| Usage | 1. Experimental instruments and materials:
Instruments: biological safety cabinet, cell incubator, horizontal centrifuge, inverted microscope, cryogenic refrigerator
Materials: 6-well cell culture plate, ultra-low adsorption 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, 50 mL sizes)
2. Use process of reagent kit:
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 bright, 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.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 Induction of post-primitive stripe (4 days)
8. D0, thaw supplement A, mix basal medium 1 and supplement A as post-primitive stripe induction medium.
9. Suction out the EB formation culture medium in the hole, and be careful not to suck out the EB.
10. Add 2mL of original stripe induction medium to each well, put it in 37 ℃, 5% CO2The culture was continued in the incubator.
11. D2 replace 2mL of the original stripe induction medium per well, as operated in 9 and 10.
③ Intermediate mesoderm induction (3 days)
12. D4, thaw the supplement B, mix the basal medium 2 and the supplement B evenly, and serve as an intermediate mesoderm induction medium. Per well
2 mL of intermediate mesoderm induction medium was replaced as operated in 9, 10.
13. Replace 2mL of intermediate mesoderm induction medium per well on D6, as operated in 9 and 10.
④ Nephrogenesis (5 days)
14. D7, thaw supplements C and D, and mix 10 mL of basal medium 3 and supplement C to form nephrogenesis medium 1. 2 mL of nephrogenesis medium 1 was replaced per well, operated as 9 and 10, and incubated in a 37 °C, 5% CO2 incubator for 1 hour.
15. After 1 hour, 30 mL of basal medium 3 and supplement D were evenly mixed to form nephrogenesis medium 2. 2 mL of nephrogenesis medium 2 was replaced per well as operated as 9, 10.
16, D9, D11, replace 2 mL of nephrogenesis medium 2 per well, as operated in 9, 10.
⑤ Renal organoid maturation (11 days)
17. Starting from D12, change the maturation medium every 2 days, 2 mL per well, operate as 9, 10, to D23. |
| Description | This product is a kit for inducing differentiation of renal organoids by hPSC. Human pluripotent stem cells hPSC can be induced and differentiated through this kit to obtain renal organoids. This kind of organ has a cell composition similar to kidney, including glomerulus and renal tubular epithelial 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 | K01 | EB forming medium | 30mL | -20 ℃, 12 months | | K01-S | EB Formation Supplement | 60uL | -20 ℃, 6 months | | Post-primitive stripe induction stage | K02 | Basal Medium 1 | 20mL | -20 ℃, 12 months | | K02-A | Supplement A | 160uL | -20 ℃, 6 months | | Intermediate mesodermal induction stage | K03 | Basal Medium 2 | 20mL | -20 ℃, 12 months | | K03-B | Supplement B | 400uL | -20 ℃, 6 months | | Stage of nephrogenesis | K04 | Basal Medium 3 | 40mL | -20 ℃, 12 months | | K04-C | Supplement C | 50uL | -20 ℃, 6 months | | K04-D | Supplement D | 600uL | -20 ℃, 6 months | | Organoid maturation stage | K05 | Maturation medium | 60mL | -20 ℃, 12 months |
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