In this presentation, we will introduce a microfluidic based eyelid perfusion system for simultaneous measurement of dynamic insulin secretion, calcium influx, and mitochondrial potential changes of pancreatic eyelets in response to insulin secrete dogs. The perfusion system is composed of a three layer microfluidic device, syringe pumps, and a fraction collector insulin. Secretagogues induced calcium influx and mitochondrial potentials are imaged using fluorescent microscope setup.
Hi, my name is Ola, a Waller from the laboratory of Dr.Val Holter in the Department of Transplant Surgery at the University of Illinois at Chicago. Hi, I'm Don Lee, also from the October's Lab. Hi, I am Tricia Hart, also from Dr.Al Holster's lab.
Today we'll show you a procedure for simultaneous eyelid imaging using a linear glucose gradient. We used this procedure in our laboratory to study eyelet visuals and function. So let's get started.
The microfluidic device used for this procedure is assembled from three layers of cured PDMS generated by standard photolithography. The first layer generated from a silicone master has the wells of the microfluidic device that will gently hold the eyelets in place, 150 micrometers deep and 500 micrometers in diameter. The second layer has channels 500 micrometers tall and two millimeters wide.
The middle of the channel is expanded to spread out the flow for better exchange of solution Inside the device, in exchange well is made by punching a hole three millimeters high and seven millimeters wide in the middle of the layer. The third layer is a thick slab of PDMS. To prepare this layer punch small holes on both sides that align with the inlet and outlet of the channel.
On the second layer, once all three layers have been generated, the first layer is bonded to a glass slide with the wells facing up. The second layer is then bonded to the first layer with the channel spacing up. Finally, the third layer is bonded to the second layer.
Once the layers have been bonded, the device is ready to use for experiments. Using a 10 milliliter syringe filled with 70%ethanol and connected to the inlet port of the device with Tai on tubing, sterilize the microfluidic device by flowing the ethanol through the device channels. Then using the same setup flow, deionized water to wash out the ethanol.
Once the device has been sterilized, place it on a heated microscope stage using tigon tubing, connect the syringe pumps containing high and low glucose solutions to a Y connector. Then connect to the inlet of the microfluidic device. Connect the outlet of the microfluidic device to a fraction collector.
Ensure that the tubing is resting on a 37 degrees Celsius hot plate. It is very important to keep the solutions at physiological temperature throughout the experiment. Next, use the lab view program to initiate the glucose gradient that will be perfused through the microfluidic network during the experiment.
This software varies the flow rate of two glucose solutions. By controlling the syringe pumps, various glucose gradient profiles can be created here, linear, bell-shaped and square shaped glucose gradient profiles. In comparison to the calculated expected values are shown in this experiment, the linear gradient of two millimolar to 25 millimolar glucose will be used with a varying flow rate of 0.01 milliliters per minute of the two glucose solutions and a constant flow rate of 0.25 milliliters per minute entering the device after the solutions are mixed.
Once the appropriate gradient profile has been selected, test the stability of the gradient. First, start the gradient system. Then start the fraction collector and collect perfuse eight in one milliliter eend orph tubes.
Then using a glucometer, test the concentration of glucose in each tube using excel. Analyze the results obtained from the glucometer. Next, replace the high glucose syringe with a syringe containing 0.5%BSA solution in Krebs ringer buffer PERFUSE 50 milliliters through the device at a rate of one milliliter per minute.
This will prevent nonspecific absorption of insulin to the microchannel walls to ensure accuracy when later measuring secreted insulin levels after perfusion with BSA. Replace it with the high glucose solution reus. Suspend 25 to 30 handpicked mouse eyelets in a two milli molar glucose Krebs ringer buffer containing the calcium indicator dye fira 2:00 AM and the mitochondrial potentials indicator dye rumine 1 2 3, incubate for 30 minutes at 37 degrees Celsius.
After incubation, disconnect the device from the perfusion system and carefully load the eyelets into the microfluidic device by inserting the tip of the pipette, containing the islets into the inlet port, and slowly dispensing the islets after loading, reconnect the device to the perfusion system. Taking caution not to introduce bubbles. Next, start the syringe pump to begin perfusing the eyelets with KRB containing two millimolar glucose.
This step will wash the excess dye from the medium, designate the excitation and emission filter sets and exposure times to be used during the experiment. Then set the fraction collector to collect at one minute intervals while para using the eyelets with the low glucose solution. Use the region of interest or ROI tool from the simple PCI imaging software to define the areas or eyelets that you want to image.
Also, circle the background area that will be subtracted after the cells have been washed for 10 minutes, start the glucose gradient fraction collector and initiate time-lapse imaging. After 30 minutes, turn off the software and high glucose syringe pump. Continue perfusing with low glucose to wash out the effect of the high glucose.
After 10 minutes, stop the flow of low glucose by turning off the syringe pump. Following the perfusion, export the data to excel for analysis. The amount of insulin secreted into the perfu eight can be determined by ELA mouse eyelets.
Were perfused with a linear gradient of two to 25 millimolar glucose as shown here. Calcium influx and insulin secretion are triggered after about 13 minutes of perfusion corresponding to six millimolar.Glucose. Changes in mitochondrial potentials are seen earlier, as expected at about 11 minutes.
This data demonstrates the advantage of using this microfluidic network to characterize eyelet physiology. We've just shown you how to use our microfluidic para fusion device for the study of eyelet physiology. When doing this procedure, it's important to remember to make sure there no bubbles in the device, as this could cause the eyelets to move during an experiment.
Also, the flury can be adjusted up to one ml per minute without disturbing the eyelets. So That's it. Thanks for watching and good luck with your experiment.
