Investigation of insulin resistance through a multiorgan microfluidic organ-on-chip.

The development of hepatic insulin resistance (IR) is a critical factor in developing type 2 diabetes (T2D), where insulin fails to inhibit hepatic glucose production but retains its capacity to promote hepatic de novo lipogenesis leading to hyperglycemia and hypertriglyceridemia. Improving insulin sensitivity can be effective in preventing and treating T2D. However, selective control of glucose and lipid synthesis has been difficult. It is known that excess white adipose tissue is detrimental to insulin sensitivity, whereas brown adipose tissue transplantation can restore it in diabetic mice. However, challenges remain in our understanding of liver-adipose communication because the confounding effects of hypothalamic regulation of metabolic function cannot be ruled out in previous studies. There is a lack ofin vitromodels that use primary cells to study cellular-crosstalk under insulin resistant conditions. Building upon our previous work on the microfluidic primary liver and adipose organ-on-chips, we report for the first time, the development of an integrated insulin resistant liver-adipose (white and brown) organ-on-chip. The design of the microfluidic device was carried out using computational fluid dynamics; the experimental studies were conducted by carrying out detailed biochemical analysis RNA-seq analysis on both cell types. Further, we tested the hypothesis that brown adipocytes (BAC) regulated both hepatic insulin sensitivity and de novo lipogenesis. Our results show that BAC effectively restored insulin sensitivity and supressed hepatic glucose production and de novo lipogenesis suggesting that the experimental platform could be useful for identifying potential therapeutics to treat IR and diabetes.

Towards an Insulin Resistant Adipose Model on a Chip.

INTRODUCTION: Adipose tissue and adipocytes are primary regulators of insulin sensitivity and energy homeostasis. Defects in insulin sensitivity of the adipocytes predispose the body to insulin resistance (IR) that could lead to diabetes. However, the mechanisms mediating adipocyte IR remain elusive, which emphasizes the need to develop experimental models that can validate the insulin signaling pathways and discover new mechanisms in the search for novel therapeutics. Currently in vitro adipose organ-chip devices show superior cell function over conventional cell culture. However, none of these models represent disease states. Only when these in vitro models can represent both healthy and disease states, they can be useful for developing therapeutics. Here, we establish an organ-on-chip model of insulin-resistant adipocytes, as well as characterization in terms of insulin signaling pathway and lipid metabolism. METHODS: We differentiated, maintained, and induced insulin resistance into primary adipocytes in a microfluidic organ-on-chip. We then characterized IR by looking at the insulin signaling pathway and lipid metabolism, and validated by studying a diabetic drug, rosiglitazone. RESULTS: We confirmed the presence of insulin resistance through reduction of Akt phosphorylation, Glut4 expression, Glut4 translocation and glucose uptake. We also confirmed defects of disrupted insulin signaling through reduction of lipid accumulation from fatty acid uptake and elevation of glycerol secretion. Testing with rosiglitazone showed a significant improvement in insulin sensitivity and fatty acid metabolism as suggested by previous reports. CONCLUSIONS: The adipose-chip exhibited key characteristics of IR and can serve as model to study diabetes and facilitate discovery of novel therapeutics.

Establishment and characterization of a primary murine adipose tissue-chip.

Better experimental models are needed to enhance our understanding of metabolic regulation which is seen in obesity and metabolic disorders, such as type 2 diabetes. In vitro models based on microfluidics enable physiological representations of tissues with several advantages over conventional culture systems, such as perfused flow to better mimic the physiological environment. Although cell lines such as 3T3-L1 have been incorporated in microfluidic devices, murine primary preadipocytes have not been differentiated and maintained for long-term monitoring in these culture systems. We describe the differentiation of these cells into white adipose depots on a perfused microfluidic chip. We compare the effects of shear flow on these cells, and show with a direct comparison of high/low shear conditions that direct shear is detrimental to the viability of preadipocytes. We further develop a dual-chamber microfluidic chip that enables perfusion while at the same time protects the cells from direct fluidic shear. We show that the dual-layer microfluidic device enables long-term culture of cells and allows stimulation of cells through perfusion-we can culture, differentiate, and maintain the differentiated adipose tissue for over multiple weeks in the device. Both triglycerides and lipolytic glycerol production increased significantly by several folds during differentiation. After successful differentiation, the adipocytes had upregulated expression of leptin and adiponectin, which are important makers of the final stage of adipogenic differentiation. In conclusion, the dual-layer microfluidic device incorporated with primary adipocytes improves the understanding of adipose differentiation under dynamic conditions and is positioned to serve as a disease model for studying obesity and other metabolic disorders.

Novel Microfluidic Colon with an Extracellular Matrix Membrane.

Collagen is a key element of basal lamina in physiological systems that participates in cell and tissue culture. Its function is for cell maintenance and growth, angiogenesis, disease progression, and immunology. The goal of our present study was to integrate a micrometer resolution membrane that is synthesized out of rat-tail type I collagen in a microfluidic device with apical and basolateral chambers. The collagen membrane was generated by lyophilization. In order to evaluate the compatibility of the resulting membrane with organs-on-chips technology, it was sandwiched between layers of polydimethylsiloxane (PDMS) that had been prepared by replica molding, and the device was used to culture human colon caco 2 cells on the top of the membrane. Membrane microstructure, transport, and cell viability in the organs-on-chips were observed to confirm the suitability of our resulting membrane. Through transport studies, we compared diffusion of two different membranes: Transwell and our resulting collagen membrane. We found that mass transport of 40 kDa dextran was an order of magnitude higher through the collagen membrane than that through the Transwell membrane. Human colon caco 2 cells were cultured in devices with no, Transwell, or ECM membrane to evaluate the compatibility of cells on the ECM membrane compared to the other two membranes. We found that caco 2 cells cultured on the collagen membrane had excellent viability and function for extended periods of time compared to the other two devices. Our results indicate a substantial improvement in establishing a physiological microenvironment for in vitro organs-on-chips.

Fabrication of Poly-l-lactic Acid/Dicalcium Phosphate Dihydrate Composite Scaffolds with High Mechanical Strength-Implications for Bone Tissue Engineering.

Scaffolds were fabricated from poly-l-lactic acid (PLLA)/dicalcium phosphate dihydrate (DCPD) composite by indirect casting. Sodium citrate and PLLA were used to improve the mechanical properties of the DCPD scaffolds. The resulting PLLA/DCPD composite scaffold had increased diametral tensile strength and fracture energy when compared to DCPD only scaffolds (1.05 vs. 2.70 MPa and 2.53 vs. 12.67 N-mm, respectively). Sodium citrate alone accelerated the degradation rate by 1.5 times independent of PLLA. Cytocompatibility of all samples were evaluated using proliferation and differentiation parameters of dog-bone marrow stromal cells (dog-BMSCs). The results showed that viable dog-BMSCs attached well on both DCPD and PLLA/DCPD composite surfaces. In both DCPD and PLLA/DCPD conditioned medium, dog-BMSCs proliferated well and expressed alkaline phosphatase (ALP) activity indicating cell differentiation. These findings indicate that incorporating both sodium citrate and PLLA could effectively improve mechanical strength and biocompatibility without increasing the degradation time of calcium phosphate cement scaffolds for bone tissue engineering purposes.