• polymer synthesis
• polymers
• synthesis and processing

• No. 52373214 National Natural Science Foundation of China (National Science Foundation of China)
• No. 2022ZYD0030 Sichuan Provincial Department of Science and Technology | Sichuan Province Science and Technology Support Program
• No. Sklpme-2023-1-01 State Key Laboratory of Polymer Materials Engineering

• Cell aggregation
• Cell reprogramming
• Induced pluripotent stem cells
• Insoluble microenvironmental cues
• Superhydrophobic microwell array chip

• Humans
• Cellular Reprogramming
• Cell Differentiation
• Induced Pluripotent Stem Cells
• Hydrophobic and Hydrophilic Interactions

• drug screening
• expansion microscopy
• high-throughput
• imaging
• microarray

• Microscopy
• Drug Evaluation, Preclinical
• Microtubules
• High-Throughput Screening Assays/methods

Patient-derived tumor organoids (PDOs) have emerged as a reliable in vitro model for drug discovery. However, RNA sequencing-based analysis of PDOs treated with drugs has not been realized in a high-throughput format due to the limited quantity of organoids. Here, we translated a newly developed pooled RNA-seq methodology onto a superhydrophobic microwell array chip to realize an assay of genome-wide RNA output unified with phenotypic data (Grouped-seq). Over 10-fold reduction of sample and reagent consumption together with a new ligation-based barcode synthesis method lowers the cost to ∼$2 per RNA-seq sample. Patient-derived colorectal cancer (CRC) organoids with a number of 10 organoids per microwell were treated with four anti-CRC drugs across eight doses and analyzed by the Grouped-seq. Using a phenotype-assisted pathway enrichment analysis (PAPEA) method, the mechanism of actions of the drugs were correctly derived, illustrating the great potential of Grouped-seq for pharmacological screening with tumor organoids.

• T cells
• acoustic droplets
• cocultures
• tumor microenvironments
• tumor organoids

• Cell phenotype maintenance
• Collagen type I coating
• In vitro microenvironment
• Macromolecular crowding
• Substrate elasticity
• Surface topography

• CRISPR
• Cas9 protein
• genome
• microfluidics
• tissue engineering

• drug screening
• high throughput screening
• microfluidic technique
• review

• Drug Evaluation, Preclinical/methods
• High-Throughput Screening Assays
• Humans
• Microfluidic Analytical Techniques
• Microfluidics

Three-dimensional cultured patient-derived cancer organoids (PDOs) represent a powerful tool for anti-cancer drug development due to their similarity to the in vivo tumor tissues. However, the culture and manipulation of PDOs is more difficult than 2D cultured cell lines due to the presence of the culture matrix and the 3D feature of the organoids. In our other study, we established a method for lung cancer organoid (LCO)-based drug sensitivity tests on the superhydrophobic microwell array chip (SMAR-chip). Here, we describe a novel in situ cryopreservation technology on the SMAR-chip to preserve the viability of the organoids for future drug sensitivity tests. We compared two cryopreservation approaches (slow freezing and vitrification) and demonstrated that vitrification performed better at preserving the viability of LCOs. Next, we developed a simple procedure for in situ cryopreservation and thawing of the LCOs on the SMAR-chip. We proved that the on-chip cryopreserved organoids can be recovered successfully and, more importantly, showing similar responses to anti-cancer drugs as the unfrozen controls. This in situ vitrification technology eliminated the harvesting and centrifugation steps in conventional cryopreservation, making the whole freeze–thaw process easier to perform and the preserved LCOs ready to be used for the subsequent drug sensitivity test.

• cryopreservation
• high throughput screening
• in situ vitrification
• patient-derived organoid

• lab-on-a-chip
• cancer models
• lung cancer

• Adenocarcinoma/drug therapy
• Adenocarcinoma/genetics
• Adenocarcinoma/metabolism
• Adenocarcinoma/pathology
• Animals
• Antineoplastic Agents/pharmacology
• Carcinoma, Squamous Cell/drug therapy
• Carcinoma, Squamous Cell/genetics
• Carcinoma, Squamous Cell/metabolism
• Carcinoma, Squamous Cell/pathology
• Cell Culture Techniques/methods
• Cell Cycle/drug effects
• Cell Line, Tumor
• Cell Proliferation/drug effects
• Drug Screening Assays, Antitumor/instrumentation
• Drug Screening Assays, Antitumor/methods
• Gefitinib/pharmacology
• Humans
• Immunohistochemistry
• Lung Neoplasms/drug therapy
• Lung Neoplasms/genetics
• Lung Neoplasms/metabolism
• Lung Neoplasms/pathology
• Male
• Mice
• Mice, Inbred NOD
• Organoids/cytology
• Organoids/drug effects
• Organoids/pathology
• Pharmaceutical Preparations
• Xenograft Model Antitumor Assays

• National Natural Science Foundation of China (National Science Foundation of China)

Although the recent advances in stem cell engineering have gained a great deal of attention due to their high potential in clinical research, the applicability of stem cells for preclinical screening in the drug discovery process is still challenging due to difficulties in controlling the stem cell microenvironment and the limited availability of high-throughput systems. Recently, researchers have been actively developing and evaluating three-dimensional (3D) cell culture-based platforms using microfluidic technologies, such as organ-on-a-chip and organoid-on-a-chip platforms, and they have achieved promising breakthroughs in stem cell engineering. In this review, we start with a comprehensive discussion on the importance of microfluidic 3D cell culture techniques in stem cell research and their technical strategies in the field of drug discovery. In a subsequent section, we discuss microfluidic 3D cell culture techniques for high-throughput analysis for use in stem cell research. In addition, some potential and practical applications of organ-on-a-chip or organoid-on-a-chip platforms using stem cells as drug screening and disease models are highlighted.

• Stem cell
• Microfluidic technology
• Three-dimensional cell culture
• High-throughput screening

Intracellular gene delivery is normally required to study gene functions. A versatile platform able to perform both chemical transfection and viral transduction to achieve efficient gene modification in most cell types is needed. Here we demonstrated that high throughput chemical transfection, virus packaging, and transduction can be conducted efficiently on our previously developed superhydrophobic microwell array chip (SMAR-chip). A total of 169 chemical transfections were successfully performed on the chip in physically separated microwells through a few simple steps, contributing to the convenience of DNA delivery and media change on the SMAR-chip. Efficiencies comparable to the traditional transfection in multi-well plates (~65%) were achieved while the manual operations were largely reduced. Two transfection procedures, the dry method amenable for the long term storage of the transfection material and the wet method for higher efficiencies were developed. Multiple transfections in a scheduled manner were performed to further increase the transfection efficiencies or deliver multiple genes at different time points. In addition, high throughput virus packaging integrated with target cell transduction were also proved which resulted in a transgene expression efficiency of >70% in NIH 3T3 cells. In summary, the SMAR-chip based high throughput gene delivery is efficient and versatile, which can be used for large scale genetic modifications in a variety of cell types.

• SMAR-chip
• high throughput gene delivery
• transduction
• transfection
• virus packaging

• cell adhesion
• cellular reprogramming
• cytoskeleton
• extracellular microenvironment
• induced pluripotency

• Animals
• Cell Communication
• Cellular Reprogramming Techniques/methods
• Chromatin/genetics
• Chromatin/metabolism
• Epigenesis, Genetic
• Humans
• Induced Pluripotent Stem Cells/cytology
• Induced Pluripotent Stem Cells/metabolism
• Solubility
• Stem Cell Niche
• Tissue Engineering/methods
• Transcription Factors/genetics
• Transcription Factors/metabolism

Microfluidic systems have been regarded as a potential platform for high-throughput screening technology in drug discovery due to their low sample consumption, high integration, and easy operation. The handling of small-volume liquid is an essential operation in microfluidic systems, especially in investigating large-scale combination conditions. Here, we develop a nanoliter centrifugal liquid dispenser (NanoCLD) coupled with superhydrophobic microwell array chips for high-throughput cell-based assays in the nanoliter scale. The NanoCLD consists of a plastic stock block with an array of drilled through holes, a reagent microwell array chip (reagent chip), and an alignment bottom assembled together in a fixture. A simple centrifugation at 800 rpm can dispense ~160 nL reagents into microwells in 5 min. The dispensed reagents are then delivered to cells by sandwiching the reagent chip upside down with another microwell array chip (cell chip) on which cells are cultured. A gradient of doxorubicin is then dispensed to the cell chip using the NanoCLD for validating the feasibility of performing drug tests on our microchip platform. This novel nanoliter-volume liquid dispensing method is simple, easy to operate, and especially suitable for repeatedly dispensing many different reagents simultaneously to microwells.

• drug screening
• high-throughput screening
• microfluidics
• microwell array
• nanoliter liquid dispensing
• superhydrophobicity

• Laplace pressure
• droplet transport
• lab-on-a-chip devices
• patterned surface
• underwater superoleophobicity

The factors leading to changes in the organization of microbial assemblages at fine spatial scales are not well characterized or understood. However, they are expected to guide the succession of community development and function toward specific outcomes that could impact human health and the environment. In this study, we put forward a combined experimental and agent-based modeling framework and use it to interpret unique spatial organization patterns of H1-Type VI secretion system (T6SS) mutants of P. aeruginosa under spatial confinement. We find that key parameters, such as T6SS-mediated cell contact and lysis, spatial localization, relative species abundance, cell density and local concentrations of growth substrates and metabolites are influenced by spatial confinement. The model, written in the accessible programming language NetLogo, can be adapted to a variety of biological systems of interest and used to simulate experiments across a broad parameter space. It was implemented and run in a high-throughput mode by deploying it across multiple CPUs, with each simulation representing an individual well within a high-throughput microwell array experimental platform. The microfluidics and agent-based modeling framework we present in this paper provides an effective means by which to connect experimental studies in microbiology to model development. The work demonstrates progress in coupling experimental results to simulation while also highlighting potential sources of discrepancies between real-world experiments and idealized models.

• Pseudomonas aeruginosa
• Type VI secretion
• agent-based modeling
• microbial organization
• microbial succession
• silicon microwell arrays
• spatial confinement

• Animals
• Lab-On-A-Chip Devices
• Lasers
• Microfluidic Analytical Techniques/methods
• Organic Chemicals
• Sf9 Cells
• Single-Cell Analysis/methods

• Animals
• CRISPR-Cas Systems
• Cell Line
• Electroporation/instrumentation
• Electroporation/methods
• Gene Editing
• Gene Expression
• Gene Transfer Techniques
• Humans
• RNA, Guide, CRISPR-Cas Systems/genetics