| Usage | 1. Experimental instruments and materials:
Instruments: biological safety cabinet, cell incubator, horizontal centrifuge, inverted microscope, cryogenic refrigerator
Materials: Cell culture plates (sizes: 6-well, 12-well, 24-well), centrifuge tubes (sizes: 15 mL and 50 mL), pipettes (sizes: 10 μL, 100 μL, 1000 μL), sterile tips (sizes: 10 μL, 200 μL, and 1000 μL), pipettes (sizes: 10 mL, 50 mL)
2. Experimental contents and methods:
1. hPSC differentiates into endodermal cells (DE)
(1) Take 5.938 mL of basal medium 1 + 60μL of supplement A + 2μL of supplement B to prepare DE differentiation medium ①, which can cultivate 3 wells of a 6-well plate at the same time;
(2) Thaw the matrigel at 4 °C, evenly mix with DMEM/F12, lay the plate, and set aside.
(3) When the confluence of hiPSC reaches 75%-85%, aspirate the culture medium and use 2mL 1x room temperature PBS (excluding Ca2+/Mg2+) The hiPSC was washed and PBS was aspirated.
(4) 1 mL of room temperature hESC/iPSC Passage Solution (human pluripotent stem cell digestion juice) (abs9409) was added and transferred to 37 °C 5% CO2In the cell incubator for 5 min to dissociate into single cells.
(5) After 5 min, aspirate the hESC/iPSC Passage Solution (human pluripotent stem cell digestion juice) (abs9409), add an equal volume of hESC/iPSC complete medium, and gently pipate the cells up and down with a P-1000 pipette tip to make a single cell suspension, count the cells with an automatic cell counter, and exclude dead cells with trypan blue. The cell single suspension was collected into a 15 mL centrifuge tube and centrifuged at 300 g for 5 min at room temperature.
(5) The coating liquid was carefully aspirated from the matrigel-coated plate without damaging the matrigel-coated surface.
(6) At the end of centrifugation, the supernatant is fully removed, the cells are resuspended with 1 mL DE differentiation medium ①, and the liquid is gently pipetted up and down to ensure the uniformity of the single cell solution.
(7) Transfer the cell suspension to the coated wells, add 1mL DE differentiation medium ①, and make the volume in the wells reach 2mL;
(8) After 24 hours (day 1), 13.86 mL of basal medium 1 + 140 μL of supplement A was prepared into DE differentiation medium ②. Three wells of a 6-well plate can be cultured at the same time. The medium was aspirated and 2mL of DE differentiation medium ② was added. Culture continuously for 2 days.
(9) On the 3rd day, before changing HS medium, a well was taken for endoderm index detection.
2. DE induced differentiation into hepatic specialized lineage cells (HS)
(1) On day 3, 19.9 mL of basal medium 2 was thoroughly mixed with 100 μL of supplement C to be formulated into HS medium, and 2 wells of a 6-well plate could be cultured simultaneously. The medium was aspirated from the culture, and each well in the 6-well plate was treated with 2 mL 1x room temperature PBS (without Ca2+/Mg2+) The cultures were washed, then PBS was removed from the wells and 2 mL of HS medium was added per well.
(2) Place the culture plate in 37 °C 5% CO2Culture in an incubator, change the medium every 24 hours, and culture continuously for 5 days.
(3) On the 8th day, a hole can be taken for HS index detection before glue embedding.
3. HS-induced differentiation into 3D liver organoids (taking 6 wells of 24-well plate as an example)
(1) On day 8, 194.1 mL of basal medium 3 +5.9 mL of supplement was thoroughly mixed to prepare 3D liver organoid medium, which was stored in separate packages.
(2) Thaw the Matrigel on ice and place the pipette tip in a-20 ℃ refrigerator in advance.
(3) Aspirate the HS medium and use 2mL 1x room temperature PBS (without Ca2+/Mg2+) washed once.
(4) Add room temperature EDTA containing 10 μM Y-27632 and add at 37 °C, 5% CO2Incubate in an incubator for 5 min; After 5 minutes, the EDTA was aspirated, and 1mL of DMEM/F12 (containing 10μM Y-27632) returned to room temperature was added to each well, and the cells were gently pipped to separate the cells from the bottom of the well.
(5) Cells were counted using an automated cell counter and dead cells were excluded using trypan blue.
(6) Collect the cell suspension into a 15 mL centrifuge tube, centrifuge at 300 g for 5 min at room temperature, aspirate the medium and resuspend the cells in cold Matrigel, embedded in 30-50 μL Matrigel at a density of 3000-10000 cells/well, and place the Matrigel on ice with the cells.
(7) 200 μL of Matrigel/cell mixture was seeded into a 24-well plate at 30 μL per well.
(8) Place the plate in a 37 °C, 5% CO2 incubator for 20-30 minutes to solidify the matrigel.
(9) Add 700 μL of 3D liver organoid medium to each well for long-term culture, change the medium every other day, and passages for 7-10 days.
4. 3D liver organoid passage
(1) Take out the frozen matrigel and thaw it at 4 °C, and place the gun tip at-20 °C for precooling for 1h in advance;
(2) Aspirate the medium, add 1mL of pre-cooled PBS to dissolve the matrigel, transfer the mixed solution to a 1.5 mL EP tube, centrifuge at 300g for 5min, and remove the upper layer of PBS and matrigel;
(3) Add 1mL of Tryple express, blow for 5-8 times, make the organoid fully contact with Tryple express, then transfer to an incubator, and let it stand for dissociation for 5 minutes;
(4) After 5 minutes, take out the EP tube, centrifuge 300g for 5 minutes, remove the supernatant, add 1ml DMEM/F12, blow the mixture in the hole for 40-50 times, completely dissociate the organoid mass into single cells, centrifuge, and remove the supernatant;
(6) Use the pre-cooling gun tip to add an appropriate amount of matrigel to the centrifuge tube in an amount of 30μL per well, blow 5-8 times, evenly mix the single cell-matrigel, and pay attention to avoid bubbles during the blowing process;
(7) Take out the 24-well plate placed in the incubator in advance, add 30μL gel droplets to the center of the well, and gradually move the gun tip upward when slowly injecting the mixed solution to make the cells evenly distributed in the gel;
(8) Place the culture plate in an incubator for 20-30min to solidify the glue droplets;
(9) Carefully add 700μL of 3D liver organoid culture medium returned to room temperature along the pore wall, and be careful not to touch glue droplets;
(10) Put the well plate in the incubator, continue to culture, and change the solution every other day (the solution can be changed on Monday, Wednesday, and Friday, and 800μL can be added on Friday, and the solution cannot be changed on Saturday and Sunday), and passaged once every week or so.
(11) After liver organoids mature, staining identification or hepatic progenitor cells/hepatic stem cells can be carried out. |
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