Abstract
BackgroundCells are continuously exposed to changes in their environment. Endocrine systems, in particular, communicate by rhythms and feedback loops. In this study, we developed an automated system to produce such conditions for cultured cells in a precisely timed manner. We utilized a programmable pair of syringe pumps for inflow and a peristaltic pump for outflow to create rhythmic pulses at 5-min intervals in solutions that mimic the endogenous patterns of insulin produced by pancreatic islets as a test case.ResultsThis perifusion system was first tested by measuring trypan blue absorbance, which was intermittently added and washed out at 3:3 and 2:3 min (in:out). Absorbance corresponded with patterns of trypan blue delivery. We then created patterns of forced oscillations in islets by intermittently switching between solutions containing 28 millimolar (mM) glucose (producing high levels of intracellular calcium ([Ca2+]i) and insulin secretion) and 28 mM glucose + calcium-channel blocker nifedipine (producing low levels of [Ca2+]i and insulin secretion). Forced perifusion effects were monitored by fura-2 AM fluorescence measurements of [Ca2+]i. Islets showed uniform oscillations in [Ca2+]i at time intervals consistent with the perifusion pattern, mimicking endogenous pulsatility.ConclusionsThis study highlights a valuable method to modify the environment of the cell culture over a period of hours to days.
Highlights
Cells are continuously exposed to changes in their environment
Trypan Blue Tests Data from the trypan blue absorbance analysis was graphed as a function of optical density vs time which demonstrates the oscillatory pattern desired from the syringe pump system
The differences between 2:3 and 3:3 patterns demonstrate that the pump system can produce subtle changes in the ‘plateau fraction’ of oscillations, which are indicative of very sensitive biological responses to small changes in nutrient load in pancreatic islets [12, 13]
Summary
Cells are continuously exposed to changes in their environment. Endocrine systems, in particular, communicate by rhythms and feedback loops. We developed an automated system to produce such conditions for cultured cells in a precisely timed manner. The fluids used can be perifused at physiologically relevant flow rates and temperatures to mimic in vivo conditions and to remove unwanted environmental variables such as osmotic and mechanical stressors [2, 4, 5]. These systems are especially useful for perifusing hormones over immobilized cells, which allows the experimenter to collect the cellular outflow for analysis. Using the SyringePumpProV1 computer system, each individual pump can be set to flow at a given rate and time, which eliminates the need for the experiment to be monitored
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