Passage method of pluripotent stem cells

A technology of pluripotent stem cells and passage, applied in the direction of non-embryo pluripotent stem cells, artificially induced pluripotent cells, animal cells, etc., can solve the problems of high cost and poor stability, and achieve increased activity, easy operation, and great application value and promotional effects

Active Publication Date: 2022-07-29
深圳市旷逸生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In view of this, the purpose of the present invention is to provide a method for subculture of pluripotent stem cells, which solves the problems of high cost and poor stability of existing subculture methods

Method used

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  • Passage method of pluripotent stem cells
  • Passage method of pluripotent stem cells
  • Passage method of pluripotent stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] A method for passage of pluripotent stem cells provided in this embodiment 1, the method is realized by the following steps:

[0043] S1. Cultivate pluripotent stem cells in a petri dish to obtain pluripotent stem cell colonies;

[0044] S2. Remove the culture medium in the culture dish from the hES that reaches 70-80% fusion, and make the passaging tool move on the surface of the culture dish containing the culture medium, from right to left, push and roll the passaging tool in turn;

[0045] The passaging tool is a spring cutter. The spring cutter includes a precision spring 1, a roller 2 and tweezers. The outer side wall of the roller 2 is provided with a slot 21. When in use, the precision The spring 1 is sleeved on the roller 2, and the end of the tweezers is embedded in the slot 21 on the outer side wall of the roller 2 for compressing the precision spring 1 and adjusting the distance between the precision spring 1; The diameter is 5-10mm; the diameter of the spr...

Embodiment 2

[0053] A method for passage of pluripotent stem cells provided in this embodiment 2, the method is realized by the following steps:

[0054] S1. Cultivate pluripotent stem cells in a petri dish to obtain pluripotent stem cell colonies;

[0055] S2. Remove the culture medium in the culture dish from the hES that reaches 70-80% fusion, and move the passaging tool on the surface of the culture dish containing the culture medium, from right to left, push and roll the passaging tool in turn; The passaging tool used was the same as in Example 1;

[0056] S3, horizontally rotating the direction of the culture dish by 85°, and repeating the process of S2 to obtain the pluripotent stem cell sheet to be cut;

[0057] S4. Add a dissociation solution composed of 1×DPBS supplemented with 2500 mg / L glucose to the culture dish, and put it into 37° C., 5% CO 2 Incubate in an incubator for 3 min to obtain shrunk pluripotent stem cell sheets;

[0058] S5, blowing the bottom of the culture di...

Embodiment 3

[0063] A method for passage of pluripotent stem cells provided in Example 3 of the present invention is achieved by the following steps:

[0064] S1. Cultivate pluripotent stem cells in a petri dish to obtain pluripotent stem cell colonies;

[0065] S2. Remove the culture medium in the culture dish from the hES that reaches 70-80% fusion, and move the passaging tool on the surface of the culture dish containing the culture medium, from right to left, push and roll the passaging tool in turn; The passaging tool used was the same as in Example 1;

[0066] S3, horizontally rotating the direction of the culture dish by 95°, and repeating the process of S2 to obtain the pluripotent stem cell sheet to be cut;

[0067] S4. Add a dissociation solution consisting of 1×DPBS supplemented with 3500 mg / L glucose to the culture dish, and put it into 37° C., 5% CO 2 Incubate in an incubator for 7 minutes to obtain shrunk pluripotent stem cell sheets;

[0068] S5, blowing the bottom of the...

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Abstract

The invention discloses a passage method of pluripotent stem cells. The method comprises a passage tool and a prepared dissociation liquid, the passage tool comprises a precision spring, a rolling shaft and tweezers, the precision spring sleeves the rolling shaft, a clamping groove is formed in the surface of the rolling shaft and can be fixed to the tip end of the tweezers, the distance between spring wires is adjusted by compressing the spring, the assembly can be used for passage of the pluripotent stem cells after being sterilized, and the tweezers are held to push and roll the precision spring during passage. The method comprises the following steps: cutting pluripotent stem cell colonies by using a precise spring wire, cutting the pluripotent stem cell colonies into small pieces with uniform sizes, and then adding dissociation liquid to enable the cut pluripotent stem cell pieces to be easily eluted and collected by pipetting, thereby completing the passage amplification of the pluripotent stem cells. The method is simple, reliable, universal, easy to implement and low in cost, integrates various advantages of an existing method, does not need digestion of an enzyme and a calcium ion chelating agent, does not damage an extracellular matrix of the pluripotent stem cells, and can efficiently carry out pluripotent stem cell passage.

Description

technical field [0001] The invention belongs to the technical field of stem cell passaging, and particularly relates to a passaging method for pluripotent stem cells. Background technique [0002] Pluripotent stem cells have the ability to self-renew and maintain multi-directional differentiation. They are the basis for drug screening, tissue and organ regeneration, and medical treatment. On the coated petri dish, typical round and polygonal colonies are formed by spreading; when the colonies are close to confluence, the pluripotent stem cells need to be passaged and expanded; the commonly used expansion method is to use enzymatic digestion, such as type IV collagenase , dispase, etc., the treatment time is 5-10 minutes, when the edge of the pluripotent stem cell colony is slightly rolled up, remove the digestive enzyme, add a certain amount of culture medium to wash, and then use a pipette or pipette to blow the culture medium to remove the pluripotent stem cells. The colo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735C12N5/074B26D1/00B26D7/26
CPCC12N5/0606C12N5/0607C12N5/0611C12N5/0696B26D1/0006B26D7/2635C12N2510/00C12N2509/00C12N2509/10B26D2001/008
Inventor 宋芸娟蒋斌温斌
Owner 深圳市旷逸生物科技有限公司
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