAPK
• Mesenchymal stem cells
• NF-kB
• Signaling

• 3D culture
• Biomaterial
• Graft-versus-host disease
• Immunomodulation
• Inflammatory bowel disease
• MSC

• 3D tissues
• biomaterials
• cellular aggregates
• spheroid engineering
• spheroids

• NRF-2020K2A9A2A08000126 National Research Foundation of Korea
• FY2020/NRF-2019R1A2C2084965 National Research Foundation of Korea
• NRF-2020R1A4A3078645 National Research Foundation of Korea
• 10050526 Technology Innovation Program
• 10050526 Ministry of Trade, Industry and Energy
• NRF-2020R1A4A3078645 Ministry of Education

Mesenchymal stromal/stem cells (MSCs), which generally expand into adherent monolayers, readily lose their proliferative and multilineage potential following repeated passages. Floating culture systems can be used to generate MSC spheroids, which are expected to overcome limitations associated with conventional adherent cultures while facilitating scaffold-free cell transplantation. However, the phenotypic characteristics of spheroids after long-term culture are unknown. In addition, regenerative therapies require new culture systems to maintain their undifferentiated state. In this study, we established a novel culture method employing three-dimensional (3D) “shaking” to generate MSC spheroids using bone marrow derived MSCs. Floating 3D cultures of mouse or human MSCs formed spheroids after shaking (85–95 rpm), within 1 month. These spheroids maintained their osteogenic-, adipogenic-, and chondrogenic-differentiation capacity. The adipogenic-differentiation capacity of adherent cultured mouse and human MSCs, which is lost following several passages, was remarkedly restored by shaking-culture. Notably, human MSC spheroids exhibited a renewable “undifferentiated MSC-pool” property, wherein undifferentiated MSCs grew from spheroids seeded repeatedly on a plastic culture dish. These data suggest that the shaking-culture method maintains and restores multipotency that is lost following monolayer expansion and thereby shows potential as a promising strategy for regenerative therapies with mesenchymal tissues.

• mesenchymal stem cell
• multipotency
• shaking-culture
• spheroid
• stem cell pool

• Cell Culture Techniques
• Chitosan
• Hydrogels
• Indoles
• Microfluidics
• Microscopy, Electron, Scanning
• Polymers

Mesenchymal stem cells are currently tested as a promising tool for the treatment of a wide range of human diseases. Enhanced therapeutic potential of spheroids formed from these cells has been proved in numerous studies, however, the fundamental basics of this effect are still being discussed. In this work, we showed that endometrial mesenchymal stem/stromal cells (eMSCs) assembled in spheroids possess a higher therapeutic efficacy compared to cells grown in monolayer in the treatment of the defects that are non-specific for eMSC tissue origin – skin wounds. With the purpose to elucidate the possible causes of superior spheroid potency, we compared the tolerance of eMSC cultivated in spheres and monolayer to the stress insults. Using genetically encoded hydrogen peroxide biosensor HyPer, we showed that three-dimensional configuration (3D) helped to shield the inner cell layers of spheroid from the external H₂O₂-induced oxidative stress. However, the viability of oxidatively damaged eMSCs in spheroids appeared to be much lower than that of monolayer cells. An extensive analysis, which included administration of heat shock and irradiation stress, revealed that cells in spheroids damaged by stress factors activate the apoptosis program, while in monolayer cells stress-induced premature senescence is developed. We found that basal down-regulation of anti-apoptotic and autophagy-related genes provides the possible molecular basis of the high commitment of eMSCs cultured in 3D to apoptosis. We conclude that predisposition to apoptosis provides the programmed elimination of damaged cells and contributes to the transplant safety of spheroids. In addition, to investigate the role of paracrine secretion in the wound healing potency of spheroids, we exploited the in vitro wound model (scratch assay) and found that culture medium conditioned by eMSC spheroids accelerates the migration of adherent cells. We showed that 3D eMSCs upregulate transcriptional activator, hypoxia-inducible factor (HIF)-1, and secret ten-fold more HIF-1-inducible pro-angiogenic factor VEGF (vascular endothelial growth factor) than monolayer cells. Taken together, these findings indicate that enhanced secretory activity can promote wound healing potential of eMSC spheroids and that cultivation in the 3D cell environment alters eMSC vital programs and therapeutic efficacy.

• apoptosis
• endometrial stem/stromal cells
• heat shock
• mesenchymal stem cells
• oxidative stress
• spheroids
• stress response
• therapeutic potential

• biomaterials
• cleft lip/palate
• extracellular matrix
• extracellular matrix remodeling
• metalloproteinases
• palatal reconstruction
• palatogenesis
• tissue bioengineering