Hydrogel Microwell Arrays Research Articles

Development and characterization of a novel poly(N-isopropylacrylamide)-based thermoresponsive photoink and its applications in DLP bioprinting.

The field of 3-dimensional (3D) bioprinting has significantly expanded capabilities in producing precision-engineered hydrogel constructs, and recent years have seen the development of various stimuli-responsive bio- and photoinks. There is, however, a distinct lack of digital light processing (DLP)-compatible photoinks with thermoresponsivity. To remedy this, this work focuses on formulating and optimizing a versatile ink for DLP printing of thermoresponsive hydrogels, with numerous potential applications in tissue engineering, drug delivery, and adjacent biomedical fields. Photoink optimization was carried out using a multifactorial study design. The optimized photoink yielded crosslinked hydrogels with strong variations in hydrophobicity (contact angles of 44.4° <LCST, 71.0° >LCST), indicating marked thermoresponsivity. Mechanical- and rheological characterization of the printed hydrogels showed significant changes above the LCST: storage- and loss moduli both increased and loss tangent and compressive modulus decreased above this temperature (P ≤ 0.01). The highly cytocompatible hydrogel microwell arrays yielded both single- and multilayer spheroids with human dermal fibroblasts (HDFs) and HeLa cells successfully. Evaluation of the release of encapsulated model macro- (bovine serum albumin, BSA) and small molecule (rhodamine B) drugs in a buffer solution showed an interestingly inverted thermoresponsive release profile with >80% release at room temperature and about 50-60% release above the gels' LCST. All told, the optimized ink holds great promise for multiple biomedical applications including precise and high-resolution fabrication of complex tissue structures, development of smart drug delivery systems and 3D cell culture.

Automated detection of patterned single-cells within hydrogel using deep learning

Single-cell analysis has been widely used in various biomedical engineering applications, ranging from cancer diagnostics, and immune response monitoring to drug screening. Single-cell isolation is fundamental for observing single-cell activities and an automatic finding method of accurate and reliable cell detection with few possible human errors is also essential. This paper reports trapping single cells into photo patternable hydrogel microwell arrays and isolating them. Additionally, we present an object detection-based DL algorithm that detects single cells in microwell arrays and predicts the presence of cells in resource-limited environments at the highest possible mAP (mean average precision) of 0.989 with an average inference time of 0.06 s. This algorithm leads to the enhancement of the high-throughput single-cell analysis, establishing high detection precision and reduced experimentation time.

Microstructured Hyaluronic Acid Hydrogel for Tooth Germ Bioengineering.

Tooth loss has been found to adversely affect not just masticatory and speech functions, but also psychological health and quality of life. Currently, teeth replacement options include dentures, bridges, and implants. However, these artificial replacement options remain inferior to biological replacements due to their reduced efficiency, the need for replacements, and the risk of immunological rejection. To this end, there has been a heightened interest in the bioengineering of teeth in recent years. While there have been reports of successfully regenerated teeth, controlling the size and shape of bioengineered teeth remains a challenge. In this study, methacrylated hyaluronic acid (MeHA) was synthesized and microstructured in a hydrogel microwell array using soft lithography. The resulting MeHA hydrogel microwell scaffold resembles the shape of a naturally developing human tooth germ. To facilitate the epithelial–mesenchymal interactions, human adult low calcium high temperature (HaCaT) cells were seeded on the surface of the hydrogels and dental pulp stem cells (DPSCs) were encapsulated inside the hydrogels. It was found that hydrogel scaffolds were able to preserve the viability of both types of cells and they appeared to favor signaling between epithelial and mesenchymal cells, which is necessary in the promotion of cell proliferation. As such, the hydrogel scaffolds offer a promising system for the bioengineering of human tooth germs in vitro.

Gastruloids generated without exogenous Wnt activation develop anterior neural tissues

SummaryWhen stimulated with a pulse from an exogenous WNT pathway activator, small aggregates of mouse embryonic stem cells (ESCs) can undergo embryo-like axial morphogenesis and patterning along the three major body axes. However, these structures, called gastruloids, currently lack the anterior embryonic regions, such as those belonging to the brain. Here, we describe an approach to generate gastruloids that have a more complete antero-posterior development. We used hydrogel microwell arrays to promote the robust derivation of mouse ESCs into post-implantation epiblast-like (EPI) aggregates in a reproducible and scalable manner. These EPI aggregates break symmetry and axially elongate without external chemical stimulation. Inhibition of WNT signaling in early stages of development leads to the formation of gastruloids with anterior neural tissues. Thus, we provide a new tool to study the development of the mouse after implantation in vitro, especially the formation of anterior neural regions.

Fabrication of PNIPAm-based thermoresponsive hydrogel microwell arrays for tumor spheroid formation

Complex three-dimensional (3D) cell cultures are being increasingly implemented in biomedical research as they provide important insights into complex cancer biology, and cell-cell and cell-matrix interactions in the tumor microenvironment. However, most methods used today for 3D cell culture are limited by high cost, the need for specialized skills, low throughput and the use of unnatural culture environments. We report the development of a unique biomimetic hydrogel microwell array platform for the generation and stress-free isolation of cancer spheroids. The poly N-isopropylacrylamide-based hydrogel microwell array (PHMA) has thermoresponsive properties allowing for the attachment and growth of cell aggregates/ spheroids at 37 °C, and their easy isolation at room temperature (RT). The reversible phase transition of the microwell arrays at 35 °C was confirmed visually and by differential scanning calorimetry. Swelling/ shrinking studies and EVOS imaging established that the microwell arrays are hydrophilic and swollen at temperatures <35 °C, while they shrink and are hydrophobic at temperatures >35 °C. Spheroid development within the PHMA was optimized for seeding density, incubation time and cell viability. Spheroids of A549, HeLa and MG-63 cancer cell lines, and human lung fibroblast (HLF) cell line generated within the PHMAs had relatively spherical morphology with hypoxic cores. Finally, using MG-63 cell spheroids as representative models, a proof-of-concept drug response study using doxorubicin hydrochloride was conducted. Overall, we demonstrate that the PHMAs are an innovative alternative to currently used 3D cell culture techniques, for the high-throughput generation of cell spheroids for disease modeling and drug screening applications.

Investigation of Cell Microenvironment Effect on Cell Behaviors Using Electrochemical Scanning Probe Microscopy

Cell microenvironment (chemical, mechanical, electric) directly effects cell functions and behaviors (biology, mechanics, electrophysiology). Study of cell microenvironment effect on cell functions and behaviors is important for understanding the pathological mechanism of diseases (heart disease, cancer). Different kinds of hydrogels, which is an ideal material to mimic 3D extracellular matrix (ECM) of cell, have been developed and used to construct 3D cell microenvironment in vitro. To understand the effect of cell microenvironment constructed by hydrogels on cell behaviors, the technique which can characterize both the property of hydrogels and the topography and multi-behaviors of cells at mm/nm scale in an in-situ, non-damage way is needed. Scanning electrochemical microscopy (SECM) and scanning ion conductance microscopy (SICM) are two kinds of electrochemical scanning probe microscopy with the capability of providing morphology and performing chemical analyses of cells in an in situ, reversible and non-destructive way. In our work, we firstly constructed cell microenvironment based on three kinds of functional hydrogels (i.e., self-healing hydrogels, magnetic hydrogels and hydrogel microwell arrays). Then SECM was applied to in-situ track the self-healing process of hydrogel, the H2O2 consumption across magnetic hydrogel nanocomposites and the oxygen permeability across hydrogel microwell arrays. The effect of mechanical microenvironment on the redox state of cardiomyocytes and cardiac fibroblasts was also characterized by SECM. To further characterize the cell morphology at nm-scale, we also home built our SICM system and used it to characterize the cell morphology and cell volume on hydrogels with different stuffiness. Figure 1

Imaging oxygen microenvironment in hydrogel microwell array

Hydrogel microwell arrays (HMAs) have been wildly used for engineering cell microenvironments by providing well-controlled biophysical and biochemical cues (e.g., three-dimensional (3-D) physical boundary, biomolecule coating) for cells. Among these cues, the oxygen microenvironment has shown great effect on the cellular physiological processes. However, it is currently technically challenging to characterize the local oxygen microenvironment within HMAs. Here, we prepared HMAs with different cross-linking concentrations to adjust the structural and physical properties of HMAs. Then we introduced a scanning electrochemical microscopy (SECM)-based electrochemical method to map the surface topography and oxygen microenvironment around HMAs. The SECM results show both the 3-D topography and the oxygen permeability of HMAs in aqueous solution. The obtained oxygen permeability of HMAs increases with increasing the cross-linking concentration, and the microwell boundaries show the highest oxygen permeability throughout HMAs. This work demonstrates that SECM offers a high spatial resolution and in situ method for characterization of the topography and the local oxygen permeability of HMAs, which can provide useful information for better engineering cell microenvironments through optimizing HMAs design.

Generation of uniform-sized multicellular tumor spheroids using hydrogel microwells for advanced drug screening

Even though in vitro co-culture tumor spheroid model plays an important role in screening drug candidates, its wide applications are currently limited due to the lack of reliable and high throughput methods for generating well-defined and 3D complex co-culture structures. Herein, we report the development of a hydrogel microwell array to generate uniform-sized multicellular tumor spheroids. Our developed multicellular tumor spheroids are structurally well-defined, robust and can be easily transferred into the widely used 2D culture substrates while maintaining our designed multicellular 3D-sphere structures. Moreover, to develop effective anti-cancer therapeutics we integrated our recently developed gold-graphene hybrid nanomaterial (Au@GO)-based photothermal cancer therapy into a series of multicellular tumor spheroid co-culture system. The multicellular tumor spheroids were harvested onto a two-dimensional (2D) substrate, under preservation of their three-dimensional (3D) structure, to evaluate the photothermal therapy effectiveness of graphene oxide (GO)-wrapped gold nanoparticles (Au@GO). From the model of co-culture spheroids of HeLa/Ovarian cancer and HeLa/human umbilical vein endothelial cell (HUVEC), we observed that Au@GO nanoparticles displayed selectivity towards the fast-dividing HeLa cells, which could not be observed to this extent in 2D cultures. Overall, our developed uniform-sized 3D multicellular tumor spheroid could be a powerful tool for anticancer drug screening applications.

Stromal cell-laden 3D hydrogel microwell arrays as tumor microenvironment model for studying stiffness dependent stromal cell-cancer interactions

Tumor properties such as growth and metastasis are dramatically dependent on the tumor microenvironment (TME). However, the diversity of the TME including the stiffness and the composition of the extracellular matrix (ECM), as well as the involvement of stromal cells, makes it extremely difficult to establish proper in vitro models for studying tumor growth and metastasis. In this research, we fabricated a stromal cell-laden microwell array system with tunable stiffness ranging from 200 Pa up to 3 kPa, which covers the stiffness range of normal and cancerous mammary tissues, to study the effect of ECM stiffness on stromal-cancer interaction. Our results showed that, tumor spheroids closely interacted with the pre-adipocyte stromal cells encapsulated within the microwell array, influencing their differentiation and maturation degree in a stiffness related manner. They inhibited adipogenesis in high stiffness tissue constructs that were at breast cancer stiffness range, while the inhibition effect diminished in the low stiffness tissue constructs that were at normal human breast tissue range. Furthermore, the 3D structure of tumor spheroids was shown to be important for the inhibition of the adipogenesis, as conditioned media from monolayer culture of cancer cells did not show any significant effect. These results show, for the first time in literature, that stromal-cancer interactions are highly dependent on ECM stiffness. The biomimetic TME platform developed here is a powerful organ-specific cancer model for studying the involvement of stromal cells in early mammary tumorigenesis and metastasis, and could be powerful platform for high-throughput drug discovery.

Uniform-sized neurosphere-mediated motoneuron differentiation in microwell arrays.

We developed the photocrosslinkable hydrogel microwell arrays for uniform-sized neurosphere-mediated motoneuron differentiation. Neural stem cells (NSCs) were obtained from embryonic cerebral cortex and spinal cord. To generate uniform-sized neurospheres in a homogeneous manner, the dissociated cells were cultured in the hydrogel microwell arrays for 3 days. Uniform-sized neurospheres harvested from microwell arrays were replated into laminin-coated substrate. In parallel, uniform-sized neurospheres cultured in microwell arrays were encapsulated by photocrosslinkable gelatin methacrylate hydrogels in a three-dimensional manner. We demonstrated the effect of hydrogel microwell sizes (e.g., 50, 100, 150μm in diameter) on motoneuron differentiation, showing that the largest uniform-sized neurospheres derived from embryonic spinal cord efficiently differentiated into motoneurons. Therefore, this hydrogel microwell array could be a powerful array to regulate the uniform-sized neurosphere-mediated motoneuron differentiation.

Isolated Reporter Bacteria in Supramolecular Hydrogel Microwell Arrays.

The combination of supramolecular hydrogels formed by low molecular weight gelator self-assembly via noncovalent interactions within a scaffold derived from polyethylene glycol (PEG) affords an interesting approach to immobilize fully functional, isolated reporter bacteria in novel microwell arrays. The PEG-based scaffold serves as a stabilizing element and provides physical support for the self-assembly of the C2-phenyl-derived gelator on the micrometer scale. Supramolecular hydrogel microwell arrays with various shapes and sizes were used to isolate single or small numbers of Escherichia coli TOP10 pTetR-LasR-pLuxR-GFP. In the presence of the autoinducer N-(3-oxododecanoyl) homoserine lactone, the entrapped E. coli in the hydrogel microwell arrays showed an increased GFP expression. The shape and size of microwell arrays did not influence the fluorescence intensity and the projected size of the bacteria markedly, while the population density of seeded bacteria affected the number of bacteria expressing GFP per well. The hydrogel microwell arrays can be further used to investigate quorum sensing, reflecting communication in inter- and intraspecies bacterial communities for biology applications in the field of biosensors. In the future, these self-assembled hydrogel microwell arrays can also be used as a substrate to detect bacteria via secreted autoinducers.

Synthesis of microgel sensors for spatial and temporal monitoring of protease activity.

Proteases are involved in almost every important cellular activity, from embryonic morphogenesis to apoptosis. To study protease activity in situ, hydrogels provide a synthetic mimic of the extracellular matrix (ECM) and have utility as a platform to study activity, such as those related to cell migration, in three-dimensions. While 3-dimensional visualization of protease activity could prove quite useful to elucidate the proteolytic interaction at the interface between cells and their surrounding environment, there has been no versatile tool to visualize local proteolytic activity in real time. Here, micron-sized gels were synthesized by inverse suspension polymerization using thiolene photo-click chemistry. The size distribution was selected to avoid cellular uptake and to lower cytotoxicity, while simultaneously allowing the integration of peptide-based FRET sensors of local cell activity. Proteolytic activity of collagenase was detected within an hour via changes in fluorescence of embedded microgels; incubation of microgel sensors with A375 melanoma cells showed upregulated MMP activity in the presence of soluble fibronectins in media. The microgel sensors were readily incorporated into both gelatin and poly(ethylene glycol) (PEG) hydrogels and used to successfully detect spatiotemporal proteolytic activity of A375 melanoma cells. Finally, a tumor model was constructed from a hydrogel microwell array that was used to aggregate A375 melanoma cells, and local variations in proteolytic activity were monitored as a function of distance from the cell aggregate center.

Production of Uniform 3D Microtumors in Hydrogel Microwell Arrays for Measurement of Viability, Morphology, and Signaling Pathway Activation.

Despite significant investments in cancer research and drug discovery/development, the rate of new cancer drug approval is ≤5% and most cases of metastatic cancer remain incurable. Ninety-five percent of new cancer drugs fail in clinical development because of a lack of therapeutic efficacy and/or unacceptable toxicity. One of the major factors responsible for the low success rate of anticancer drug development is the failure of preclinical models to adequately recapitulate the complexity and heterogeneity of human cancer. For throughput and capacity reasons, high-throughput screening growth inhibition assays almost exclusively use two-dimensional (2D) monolayers of tumor cell lines cultured on tissue culture-treated plastic/glass surfaces in serum-containing medium. However, these 2D tumor cell line cultures fail to recapitulate the three-dimensional (3D) context of cells in solid tumors even though the tumor microenvironment has been shown to have a profound effect on anticancer drug responses. Tumor spheroids remain the best characterized and most widely used 3D models; however, spheroid sizes tend to be nonuniform, making them unsuitable for high-throughput drug testing. To circumvent this challenge, we have developed defined size microwell arrays using nonadhesive hydrogels that are applicable to a wide variety of cancer cell lines to fabricate size-controlled 3D microtumors. We demonstrate that the hydrogel microwell array platform can be applied successfully to generate hundreds of uniform microtumors within 3-6 days from many cervical and breast, as well as head and neck squamous cell carcinoma (HNSCC) cells. Moreover, controlling size of the microwells in the hydrogel array allows precise control over the size of the microtumors. Finally, we demonstrate the application of this platform technology to probe activation as well as inhibition of epidermal growth factor receptor (EGFR) signaling in 3D HNSCC microtumors in response to EGF and cetuximab treatments, respectively. We believe that the ability to generate large numbers of HNSCC microtumors of uniform size and 3D morphology using hydrogel arrays will provide more physiological in vitro 3D tumor models to investigate how tumor size influences signaling pathway activation and cancer drug efficacy.

New application of scanning electrochemical microscopy incharac-terization of hydrogel microwell arrays

Hydrogel microwell arrays are new soft substrates for cell cultivation. The microwell morphology has direct influence on the behavior of cells cultured within microwells. But the conventional morphology characterization techniques of hydrogel microwell arrays can not in situ characterize hydrogel microwell arrays in aqueous solution and also not in a reversible way. Using the oxygen dissolved in water as the redox mediator, scanning electrochemical microscopy (SECM) was applied to in situ characterize the morphology of microwell array made of polyethylene glycol dimethacrylate (PEGDMA) hydrogel in aqueous solution. Both the two-dimensional and three-dimensional surface morphology images of PEGDMA microwell arrays were obtained. This work demonstrates that SECM is a new method of in situ characterization of hydrogel microwell arrays in a non-destructive and reversible way with providing three-dimensional surface morphology information.

Microwell arrays for uniform-sized embryoid body-mediated endothelial cell differentiation

Embryonic stem (ES) cell is of great interest cell source in regenerating tissue constructs. We hypothesized that the interaction of cell-extracellular matrices (ECMs) would enable the control of ES cell differentiation pathway. We fabricated the hydrogel microwell array system to regulate uniform-sized embryoid bodies (EBs) and replate into various ECM components (e.g., gelatin, collagen I, fibronectin, laminin, and Matrigel). We demonstrated that collagen I and laminin largely induced ES cell-derived endothelial cell differentiation compared to gelatin. We also characterized ECMs-dependent endothelial cell differentiation by evaluating the endothelial gene expression, showing that Flk1 endothelial gene was highly expressed on collagen I. We also demonstrated the effect of the integrin on uniform-sized EBs-derived endothelial cell differentiation, showing that integrin α1 was largely expressed on laminin. Therefore, the cell-ECM interaction could be potentially powerful for controlling the uniform-sized EBs-derived endothelial cell differentiation.

Inverting microwell array chip for the cultivation of human induced pluripotent stem cells with controlled aggregate size and geometrical arrangement.

We present a novel cell culture chip, namely, "inverting microwell array chip," for cultivation of human induced pluripotent stem cells. The chip comprises a lower hydrogel microwell array and an upper polystyrene culture surface. We demonstrate the formation of uniform cellular aggregates in the microwell array, and after inversion, a culture with controlled aggregate size and geometrical arrangement on the polystyrene surface. Here, we report effects of cell concentrations on a cultivation sequence in the chip.

Microwell-mediated micro cartilage-like tissue formation of adipose-derived stem cell

In cartilage tissue engineering, various technical approaches using postnatal stem cells, three-dimensional (3D) scaffolds composed of synthetic or natural polymers, and culture systems have been applied to develop 3D cartilage-like tissue. In this study, scaffold-free 3D micro-cartilage-like tissue was developed via microwell-mediated cell spheroid formation and 3D dynamic chondrogenic culture in a bioreactor. First, homogenous micro-cell spheroids were generated by the self-condensation of adipose tissue-derived stem cells (ADSCs) in microfabricated poly(ethylene glycol) (PEG) hydrogel microwells. Next, chondrogenic differentiation of the micro-ADSC spheroids was induced in the presence of transforming growth factor-beta 3 under dynamic 3D culture conditions using a high aspect ratio vessel bioreactor. Several hundred viable ADSC spheroids could be generated at a time from ADSC culture in PEG microwells. The 3D dynamic chondrogenic culture of ADSCs in the bioreactor facilitated the chondrogenic mRNA expression of proteins such as sox-9, runx2, osterix, type II collagen, and aggrecan, and the well deposition of glycosaminoglycan and type II collagen, which finally generated micro-cartilage-like tissue. Therefore, the hydrogel microwell arrays could be useful for efficiently deriving initial cell condensation-mediated chondrogenic differentiation, and for developing 3D cell-based micro-cartilage-like tissue with stem cells in a controlled manner.

Controlled local presentation of matrix proteins in microparticle–laden cell aggregates

Multi-cellular aggregates are found in healthy and diseased tissues, and while cell-cell contact is important for regulating many cell functions, cells also interact, to varying degrees, with extra-cellular matrix (ECM) proteins. Islets of Langerhans are one such example of cell aggregates in contact with ECM, both at the periphery of the cluster and dispersed throughout. While several studies have investigated the effect of reintroducing contact with ECM proteins on islet cell survival and function, the majority of these experiments only allow contact with the exterior cells. Thus, cell-culture platforms that enable the study of ECM-cell interactions throughout multi-cellular aggregates are of interest. Here, local presentation of ECM proteins was achieved using hydrogel microwell arrays to incorporate protein-laden microparticles during formation of MIN6 β-cell aggregates. Varying the microparticle seeding density reproducibly controlled the number of microparticles incorporated within three-dimensional aggregates (i.e., total amount of protein). Further, a relatively uniform spatial distribution of laminin- and fibronectin-coated microparticles was achieved throughout the x-, y-, and z-directions. Multiple ECM proteins were presented to β-cells in concert by incorporating two distinct populations of microparticles throughout the aggregates. Finally, scaling the microwell device dimensions allowed for the formation of two different sized cell-particle aggregates, ∼80 and 160 µm in diameter. While the total number of microparticles incorporated per aggregate varied with size, the fraction of the aggregate occupied by microparticles was affected only by the microparticle seeding density, indicating that uniform local concentrations of proteins can be preserved while changing the overall aggregate dimensions.

Hydrogel Microwell Arrays Allow the Assessment of Protease-Associated Enhancement of Cancer Cell Aggregation and Survival.

Current routine cell culture techniques are only poorly suited to capture the physiological complexity of tumor microenvironments, wherein tumor cell function is affected by intricate three-dimensional (3D), integrin-dependent cell-cell and cell-extracellular matrix (ECM) interactions. 3D cell cultures allow the investigation of cancer-associated proteases like kallikreins as they degrade ECM proteins and alter integrin signaling, promoting malignant cell behaviors. Here, we employed a hydrogel microwell array platform to probe using a high-throughput mode how ovarian cancer cell aggregates of defined size form and survive in response to the expression of kallikreins and treatment with paclitaxel, by performing microscopic, quantitative image, gene and protein analyses dependent on the varying microwell and aggregate sizes. Paclitaxel treatment increased aggregate formation and survival of kallikrein-expressing cancer cells and levels of integrins and integrin-related factors. Cancer cell aggregate formation was improved with increasing aggregate size, thereby reducing cell death and enhancing integrin expression upon paclitaxel treatment. Therefore, hydrogel microwell arrays are a powerful tool to screen the viability of cancer cell aggregates upon modulation of protease expression, integrin engagement and anti-cancer treatment providing a micro-scaled yet high-throughput technique to assess malignant progression and drug-resistance.

Confined 3D microenvironment regulates early differentiation in human pluripotent stem cells

The therapeutic potential of human pluripotent stem (hPS) cells is threatened, among various problems, by the difficulty to homogenously direct cell differentiation into specific lineages. The transition from hPSC into committed differentiated cells is accompanied by secretome activity, remodeling of extracellular matrix and self-organization into germ layers. In this work, we aimed to investigate how different three-dimensional microenvironments regulate the early differentiation of the three germ layers in human embryonic stem (hES) cells derived embryoid bodies. In particular, a permeable, biocompatible, hydrogel microwell array was specifically designed for recreating a confined niche in which EB secreted molecules accumulate in accordance with hydrogel diffusional cut-off. Fluorescence recovery after photobleaching technique was performed to accurately evaluate hydrogel permeability, mesh size and diffusional cutoff for soluble molecules. Three different culture conditions of EB culture were analyzed: suspension, confinement in microwells of width/depth ratio 1:1 and 1:2. Results show that EBs cultured in microwells are viable and have comparable average size after 8 days culture. Whole genome microarrays show that significative differential gene expression was observed between suspension and confined EBs culture. In particular, EBs culture in microwells promotes the expression of genes involved in pattern specification processes, brain development, ectoderm and endoderm differentiation. On the contrary, suspension EBs express instead genes involved in mesoderm specification and heart development. These results suggest that local accumulation of EBs secreted molecules drives differentiation patterns, as confirmed by immunofluorescence of germ layer markers, in hydrogel confined EB culture from both hES cells and human induced pluripotent stem (hiPS) cells. Our findings highlight an additional potential role of biomaterial in controlling hPSC differentiation through secreted factor niche specification.