Microfluidic Assays for Perfusion Culture and Chemical Monitoring of Living Cells

Abstract

Insulin is secreted from islets of Langerhans to control glucose homeostasis. Deficient insulin secretion is a hallmark of type 2 diabetes. New tools for studying insulin secretion are of interest to better understand this process. An advanced method for measuring insulin secretion is based on using electrophoresis. In the method, a single islet is incubated on a microfluidic device or “chip”. Fluid around the islet is sampled by electroosmotic flow and reagents for a competitive immunoassay added to the sample stream. The resulting stream is analyzed at 5-10 s intervals by using electrophoresis to separate antibody bound and free insulin. In this work, this system was improved by adding more automated control of the islet environment, adapting to measure other peptides, and performing co-culture experiments to elucidate how adipocytes affect insulin secretion. To provide further automation, two computer-controlled syringe pumps were used to deliver programmed glucose gradients to the islet. Automatic online calibration to improve quantification was also added. The device performed an electrophoresis-based competitive immunoassay every 8 s and operated for 24 h, resulting in the completion of 14,000 assays. It was applied to several insulin secretion studies including an investigation of glucose pretreatment on glucose sensitivity. A modification of the chip was applied to measure the secretory kinetics of C-peptide. The kinetics were compared with insulin and used to validate C-peptide as indicator of insulin secretion. The chip was further modified to enable study of the impact of adipocytes on islet insulin secretion by co-culture both cells on the same chip. Adipocytes are fat-storing cells that secrete glycerol, non-esterified fatty acids and adipokines. The adipocytes strongly potentiated insulin secretion from islets. The effect appeared to be due to multiple chemicals released by the adipocytes illustrating the potential for observing complex interactions. A second microfluidic chip was developed to perform intracellular [Ca2+] measurements. The chip was used to investigate the role of X-Box Binding Protein 1 in insulin regulated pancreatic α-cell function. The results suggested that [Ca2+] alterations did not underlie the dysregulation of glucagon secretion in XBP1 deficient α-cells.