| Usage | 1. Experimental instruments and materials:
Instruments: biological safety cabinet, cell incubator, horizontal shaker, inverted microscope, low temperature refrigerator
Materials: 6-well cell culture plate, ultra-low sorption 6-well plate, centrifuge tubes (15 mL and 50 mL sizes), pipettes (10 μL, 100 μL, 1000 μL sizes), sterile tips (10 μL, 200 μL, and 1000 μL sizes), pipettes (10 mL sizes, 50 mL sizes)
2. Kit usage process:
① EB formation (2 days)
1. hPSCs were grown to 70-80% confluence in one well of a matrigel-coated six-well plate.
2. Aspirate the culture medium.
3. Add 1mL PBS (no calcium and magnesium ions) to the wells, wash and aspirate.
4. Add 1mL accutase to the well and digest at 37 °C for 3min.
5. Take 11mL of EB forming medium and mix 22μL of EB forming supplement to obtain EB forming complete medium, and add 2mL of EB to each well of 3 wells of an ultra-low adsorption 6-well plate to form complete medium.
6. When you see that the clones are white and shiny, gently pat the side of the plate (twice on each side), pat the cells down, and then add 1mL of EB to form a complete medium in the biological safety cabinet to terminate digestion. Suck this 2mL system into a 15mL centrifuge tube filled with 3mL of EB to form a complete medium and centrifuge 160g for 5min, discard the supernatant until the remaining tens of microliters.2Overnight.
7. On the next day (D-1), it can be seen that a large number of uniform EB is formed under the microscope. Half of the solution is changed, 50% of the EB formation medium (without supplements) is supplemented, and it is placed on a horizontal shaker for culture. (Note: The culture medium used should be placed on the workbench for 30min in advance to restore room temperature, and the same is true for the following operations.)
② DE induction (5 days)
8. D0, thaw supplement A, mix basal medium 1 and supplement A as DE induction medium, and restore room temperature.
9. Suction out the EB formation culture medium in the hole, and be careful not to suck out the EB.
10. Add 2mL DE induction medium to each well, put it in 37 ℃, 5% CO2The culture was continued in the incubator.
11. Change the DE induction medium every day for 5 days.
③ PE induction (6 days)
12. D5, thaw supplement B, mix basal medium 2 and supplement B as PE induction medium. 2 mL of PE induction medium was replaced daily per well for 6 days as operated as 9, 10.
④ EP induction (5 days)
13. D11, thaw supplement C, mix basal medium 3 and supplement C as EP induction medium. 2 mL of EP induction medium was replaced daily per well for 5 days as operated as 9, 10.
⑤ Islet organoid maturation stage (taking 20 days as an example)
14. D16, thaw the supplement D, mix the basal medium 4 and the supplement D evenly, and use it as the islet organoid maturation medium. 2 mL of maturation medium was changed per well daily for 20 days, as operated 9, 10. |
| Description | This product is a kit for inducing differentiation of islet organoids by PSC. Human pluripotent stem cells hPSC can be induced to differentiate through this kit to obtain islet organoids. This kind of organ has a cell composition similar to islet and has mature α and β cells.
This kit requires the operator to have experience in hPSC culture and some knowledge of organoids.
Product composition: | Cultivation stage | serial number | Name | Specifications | Save | | EB formation stage | I01 | EB forming medium | 30mL | -20 ℃, 12 months | | I01-S | EB Formation Supplement | 60uL | -20 ℃, 6 months | Definite endoderm (DE)
Induction phase | I02 | Basal Medium 1 | 50mL | -20 ℃, 12 months | | I02-A | Supplement A | 250uL | -20 ℃, 6 months | Pancreatic endoderm (PE)
Induction phase | I03 | Basal Medium 2 | 60mL | -20 ℃, 12 months | | I03-B | Supplement B | 30uL | -20 ℃, 6 months | Endocrine progenitor cells
(EP) induction phase | I04 | Basal Medium 3 | 50mL | -20 ℃, 12 months | | I04-C | Supplement C | 650uL | -20 ℃, 6 months | Islet organoid maturation
Stage | I05 | Basal Medium 4 | 200mL | -20 ℃, 12 months | | I05-D | Supplement D | 600uL | -20 ℃, 6 months |
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