Concave Microwell Arrays Research Articles

Reflow-molded deep concave microwell arrays for robust and large-scale production of embryoid bodies.

Embryonic stem cell (ESC)-derived aggregates, called embryoid bodies (EBs), are powerful in vitro models used to study human development and disease. However, the cost-effective and large-scale production of homogeneous EBs still remains a challenge. Here, we report a rapid, straightforward method for fabricating closely arrayed deep concave microwells, enabling the mass production of uniform EBs from single cell suspensions. By simply combining micromilling, caramel replica molding, and thermal reflow, we generate convex micromolds with high aspect ratios and excellent surface smoothness. Benefitting from the nature of reflow, this method can produce rounded bottom polydimethylsiloxane (PDMS) microwells, which are not easily achieved with standard soft lithography techniques but critical to producing spherical EBs. To achieve optimal concave microwells, we investigated the effect of thermal reflow temperature and time on the surface smoothness and roundness of the finished microwells. In addition, to further improve the utility of this method, we also investigated the effect of microwell aspect ratio (AR) on the loss of EBs during medium manipulation. The capability of this deep concave microwell system was validated by rapidly generating a large number of human embryonic stem cell (hESC)-derived EBs and then efficiently differentiating them into a cardiac lineage. The proposed fabrication method and deep concave microwell platform are highly practical, and thus will benefit the mass production of EBs for potential tissue regeneration and cell therapy applications.

Flow-Based Three-Dimensional Co-Culture Model for Long-Term Hepatotoxicity Prediction

We developed concave microwell arrays to establish a size-controllable 3-D co-culture liver model for in vitro drug toxicity testing, to predict hepatotoxicity. The interaction of hepatocytes with hepatic stellate cells (HSCs) was investigated by co-culturing primary 3-D hepatocyte spheroids and HSCs (heterosphere), using 3-D liver-on-a-chip. The effect of HSCs was investigated during spheroid formation; they were involved in controlling the organization of spheroidal aggregates and the formation of tight cell–cell contacts. Scanning electron microscopy (SEM) images showed that co-cultured spheroids with smoother surfaces in the flow chip aggregated more tightly and rapidly, compared to mono-cultured spheroids, until 13 days. Metabolic function analysis revealed that heterospheres secreted 40% more albumin and urea than hepatospheres on day 13. Additionally, an acetaminophen (AAP) and isoniazid (INH) concentration-dependent increase in CYP3A4 expression was detected in the 3-D cultures, and an increase in Lactate dehydrogenase (LDH) release after AAP and INH treatment was observed. CYP1A2, Mrp1 and UGT1A5 mRNA expression levels in the heterospheres and hepatospheres were evaluated from days 3 to 13. To examine the potential for toxicity testing in the flow-conditioned culture of the heterospheres, we evaluated cytotoxicity using the endpoint LDH release in the heterospheres and hepatospheres. IC50 values for AAP and INH after 24 h of exposure were calculated from the dose–response curves of the compounds. Flow-conditioned heterosphere culture results suggest that it may be suitable for long-term culture and cytotoxicity testing. Thus, our co-culture system closely resembles the in vivo environment and allows long-term in vitro hepatotoxicity prediction.

A cell-loss-free concave microwell array based size-controlled multi-cellular tumoroid generation for anti-cancer drug screening.

The 3D multi-cellular tumoroid (MCT) model is an in vivo-like, avascular tumor model that has received much attention as a refined screening platform for drug therapies. Several types of research have been efforted to improve the physiological characteristics of the tumor microenvironment (TME) of the in vivo-like MCTs. Size-controlled MCTs have received much attention for obtaining highly reproducible results in drug screening assays and achieving a homogeneous and meaningful level of biological activities. Here, we describe an effective method for fabricating the size-controlled in vivo-like MCTs using a cell-loss-free (CLF) microwell arrays. The CLF microwell arrays was fabricated by using the simple operation of laser carving of a poly (methyl methacrylate) (PMMA) master mold. We also demonstrated the biophysicochemical effect of tumor microenvironment (TME) resident fibroblasts through the expression of TGFβ, αSMA, Type I-, IV collagen, angiogenesis related markers on tumorigenesis, and confirmed the drug response of MCTs with anti-cancer agents. This technology for the fabrication of CLF microwell arrays could be used as an effective method to produce an in vitro tumor model for cancer research and drug discovery.

Fabrication of agarose concave petridish for 3D-culture microarray method for spheroids formation of hepatic cells.

Liver is one of the most important organ in the body. But there are many limitations about liver transplantation for liver failure. It is quite important to develop the xenogeneic biological liver for providing an alternation to transplantation or liver regeneration. In this paper, we proposed a method to construct a novel kind of agarose 3D-culture concave microwell array for spheroids formation of hepatic cells. Using the 3D printing method, the microwell array was fabricated with an overall size of 6.4 mm × 6.4 mm, containing 121 microwells with 400 μm width/400 μm thickness. By exploiting the Polydimethylsiloxane (PDMS) membranes as a bridge, we finally fabricated the agarose one. We co-cultured three types of liver cells with bionics design in the microwell arrays. Using the methods described above, the resulting co-formed hepatocyte spheroids maintained the high viability and stable liver-specific functions. This engineered agarose concave microwell array could be a potentially useful tool for forming the elements for biological liver support. After developing the complete system, we also would consider to scale up the application of this system. It will be not only applied to the therapy of human organ damage, but also to the development of disease models and drug screening models.

Construction of neurospheroids via surface modified concave microwells

Developing a three-dimensional (3D) neural tissue model is important to comprehensively understand neural development and neuronal degeneration associated with various neurological disorders such as axonopathy and neuronopathy. Here, a new microplatform suitable for constructing neuronal spheroids (neurospheroids) was developed by modulating cell–surface interactions. The inner surface of a polydimethylsiloxane (PDMS) concave microwell array extensively used in in vitro cell aggregation was modified with typical extracellular matrix (ECM) molecules or carbon nanotubes to control neural spheroid formation. Modulating neuronal cell–ECM interactions could tune 3D intercellular interactions and spheroidal functionality. Neurite outgrowth, a neuronal marker for complex interneuronal signaling, was found to be tightly regulated by cell–ECM interactions in a confined space. Furthermore, amyloid-β (Aβ)-induced axonopathy representing a pathological feature of neurodegenerative diseases in vivo was examined in this study to monitor the degeneration of neurite outgrowth and alteration of neuronal morphology in these neurospheroids. The proposed neural tissue model could be used to study various neurodegenerative diseases in the future.

Networked concave microwell arrays for constructing 3D cell spheroids

The engineered three-dimensional (3D) cell cultivation system for the production of multicellular spheroids has attracted considerable attention due to its improved in vivo relevance to cellular communications compared with the traditional two-dimensional (2D) cell culture platform. The formation and maintenance of cell spheroids in a healthy condition is the critical factor for tissue engineering applications such as the repair of damaged tissues, the development of organ replacement parts and preclinical drug tests. However, culturing spheroids in conventional isolated single wells shows limited yield and reduced maintenance periods due to the lack of proper supplies of nutrition as well as intercellular chemical signaling. Here, we develop novel networked concave microwell arrays for the effective construction of 3D multi-cellular spheroids. The proposed method provides a suitable structure for the diffusion of oxygen, water-soluble nutrients and cytokines for cell–cell interactions between the spheroids in neighboring microwells. We have further demonstrated that hepatocyte spheroid cultured networked concave microwells show enhanced cell viability and albumin secretion compared to the un-networked control group over two weeks. Our results reveal that multi-cellular functionality can be tuned up by networking individual 3D spheroids without supplying additional chemicals or biological supplements. We anticipate our result to be useful in high-throughput cellular screening platforms to study cell–cell interactions, in response to diverse chemical stimuli as well as the development of the in vivo mimicking of the customized 3D tissue culture system.

Enhanced oxygen permeability in membrane-bottomed concave microwells for the formation of pancreatic islet spheroids.

In this study, we present a noble design to control the oxygen distribution in concave microwell arrays for the formation of highly functional pancreatic islet spheroids by engineering the bottom of the microwells. Our new platform significantly enhanced oxygen permeability that turned out to improve cell viability and spheroidal functionality compared to the conventional thick-bottomed 3-D culture system. Therefore, we believe that this could be a promising medical biotechnology platform to further develop high-throughput tissue screening system as well as in vivo-mimicking customised 3-D tissue culture systems.

Reproducible Construction of Surface Tension-Mediated Honeycomb Concave Microwell Arrays for Engineering of 3D Microtissues with Minimal Cell Loss.

The creation of engineered 3D microtissues has attracted prodigious interest because of the fact that this microtissue structure is able to mimic in vivo environments. Such microtissues can be applied extensively in the fields of regenerative medicine and tissue engineering, as well as in drug and toxicity screening. Here, we develop a novel method of fabricating a large number of dense honeycomb concave microwells via surface tension-mediated self-construction. More specifically, in order to control the curvature and shape of the concavity in a precise and reproducible manner, a custom-made jig system was designed and fabricated. By applying a pre-set force using the jig system, the shape of the honeycomb concave well was precisely and uniformly controlled, despite the fact that wells were densely packed. The thin wall between the honeycomb wells enables the minimization of cell loss during the cell-seeding process. To evaluate the performance of the honeycomb microwell array, rat hepatocytes were seeded, and spheroids were successfully formed with uniform shape and size. Liver-specific functions such as albumin secretion and cytochrome P450 were subsequently analyzed. The proposed method of fabricating honeycomb concave wells is cost-effective, simple, and reproducible. The honeycomb well array can produce multiple spheroids with minimal cell loss, and can lead to significant contributions in tissue engineering and organ regeneration.

Local delivery of stem cell growth factors with injectable hydrogels for myocardial therapy

Event Abstract Back to Event Local delivery of stem cell growth factors with injectable hydrogels for myocardial therapy Renae Waters1, 2, Settimio Pacelli1, Ryan Maloney1, 2 and Arghya Paul1, 2 1 University of Kansas, Chemical and Petroleum Engineering, United States 2 School of Engineering, University of Kansas, Bioengineering Graduate Program, United States Introduction: Stem cell therapy offers a promising approach to regenerate damaged cardiac tissue after acute myocardial infarction. Recent evidences have attributed such improvement to the broad repertoire of angiogenic paracrine factors (growth factors, chemokines and exosomes) secreted by the stem cells, known as secretome[1]. Here, we intend (i) to efficiently bio-manufacture cell secretome from 3D stem cell aggregates using a microfluidic system and (ii) efficiently deliver the harnessed secretome for cardiac repair and revascularization applications using a hydrogel-based injectable delivery system. Experiments and Results: As a first step, a microfluidic device consisting of deep concave microwell arrays was fabricated using soft lithography microscale techniques. Results demonstrate that bone marrow derived human mesenchymal stem cell (hMSC) suspensions, injected to the microfluidic chip through inlet port, form uniformly sized aggregates (~220μm diameter) within 2 days. Computational analysis confirmed rapid oxygen diffusion in the microwells. 3 days post-incubation, hMSC secretome (4.5-9 times increase in angiogenic protein concentrations compared to 2D culture including VEGF, FGF, Angiogenin) was collected, leaving behind the hMSC aggregates at the bottom of the well. As a next step, a physically cross-linked injectable nanocomposite hydrogel, comprising of silicate nanoplatelets and gelatin, was formulated to deliver the harnessed hMSC secretome to the target site. In vitro studies confirmed controlled release of bioactive hMSC growth factors from the hydrogel which induced proliferation of human endothelial cells (HUVEC). For in vivo studies rat model with acute myocardial infarction (n=5) was used. The secretome carrying hydrogel was injected intramyocardially at the per-infarct region of the heart, post infarction as mentioned in earlier work[2]. After 3 weeks, the animals were sacrificed and the injected tissue regions were examined for histological analysis. Masson’s Trichome staining confirmed reduced scar area in the treated Secretome+ group (3D cell aggregates) compared to control group (secretome from 2D cell culture). Immunostaining with CD31 antibodies also confirmed significantly high local myocardial angiogenesis at the injected site in Secretome+ group. These data confirms the angiogenic therapeutic potential of the developed hydrogel in vitro and in vivo. Conclusion: Take together, this study reports a novel strategy to utilize cell secretome, as an alternate to traditional cell therapy, for myocardial regeneration therapy. The strategy can also be used for other biomedical applications, such as wound healing and vascular tissue engineering. Arghya Paul likes to acknowledge the Institutional Development Award (IDeA) from the National Institute of General Medical Sciences, National Institutes of Health (NIH), under Award Number P20GM103638.

A concave microwell array fabricated using the ommatidium of the common fruit fly for efficient cell culture

Top left: SEM of compound eye ofDrosophila melanogasterreplica in PDMS. Bottom left: SEM of MCF-7 cell grown in the micro well. Bottom right: confocal of the MCF-7 cells grown for 72 h.

Three-dimensional brain-on-a-chip with an interstitial level of flow and its application as an in vitro model of Alzheimer's disease.

There has been a growing need for in vitro models of neurodegenerative diseases such as Alzheimer's disease that would enable a better understanding of etiology and faster development of treatment strategies. However, meeting this demand has been held back by the limited ability to mimic the in vivo microenvironment in an in vitro system. Here, we developed a microfluidic chip based on three-dimensional (3D) neurospheroids that more closely mimics the in vivo brain microenvironment by providing a constant flow of fluid that is readily observed in the interstitial space of the brain. Uniform neurospheroids, with cell-cell interactions and contacts in all directions, were formed in concave microwell arrays, and a slow interstitial level of flow was maintained using an osmotic micropump system. Using this platform, we investigated the effect of flow on neurospheroid size, neural network, and neural differentiation. Neurospheroids cultured with flow were larger and formed more robust and complex neural networks than those cultured under static conditions, suggesting an effect of the interstitial level of slow and diffusion-dominant flow on continuous nutrient, oxygen, and cytokine transport and removal of metabolic wastes. We also tested the toxic effects of amyloid-β, which is generally considered to be the major contributor in Alzheimer's disease. Amyloid-β treatment via an osmotic micropump significantly reduced the viability of neurospheroids and caused a significantly more destruction of neural networks, compared to the amyloid-β treatment under static conditions. By adding in vivo-like microenvironments, we propose this 3D culture-based microfluidic chip as an in vitro brain model for neurodegenerative disease and high-throughput drug screening.

Networked neuro-spheres formed by topological attractants for engineering of 3-dimensional nervous system

The nervous systems including central and peripheral nervous system have an important role in transmitting signals from brain to organs and tissues. Due to such critical function of nervous system, considerable effort has been tried to establish in vitro nervous model. In this paper, the neuro-spheres networked by the nerve-like structure were created using concave well arrays connected by the hemicylindrical channels. The concave microwells and the hemicylindrical channels were fabricated using the surface tension of viscose liquid PDMS prepolymer for the creation of neuro-spheres-networking (NSN). To investigate the topological effect of the concave-well-hemicylindrical-channel-networking (CWHCN), comparative experiments were conducted on a conventional cylindrical-wells-rectangular-channel-networking (CWRCN). Neuro-progenitor cells from the rat were seeded on the concave well arrays connected by the hemicylindrical channels and cultured for 10 days. Small neuro-pehroids consisting of neurons and glia cells were autonomously formed in the concave microwell arrays. These neuro-spheres were networked by the nerve-like structures formed along the CWHCN. In order to confirm the interconnection of the neurites in the NSN, a calcium imaging experiment was performed to measure the calcium flux along the nerve-like networking. To demonstrate further use of the networked neuro-networking for brain regeneration, we transferred the NSN onto hydrogel maintaining approximately 90% viability, and this model is expected to be used for the regeneration of a damaged brain.

Advanced micromachining of concave microwells for long term on-chip culture of multicellular tumor spheroids.

A novel approach based on advanced micromachining is demonstrated to fabricate concave microwell arrays for the formation of high quality multicellular tumor spheroids. Microfabricated molds were prepared using a state-of-the-art CNC machining center, containing arrays of 3D convex micropillars with size ranging from 150 μm to 600 μm. Microscopic imaging of the micropillars machined on the mold showed smooth, curved microfeatures of a dramatic 3D shape. Agarose microwells could be easily replicated from the metallic molds. EMT-6 tumor cells seeded in the primary macrowell sedimented efficiently to the bottom of the concave microwells and formed multicellular spheroids within 48 h. Dense and homogeneous multicellular spheroids were obtained after 10 days of culture, confirming the suitability of the proposed approach. To facilitate long term spheroid culture and reliable on-chip drug assay, polydimethylsiloxane microwells were also replicated from the metallic molds. A solvent swelling method was adapted and optimized to Pluronic F127 towards physically entrapping the block copolymer molecules within the polydimethylsiloxane network and in turn to improve long term cell-binding resistance. Homogeneous multicellular spheroids were efficiently formed in the concave microwells and on-chip drug assays could be reliably carried out using curcumin as a model anti-cancer drug. Advanced micromachining provides an excellent technological solution to the fabrication of high quality concave microwells.

3D co-culturing model of primary pancreatic islets and hepatocytes in hybrid spheroid to overcome pancreatic cell shortage

Here, a spheroidal 3D co-culture model of primary (rat) pancreatic islets and hepatocytes with uniform size and shape was developed using hemispheric concave microwell arrays. We conducted morphological and functional analyses of hybrid spheroids versus mono-cultures of islets or hepatocytes (controls). For the establishment of a 3D hybrid model, a broad range of cell ratios – 1:1, 1:3, 1:5, 1:7, 3:1, 5:1 and 7:1 mixture – of hepatocytes and pancreatic islets were used. As control, each hepatocyte and pancreatic islet were mono-cultured forming 3D spheroids. The transient morphology of spheroid formation in 9 culture models was observed using optical microscopy. Cell viability under these culture environments was assessed, and the morphologies of the outer and inner porous cell-spheroid structures were investigated using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and imaging of stained spheroid sections. The pancreatic islet-specific function of hybrid spheroids was evaluated by measuring insulin secretion and in vivo test by xenotransplantation of encapsulated spheroids in microfibers with a consistent maintenance of normal blood glucose levels over 4 weeks, while liver-specific functions were measured in terms of albumin secretion, urea secretion and cytochrome P450 activity. These diverse observations and evaluations validated the positive and bidirectional effects of co-cultured 3D spheroids. The proposed 3D co-culture model demonstrated that both cells appeared to support each other's functions strongly in spheroids, even though smaller proportions of each cell type was evaluated compared to mono-culture models, suggesting that the proposed model could help overcome the problem of cell shortages in clinical applications.

Size-controllable networked neurospheres as a 3D neuronal tissue model for Alzheimer's disease studies

Intensive in vitro studies on the neurotoxicity of amyloid beta have been conducted for decades; however, a three-dimensional neuronal tissue model for Alzheimer's disease has not yet been achieved. In this study, we developed size-controllable networked neurospheres comprised of cerebral cortical neuronal cells that mimics the cytoarchitecture of the cortical region of the brain. The toxicity of amyloid beta on the neurosphere model was assessed quantitatively and qualitatively. Decreased cell viability after amyloid beta exposure was demonstrated using MTT and live/dead assays. Neurite degeneration after amyloid beta exposure was evident in both SEM and fluorescence images. Ultrastructural features of apoptotic neurons were analyzed and quantitative analysis of synapsin II concentration and an acetylcholine assay were also performed. The three-dimensional neurospheres, produced using a concave microwell array, are a potential in vitro model for Alzheimer's disease studies.

Functional clustering of pancreatic islet cells using concave microwell array

Pancreatic islet transplantation is an ideal therapy for type I diabetes. Interestingly, small size of islets may exhibit superior performance in transplantation in terms of function and hypoxia. To this end, large size of islets was dissociated to generate islet single cells and they were re-aggregated to make small size of islet-cell-cluster (ICC). However, the function of insulin secretion from ICCs was damaged due to weak cell-to-cell interaction. To overcome this limitation, here the dispersed islet single cells were re-aggregated at the concave microwell array that was fabricated with poly(dimethylsiloxane) (PDMS) by using soft-lithography and mold-replication technology. The aggregated ICCs had uniform size, and their insulin secretion in response to the change of low and high glucose concentration was similar to that of intact islets since the cell-cell interaction of islet single cells was strongly induced due to the shape of concave microwell. Also, after cultivation for 2 weeks, the expression of hypoxia-induced factor- 1α(HIF-1α; a hypoxia marker protein) in the ICCs was rare; meaning that hypoxic state in the central area of ICCs was attenuated. On the other hand, the ICCs generated in flat-bottomed wells had abnormal insulin secretion caused by weak cell-to-cell interaction. In addition, after cultivation for 2 weeks, the generated ICCs in flat-bottomed well had weak signal of HIF-1α, while intact islets had its strong signal. Therefore, new culture system using concave microwell array may be a promising alternative to success fully prepare functional ICCs. Open image in new window

In situ formation and collagen-alginate composite encapsulation of pancreatic islet spheroids

In this study, we suggest in situ islet spheroid formation and encapsulation on a single platform without replating as a method for producing mono-disperse spheroids and minimizing damage to spheroids during encapsulation. Using this approach, the size of spheroid can be controlled by modulating the size of the concave well. Here, we used 300 μm concave wells to reduce spheroid size and thereby eliminating the central necrosis caused by large volume. As the encapsulation material, we used alginate and collagen-alginate composite (CAC), and evaluated their suitability through diverse in vitro tests, including measurements of viability, oxygen consumption rate (OCR), hypoxic damage to encapsulated spheroids, and insulin secretion. For in situ encapsulation, alginate or CAC was spread over a concave microwell array containing spheroids, and CaCl 2 solution was diffused through a nano-porous dialysis membrane to achieve uniform polymerization, forming convex structures. By this process, the formation of uniform-size islet spheroids and their encapsulation without an intervening replating step was successfully performed. As a control, intact islets were evaluated concurrently. The in vitro test demonstrated excellent performance of CAC-encapsulated spheroids, and on the basis of these results, we transplanted the islet spheroids-encapsulated with CAC into the intraperitoneal cavity of mice with induced diabetes for 4 weeks, and evaluated subsequent glucose control. Intact islets were also transplanted as control to investigate the effect of encapsulation. Transplanted CAC-encapsulated islet spheroids maintained glucose levels below 200 mg/dL for 4 weeks, at which they were still active. At the end of the implantation experiment, we carried out intraperitoneal glucose tolerance test (IPGTT) in mice to investigate whether the implanted islets remained responsive to glucose. The glucose level in mice with CAC-encapsulated islet spheroids dropped below 200 mg/dL 60 min after glucose injection and was stably maintained. In conclusion, the proposed encapsulation method enhances the viability and function of islet spheroids, and protects these spheroids from immune attack.

Concave microwell based size-controllable hepatosphere as a three-dimensional liver tissue model

We have developed a size-controllable spheroidal hepatosphere and heterosphere model by mono-culturing of primary hepatocytes and by co-culturing primary hepatocytes and hepatic stellate cells (HSCs). We demonstrated that uniform-sized heterospheres, which self-aggregated from primary hepatocytes and HSCs, formed within concave microwell arrays in a rapid and homogeneous manner. The effect of HSCs was quantitatively and qualitatively investigated during spheroid formation, and HSC played an important role in controlling the organization of the spheroidal aggregates and formation of tight cell–cell contacts. An analysis of the metabolic function showed that heterospheres secreted 30% more albumin than hepatospheres on day 8. In contrast, the urea secretion from heterospheres was similar to that of hepatospheres. A quantitative cytochrome P450 assay showed that the enzymatic activity of heterospheres cultured for 9 days was higher as compared with primary hepatospheres. These size-controllable heterospheres could be mass-produced using concave plate and be useful for creating artificial three-dimensional hepatic tissue constructs and regeneration of failed liver.

Controlled-size embryoid body formation in concave microwell arrays

Embryonic stem (ES) cells hold great potential as a renewable cell source for regenerative medicine and cell-based therapy. Despite the potential of ES cells, conventional stem cell culture methods do not enable the control of the microenvironment. A number of microscale engineering approaches have been recently developed to control the extracellular microenvironment and to direct embryonic stem cell fate. Here, we used engineered concave microwell arrays to regulate the size and shape of embryoid bodies (EBs)—cell aggregate intermediates derived from ES cells. Murine ES cells were aggregated within concave microwells, and their aggregate sizes were controlled by varying the microwell widths (200, 500, and 1000 μm). Differentiation of murine ES cells into three germ layers was assessed by analyzing gene expression. We found that ES cell-derived cardiogenesis and neurogenesis were strongly regulated by the EB size, showing that larger concave microwell arrays induced more neuronal and cardiomyocyte differentiation than did smaller microwell arrays. Therefore, this engineered concave microwell array could be a potentially useful tool for controlling ES cell behavior.