Method for preparing induced pluripotent stem cells through reprogramming of somatic cells

A pluripotent stem cell and reprogramming technology, applied in the field of induced pluripotent stem cells, can solve the problems of low reprogramming efficiency, difficult to meet clinical needs, time-consuming, etc., and achieve the effect of reducing tumorigenicity and improving reprogramming efficiency.

Inactive Publication Date: 2021-02-02
ZHEJIANG HUODE BIOENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At the same time, multiple small molecule compounds have been found to induce iPSCs, but the reprogramming efficiency is low, and it is time-consuming, which is difficult to meet clinical needs

Method used

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  • Method for preparing induced pluripotent stem cells through reprogramming of somatic cells
  • Method for preparing induced pluripotent stem cells through reprogramming of somatic cells
  • Method for preparing induced pluripotent stem cells through reprogramming of somatic cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] 1.1 Put human-derived skin tissue into a petri dish, and rinse with 4°C pre-cooled phosphate buffered saline solution (PBS) rapidly and repeatedly for 4 times.

[0030] 1.2 Treat the skin tissue with sterilized ophthalmic scissors and a scalpel to remove the white subcutaneous fat, remove the epidermis and subcutaneous tissue, leave the dermis, and obtain the pretreated skin tissue. After the pretreated skin tissue was transferred into the culture medium, it was cut into small pieces to obtain skin tissue blocks with neat edges.

[0031] 1.3 Take 3mL of fetal bovine serum (FBS) and add evenly to each well of the 6-well cell culture plate, put the skin tissue block into the incubator and incubate for 0.5-1h, so that the tissue sticks to the bottom of the culture plate.

[0032] 1.4 Add 1 mL of DMEM (Gibco) medium containing 20% ​​(v / v) FBS to the 6-well cell culture plate, and put it back into the incubator for culture.

[0033] 1.5 Change the medium when fibroblasts cr...

Embodiment 2

[0041] 2.1 Put human-derived skin tissue into a petri dish, and rinse with 4°C pre-cooled phosphate buffered saline solution (PBS) rapidly and repeatedly for 4 times.

[0042] 2.2 Treat the skin tissue with sterilized ophthalmic scissors and a scalpel to remove the white subcutaneous fat, remove the epidermis and subcutaneous tissue, leave the dermis, and obtain the pretreated skin tissue. After the pretreated skin tissue was transferred into the culture medium, it was cut into small pieces to obtain skin tissue blocks with neat edges.

[0043] 2.3 After adding 3mL of fetal bovine serum (FBS) evenly to each well of the 6-well cell culture plate, put the skin tissue block into the incubator and incubate for 0.5-1h to make the tissue stick to the bottom of the culture plate.

[0044] 2.4 Add 1 mL of DMEM (Gibco) medium containing 20% ​​(v / v) FBS to the 6-well cell culture plate, and put it back into the incubator for culture.

[0045] 2.5 Change the medium when fibroblasts craw...

Embodiment 3

[0053] 3.1 Put the human skin tissue into a petri dish, and rinse it with 4°C pre-cooled phosphate buffered saline solution (PBS) rapidly and repeatedly for 4 times.

[0054] 3.2 Treat the skin tissue with sterilized ophthalmic scissors and a scalpel to remove the white subcutaneous fat, remove the epidermis and subcutaneous tissue, leave the dermis, and obtain the pretreated skin tissue. After the pretreated skin tissue was transferred into the culture medium, it was cut into small pieces to obtain skin tissue blocks with neat edges.

[0055] 3.3 After adding 3mL of fetal bovine serum (FBS) evenly to each well of the 6-well cell culture plate, put the skin tissue block into the incubator and incubate for 0.5-1h to make the tissue stick to the bottom of the culture plate.

[0056] 3.4 Add 1 mL of DMEM (Gibco) medium containing 20% ​​(v / v) FBS to the 6-well cell culture plate, and put it back into the incubator for culture.

[0057] 3.5 Change the medium when fibroblasts crawl...

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Abstract

The invention provides a method for preparing induced pluripotent stem cells through the reprogramming of somatic cells, and the induced pluripotent stem cells obtained by the method. The method includes the following steps: taking factor Oct4 and Nanog as reprogramming inducing factors to induce into the somatic cells so as to perform reprogramming; and then, cultivating the partially or fully reprogrammed somatic cells in a culture medium containing a specific chemical inducer to obtain the induced pluripotent stem cells. Through the combination of the reprogrammed inducing factors existingin different forms and three small molecular compounds as the specific chemical inducer, the reprogramming efficiency of human somatic cells can be significantly enhanced, and the tumorigenicity of the induced pluripotent stem cells can be reduced.

Description

technical field [0001] The invention belongs to the technical field of stem cells, and in particular relates to a method for preparing induced pluripotent stem cells through somatic cell reprogramming and induced pluripotent stem cells obtained therefrom. Background technique [0002] In 2006, Yamanaka's team invented a cocktail consisting of four genes, Oct4, Sox2, Klf4 and c-Myc, which could successfully reprogram terminally differentiated skin fibroblasts into induced pluripotent stem cells (induced pluripotent stem cells) cells, iPSCs). This method is relatively simple and stable, breaks through the moral and ethical restrictions on the use of human embryonic stem cells in medicine, can solve the immune rejection problem in cell transplantation therapy, and greatly expands the application potential of stem cell technology in clinical medicine. [0003] In addition, iPSCs technology has great potential value in cell replacement therapy, research on pathogenesis, and scre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/86C12N5/10
CPCC12N15/85C12N15/86C12N5/0696C12N2760/18843C12N2501/603C12N2501/605C12N2501/01C12N2501/15C12N2510/00C12N2820/60C12N2506/11C12N2506/1307C12N2501/727C12N2501/415C12N2533/52C12N2533/90C07K14/4705C07K14/71C12N9/1205C12N5/10
Inventor 范靖任芳王安欣
Owner ZHEJIANG HUODE BIOENG CO LTD
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