A kind of embryonic stem cell culture medium and its application

A technology of embryonic stem cells and culture medium, applied in the field of stem cells, can solve the problems of slow growth of embryonic stem cells, affect cell expansion and application, and be prone to aging, etc., achieve high safety, broad application prospects, and ensure the effect of differentiation ability

Active Publication Date: 2020-11-17
MUDANJIANG MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Embryonic stem cells have great application prospects. Whether it is scientific research or clinical use, a large number of embryonic stem cells are needed. However, in the process of culturing cells in culture medium, embryonic stem cells grow slowly, are prone to aging, and appear prematurely. Differentiation characteristics, thereby affecting the bulk expansion and application of cells

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  • A kind of embryonic stem cell culture medium and its application

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Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1, the cultivation of human embryonic stem cells (H1 cell line)

[0018] In order to verify the effect of the differentiation-inhibiting short peptide (as shown in SEQ ID NO.1) of the invention of the present application, the embodiment sets up an experimental group and a control group in particular, and the experimental group and the control group use the same method for culturing. The only difference between the two is: The culture medium used in the control group does not contain the short differentiation-inhibiting peptide.

[0019] Specifically, the medium components used in the experimental group are: N2 20mL, B27 20mL, glutamine 0.2g, β-mercaptoethanol 5μl, taurine: 0.2g, basic fibroblast growth factor 100mg, calf 0.5 g of serum albumin; 0.3 g of short differentiation inhibitory peptide (as shown in SEQ ID NO.1); dilute to 1000 mL with DMEM / F12.

[0020] The medium components used in the control group are: N2 20mL, B27 20mL, glutamine 0.2g, β-mercapto...

Embodiment 2

[0026] Example 2. Detecting the maintenance of the undifferentiated state of human embryonic stem cells

[0027] Oct4 and Tra-1-81 are commonly used indicators for detecting whether embryonic stem cells are in an undifferentiated state; by detecting the expression of the cells, it can be shown whether human embryonic stem cells are in an undifferentiated state. In this example, the method of cell immunofluorescence staining (Elisa double-antibody sandwich method) was used to further detect the expression of the genes Oct4 and Tra-1-81 in the 10th generation H1 cells, wherein rabbit anti-Oct4 and mouse anti-Tra-1 -81 was purchased from American Chemicon Company.

[0028]The test results showed that the expression of Oct4 and Tra-1-81 could be detected in the experimental group at the 6th, 8th, and 10th generations, and the detection intensity did not change; while the control group was at the 6th and 8th generations At the same time, the expression of Oct4 and Tra-1-81 could a...

Embodiment 3

[0033] Example 3. Totipotency Detection of Human Embryonic Stem Cells

[0034] In order to further evaluate the properties of the passaged embryonic stem cells of the present invention, the totipotency of the cultured human embryonic stem cells is now detected, and the differentiation ability of stem cells into keratinocytes is detected with reference to the method of patent CN104651298B. When cultured to the 40th day, the high expression of keratinocyte markers p63 protein, keratin 14 and 15 was detected in the passaged stem cells of the experimental group, and the cell morphology was close to that of human keratinocytes; while the expression of the above proteins in the control group was significantly lower than that of the experimental group. Group. As detected by flow cytometry, the expression rate of keratin 14 in the experimental group was as high as 70.31%, while that in the control group was only 22.62%.

[0035]

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Abstract

The invention relates to an ESC (embryonic stem cell) culture medium and an application thereof. The culture medium contains one differential inhibition short-chain polypeptide capable of inhibiting differentiation of ESCs, wherein the short-chain polypeptide can inhibit differentiation of the ESCs to nerve cells. The culture medium has the advantages of being rapid in multiplication, safe, stableand long in passage time when used for multiplication culture on the ESCs; and a large quantity of passaged and multiplied ESCs can be obtained, the obtained ESCs can keep an undifferentiated state,and the totipotency of cell differentiation can be kept.

Description

technical field [0001] The invention belongs to the technical field of stem cells, and in particular relates to a cell culture medium and a method for culturing embryonic stem cells. Background technique [0002] Embryonic stem cells (ESCs, referred to as ES, EK or ESC cells) are a type of cells isolated from early embryos (before the gastrula stage) or primitive gonads, which have unlimited proliferation, self-renewal and multidirectional differentiated characteristics. Whether in vitro or in vivo, embryonic stem cells can be induced to differentiate into almost all cell types in the body, and have broad application prospects. However, the current culture system of embryonic stem cells still has problems such as slow cell proliferation, unstable state, and easy differentiation. . Therefore, further research and development of the humanized culture system of human embryonic stem cells, long-term maintenance of the biological characteristics and multi-lineage differentiatio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0735C07K14/47
CPCC07K14/4703C12N5/0606C12N2500/32C12N2500/44C12N2501/115C12N2501/999C12N2533/52
Inventor 穆寅东周福波李瑶闫磊梁军崔红李红卫冬宋铁军周鸿锁
Owner MUDANJIANG MEDICAL UNIV
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