A vascularized 3D model of the human pancreatic islet forex vivostudy of immune cell-islet interaction.

Insulin is an essential regulator of blood glucose homeostasis that is produced exclusively byßcells within the pancreatic islets of healthy individuals. In those affected by diabetes, immune inflammation, damage, and destruction of isletßcells leads to insulin deficiency and hyperglycemia. Current efforts to understand the mechanisms underlyingßcell damage in diabetes rely onin vitro-cultured cadaveric islets. However, isolation of these islets involves removal of crucial matrix and vasculature that supports islets in the intact pancreas. Unsurprisingly, these islets demonstrate reduced functionality over time in standard culture conditions, thereby limiting their value for understanding native islet biology. Leveraging a novel, vascularized micro-organ (VMO) approach, we have recapitulated elements of the native pancreas by incorporating isolated human islets within a three-dimensional matrix nourished by living, perfusable blood vessels. Importantly, these islets show long-term viability and maintain robust glucose-stimulated insulin responses. Furthermore, vessel-mediated delivery of immune cells to these tissues provides a model to assess islet-immune cell interactions and subsequent islet killing-key steps in type 1 diabetes pathogenesis. Together, these results establish the islet-VMO as a novel,ex vivoplatform for studying human islet biology in both health and disease.

Modeling of three-dimensional innervated epidermal like-layer in a microfluidic chip-based coculture system.

Reconstruction of skin equivalents with physiologically relevant cellular and matrix architecture is indispensable for basic research and industrial applications. As skin-nerve crosstalk is increasingly recognized as a major element of skin physiological pathology, the development of reliable in vitro models to evaluate the selective communication between epidermal keratinocytes and sensory neurons is being demanded. In this study, we present a three-dimensional innervated epidermal keratinocyte layer as a sensory neuron-epidermal keratinocyte co-culture model on a microfluidic chip using the slope-based air-liquid interfacing culture and spatial compartmentalization. Our co-culture model recapitulates a more organized basal-suprabasal stratification, enhanced barrier function, and physiologically relevant anatomical innervation and demonstrated the feasibility of in situ imaging and functional analysis in a cell-type-specific manner, thereby improving the structural and functional limitations of previous coculture models. This system has the potential as an improved surrogate model and platform for biomedical and pharmaceutical research.

Nutrient regulation of the islet epigenome controls adaptive insulin secretion.

Adaptation of the islet ß cell insulin-secretory response to changing insulin demand is critical for blood glucose homeostasis, yet the mechanisms underlying this adaptation are unknown. Here, we have shown that nutrient-stimulated histone acetylation plays a key role in adapting insulin secretion through regulation of genes involved in ß cell nutrient sensing and metabolism. Nutrient regulation of the epigenome occurred at sites occupied by the chromatin-modifying enzyme lysine-specific demethylase 1 (Lsd1) in islets. ß Cell-specific deletion of Lsd1 led to insulin hypersecretion, aberrant expression of nutrient-response genes, and histone hyperacetylation. Islets from mice adapted to chronically increased insulin demand exhibited shared epigenetic and transcriptional changes. Moreover, we found that genetic variants associated with type 2 diabetes were enriched at LSD1-bound sites in human islets, suggesting that interpretation of nutrient signals is genetically determined and clinically relevant. Overall, these studies revealed that adaptive insulin secretion involves Lsd1-mediated coupling of nutrient state to regulation of the islet epigenome.

High performance inkjet printed embedded electrochemical sensors for monitoring hypoxia in a gut bilayer microfluidic chip.

Sensing devices have shown tremendous potential for monitoring state-of-the-art organ chip devices. However, challenges like miniaturization while maintaining higher performance, longer operating times for continuous monitoring, and fabrication complexities limit their use. Herein simple, low-cost, and solution-processible inkjet dispenser printing of embedded electrochemical sensors for dissolved oxygen (DO) and reactive oxygen species (ROS) is proposed for monitoring developmental (initially normoxia) and induced hypoxia in a custom-developed gut bilayer microfluidic chip platform for 6 days. The DO sensors showed a high sensitivity of 31.1 nA L mg-1 with a limit of detection (LOD) of 0.67 mg L-1 within the 0-9 mg L-1 range, whereas the ROS sensor had a higher sensitivity of 1.44 nA µm-1 with a limit of detection of 1.7 µm within the 0-300 µm range. The dynamics of the barrier tight junctions are quantified with the help of an in-house developed trans-epithelial-endothelial electrical impedance (TEEI) sensor. Immunofluorescence staining was used to evaluate the expressions of HIF-1α and tight junction protein (TJP) ZO-1. This platform can also be used to enhance bioavailability assays, drug transport studies under an oxygen-controlled environment, and even other barrier organ models, as well as for various applications like toxicity testing, disease modeling and drug screening.

Three-Dimensional In Vitro Lymphangiogenesis Model in Tumor Microenvironment.

Lymphangiogenesis is a stage of new lymphatic vessel formation in development and pathology, such as inflammation and tumor metastasis. Physiologically relevant models of lymphatic vessels have been in demand because studies on lymphatic vessels are required for understanding the mechanism of tumor metastasis. In this study, a new three-dimensional lymphangiogenesis model in a tumor microenvironment is proposed, using a newly designed macrofluidic platform. It is verified that controllable biochemical and biomechanical cues, which contribute to lymphangiogenesis, can be applied in this platform. In particular, this model demonstrates that a reconstituted lymphatic vessel has an in vivo-like lymphatic vessel in both physical and biochemical aspects. Since biomechanical stress with a biochemical factor influences robust directional lymphatic sprouting, whether our model closely approximates in vivo, the initial lymphatics in terms of the morphological and genetic signatures is investigated. Furthermore, attempting an incorporation with a tumor spheroid, this study successfully develops a complex tumor microenvironment model for use in lymphangiogenesis and reveals the microenvironment factors that contribute to tumor metastasis. As a first attempt at a coculture model, this reconstituted model is a novel system with a fully three-dimensional structure and can be a powerful tool for pathological drug screening or disease model.

In vivo-mimicking microfluidic perfusion culture of pancreatic islet spheroids.

Native pancreatic islets interact with neighboring cells by establishing three-dimensional (3D) structures, and are surrounded by perfusion at an interstitial flow level. However, flow effects are generally ignored in islet culture models, although cell perfusion is known to improve the cell microenvironment and to mimic in vivo physiology better than static culture systems. Here, we have developed functional islet spheroids using a microfluidic chip that mimics interstitial flow conditions with reduced shear cell damage. Dynamic culture, compared to static culture, enhanced islet health and maintenance of islet endothelial cells, reconstituting the main component of islet extracellular matrix within spheroids. Optimized flow condition allowed localization of secreted soluble factors near spheroids, facilitating diffusion-mediated paracrine interactions within islets, and enabled long-term maintenance of islet morphology and function for a month. The proposed model can aid islet preconditioning before transplantation and has potential applications as an in vitro model for diabetic drug testing.

3D-cultured human placenta-derived mesenchymal stem cell spheroids enhance ovary function by inducing folliculogenesis.

Placenta-derived mesenchymal stem cells (PD-MSCs) have numerous advantages over other adult MSCs that make them an attractive cell source for regenerative medicine. Here, we demonstrate the therapeutic effect of PD-MSCs in ovariectomized (Ovx) rats and compare their efficacy when generated via a conventional monolayer culture system (2D, naïve) and a spheroid culture system (3D, spheroid). PD-MSC transplantation significantly increased the estradiol level in Ovx rats compared with the non-transplantation (NTx) group. In particular, the estradiol level in the Spheroid group was significantly higher than that in the Naïve group at 2 weeks. Spheroid PD-MSCs exhibited a significantly higher efficiency of engraftment onto ovarian tissues at 2 weeks. The mRNA and protein expression levels of Nanos3, Nobox, and Lhx8 were also significantly increased in the Spheroid group compared with those in the NTx group at 1 and 2 weeks. These results suggest that PD-MSC transplantation can restore ovarian function in Ovx rats by increasing estrogen production and enhancing folliculogenesis-related gene expression levels and further indicate that spheroid-cultured PD-MSCs have enhanced therapeutic potential via increased engraftment efficiency. These findings improve our understanding of stem-cell-based therapies for reproductive systems and may suggest new avenues for developing efficient therapies using 3D cultivation systems.

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

Oxygen availability is a critical factor in regulating cell viability that ultimately contributes to the normal morphogenesis and functionality of human tissues. Among various cell culture platforms, construction of 3D multicellular spheroids based on microwell arrays has been extensively applied to reconstitute in vitro human tissue models due to its precise control of tissue culture conditions as well as simple fabrication processes. However, an adequate supply of oxygen into the spheroidal cellular aggregation still remains one of the main challenges to producing healthy in vitro spheroidal tissue models. Here, we present a novel design for controlling the oxygen distribution in concave microwell arrays. We show that oxygen permeability into the microwell is tightly regulated by varying the poly-dimethylsiloxane (PDMS) bottom thickness of the concave microwells. Moreover, we validate the enhanced performance of the engineered microwell arrays by culturing non-proliferated primary rat pancreatic islet spheroids on varying bottom thickness from 10 µm to 1050 µm. Morphological and functional analyses performed on the pancreatic islet spheroids grown for 14 days prove the long-term stability, enhanced viability, and increased hormone secretion under the sufficient oxygen delivery conditions. We expect our results could provide knowledge on oxygen distribution in 3-dimensional spheroidal cell structures and critical design concept for tissue engineering applications. STATEMENT OF SIGNIFICANCE: 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.

Electrospinning versus microfluidic spinning of functional fibers for biomedical applications.

Micro- or nanofiber-based materials have extensive applications in biomedical fields due to their capability to mimic many aspects of physiological microenvironment in vivo. Fabricating micro- or nanofibers using biocompatible and biodegradable materials is becoming of great interest in the area of biomaterials and tissue engineering. Among the various technologies, electrospinning and microfluidic spinning are the two promising approaches to produce fibers at micro- and nano-scale. Choosing an appropriate spinning method is critical important for a specific application. Although some review papers on each spinning method have been published, a review comparing these two methods has not been reported yet. In this review, we present an overview of the two spinning methods including the spinning principle, their unique features and materials selections. Several applications of fibers spun by both methods, especially in tissue engineering, organ function regeneration and drug delivery are introduced. The current challenges, future directions and potential applications of these approaches are discussed as well.

Biomimetic spinning of silk fibers and in situ cell encapsulation.

In situ embedding of sensitive materials (e.g., cells and proteins) in silk fibers without damage presents a significant challenge due to the lack of mild and efficient methods. Here, we report the development of a microfluidic chip-based method for preparation of meter-long silk fibroin (SF) hydrogel fibers by mimicking the silkworm-spinning process. For the spinning of SF fibers, alginate was used as a sericin-like material to induce SF phase separation and entrap liquid SFs, making it possible to shape the outline of SF-based fibers under mild physicochemical conditions. L929 fibroblasts were encapsulated in the fibric hydrogel and displayed excellent viability. Cell-laden SF fibric hydrogels prepared using our method offer a new type of SF-based biomedical device with potential utility in biomedicine.

Scaffold-free parathyroid tissue engineering using tonsil-derived mesenchymal stem cells.

To restore damaged parathyroid function, parathyroid tissue engineering is the best option. Previously, we reported that differentiated tonsil-derived mesenchymal stem cells (dTMSC) restore in vivo parathyroid function, but only if they are embedded in a scaffold. Because of the limited biocompatibility of Matrigel, however, here we developed a more clinically applicable, scaffold-free parathyroid regeneration system. Scaffold-free dTMSC spheroids were engineered in concave microwell plates made of polydimethylsiloxane in control culture medium for the first 7days and differentiation medium (containing activin A and sonic hedgehog) for next 7days. The size of dTMSC spheroids showed a gradual and significant decrease up to day 5, whereafter it decreased much less. Cells in dTMSC spheroids were highly viable (>80%). They expressed high levels of intact parathyroid hormone (iPTH), the parathyroid secretory protein 1, and cell adhesion molecule, N-cadherin. Furthermore, dTMSC spheroids-implanted parathyroidectomized (PTX) rats revealed higher survival rates (50%) over a 3-month period with physiological levels of both serum iPTH (57.7-128.2pg/mL) and ionized calcium (0.70-1.15mmol/L), compared with PTX rats treated with either vehicle or undifferentiated TMSC spheroids. This is the first report of a scaffold-free, human stem cell-based parathyroid tissue engineering and represents a more clinically feasible strategy for hypoparathyroidism treatment than those requiring scaffolds. STATEMENT OF SIGNIFICANCE: Herein, we have for the first time developed a scaffold-free parathyroid tissue spheroids using differentiated tonsil-derived mesenchymal stem cells (dTMSC) to restore in vivo parathyroid cell functions. This new strategy is effective, even for long periods (3months), and is thus likely to be more feasible in clinic for hypoparathyroidism treatment. Development of TMSC spheroids may also provide a convenient and efficient scaffold-free platform for researchers investigating conditions involving abnormal calcium homeostasis, such as osteoporosis.

Microchip-based engineering of super-pancreatic islets supported by adipose-derived stem cells.

Type 1 diabetes mellitus (T1DM) is a chronic disorder characterized by targeted autoimmune-mediated destruction of the ß cells of Langerhans within pancreatic islets. Currently, islet transplantation is the only curative therapy; however, donor shortages and cellular damage during the isolation process critically limit the use of this approach. Here, we describe a method for creating viable and functionally potent islets for successful transplantation by co-culturing single primary islet cells with adipose-derived stem cells (ADSCs) in concave microwells. We observed that the ADSCs segregated from the islet cells, eventually yielding purified islet spheroids in the three-dimensional environment. Thereafter, the ADSC-exposed islet spheroids showed significantly different ultrastructural morphologies, higher viability, and enhanced insulin secretion compared to mono-cultured islet spheroids. This suggests that ADSCs may have a significant potential to protect islet cells from damage during culture, and may be employed to improve islet cell survival and function prior to transplantation. In vivo experiments involving xenotransplantation of microfiber-encapsulated spheroids into a mouse model of diabetes revealed that co-culture-transplanted mice maintained their blood glucose levels longer than mono-culture-transplanted mice, and required less islet mass to reverse diabetes. This method for culturing islet spheroids could potentially help overcome the cell shortages that have limited clinical applications and could possibly be developed into a bioartificial pancreas.

Microfluidic spinning of micro- and nano-scale fibers for tissue engineering.

Microfluidic technologies have recently been shown to hold significant potential as novel tools for producing micro- and nano-scale structures for a variety of applications in tissue engineering and cell biology. Over the last decade, microfluidic spinning has emerged as an advanced method for fabricating fibers with diverse shapes and sizes without the use of complicated devices or facilities. In this critical review, we describe the current development of microfluidic-based spinning techniques for producing micro- and nano-scale fibers based on different solidification methods, platforms, geometries, or biomaterials. We also highlight the emerging applications of fibers as bottom-up scaffolds such as cell encapsulation or guidance for use in tissue engineering research and clinical practice.

Microfluidics-generated pancreatic islet microfibers for enhanced immunoprotection.

Pancreatic islet transplantation is a promising method for treatment of type 1 diabetes mellitus. However, transplanted islets can be destroyed due to host immune reactions. To immunologically protect transplanted islets, here an immunoprotective microfiber including islets by using a polydimethylsiloxane (PDMS)-based microfluidic device is newly designed. A cylindrical-flow channel in the microfluidic platform is used for producing collagen-alginate composite (CAC) fibers. This enables mass production and uniform diameter distribution (<250 µm) without protruding islets. Collagen, which is the main extracellular matrix component, is added to alginate to mimic the native islet microenvironment. Compared to free islets (control) and alginate-fiber-encapsulated islets, CAC-fiber-encapsulated islets show higher viability and normal insulin secretion. When CAC-fiber-encapsulated islets (1200 islet equivalent) are implanted into the intraperitoneal cavity of streptozotocin-induced diabetic BALB/C mice, the blood glucose levels of all mice return to normoglycemia. Moreover, intraperitoneal glucose tolerance tests demonstrate that islets in the CAC-fiber have similar glucose responsiveness to those of non-diabetic normal mice. These results are attributed to the immunoprotection of the transplanted islets from host immune reactions. On the other hand, all free islets are completely rejected within a week due to severe immune responses. Collectively, fabrication of CAC fibers using microfluidic devices can be used for successful islet transplantation.

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.

Surface tension-mediated, concave-microwell arrays for large-scale, simultaneous production of homogeneously sized embryoid bodies.

Embryonic stem cells (ESCs) are pluripotent and capable of self-renewal. ESC aggregates, termed embryoid bodies (EBs), have been widely adopted as an in vitro differentiation model. However, the mass production of uniform size and shaped EBs has been challenging. Herein is described the development of a culture plate containing a large number of concave microwells with minimal use of tools, labor, skill, and cost, enabling the production of a large number of homogeneous EBs simultaneously using the culture plate. The large number of concave well structures is self-constructed through the surface tension of the viscoelastic PDMS prepolymer. Murine ESCs (mESCs) are then seeded onto the concave wells for mass production of monodisperse EBs. It is observed that the EBs produced over a large area are uniform in shape and size regardless of microwell position and differences in cell seeding densities, and whether their phenotype is maintained. The capability to differentiate into adult cells (neuron and endothelial cells) from EBs is also evaluated and the neural spikes from differentiated neuron cells are measured to observe their function. Uniform size and shape EBs are successfully generated in large scale and their pluripotency is maintained similar to other methods.

Meniscus induced self organization of multiple deep concave wells in a microchannel for embryoid bodies generation.

Embryonic stem cells (ESCs) have attracted great interest in the fields of tissue engineering, regenerative medicine, and organogenesis for their pluripotency and ability to self-renew. ESC aggregation, which produces an embryoid body (EB), has been widely utilized as a trigger of in vitro directed differentiation. In this paper, we propose a novel method for constructing large numbers of deep concave wells in PDMS microfluidic chips using the meniscus induced by the surface tension of a liquid PDMS prepolymer, and applied this chip for the mass production of uniform sized EBs. To investigate if the microenvironment in the deep concave well is suitable for ES cells, the oxygen diffusion to the deep concave well was analyzed by CFD simulation. Murine EBs were successfully formed in the deep concave wells without loss of cells and laborious careful intervention to refresh culture media. The size of the EBs was uniform, and retrieving of EBs was done just by flipping over the chip. All the processes including EB formation and harvest are easy and safe to cells, and their viability after completion of all processes was over 95%. The basic properties of the EBs were generated and their capacity to differentiate into 3 germ layers was investigated by analyzing the gene expression profile. The harvested EBs were found to differentiate into cardiac cells and neurons, and neurofilaments formed branches of elongated extensions more than 1.0 mm in length.

Development of a multi-layer microfluidic array chip to culture and replate uniform-sized embryoid bodies without manual cell retrieval.

We have developed a multi-layer, microfluidic array platform containing concave microwells and flat cell culture chambers to culture embryonic stem (ES) cells and regulate uniform-sized embryoid body (EB) formation. The main advantage of this platform was that EBs cultured within the concave microwells of a bottom layer were automatically replated into flat cell culture chambers of a top layer, following inversion of the multi-layer microfluidic array platform. This allowed EB formation and EB replating to be controlled simultaneously inside a single microfluidic device without pipette-based manual cell retrieval, a drawback of previous EB culture methods.