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Method for culturing pluripotent stem cells

A technology of pluripotent stem cells and culture methods, applied in the field of pluripotent stem cell culture, can solve the problems of inability to accurately evaluate cell growth and differentiation, inability to carry out large-scale drug screening, and high cost of suspension culture systems, so as to reduce the differences between experiments , saving cost, and stabilizing multiplication effect

Active Publication Date: 2021-06-29
SUNSHINE LAKE PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method for cultivating pluripotent stem cells provided by the present invention overcomes the shortcomings of the prior art: the differentiation efficiency of the planar culture differentiation system in the existing screening system is low, the growth and differentiation of cells cannot be accurately evaluated, and the large-system suspension culture High system cost, unable to carry out large-scale drug screening, etc.

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  • Method for culturing pluripotent stem cells
  • Method for culturing pluripotent stem cells
  • Method for culturing pluripotent stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] In this example, the dynamic suspension culture of human embryonic stem cells H9 was carried out, and relevant detection was carried out on the cultured cells.

[0071] Dynamic suspension culture of pluripotent stem cells:

[0072] (1) with 5.5×10 5 Inoculation density of cells / mL Human embryonic stem cells H9 were inoculated in a 48-well plate, and dynamic suspension culture was carried out on an orbital shaker. ±5%, 5% CO 2 (v / v); fresh medium was replaced every 24 hours of culture, co-cultured for 4 days, the particle size of cell clusters was 300-400 μm, and the cell aggregation morphology was shown in figure 1 shown.

[0073] (2) Take out the cell suspension, pass through a 37 μm reversible filter to enrich the cell cluster (remove single cells), use 16 mL of Accutase (StemCell) to backwash the cell cluster into a centrifuge tube and place it in a 37°C water bath for 15 min. Pipet once every 5 minutes until cell flocculation appears, add 2 times the volume of m...

Embodiment 2

[0082] In this example, induced pluripotent stem cells (iPS) are dynamically suspended and cultured, and relevant tests are performed on the cultured cells. Except for the step (1) in the suspension culture, other steps and detection methods are the same as in Example 1. Step (1) in the present embodiment suspension culture is:

[0083] Take 5.5×10 5 Inoculation density of cells / mL Inoculated induced pluripotent stem cells (iPS) in 48-well plates, and carried out dynamic suspension culture on an orbital shaker, the medium was mTeSR1 complete medium, the culture system was 600 μL, the rotation speed was 180 rpm, and the temperature was 37°C , relative humidity 90±5%, 5% CO 2 (v / v); fresh medium was replaced every 24 hours of culture, and co-cultivated for 4 days.

[0084] The aggregation morphology of the cells obtained through step (1) culture Figure 5 Shown; flow detection results see Image 6 shown, according to Image 6 , after being cultured by the method of the pre...

Embodiment 3

[0086] In this example, the dynamic suspension culture of human embryonic stem cells H9 was carried out, and relevant tests were carried out on the cultured cells:

[0087] Take 4.5×10 5 Inoculation density of cells / mL Human embryonic stem cells H9 were inoculated in a 48-well plate, and dynamic suspension culture was carried out on an orbital shaker. ±5%, 5% CO 2 (v / v); fresh medium was replaced every 24 hours of culture, and co-cultivated for 4 days.

[0088] The aggregation morphology of cultured cells is shown in Figure 8 As shown, it can be seen that the cell aggregates are uniform in size and round in shape.

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Abstract

The invention relates to a method for culturing pluripotent stem cells. The method adopts a 48-pore plate as a culture container, the cell inoculation density is 4.5-6.5 * 10 < 5 > cells / mL, the culture volume is 0.4-0.6 mL, and the rotating speed of a shaking table is 170-190 rpm. According to the method, a 48-pore plate suspension culture system suitable for growth and differentiation of the pluripotent stem cells is established from nothing to thing, and the culture system can be used for efficiently screening a differentiation inducer of the pluripotent stem cells. The culture method provided by the invention has the advantages of low cost, high efficiency, capability of maintaining better cellular morphology, stable proliferation multiple, capability of maintaining better karyotype and phenotype, smaller difference between experiments in the differentiation process and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for culturing pluripotent stem cells. Background technique [0002] Stem cells are a special cell group with self-renewal and differentiation potential. Under appropriate culture conditions in vitro, stem cells can be massively expanded and differentiated into cells with specific functions. Therefore, stem cells can provide cells needed for transplantation for clinical disease treatment. At the same time, because drug screening and safety testing are not directly carried out in the human body, human stem cells have also become an ideal model for large-scale new drug screening and drug research. [0003] Stem cells derived from different developmental stages and different tissues and organs have great differences in gene expression regulation, epigenetic state, in vitro proliferation and differentiation potential, etc. Generally speaking, stem cells can be divided into tot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735C12N5/074C12Q1/02
CPCC12N5/0606C12N5/0696G01N33/5005C12N2503/02
Inventor 宋益哲梁德灿唐婷婷陈晓倩欧镇生李景秋邢佩雯刘靖张世都郭蕾蕾郑清炼叶群瑞陈小锋李文佳
Owner SUNSHINE LAKE PHARM CO LTD
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