3D Microwell Research Articles

A rapid and low-cost method to fabricate well of the well (WOW) dishes with arbitrary 3D microwell shapes for improved embryo culture

Despite its high cost, the success rate for in vitro fertilization (IVF) remains < 33% in humans, driving the need for new techniques to improve embryo culture outcomes. The well-of-the-well (WOW) culture system is a platform for in-vitro mammalian embryo culture that has been shown to enhance the developmental competence of embryos and clinical pregnancy rates in humans. However, discovery and testing of the best design for optimal embryo culture quality is hindered by the lack of a method to flexibly produce WOW dishes of various designs. Here, we present a low-cost and simple method to fabricate WOW dishes with microwells of arbitrary shapes and dimensions. We use a low-cost 3D printing service to fabricate a poly(dimethylsiloxane) (PDMS)-based WOW insert that can be paired with a standard in vitro fertilization (IVF) dish to create WOW dishes with new microwell shapes, including pyramidal and hemispherical designs. We validate the fabrication quality of the WOW inserts and demonstrate the utility of the assembled WOW dishes for observation and grading of mouse embryo quality. Moreover, our results indicate that WOW dishes with hemispherical microwells result in better culture outcomes than traditional flat-bottomed IVF dishes and those with other microwell shapes, including the semi-elliptical microwells used in commercial WOW dishes. The proposed fabrication strategy thus provides a way to rapidly fabricate and test new WOW dishes that may bolster IVF success rates.

Scalable production of homogeneous cardiac organoids derived from human pluripotent stem cells

Three-dimensional (3D) cultures are known to more closely mimic invivo conditions compared with 2D cultures. Cardiac spheroids (CSs) and organoids (COs) are useful for 3D tissue engineering and are advantageous for their simplicity and mass production for regenerative therapy and drug discovery. Herein, we describe a large-scale method for producing homogeneous human induced pluripotent stem cell (hiPSC)-derived CSs (hiPSC-CSs) and COs without scaffolds using a porous 3D microwell substratum with a suction system. Our method has many advantages, such as increased efficiency and improved functionality, homogeneity, and sphericity of hiPSC-CSs. Moreover, we have developed a substratum on a clinically relevant large scale for regenerative therapy and have succeeded in producing approximately 40,000 hiPSC-CSs with high sphericity at once. Furthermore, we efficiently produced a fused CO model consisting of hiPSC-derived atrial and ventricular cardiomyocytes localized on opposite sides of one organoid. This method will facilitate progress toward hiPSC-based clinical applications.

Direct single-cell antimicrobial susceptibility testing of Escherichia coli in urine using a ready-to-use 3D microwell array chip.

Empirical antibiotic therapies are prescribed for treating uncomplicated urinary tract infections (UTIs) due to the long turnaround time of conventional antimicrobial susceptibility testing (AST), leading to the prevalence of multi-drug resistant pathogens. We present a ready-to-use 3D microwell array chip to directly conduct comprehensive AST of pathogenic agents in urine at the single-cell level. The developed device features a highly integrated 3D microwell array, offering a dynamic range from 102 to 107 CFU mL-1, and a capillary valve-based flow distributor for flow equidistribution in dispensing channels and uniform sample distribution. The chip with pre-loaded reagents and negative pressure inside only requires the user to initiate AST by loading samples (∼3 s) and can work independently. We demonstrate an accessible sample-to-result workflow, including syringe filter-based bacteria separation and rapid single-cell AST on chip, which enables us to bypass the time-consuming bacteria isolation and pre-culture, speeding up the AST in ∼3 h from 2 days of conventional methods. Moreover, the bacterial concentration and AST with minimum inhibitory concentrations can be assessed simultaneously to provide comprehensive information on infections. With further development for multiple antibiotic conditions, the Dsc-AST assay could contribute to timely prescription of targeted drugs for better patient outcomes and mitigation of the threat of drug-resistant bacteria.

All-In-One Escherichia coli Viability Assay for Multi-dimensional Detection of Uncomplicated Urinary Tract Infections.

The urinary tract infections by antibiotic-resistant bacteria have been a serious public health problem and increase the healthcare costs. The conventional technologies of diagnosis and antimicrobial susceptibility testing (AST) relying on multiple culture-based assays are time-consuming and labor-intensive and thus compel the empirical antimicrobial therapies to be prescribed, fueling the prevalence of antimicrobial resistance. Herein, we propose an all-in-one Escherichia coli viability assay in an enclosed 3D microwell array chip, termed digital β-d-glucuronidase (GUS)-AST assay. It employs GUS, a specific metabolism-related enzyme, to convert the presence of E. coli into bright fluorescence. The random distribution of single bacteria in microwell array enables to quantify the E. coli concentrations by counting the positive microwells. We incorporate the most probable number with digital quantification to lower the limit of detection and expand the dynamic range to 7 orders. The digital GUS-AST assay is able to indicate the potency of antibiotics and determine the minimum inhibitory concentrations. A streamlined procedure of urine removal, bacterial separation, and digital GUS-AST is established to perform the direct analysis of bacteria population in urine. The sample-to-result workflow can be finished in 4.5 h with a limit of detection of 39 CFU/mL. With further development for additional pathogens and multiple antibiotic conditions, the digital GUS-AST assay could help physicians to prescribe timely targeted therapies for better patient outcomes and the minimum emergence of resistant bacteria.

Scaffold-free 3D culturing enhance pluripotency, immunomodulatory factors, and differentiation potential of Wharton’s jelly-mesenchymal stem cells

Mesenchymal stem cells (MSCs) show a decline in pluripotency and differentiation with increased cell culture passages in 2D cultures. The 2D monolayer culture fails to correctly imitate the architecture and microenvironments of in-vivo cell models. Alternatively, 3D culture may improve the simulations of in-vivo cell microenvironments with wide applications in cell culture and drug discovery. In the present study, we compared various 3D culturing techniques such as 3D micro-well (3D-S), hanging drop (HD), and ultra-low attachment (ULA) plate-based spheroid culture to study their effect on morphology, viability, pluripotency, cell surface markers, immunomodulatory factors, and differentiation capabilities of Wharton’s jelly-mesenchymal stem cells (WJ-MSCs). The cell morphology, viability, and senescence of 3D cultured WJ-MSCs were comparable to cells in 2D culture. The expression of pluripotency markers (OCT4, SOX2, and NANOG) was enhanced upto 2–8 fold in 3D cultured WJ-MSCs when compared to 2D culture. Moreover, the immunomodulatory factors (IDO, IL-10, LIF, ANG1, and VEGF) were significantly elevated in ULA based 3D cultured WJ-MSCs. Furthermore, significant enhancement in the differentiation potential of WJ-MSCs towards adipocyte (ADP and C/EBP-α), osteocyte (OPN and RUNX2), and definitive endodermal (SOX17, FOXA2, and CXCR4) lineages in 3D culture conditions were observed. Additionally, the osteogenic and adipogenic differentiation potential of WJ-MSCs over the time points 7 days, 14 days, and 28 days was also significantly increased in 3D culture groups. Our study demonstrates that stemness properties of WJ-MSCs were significantly enhanced in 3D cultures and ULA-based culture outperformed other methods with high pluripotency gene expression and enhanced differentiation potential. This study indicates the efficacy of 3D cultures to bridge the gap between 2D cell culture and animal models in regenerative medicine.

402.1: Towards a GMP-Compliant Protocol for the Differentiation of Human Pluripotent Stem Cells to Beta-like Cells for the Treatment of Type 1 Diabetes

Background: β-cell replacement therapy by allogeneic pancreas or islet transplantation is a promising approach for patients with type 1 diabetes. However, there is a scarcity of organ donors. The generation of insulin-producing pancreatic β-cells from human pluripotent stem cells (hPSC) in vitro would provide an unlimited cell source for drug discovery and cell replacement therapy for diabetes. Here we aim to generate β-cells under GMP (Good Manufacturing Practice)-compliant conditions that can be used for clinical application. Methods: We applied a modified seven-stage (30-day) differentiation protocol to generate hPSC-derived insulin-producing β-like cells in a 3D microwell culture system. Next, in order to scale up the production of hPSC-derived β-cells, we adapted the protocol to spinner flask culture. Differentiation was assessed by qPCR, immunocytochemistry and flowcytometry for stage-specific marker expression. β-cell function was evaluated in vitro by GSIS (Glucose-Stimulated Insulin Secretion), and in vivo by intraperitoneal glucose tolerance tests after transplantation of stage-7 cell clusters in immunodeficient mice and human C-peptide secretion was determined by ELISA. Results: In the microwell set-up, cells acquired a definitive endoderm phenotype at stage 1 (day 3), characterized by expression of SOX17 (87.2% ± 4.6; n=5), FOXA2 (82.8% ± 4.6; n=3), co-expression of c-KIT and CXCR4 (93.3% ± 5.6; n=4), and silencing of pluripotency marker OCT4 (2.8% ± 6; n=5). At the end of stage 4 (day 12), pancreatic progenitor cells were identified by the co-expression of PDX1/NKX6.1 (46.7% ± 3.5; n=3). Finally, at the end of stage 7 (day 30), C-peptide-positive β-like cells (49.5% ± 10.5; n=3) and glucagon-positive α-like cells (18.4%± 6.1; n=3) were present. The stimulation index upon exposure to glucose was 4.4 ± 3.1(n=5). Following transplantation of day-30 clusters into mice, stimulated human C-peptide levels reached 46 pmol/L (n=21), 70.5 pmol/L (n=19) and 327.4 pmol/L (n=11) at day 14, 28 and 60 respectively, indicating further maturation of the cells in vivo after transplantation. Next, we scaled up the cell culture in disposable spinner flasks, and obtained comparable differentiation efficiency. Conclusion: We can generate functional hPSCs-derived β-like cells in GMP-compliant conditions in vitro. We will apply these protocols on a clinical-grade PSC line that is currently in expansion (banking) in the GMP facility of our institute. hPSCs-derived β-like cells are a promising future alternative to donor islets for the treatment of type 1 diabetes.

Multiplexing physical stimulation on single human induced pluripotent stem cell-derived cardiomyocytes for phenotype modulation

Traditional in vitro bioengineering approaches whereby only individual biophysical cues are manipulated at any one time are highly inefficient, falling short when recapitulating the complexity of the cardiac environment. Multiple biophysical cues are present in the native myocardial niche and are essential during development, as well as in maintenance of adult cardiomyocyte (CM) phenotype in both health and disease. This study establishes a novel biofabrication workflow to study and manipulate hiPSC-CMs and to understand how these cells respond to a multiplexed biophysical environment, namely 3D shape and substrate stiffness, at a single cell level. Silicon masters were fabricated and developed to generate inverse patterns of the desired 3D shapes in bas relief, which then were used to mold the designed microwell arrays into a hydrogel. Polyacrylamide (PAAm) was modified with the incorporation of acrylic acid to provide a carboxylic group conjugation site for adhesion motifs, without compromising capacity to modulate stiffness. In this manner, two individual parameters can be finely tuned independently within the hydrogel: the shape of the 3D microwell and its stiffness. The design allows the platform to isolate single hiPSC-CMs to study solely biophysical cues in the absence of cell-cell physical interaction. Under physiologic-like physical conditions (3D shape resembling that of adult CM and 9.83 kPa substrate stiffness that mimics muscle stiffness), isolated single hiPSC-CMs exhibit increased Cx-43 density, cell membrane stiffness and calcium transient amplitude; co-expression of the subpopulation-related MYL2-MYL7 proteins; and higher anisotropism than cells in pathologic-like conditions (flat surface and 112 kPa substrate stiffness). This demonstrates that supplying a physiologic or pathologic microenvironment to an isolated single hiPSC-CM in the absence of any physical cell-to-cell communication in this biofabricated platform leads to a significantly different set of cellular features, thus presenting a differential phenotype. Importantly, this demonstrates the high plasticity of hiPSC-CMs even in isolation. The ability of multiple biophysical cues to significantly influence isolated single hiPSC-CM phenotype and functionality highlights the importance of fine-tuning such cues for specific applications. This has the potential to produce more fit-for-purpose hiPSC-CMs. Further understanding of human cardiac development is enabled by the robust, versatile and reproducible biofabrication techniques applied here. We envision that this system could be easily applied to other tissues and cell types where the influence of cellular shape and stiffness of the surrounding environment is hypothesized to play an important role in physiology.

3D Microwell Platforms for Control of Single Cell 3D Geometry and Intracellular Organization.

Cell structure and migration is impacted by the mechanical properties and geometry of the cell adhesive environment. Most studies to date investigating the effects of 3D environments on cells have not controlled geometry at the single-cell level, making it difficult to understand the influence of 3D environmental cues on single cells. Here, we developed microwell platforms to investigate the effects of 2D vs. 3D geometries on single-cell F-actin and nuclear organization. We used microfabrication techniques to fabricate three polyacrylamide platforms: 3D microwells with a 3D adhesive environment (3D/3D), 3D microwells with 2D adhesive areas at the bottom only (3D/2D), and flat 2D gels with 2D patterned adhesive areas (2D/2D). We measured geometric swelling and Young's modulus of the platforms. We then cultured C2C12 myoblasts on each platform and evaluated the effects of the engineered microenvironments on F-actin structure and nuclear shape. We tuned the mechanical characteristics of the microfabricated platforms by manipulating the gel formulation. Crosslinker ratio strongly influenced geometric swelling whereas total polymer content primarily affected Young's modulus. When comparing cells in these platforms, we found significant effects on F-actin and nuclear structures. Our analysis showed that a 3D/3D environment was necessary to increase actin and nuclear height. A 3D/2D environment was sufficient to increase actin alignment and nuclear aspect ratio compared to a 2D/2D environment. Using our novel polyacrylamide platforms, we were able to decouple the effects of 3D confinement and adhesive environment, finding that both influenced actin and nuclear structure.

3D Microwell Platform for Cardiomyocyte Differentiation of Human Pluripotent Stem Cells.

The generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs) represents a valuable tool for a myriad of in vitro applications, including drug screening, disease modeling and regenerative medicine. However, the success of these applications is dependent on the establishment of reliable, efficient, simple, and cost-effective differentiation methods. In this chapter, we describe an efficient and robust 3D platform for the generation of hPSC-CMs based on the use of a microwell culture system, which can be applied in any laboratory environment. Additionally, we will also describe protocols for the structural and functional characterization of the obtained CMs for further quality control upon differentiation.

Organotypic 3D Culture in Nanoscaffold Microwells Supports Salivary Gland Stem-Cell-Based Organization.

The self-organizing properties of stem cells have been exploited to generate organoids, organ-specific, cell-containing, three-dimensional (3D) structures. The present study aimed to introduce a novel bioengineering technique for driving the effective organization of adult tissue stem cells via niche-independent 3D microwell culture. Microwells were fabricated by photopatterning poly(ethylene glycol) hydrogel in the presence of an electrospun polycaprolactone nanofibrous scaffold. Human single clonal salivary gland stem cells (SGSCs) were cultured in nanofibrous microwells through two simple steps, priming and differentiation. Before the induction of 3D organization, single clonal SGSCs were preconditioned to aggregate to form 3D spheroids in different matrices, such as Matrigel, floating dish, and microwells. Expression of salivary stem cell markers and pluripotency markers was greater in 3D spheroid cultures than in 2D plastic culture. Lobular structures were organized by changing media, and those in microwells exhibited higher salivary acinar, ductal, and tight junction marker levels and decreased stem-cell marker levels relative to other 3D cultures. Furthermore, higher α-amylase secretion and intracellular calcium levels were observed in the presence of adrenergic or cholinergic agonists, respectively, along with an increased intracellular pH, suggesting more functional salivary organoid formation. These microwell-driven organoids also engrafted successfully into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Our results showed that microwell-cultured SGSCs organize into salivary structures and that this biomimetic 3D culture technique can promote effective generation of niche-independent single stem-cell-based 3D organoids.

Human liver organoids generated with single donor-derived multiple cells rescue mice from acute liver failure

BackgroundAcute liver failure (ALF) is a life-threatening disease with a high mortality rate. However, there are limited treatments or devices available for ALF therapy. Here, we aimed to develop a new strategy for ALF treatment by transplanting functional liver organoids (LOs) generated from single donor-derived human induced pluripotent stem cell (hiPSC) endoderm, endothelial cells (ECs), and mesenchymal cells (MCs).MethodsFirst, we isolated ECs and MCs from a single donor umbilical cord (UC) through enzyme digestion and characterized the UC-ECs and UC-MCs by flow cytometry. Second, using a nonviral reprogramming method, we generated same donor-derived hiPSCs from the UC-ECs and investigated their hepatic differentiation abilities. Finally, we simultaneously plated EC-hiPSC endoderm, UC-ECs, and UC-MCs in a three-dimensional (3D) microwell culture system, and generated single donor cell-derived differentiated LOs for ALF mouse treatment.ResultsWe obtained ECs and MCs from a single donor UC with high purity, and these cells provided a multicellular microenvironment that promoted LO differentiation. hiPSCs from the same donor were generated from UC-ECs, and the resultant EC-hiPSCs could be differentiated efficiently into pure definitive endoderm and further into hepatic lineages. Simultaneous plating of EC-hiPSC endoderm, UC-ECs, and UC-MCs in the 3D microwell system generated single donor cell-derived LOs (SDC-LOs) that could be differentiated into functional LOs with enhanced hepatic capacity as compared to that of EC-hiPSC-derived hepatic-like cells. When these functional SDC-LOs were transplanted into the renal subcapsules of ALF mice, they rapidly assumed hepatic functions and improved the survival rate of ALF mice.ConclusionThese results demonstrate that functional LOs generated from single donor cells can improve the condition of ALF mice. Functional SDC-LO transplantation provides a promising novel approach for ALF therapy.

Spheroid Coculture of Hematopoietic Stem/Progenitor Cells and Monolayer Expanded Mesenchymal Stem/Stromal Cells in Polydimethylsiloxane Microwells Modestly Improves In Vitro Hematopoietic Stem/Progenitor Cell Expansion.

While two-dimensional (2D) monolayers of mesenchymal stem/stromal cells (MSCs) have been shown to enhance hematopoietic stem/progenitor cell (HSPC) expansion in vitro, expanded cells do not engraft long term in human recipients. This outcome is attributed to the failure of 2D culture to recapitulate the bone marrow (BM) niche signal milieu. Herein, we evaluated the capacity of a novel three-dimensional (3D) coculture system to support HSPC expansion in vitro. A high-throughput polydimethylsiloxane (PDMS) microwell platform was used to manufacture thousands of uniform 3D multicellular coculture spheroids. Relative gene expression in 3D spheroid versus 2D adherent BM-derived MSC cultures was characterized and compared with literature reports. We evaluated coculture spheroids, each containing 25–400 MSCs and 10 umbilical cord blood (CB)-derived CD34+ progenitor cells. At low exogenous cytokine concentrations, 2D and 3D MSC coculture modestly improved overall hematopoietic cell and CD34+ cell expansion outcomes. By contrast, a substantial increase in CD34+CD38− cell yield was observed in PDMS microwell cultures, regardless of the presence or absence of MSCs. This outcome indicated that CD34+CD38− cell culture yield could be increased using the microwell platform alone, even without MSC coculture support. We found that the increase in CD34+CD38− cell yield observed in PDMS microwell cultures did not translate to enhanced engraftment in NOD/SCID gamma (NSG) mice or a modification in the relative human hematopoietic lineages established in engrafted mice. In summary, there was no statistical difference in CD34+ cell yield from 2D or 3D cocultures, and MSC coculture support provided only modest benefit in either geometry. While the high-throughput 3D microwell platform may provide a useful model system for studying cells in coculture, further optimization will be required to generate HSPC yields suitable for use in clinical applications.

3D hydrogel-based microwell arrays as a tumor microenvironment model to study breast cancer growth

The tumor microenvironment (TME) is distinctly heterogeneous and is involved in tumor growth, metastasis, and drug resistance. Mimicking this diverse microenvironment is essential for understanding tumor growth and metastasis. Despite the substantial scientific progress made with traditional cell culture methods, microfabricated three-dimensional (3D) cell cultures that can be precisely controlled to mimic the changes occur in the TME over tumor progression are necessary for simulating organ-specific TME in vitro. In this research, to simulate the breast cancer TME, microwell arrays of defined geometry and dimensions were fabricated using photo-reactive hydrogels for a cancer cell line and primary explant tissue culture. Microwell arrays fabricated from 4-arm polyethylene glycol acrylate and methacrylated gelatin with different degrees of methacrylation for controlled cell–matrix interactions and tunable stiffness were used to create a platform for studying the effects of distinct hydrogel compositions and stiffness on tumor formation. Using these microwell arrays, size-controlled spheroids of human breast cancer cell line HCC1806 were formed and the cell attachment properties, viability, metabolic activity, and migration levels of these spheroids were examined. In addition, primary mammary organoid tissues explanted from mice were successfully cultured in these hydrogel-based microwell arrays and the organoid morphology and viability, as well as organoid branching were studied. The microwell array platform developed and characterized in this study could be useful for generating a tissue-specific TME for in vitro high throughput studies of breast cancer development and progression as well as in drug screening studies for breast cancer treatment.

Hydrogel-encapsulated 3D microwell array for neuronal differentiation

We developed a photo-crosslinkable hydrogel-encapsulated three-dimensional (3D) microwell array for studying embryonic stem (ES) cell-derived neuronal differentiation. ES cells were cultured for 5 d in microwells and were subsequently encapsulated by photo-crosslinkable gelatin methacrylate (GelMA) and polyethylene glycol (PEG) hydrogels for an additional 7 d. We observed that ES cells cultured in PEG microwells became uniform-sized embryoid bodies (EBs) compared to those in GelMA microwells. Although ES cells were encapsulated by photo-crosslinkable GelMA and PEG hydrogels, they were highly viable. We demonstrated that uniform-sized EBs encapsulated by GelMA hydrogels in PEG microwells are largely differentiated into neuronal cells. It was revealed that neurites at the periphery of EBs in PEG microwells largely extended into the interface between GelMA hydrogels and PEG microwells for generating neuronal networks. Therefore, this photo-crosslinkable GelMA hydrogel-encapsulated PEG microwell array could be a potentially powerful tool for neurodegenerative disease applications.

Effects of 3D microwell culture on initial fate specification in human embryonic stem cells.

Several studies have demonstrated that 3D culture systems influence human embryonic stem cell (hESC) phenotypes and fate choices. However, the effect that these microenvironmental changes have on signaling pathways governing hESC behaviors is not well understood. Here, we have used a 3D microwell array to investigate differences in activation of developmental pathways between 2D and 3D cultures of both undifferentiated hESCs and hESCs undergoing initial differentiation in embryoid bodies (EBs). We observed increased induction into mesoderm and endoderm and differences in expression of genes from multiple signaling pathways that regulate development, including Wnt/β-catenin, TGF-β superfamily, Notch and FGF during EB-mediated differentiation, in 3D microwells as compared with the 2D substrates. In undifferentiated hESCs, we also observed differences in epithelial-mesenchymal transition phenotypes and the TGFβ/BMP pathway between cultures in 3D and 2D. These results illustrate that 3D culture influences multiple pathways that may regulate the differentiation trajectories of hESCs.

Microwell Regulation of Pluripotent Stem Cell Self-Renewal and Differentiation

The fates of pluripotent stem cells (PSCs), including survival, self-renewal, and differentiation, are regulated by chemical and mechanical cues presented in the three-dimensional (3D) microenvironment. Most PSC studies have been performed on two-dimensional substrates. However, 3D culture systems have demonstrated the importance of intercellular interactions in regulating PSC self-renewal and differentiation. Microwell culture systems have been developed to generate homogenous PSC colonies of defined sizes and shapes and to study how colony morphology affects cell fate. Using microwells, researchers have demonstrated that PSCs remain in a self-renewing undifferentiated state as a result of autocrine and paracrine signaling. Other studies have shown that microwell regulation of embryoid body size affects lineage commitment during differentiation via cell-cell contact and expression of soluble signals. In this review, we discuss recent advances in the design and utilization of 3D microwell platforms for studying intercellular regulation of PSC cell fate decisions and the underlying molecular mechanisms.

Effects of 3D microwell culture on growth kinetics and metabolism of human embryonic stem cells

Human embryonic stem cells (hESCs) hold potential in the field of tissue engineering, given their capacity for both limitless self-renewal and differentiation to any adult cell type. However, several limitations, including the ability to expand undifferentiated cells and efficiently direct differentiation at scales needed for commercial cell production, prevent realizing the potential of hESCs in tissue engineering. Numerous studies have illustrated that three-dimensional (3D) culture systems provide microenvironmental cues that affect hESC pluripotency and differentiation fates, but little is known about how 3D culture affects cell expansion. Here, we have used a 3D microwell array to model the differences in hESC growth kinetics and metabolism in two-dimensional (2D) versus 3D cultures. Our results demonstrated that 3D microwell culture reduced hESC size and proliferative capacity, and impacted cell cycle dynamics, lengthening the G1 phase and shortening the G2/M phase of the cell cycle. However, glucose and lactate metabolism were similar in 2D and 3D cultures. Elucidating the effects of 3D culture on growth and metabolism of hESCs may facilitate efforts for developing integrated, scalable cell expansion and differentiation processes with these cells.

Three-dimensional microwell arrays for cell culture

We propose the concept of three-dimensional (3D) microwell arrays for cell culture applications and highlight the importance of oxygen diffusion through pores in all three dimensions to enhance cell viability.

Stem cell plasticity, osteogenic differentiation and the third dimension

Different cues present in the cellular environment control basic biological processes. A previously established 3D microwell array was used to study dimensionality-related effects on osteogenic differentiation and plasticity of marrow stromal cells. To enable long-term culture of single cells in the array a novel surface functionalization technique was developed, using the principle of subtractive micro contact printing of fibronectin and surface passivation with a triblock-copolymer. Immunohistochemical stainings showed that when cultivated in 3D microenvironments, marrow stromal cells can be maintained in the wells for up to 7 days and be induced to commit to the osteogenic lineage. In conclusion, this work shows the modification of a 3D microwell array allowing the long term study of single stem cell plasticity and fate in controlled microenvironments.