Lab-on-a-Chip System for Developing and Fluorescence Imaging a Three-Dimensional Model of Pancreatic Islets Under Flow Conditions
Authors: Brzozka Zbigniew , Sokołowska Patrycja , Zukowski Kamil , Janikiewicz Justyna , Jastrzebska Ekzbieta , Dobrzyn Agnieszka 
Published: July 2021

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]. In 1980s, there were approximately over 100 million diabetic adults in the world. Nowadays this number is 4 times bigger and by 2030 it will probably reach 1 billion [2]. The most common form of the disease is diabetes mellitus type 2 (insulin-dependent), which is diagnosed in about 80% of all patients. In patients with this type of diabetes, both the efficiency and the secretion of insulin are disturbed [3]. The most important cells for this disease are insulin secreting β-cells and their antagonist - glucagon-secreting α-cells. The pancreatic islet consists of 5 cell types, the largest percentage is occupied by β-cells, constitute about 60% (in the core) and α-cells constitute about 25% (located on the periphery) of all cells in the islet [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 7 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), which are a model cell lines for the study of diabetes mellitus type 2. 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. 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. Cells proliferation was measured using spectrofluorimetric multi-well plate reader after 1 hour incubation with AlamarBlue solution. Moreover, to confirm functionality of the α- and β-cells aggregates, insulin secretion tests were performed 24 h after introducing the cells into the microfluidic system. The ELISA test was performed after stimulating the aggregates with low (2.75 mM) and high (16.5 mM) glucose concentrations.

Results and Conclusions

It was confirmed that 15 spherical α and β cell aggregates with diameters of 160-180 µm were obtained 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 also confirmed by the use of immunostaining method. Thanks to this, it was confirmed that the obtained three-dimensional models correspond to the distribution/arrangement and cellular composition of the pancreatic islet located in the body. Moreover, the compatibility of the designed microsystem with the multi-well plate reader allows to monitor cell proliferation and viability in situ during the cultures. Due to using Calcein AM and Propidium Iodide dye 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, functionality of the α- and β-cells aggregates was confirmed. The level of insulin secretion in the developed microfluidic system was 1.38 ng per pseudoislet after low glucose stimulation. For high glucose, we detected 2.75 ng insulin per aggregate. At this stage, a research model corresponding to the model in vivo conditions was obtained. 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.

References

[1] Selby J.V., Ray G.T., Zhang D., Colby C.J., Excess costs of medical care for patients with diabetes in a managed care population. Diabetes Care 1997; 20: 1396–1402.
[1] World Health Organization. Global report on diabetes. World Health Organization, 2016.
[2] American diabetes association Diagnosis and Classification of Diabetes Mellitus. Diabetes care 2008, 31:1
[3] Brissova, M., Fowler, M. J., Nicholson, W. E., Chu, A., Hirshberg, B., Harlan, D. M. & Powers, A. C.Assessment of human pancreatic islet architecture and composition by laser scanning confocal microscopy. J.Histochem. Cytochem. 2005; 53:1087–1097.