Method for inducing differentiation of iPS cells to mammary glands in vitro and special culture medium for method

A technology for differentiation medium and culture medium, applied in the fields of developmental biology and biomedical engineering, can solve the problems of complicated steps, low differentiation efficiency, lack of iPS cells, etc., and achieve the effects of broad application prospects, high differentiation efficiency and simple operation.

Active Publication Date: 2020-06-05
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

In 2017, Qu et al. achieved 3D differentiation of human iPS cells into mammary gland-like structures. However, this method of inducing differentiation in vitro has certain defects in the application of tissue engineering and disease model establishment.
This differentiation method has the following deficiencies: 1. In the process of inducing differentiation in vitro, there is not enough human intervention on cell fate, resulting in low differentiation efficiency
3. In view of the above two technical characteristics, the steps of this differentiation method are cumbersome, the yield of mammary gland cells is low, and it is difficult to obtain a large number of single mammary gland cells at different differentiation stages
[0005] In summary, due to the lack of effective methods for inducing mammary gland differentiation of iPS cells, the application of stem cells in the field of breast diseases is greatly limited, and the development of new iPS cells for mammary gland differentiation induction The method is significant

Method used

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  • Method for inducing differentiation of iPS cells to mammary glands in vitro and special culture medium for method
  • Method for inducing differentiation of iPS cells to mammary glands in vitro and special culture medium for method
  • Method for inducing differentiation of iPS cells to mammary glands in vitro and special culture medium for method

Examples

Experimental program
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Effect test

Embodiment 1

[0053] Example 1 Induction of directed differentiation of human iPS cells into mammary gland cells in vitro

[0054] 1, use following culture medium in the embodiment:

[0055] iPS cell maintenance medium: mTeSR medium and 10 μM Y27632;

[0056] Differentiation medium I: E6 medium, 10 ng / mL BMP4, 10 μM SB431542 and 10 μM SU5402;

[0057] Differentiation medium II: E6 medium, 10 ng / mL BMP4, 10 μM SB431542;

[0058] Differentiation medium III: including MammoCult™ Human Medium Kit medium and 5 ng / mL BMP4;

[0059] Differentiation medium IV: MammoCult™ Human Medium Kit medium.

[0060] 2. A method for inducing human iPS cells to differentiate into mammary gland cells in vitro, specifically comprising the following steps:

[0061] S1. Take human iPS cells with a confluence of 70%-80%, digest them with accutase for 2-5 minutes, collect single cells by centrifugation, wash with PBS, and obtain seed cells; seed cells at 350,000-400,000 cells / cm 2 The density was inoculated on th...

Embodiment 2

[0066] Example 2 Induction of directed differentiation of human iPS cells into mammary gland cells in vitro

[0067] 1, use following culture medium in the embodiment:

[0068] iPS cell maintenance medium: mTeSR medium and 10 μM Y27632;

[0069] Differentiation medium I: E6 medium, 10 ng / mL BMP4, 10 μM SB431542 and 10 μM SU5402;

[0070] Differentiation medium II: E6 medium, 10 ng / mL BMP4, 10 μM SB431542;

[0071] Differentiation medium III: including MammoCult™ Human Medium Kit medium and 10 ng / mL BMP4;

[0072] Differentiation medium IV: MammoCult™ Human Medium Kit medium.

[0073] 2. A method for inducing human iPS cells to differentiate into mammary gland cells in vitro, specifically comprising the following steps:

[0074] S1. Take human iPS cells with a confluence of 70%-80%, digest them with accutase for 2-5 minutes, collect single cells by centrifugation, wash with PBS, and obtain seed cells; seed cells at 350,000-400,000 cells / cm 2 The density was inoculated on t...

Embodiment 3

[0079] Example 3 Induction of directed differentiation of human iPS cells into mammary gland cells in vitro

[0080] 1, use following culture medium in the embodiment:

[0081] Differentiation medium I: mTeSR medium and 15 μM Y27632;

[0082] Differentiation medium II: E6 medium, 15 ng / mL BMP4, 10 μM SB431542 and 10 μM SU5402;

[0083] Differentiation medium III: including MammoCult™ Human Medium Kit medium and 10 ng / mL BMP4;

[0084] Differentiation medium IV: MammoCult™ Human Medium Kit medium.

[0085] 2. A method for inducing human iPS cells to differentiate into mammary gland cells in vitro, specifically comprising the following steps:

[0086] S1. Take human iPS cells with a confluence of 70%-80%, digest them with accutase for 2-5 minutes, collect single cells by centrifugation, wash with PBS, and obtain seed cells; seed cells at 350,000-400,000 cells / cm 2 The density was inoculated on the Matregel-coated culture plate for 30 min, and the cells were inserted into iPS...

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Abstract

The invention discloses a method for inducing differentiation of iPS cells to mammary glands in vitro and a special culture medium for the method. The in-vitro induction method comprises the followingsteps: dissociating the iPS cell into single cells, inoculating an iPS cell maintaining culture medium with the single cells as seed cells, and obtaining high-density monolayer cells; culturing the monolayer iPS cells in a differentiation culture medium I, and changing solutions for the cells every day; discarding the culture medium, culturing the cells with a differentiation culture medium II, and changing solutions for the cells every other day; discarding the culture medium, culturing the cells with a differentiation culture medium III, and changing solutions for the cells every other day;and discarding the culture medium, and continuously culturing the cells with a differentiation culture medium IV. The method can obtain a large quantity of single mammary gland cells at different differentiation stages, has the advantages of being simple to operate, low in cost, high in differentiation efficiency and the like, and has broad application prospects.

Description

technical field [0001] The invention belongs to the technical fields of developmental biology and biomedical engineering. Specifically, it relates to a method for inducing iPS cells to differentiate in the direction of mammary glands in vitro and its special medium; more specifically, it relates to a method based on in vitro directional induction of differentiation, transforming iPS cells into mammary gland cells, and obtaining a large number of single cells in Methods for different differentiation stages of mammary gland cells and their special media. Background technique [0002] Stem cells have attracted much attention in various fields such as life sciences, biology, and medicine because of their self-renewal and multi-directional differentiation potential. At present, research on stem cells is emerging in an endless stream around the world. From blood diseases to tissue damage repair to anti-aging, stem cell technology is gradually transitioning from clinical basic res...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10
CPCC12N5/0631C12N2510/00C12N2506/45C12N2501/155C12N2501/727
Inventor 丁俊军刘静馨赵偲
Owner SUN YAT SEN UNIV
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