This protocol uses a static suspension culture strategy for differentiating pancreatic progenitors into islet clusters, reduces the stresses that cells experience during shaking culture, and greatly improves differentiation success. The hardware protocol is beginner-friendly, and save time for optimization of the first four stages. Personnel with basic stem cell culture techniques can reproduce this protocol with a good chance of generating functional islet-like clusters.
To begin, precoat culture wells with hESC-qualified matrigel diluted in ice-cold DMEM/F12 and place the plate in a humidified 37-degrees-Celsius 5%carbon dioxide incubator for 30 minutes. Aspirate the spent medium from the hPSC culture and rinse once with DPBS. Dissociate hPSC culture into single cells with cell dissociation enzyme at 37 degrees Celsius for three to five minutes.
After dissociation, rinse the cells by adding warm DMEM/F12 medium and transfer them into a 15-or 50-milliliter conical tube. Centrifuge the cell suspension at 300 G for five minutes and resuspend the pellet in a stem cell complete medium containing 10-micromolar Y-27632. Then, perform live cell counting using a hemocytometer.
After counting aspirate diluted matrigel and immediately seed live cells at 1.6 to 1.8 times 10 to the five per square centimeter density on matrigel-coated wells. Incubate the cells in a humidified incubator for 24 hours. Next, on stage one, day one, prepare stage 1A medium.
Replace the spent medium with stage 1A medium and incubate for 24 hours. The next day, replace the spent medium from the wells with freshly prepared stage 1B medium. On stage two, day one, prepare stage 2A medium and replace the medium from wells with stage 2A medium.
Incubate for 24 hours before adding stage 2B medium into the wells;An stage three, day one, aspirate the spent medium from wells, then add freshly prepared stage 3 medium and incubate in a humidified incubator. On stage four, day one, replace the spent medium with freshly prepared stage 4 medium and incubate in a humidified incubator. On stage four, day five, pretreat the microwells with 500 microliters of anti-adherence rinsing solution per well.
Centrifuge the microwell plate at 1, 300 G for five minutes and observe under a microscope to ensure no air bubbles are trapped in microwells. Next, aspirate the rinsing solution from the wells and rinse once with one milliliter of DPBS. Then, add one milliliter of aggregation medium to each well.
After removing the spent medium from the pancreatic progenitor cultures, wash the culture once with DPBS before dissociating into single cells with dissociation reagent at 37 degrees Celsius for 10 to 12 minutes. Rinse the cells with warm DMEM/F12 and transfer them into a tube for centrifugation at 300 G for five minutes. Resuspend the pellet in the aggregation medium and perform live cell counting.
Seed 2.4 to 3.6 million cells per well and add an aggregation medium to each well to achieve a final volume of two milliliters per well. Using a P-1000 pipette tip, gently pipette the cells up and down for even distribution. Centrifuge the cell suspension at 300 G for five minutes to capture the cells in the microwells and incubate in a humidified incubator for 24 hours to initiate aggregation.
After aggregation, gently pipette the aggregates up and down several times to float any non-aggregated cells. Once the aggregates settle, carefully remove the spent medium containing floating non-aggregated cells without aspirating aggregates. Dispense fresh stage 5 medium onto the surface of the microwell plate to dislodge aggregates from the microwells, and transfer them into the ultra low-attachment six-well plate.
After collecting all aggregates in the ultra low-attachment six wells, resuspend them in stage 5 medium to adjust the density to 20 clusters per 100 microliters. Then, using a multichannel pipette, dispense 50 microliters of stage 5 medium into each well of an ultra-low attachment ULA flat-bottom 96-well plate. Fill the corner and edge wells with 200 microliters of DPBS.
Gently mix cluster resuspension every time before dispensing. Then, add 100 microliters of the cluster resuspension into each well of the ULA flat-bottom 96-well plate. Place the culture plates on a level surface in a humidified incubator for 24 hours.
The next day or on stage five, day two, using a multi-channel pipette, gently remove 90 microliters of the spent medium from each well and refresh with 100 microliters of stage 5 medium. On stage six, day one, replace the 90 microliters of spent medium with 100 microliters of stage 6 medium per well. On stage seven, day one, remove the spent medium and add 100 microliters of stage 7 medium per well.
In this study, using microwell plates, thousands of pancreatic progenitor clusters with uniform size and morphology were generated at a time, and the average aggregation efficiency was around 74%Importantly, expression of PDX1 and NKX6.1 was maintained at high levels in these clusters post-aggregation. Insulin expressing cells were gradually induced within endocrine clusters, as indicated by increased insulin GFP signals in living cultures over time. Cluster size increases from an average diameter of 150 microns to 220 microns between stages five and seven.
Characterization of cell composition shows that stage seven hPSC islets generated by the hybrid protocol were primarily endocrine, and comprised of four major islet cell types. This hybrid protocol generated largely monohormonal islet cells and a minority of bihormonal cells. Differentiating beta cells express NKX6.1, MAFA, NEUROD1, PDX1, and GLUT1.
Furthermore, 60%insulin and PDX1 double-positive cells and 50%C-peptide and NKX6.1 double-positive cells were induced in stage seven cultures. In vitro, static glucose-stimulated insulin secretion assays demonstrated that stage seven hPSC islets secrete high insulin levels in response to high glucose and direct depolarization. The total insulin content of hPSC islets is approximately half the amount of cadaveric islets.
The multi-well-based static culture system for generating stem cell-derived islets facilitates various in situ assays, and can serve as a suitable platform for protocol development and small-scale screening studies. To minimize cluster fusion during a static culture in 96-well plate, do not tilt the plate when performing the medium change. Always keep the plate horizontal on a level surface.
