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Multi-potent stem cell culture medium

A pluripotent stem cell and culture medium technology, applied in the field of pluripotent stem cell culture medium, can solve the problems of high price, limited scientific research, and high cost, and achieve the effects of reducing production costs, reducing the risk of disease, and ensuring normal proliferation

Active Publication Date: 2016-08-31
广州杰尔克生物技术有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 1. Human apotransferrin in E8 medium is usually a product of genetic engineering, and its high price severely limits scientific research, so it is urgent to find alternatives;
[0007] 2. If small molecular compounds or β-mercaptoethanol that promote reprogramming are added to the E8 medium, cells will exhibit obvious cytotoxicity, and cannot be used in the process of efficiently inducing reprogramming, especially in the process of extreme condition reprogramming, which will affect the addition of different Differentiation experiments of various small molecular compounds also limit the cultivation of hiPSCs under low-density conditions;
[0008] 3. The concentration of cytokines contained in E8 is very high, and the required cost is still high

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] A kind of pluripotent stem cell culture medium, this culture comprises basal medium and following added components: L-ascorbic acid-2 phosphate magnesium, sodium selenite, human insulin, ferric citrate, basic fibroblast growth factor (abbreviated bFGF ) and transforming growth factor beta1 (abbreviated as TGF-β1), the distribution ratio range of medium components is shown in the table below;

[0060] Table 3 The components and contents of the medium in the examples

[0061] components

Dosage range

Magnesium L-ascorbic acid-2 phosphate (ug / mL)

64

Sodium selenite (ng / mL)

13.6

Human insulin (ug / mL)

20

Ferric citrate (ng / mL)

40

Basic fibroblast growth factor (ng / mL)

20

Transforming growth factor beta1(ng / mL)

0.5

Sodium chloride (adjust osmotic pressure mosmol / L)

280～350

DMEM / F12 medium

Make up to 1L

[0062] The specific experimental operation of MTT detection is as follows:...

Embodiment 2

[0076] In this example, experiments were carried out using BioCI medium and hPLWI medium to compare the effect of ferric citrate on cell proliferation.

[0077] In this example, 1 copy of BioCI medium, 1 copy of control medium, and 11 copies of hPLWI medium (named No. 1-11 respectively) were used, and their components and osmotic pressure information are shown in the following table:

[0078] Table 5 The composition and osmotic pressure information of embodiment 2 culture medium

[0079]

[0080] Note: a) Use sodium chloride to adjust the osmotic pressure.

[0081] The content and osmotic pressure of ferric citrate in No. 1-11 hPLWI medium are shown in the table below:

[0082] Table 6 Information of hPLWI medium No. 1-11

[0083]

[0084]

[0085] The specific experimental operation of the MTT detection is as follows:

[0086] 1) Pre-culture: iPSCs were coated with Matrigel and cultured in BioCI medium for 3-15 days;

[0087] 2) Sampling: Use a 96-well plate, wit...

Embodiment 3

[0104] According to the results of Example 2, we selected the optimal ferric citrate concentration of 40 ng / mL, and carried out culture experiments on induced pluripotent stem cell iPSC and human embryonic stem cell H1 coated with Matrigel and cultured in BioCI medium for 3-15 days , seeded into the plate wells containing the medium shown in the table below, cultured and AP staining detected respectively.

[0105] Table 9 Components and content information of the culture medium in Example 3

[0106]

[0107]

[0108] The AP staining test results of induced pluripotent stem cells are shown in the table below, and the functional relationship between the test time and the proliferation multiple is as follows: figure 1 as shown,

[0109] Table 10 Proliferation of iPSCs under different cultures

[0110]

[0111]

[0112] Depend on figure 1 Compared with the results in Table 10, we can know that the proliferation rate of iPSC in BioCI medium and hPLWI medium is basica...

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Abstract

The invention discloses a multi-potent stem cell culture medium. The multi-potent stem cell culture medium comprises a basic culture medium and following added components: L-ascorbic acid-2-magnesium phosphate, sodium selenite, human insulin, ferric citrate, an alkaline basic fibroblast growth factor and a transforming growth factor beta1. The sodium selenite is successfully introduced into a multi-potent stem cell culture medium system to replace human apo-transferrin, so that the production cost of the multi-potent stem cell culture medium can be reduced; exogenous pathogenic risks are reduced; the multi-potent stem cell culture medium can guarantee normal proliferation of cells in a multi-potent stem cell culture process and the multi-potent property of the stem cells can also be maintained.

Description

technical field [0001] The invention relates to a culture medium for pluripotent stem cells, belonging to the technical field of bioengineering. Background technique [0002] Pluripotent stem cells (PSCs) have self-renewal ability and multipotency, and they can differentiate into any tissue and cells of the three embryonic germ layers (ectoderm, mesoderm, endoderm). Therefore, PSCs are used in regenerative medicine, Developmental biology and other fields play an important role. PSC includes embryonic stem cell (Embryonic stem cell, ESC) and induced pluripotent stem cell (induced pluripotency stem cell, iPSC). Both ESCs and iPSCs have similar gene expression profiles, self-renewal capabilities, pluripotency, and epigenetic profiles. and purified pluripotent stem cells. [0003] The human iPSC culture process has gone through three stages: using feeder cell culture, feeder-free cell culture, and animal-derived feeder-free cell culture. Feeder layer cells are usually derive...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735C12N5/071
CPCC12N5/0606C12N5/0696C12N2500/12C12N2500/25C12N2500/34C12N2500/42C12N2500/90C12N2501/115C12N2501/15C12N2501/727C12N2501/91C12N2501/998
Inventor 王淋立陈月花宋立兵关春燕
Owner 广州杰尔克生物技术有限公司
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