Lab-on-a-Chip System for Developing and Fluorescence Imaging a Three-Dimensional Model of Pancreatic Islets Under Flow Conditions

Abstract

Introduction Diabetes mellitus is a group of metabolic diseases characterized by elevated blood sugar resulting from a defect in the production of insulin by pancreatic β-cells or tissue insensitivity for this peptide hormone [1]. The most common form of the disease is diabetes mellitus type 2 (insulin-dependent), which is diagnosed in about 80% of all patients [2]. In patients with this type of diabetes, both the efficiency and the secretion of insulin are disturbed [3]. For this reason, it is very important to thoroughly understand the kinetics of insulin release in response to various conditions, which can help develop effective therapy. The most important cells for this disease are insulin secreting β-cells and their antagonist - glucagon-secreting α-cells [4]. In our research, we focused on imitate the pancreatic islet structure, which can be a universal model for testing the impact of various environmental factors on disease development. The idea of using a Lab-on-a-chip system for three-dimensional islet cells culture came from the possibility of mimicking in vivo conditions. This can give a wider and more accurate view of the pancreatic islets function than the cell culture in static conditions. Lab-on-a-chip system In our research we present a Lab-on-a-chip system in which a pancreatic “pseudoislet” model will be developed. This system is composed of biocompatible, non-toxic and transparent materials, such as: poly(dimethylsiloxane) (PDMS) and thin glass. Thanks to this, it is possible to conduct cell culture and observe the results in various types of microscopes. The geometry of Lab-on-a-chip system consist of two elliptical cell culture chambers, one for the test sample and second for the reference sample. In in each of the chambers there are 15 round microtraps. Each of the microtraps is made of 6 micropillars. This solution forces the dense packing of cells and their aggregations. The geometry of the developed system is consistent with the culture wells on a standard multi-well plate, which allows measurements in a multi-well plate reader. Methods All experiments were performed using two commercially purchased pancreatic islets cell lines: β-cells (INS-1E) and α-cells (α-TC1-6). Appropriate α-cells and β-cells ratios were selected to reflect the morphology and composition of the pancreatic islet found in the body. The cells were introduced through the inlet to the Lab-on-a-chip system and placed for 24h in an incubator to form spherical aggregates (“pseudoislets”). In the next stage immunostaining protocol using primary and secondary antibody solutions was developed. Due to the use this method and confocal microscope it was possible to confirm the correct distribution of cells in the obtained aggregate. To confirm cells viability previously prepared solution of Calcein AM and Propidium Iodide were introduced through the microsystem inlet with a flow rate 5µL/min for 10 min. After 10 min incubation (37oC, 5% CO2) the viability of the cells was determined using fluorescence microscope. Proliferation of the ells was measured using spectrofluorimetric multi-well plate reader after incubation with AlamarBlue solution. The “pseudoislets” were then stimulated with various glucose concentrations (imitation of health and disease state). After this, the standard ELISA test procedure was adapted to the microfluidic conditions to examine the insulin secretion profile from the obtained “pseudoislets”. Results and Conclusions It was confirmed that 15 spherical α and β-cell aggregates with diameters of 160-180 µm were obtained in one culture chamber using the method described earlier. As was expected the participation of β-cells in the core and α-cells on the periphery of the aggregate was confirmed by the use of immunostaining method. Moreover, the compatibility of the designed microsystem with the multi-well plate reader allowed to monitor cell proliferation and viability in situ during the culture. A high viability after 24 and 48 hours of culture in the microchip was confirmed and was 97% and 95% respectively. The cells maintained their morphology, function and high level of proliferation for up to 48 hours of culture. Moreover, in the developed model health and disease conditions were simulated and the level of insulin secreted from this model was determined using the ELISA test. At this stage, a research model corresponding to the model in vivo conditions was obtained. The next step will be to develop a method for analyzing insulin secretion in real time (such as Surface Plasmon Resonance). This study presents basic research and in the future, this model could be utilized to simulate diabetes, testing new drugs and therapy in diabetes mellitus treatment.