Our lab is working to understand, prevent, and reverse beta cell dysfunction and other islet abnormalities in Type 1 and Type 2 diabetes. Our studies focus on the pathogenesis of human diabetes by integrating studies of the human pancreas and islet biology and health and disease. Human islets are 3D spherical structures, and accessing cells throughout the entire islet presents some experimental challenges, for example, when trying to introduce biosensors to understand islet cell signaling.
Our pseudoislet protocol overcomes this challenge by performing genetic manipulations in the single-cell state and reaggregating these transduced human islet cells into pseudoislets for downstream studies. Our pseudoislet system allows us to express biosensors throughout the entire islet rather than just the islet surface. Combined with our lifestyle imaging and microperfusion system, we can both measure and co-register dynamic intracellular processes with downstream hormone secretion.
This protocol could be applied to answer a variety of important scientific questions. For example, gene-silencing strategies could be combined with this approach to understand the impact of a gene of interest on islet function and associated signaling pathways either globally or in a cell-specific manner. Start by connecting the tubing of the microperfusion apparatus to the chip holder.
Flush the line with a baseline secretagogue, like 2-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 5 microliters of DMEM. Pipette around the outer well edges to wet the crevices. Take bright field and dark field images of the cultured human pseudoislets isolated from pancreatic primary human islets.
Using a pre-wetted pipette, collect 30 to 32 pseudoislets in 23 microliters of medium, and dispense them slowly into the microchip well center. Ensure the pipette tip has no pseudoislets left attached. Use a gel-loading tip to gently maneuver the pseudoislets into the well center for maximum field view.
Capture a stereoscope image of the pseudoislets in the microchip to adjust for pseudoislet loss. If all pseudoislets from the plate are not loaded into the microchip, adjust the islet equivalent quantification accordingly. Place the microchip bottom into the holder.
Then, carefully place the microchip top on with the 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 ferial, then screw the tubing into the debubbler 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 pseudoislets 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 UPlan fluoride 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 pseudoislets 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 4 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 debubbler and fraction collector. Close all doors of the environmental chamber to maintain the temperature.
Store the perfusates at minus 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 pseudoislets in the microchip before removal.
to adjust the islet extract IEQ. Using a pipette and a microscope, transfer the pseudoislets from the well to a 1.5-milliliter tube. Ensure that all of the pseudoislets have been collected with the help of the microscope.
Wash twice with 500 microliters of PBS. Then, spin the pseudoislets for one minute at 94 G between each wash. After aspirating the supernatant, add 200 microliters of acid ethanol and store at 4 degrees Celsius overnight.
The next day, centrifuge the extracts, then aliquot 45 microliters of the supernate into three new tubes. Store the tubes at minus 80 degrees Celsius for hormone content analysis. The transduced human islet cells showed reaggregation over time with fully-formed pseudoislets 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 pseudoislets 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 2-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 pseudoislets to 2-millimolar glucose and epinephrine increased the intracellular cAMP concentration, which was associated with increased glucagon.
