A High-Throughput Automated Microfluidic Platform for Calcium Imaging of Taste Sensing

Abstract

Abstract:The human enteroendocrine L cell line NCI-H716, expressing taste receptors and taste signaling elements, constitutes a unique model for the studies of cellular responses to glucose, appetite regulation, gastrointestinal motility, and insulin secretion. Targeting these gut taste receptors may provide novel treatments for diabetes and obesity. However, NCI-H716 cells are cultured in suspension and tend to form multicellular aggregates, preventing high-throughput calcium imaging due to interferences caused by laborious immobilization and stimulus delivery procedures. Here, we have developed an automated microfluidic platform that is capable of trapping more than 500 single cells into microwells with a loading efficiency of 77% within two minutes, delivering multiple chemical stimuli and performing calcium imaging with enhanced spatial and temporal resolutions when compared to bath perfusion systems. Results revealed the presence of heterogeneity in cellular responses to the type, concentration, and order of applied sweet and bitter stimuli. Sucralose and denatonium benzoate elicited robust increases in the intracellular Ca 2+ concentration. However, glucose evoked a rapid elevation of intracellular Ca 2+ followed by reduced responses to subsequent glucose stimulation. Using Gymnema sylvestre as a blocking agent for the sweet taste receptor confirmed that different taste receptors were utilized for sweet and bitter tastes. This automated microfluidic platform is cost-effective, easy to fabricate and operate, and may be generally applicable for high-throughput and high-content single-cell analysis and drug screening.

Development of innovative tools for investigation of nutrient-gut interaction

“…The application of the “intestine-on-a-chip” model for gut hormone secretion study is in its infancy. In 2016, Hsiao et al[ 106 ] developed a high-throughput automated microfluidic platform to assess the response of NCL-H716 cells to sweet and bitter stimuli. Although gut hormones were not measured in the study, the microfluidic system recorded the dynamic changes in intracellular Ca 2+ in over 500 single NCI-H716 cells trapped in each micro-well.…”

PiFlow: A Biocompatible Low-Cost Programmable Dynamic Flow Pumping System Utilizing a Raspberry Pi Zero and Commercial Piezoelectric Pumps

“…MICCS can reliably produce any dynamic flow profile (<3,000 µL/min peak flow rate) by loading a custom ".csv" file containing the desired flow rates and time intervals. We chose here to demonstrate the improved functionality of MICCS by incorporating a microfluidic static mixer to create dynamic concentration profiles useful in many cell stimuli studies 13 whether the stimulus be drugs, glucose 49,50 , hormones or growth factors 42,44 . By connecting one pump to a reservoir containing a concentrated solution and the other pump to a reservoir containing buffer solution, dynamically changing the ratio of the flow rate of one pump to the other, and mixing the flow from both pumps, we can dynamically control the concentration of a solute while keeping the flow rate constant (Fig.…”