Passage method of pluripotent stem cells and application thereof
A technology for pluripotent stem cells and uses, which is applied in the field of subculture of pluripotent stem cells, can solve problems such as not being suitable for subculture of pluripotent stem cells in suspension culture, reducing the feasibility of directed differentiation, and chromosomal variation of pluripotent stem cells, so as to improve the formation of clonal balls ability, preventing mutual aggregation into clusters, and improving the quality of stem cells
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[0049] Example one
[0050] Passaging methods of pluripotent stem cells, such as figure 1 As shown, including the following steps:
[0051] Step 101: Move multiple pluripotent stem cell pellets into the culture medium and mix them evenly;
[0052] Step 102: Use a pipette to suck the pluripotent stem cell pellet mixed in the culture medium, and use the pressure generated by the pipette to squeeze the pluripotent stem cell pellet mixed in the culture medium to pass through 30-70 μm The sieve of the cell sieve forms small clumps of pluripotent stem cells mixed in the culture medium;
[0053] Step 103: Transfer the small clumps of pluripotent stem cells mixed in the culture medium into a new petri dish, continue to culture, and complete the passage;
[0054] The pluripotent stem cell pellets are spherical aggregates of pluripotent stem cells formed by suspension culture (non-adherent), and the diameter of the pluripotent stem cell pellets is 240±40 μm.
[0055] Compared with the existing pa...
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[0076] Example two
[0077] The pluripotent stem cells used in the following test examples are all human embryonic stem cells BG02.
[0078] The suspension cultured pluripotent stem cells were subcultured using a cell sieve when the average diameter reached 240 μm.
[0079] Step 1: Gently shake the petri dish to make the suspension cultured pluripotent stem cell pellets gather in the center of the petri dish.
[0080] Step 2: Use a 200μl pipette to pick up a small amount of pluripotent stem cell pellets and place them in a well of a 96-well plate.
[0081] The third step: Count under the microscope and adjust the number of balls to 70-90.
[0082] Step 4: Transfer the pluripotent stem cell pellets in the small wells to 3ml of 37℃ pre-warmed medium (containing 10μmol / L Rock inhibitor Y-27632).
[0083] Step 5: Place a cell sieve with a diameter of 40 μm on a clean tube.
[0084] Step 6: Use a pipette to gently pipette the pluripotent stem cell pellet in step 4, and use a 1ml pipette tip wit...
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