Start by connecting the tubing of the microperfusion apparatus to the chip holder. Flush the line with a baseline secretagogue, like two-millimolar glucose, for five minutes. Aspirate any extra fluid from the top and bottom valves of the microchip.
The top half of the microchip ensures adequate sealing of wells, while the bottom half contains the wells with a glass cover slip attached. Next, pre-wet a well with five microliters of DMEM. Pipette around the outer well edges to wet the crevices.
Take brightfield and darkfield images of the cultured human pseudo eyelets isolated from pancreatic primary human eyelets. Using a pre-wetted pipette, collect 30 to 32 pseudo eyelets in 23 microliters of medium, and dispense them slowly into the microchip well center. Ensure the pipette tip has no pseudo eyelets left attached.
Use a gel loading tip to gently maneuver the pseudo eyelets into the well center for maximum field view. Capture a stereoscope image of the pseudo eyelets in the microchip to adjust for pseudo eyelet loss. If all pseudo eyelets from the plate are not loaded into the microchip, adjust the eyelet equivalent quantification accordingly.
Place the microchip bottom into the holder, then carefully place the microchip top on with a green gasket facing down. While holding the microchip in place, close the holder to clamp the microchip together. Transfer the secretagogues, pump, and microchip in the holder into the environmental chamber fitted to the confocal microscope and direct the efflux tubing out of the chamber to the fraction collector.
Place the buffers in 15-milliliter conical tubes with openings drilled into their caps to prevent tubing drift. Separate the nut and ferrule. Then screw the tubing into the de-bubbler to prevent line twisting.
Next, set the fraction collector to rotate every two minutes. Then load it with the appropriate number of tubes, accounting for washes and experimental fractions. Start the pump to deliver the baseline glucose medium at 100 microliters per minute.
Watch for droplets at the end of the fraction collector spout, indicative of system flow through. Decreased system efflux, as seen in the tube labeled with the red X, is indicative of system blockage or leak. Once a steady medium flow has been established, rotate the fraction collector head to dispense into the tubes and start the fraction collector.
Collect the first 15 wash fractions to allow the pseudo eyelets to equilibrate. After ensuring continual and accurate medium flow through, discard the washes. While the washes are being collected, set up the microscope for live cell imaging.
Set the objective lens to U Plan Fluorite 20X with 1X zoom. The fluorescence channels to EGFP with emission set to 510 nanometers. Laser wavelength to 488.
And detection wavelength to 500 to 600 nanometers. Set the time series to image acquired every two seconds for the entirety of the experiment and the sampling speed to two microseconds per pixel. Now identify the bottom of the pseudo eyelets in the field of view and adjust the focal plane to 15 micrometers above this position, a frame to be used throughout imaging.
Once the wash is complete, press Start on the imaging software when the fraction collector moves to the first fraction and collect the effluent into 1.5 milliliter tubes. At an appropriate time, switch the tubing from the baseline buffer to the new stimulus buffer tube manually. Place the first 10 collections at four degrees Celsius to prevent hormone degradation and continue to monitor the system flow by checking the effluent volume in each tube.
Once the experiment is complete, switch back to the baseline medium, allowing the pump to wash out the stimulus buffer for five minutes. Stop the pump and disconnect the microchip holder tubing at the de-bubbler and fraction collector. Close all doors of the environmental chamber to maintain the temperature.
Store the perfusates at 80 degrees Celsius for subsequent hormone analysis. Carefully open the microchip holder and lift off the top of the microchip. Take a final microscopic image of the pseudo eyelets in the microchip before removal to adjust the eyelet extract IEQ.
Using a pipette and a microscope, transfer the pseudo eyelets from the well to a 1.5-milliliter tube. Ensure that all of the pseudo eyelets have been collected with the help of the microscope. Wash twice with 500 microliters of PBS.
Then spin the pseudo eyelets for one minute at 94G between each wash. After aspirating the supernatant, add 200 microliters of acid ethanol and store at four degrees Celsius overnight. The next day, centrifuge the extracts.
Then aliquot 45 microliters of the supernatant into three new tubes. Store the tubes at 80 degrees Celsius for hormone content analysis. The transduced human eyelet cells showed reaggregation over time with fully-formed pseudo eyelets formed after six culture days.
The cells began to show visible cADDis fluorescence within 48 hours, and there was high biosensor expression in transduced cells by the end of the culture period. The pseudo eyelets displayed an average transduction efficiency of 60%in alpha cells and 95%in beta cells. The live cell imaging and microperfusion setup facilitated the synchronous collection of the intracellular cAMP dynamics through cADDis fluorescence and hormone secretion.
Exposure to two-millimolar glucose resulted in low and stable relative cADDis intensity and insulin secretion. A robust increase in the intracellular cAMP concentration was seen when the cells were exposed to 20-millimolar glucose and IBMX. This was accompanied by increased insulin and glucagon.
The exposure of the pseudo eyelets to two-millimolar glucose and epinephrine increased the intracellular cAMP concentration, which was associated with increased glucagon.
