Reprogrammed culture medium and culture method of reprogrammed induced multipotential stem cell

A technology for pluripotent stem cells and a culture method, which is applied to the culture field of reprogramming medium and reprogramming induced pluripotent stem cells, can solve the problems of poor practicability, complicated operation process, low reprogramming efficiency, etc., so as to improve safety, overcome the Low efficiency, high efficiency effect

Active Publication Date: 2019-04-16
IREGENE THERAPEUTICS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] (1) Virus method: It includes viral vectors (e.g. lentivirus) and non-genomic insertion vectors (e.g. Sendai virus) with genome random insertion. The disadvantage of this type of method is the potential risk of random genome insertion, or lengthy Unchanged by the virus dilution process
[0005] (2) DNA method: various plasmid types including Piggy Bac, Sleeping Beaμty and Episomal, which are characterized by low reprogramming efficiency
[0006] (3) RNA method and protein method: the operation process of this kind of method is complicated, and the practicability in industrial application is poor
[0007] (4) Compound small molecule method: This is a reprogramming method that is more and more widely used at present, but there are large individual differences in its reprogramming efficiency
[0013] However, the culture systems used in these two culture methods all use animal serum components as an important raw material for maintaining cell viability, which makes this type of medium contain a large amount of protein from animal and plant sources, and the chemical composition of the added substances is not clear enough, which will lead to It has adverse effects on subsequent processing applications, such as difficulties in the separation and purification of target proteins, and the cost of culture media is also high

Method used

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  • Reprogrammed culture medium and culture method of reprogrammed induced multipotential stem cell
  • Reprogrammed culture medium and culture method of reprogrammed induced multipotential stem cell
  • Reprogrammed culture medium and culture method of reprogrammed induced multipotential stem cell

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Embodiment 1

[0041] This embodiment provides a serum-free transferrin-free reprogramming medium, which is composed of DMEM-F12 basal medium and additional components, the additional components are: 80 μg / mL of L-ascorbic acid, 50 μmol / L of hydrocortisone , 20ng / mL of sodium selenite, 10μmol / L of Optiferrin, 4μmol / L of retinyl acetate, 50ng / mL of plant-derived recombinant human basic growth factor (OsrbFGF), 10μg / mL of IGF, 0.4 μg / mL of A-83, 4 μmol / L of CHIR99021 and 400 μmol / L of sodium butyrate.

Embodiment 2

[0043] This embodiment provides a culture method for reprogramming adult cells into human induced pluripotent stem cells, comprising the following steps:

[0044] Matrigel (STEMCELL Technologies) was used to coat the 6-well culture plate, and after plating, it was placed in a 37°C incubator and incubated for more than one hour.

[0045] Epi5 Reprogramming Kit was used for reprogramming (see the kit instruction manual for specific steps), and Neon electroporation kit (Thermo Fisher) was used to operate various human adult cells. The operation steps were as follows: umbilical cord-derived human stromal cells, human After CD34+ cells, human skin fibroblasts and human adult cells are centrifuged and washed, they are resuspended in the Resuspension Buffer provided in the electroporation kit, and electroporation is performed according to the following procedure: PulseVoltage: 1650V, PulseWidth: 10ms, PulseNumber: 3, electroporation ends , to obtain cells after electroporation.

[0...

Embodiment 3

[0066] This embodiment provides a culture method for reprogramming adult cells into human induced pluripotent stem cells, comprising the following steps:

[0067] Matrigel (STEMCELL Technologies) was used to coat the 6-well culture plate, and after plating, it was placed in a 37°C incubator and incubated for more than one hour. Reprogramming using CytoTune TM -iPS 2.0 Sendai Reprogramming Kit (see the kit manual for specific steps), according to 2×10 6 The cells were transduced with the virus at a ratio of MOI=5. After the virus was added, the cells were placed in an environment of 37° C. and 5% carbon dioxide and incubated for 12-16 hours. On the second day, the medium was replaced with the reprogramming medium of Example 1. After culturing until the 28th day, a large number of human induced pluripotent stem cell clones appeared in the culture plate.

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Abstract

The invention relates to a reprogrammed culture medium and a culture method of a reprogrammed induced multipotential stem cell. The reprogrammed culture medium is composed of a DMEM-F12 basal culturemedium and additive components, wherein the additive components comprise 60-180 microgram/mL of L-ascorbic acid, 5.3-74 micromole/L of hydrocortisone, 3-89 nanogram/mL of sodium selenite, 8-23 micromole/L of Optiferrin, 0.5-7.4 micromole/L of retinyl acetate, 40-60 nanogram/mL plant-derived recombinant human alkaline growth factors, 8-12 microgram/mL of IGF, 0.2-0.6 microgram/mL A-83, 2-6 micromole/L of CHIR99021 and 100-450 micromole/L of sodium butyrate. The use of the reprogrammed culture medium not only can improve the safety of clinical use, but also can improve the efficiency of adult cell reprogramming to induce the multipotential stem cell.

Description

technical field [0001] The invention relates to the biological field, in particular to a reprogramming medium and a method for culturing reprogrammed induced pluripotent stem cells. Background technique [0002] In 2006, Shinya Yamanaka's team invented a "cocktail" method consisting of four transcription factors, OCT4, SOX2, KLF4 and c-Myc, which can successfully reprogram terminally differentiated skin fibroblasts into differentiated pluripotent cells These stem cells are called induced pluripotent stem cells (induced plμripotentstem cells, referred to as "iPSC"). These stem cells have similar differentiation potential to embryonic stem cells, and can form the three most basic germ layers of human development, and eventually form a variety of adult cells. This method breaks through the ethical limitations of using human embryonic stem cells in medicine, can solve the immune rejection problem in cell transplantation therapy, and greatly expands the application potential of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/074C12N5/079
Inventor 魏君
Owner IREGENE THERAPEUTICS LTD
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