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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

Active Publication Date: 2013-06-12
吉林省中科生物工程股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Enzyme digestion combined with pipette gun pipetting compared with enzyme digestion alone, although the digestion is relatively uniform, it is still difficult to grasp and control when passaged, resulting in uneven size of the prepared small clumps, unable to inhibit spontaneous differentiation, and reduce the feasibility of directed differentiation
And long-term enzyme digestion can easily cause chromosomal variation or deletion in pluripotent stem cells
[0008] It can be seen that the existing cell subculture methods are not suitable for the subculture of suspension cultured pluripotent stem cells

Method used

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  • Passage method of pluripotent stem cells and application thereof
  • Passage method of pluripotent stem cells and application thereof
  • Passage method of pluripotent stem cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Methods for subculture of pluripotent stem cells, such as figure 1 shown, including the following steps:

[0051] Step 101: moving multiple pluripotent stem cell pellets into the culture medium and mixing them evenly;

[0052] Step 102: Use a pipette to suck up the pluripotent stem cell pellets mixed in the culture medium, and use the pressure generated by the pipette to squeeze the said pluripotent stem cell pellets mixed in the culture medium to pass through a 30-70 μm The sieve of the cell sieve, forming small clumps of pluripotent stem cells mixed with the culture medium;

[0053] Step 103: Transfer the small clumps of pluripotent stem cells mixed in the culture medium into a new culture dish, continue culturing, and complete passage;

[0054]The pluripotent stem cell spheres are spherical aggregates of pluripotent stem cells formed in suspension culture (non-adherent), and the diameter of the pluripotent stem cell spheres is 240±40 μm.

[0055] Compared with exi...

Embodiment 2

[0077] The pluripotent stem cells used in the following test examples are human embryonic stem cells BG02.

[0078] The pluripotent stem cells cultured in suspension were passaged using a cell sieve when the average diameter reached 240 μm.

[0079] Step 1: Gently shake the culture dish to make the suspension-cultured pluripotent stem cell pellets gather in the center of the culture dish.

[0080] Step 2: Use a 200 μl pipette gun to suction a small amount of pluripotent stem cell pellets without selection and place them in a well of a 96-well plate.

[0081] Step 3: count under a microscope, and adjust the number of small balls to 70-90.

[0082] Step 4: Transfer the pluripotent stem cell pellets in the small wells to 3 ml of 37°C preheated medium (containing 10 μmol / L Rock inhibitor Y-27632).

[0083] Step 5: Place a cell sieve with a diameter of 40 μm on a clean small tube.

[0084] Step 6: Gently blow and suck the pluripotent stem cell pellet in step 4 with a pipette gun...

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Abstract

The invention relates to the field of biotechnologies, in particular to a passage method of pluripotent stem cells and application thereof. The method includes: shifting quantitative pluripotent stem cell balls into a medium, and mixing them evenly; making use of a liquid shifter to suck the pluripotent stem cell balls mixed in the medium, and making use of the pressure generated by the liquid shifter to perform extrusion so as to make the balls pass a cell sieve of 30-70 micrometers, thus forming more pluripotent stem cell small block masses mixed in the medium; and transferring the pluripotent stem cell small block masses mixed in the medium into a new culture dish, thus completing passage. The method is used for passage of vertebrate embryonic stem cells and induced pluripotent stem cells. The method provided in the invention is suitable for pluripotent stem cell balls formed by suspension culture, and is a novel passage method with no need for enzymatic digestion. After passage by the method, pluripotent stem cell cloned balls have a high survival rate up to 90%, and each passage ratio can reach 1:8-1:10, so that the method can meet the large-scale volume production needs of stem cells.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a passage method and application of pluripotent stem cells. Background technique [0002] Pluripotent Stem Cells (PSCs) can proliferate rapidly under suitable conditions in vitro without differentiation, and after a large amount of expansion in vitro, they can be treated with specific conditions, such as induced by a combination of proteins or small molecular compounds, can Make it oriented to differentiate into cells with specific functions, and collect these specific cells that lose pluripotency after differentiation and can be used to treat human related diseases. For example, pluripotent stem cells can be induced into nerve cells and injected into the brain for use For the treatment of Alzheimer's disease or Huntington's disease caused by neuronal defects. [0003] In industrial production, the common proliferation and culture methods of pluripotent stem cells are adherent cultu...

Claims

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Application Information

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IPC IPC(8): C12N5/0735C12N5/02C12N5/071
Inventor 蒋斌李天晴季维智牛昱宇
Owner 吉林省中科生物工程股份有限公司
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