A culture medium for maintaining the pluripotency of human amniotic epithelial stem cells

A technology of culture medium and stem cells, applied in cell culture active agent, culture process, animal cells, etc., can solve the problems of cell demand, influence of cell pluripotency, limitation of transplantation treatment, etc., to achieve induced proliferation, high pluripotency, etc. , the effect of avoiding damage

Active Publication Date: 2019-05-14
COBAXER BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Human amniotic epithelial cells are derived from placental amniotic tissue. Although they express stem cell marker molecules and related genes, they do not express telomerase gene, and their in vitro expansion is limited. They can only be cultured for 4 to 5 generations in vitro. After more than ten days of culture The cells began to shrink, and the pluripotency of the cells was also greatly affected. It has been reported that the relevant culture systems adjusted the concentration of the growth factor EGF, but there was no significant change in the maintenance of the pluripotency of hAECs. The cells also Cannot be maintained for long periods of time
Although we can obtain more hAECs from human amniotic membrane, there will be great limitations for in vitro research and transplantation therapy. Therefore, a stable hAECs pluripotent culture system is of great significance, which can be maintained in long-term in vitro culture. The stability of pluripotency also solves the problem of acute treatment cell demand

Method used

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  • A culture medium for maintaining the pluripotency of human amniotic epithelial stem cells
  • A culture medium for maintaining the pluripotency of human amniotic epithelial stem cells
  • A culture medium for maintaining the pluripotency of human amniotic epithelial stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Take the basal culture medium DMEM / F12, first add serum substitutes and non-essential amino acids to it, mix well and wait until the solution is stable, then add the essential amino acids L-glutamine, dithiothreitol, growth factors and system stability factors in sequence Mix evenly; Expressed by volume fraction and substance molar volume ratio, the contents of various components in the prepared culture solution are:

[0054] Serum Replacement 15%; pH 7.4

[0055] Non-essential amino acid reagent 1%;

[0056] Essential amino acid 2mmol / L;

[0057]Dithiothreitol 0.05mmol / L;

[0058] Basic fibroblast growth factor 5ng / ml;

[0059] Epidermal growth factor 15ng / ml;

[0060] Hepatocyte growth factor 30ng / ml;

[0061] Keratinocyte growth factor-2 10ng / ml;

[0062] System stability factor 1%.

[0063] Among them, the distribution ratio of each component of the system stability factor is:

[0064] β-taurine 10%;

[0065] Arginine 20%;

[0066] Transforming Growth Fact...

Embodiment 2

[0070] Take the basal culture medium DMEM / F12, first add serum substitutes and non-essential amino acids to it, mix well and wait until the solution is stable, then add the essential amino acids L-glutamine, dithiothreitol, growth factors and system stability factors in sequence Mix evenly; Expressed by volume fraction and substance molar volume ratio, the contents of various components in the prepared culture solution are:

[0071] Serum Replacement 20%

[0072] Non-essential amino acid reagent 0.8%;

[0073] Essential amino acid 3mmol / L;

[0074] Dithiothreitol 0.08mmol / L;

[0075] Basic fibroblast growth factor 8ng / ml;

[0076] Epidermal growth factor 18ng / ml;

[0077] Hepatocyte growth factor 30ng / ml;

[0078] Keratinocyte Growth Factor-2 15ng / ml;

[0079] The system stability factor is 1.2%.

[0080] Among them, the distribution ratio of each component of the system stability factor is:

[0081] β-taurine 15%;

[0082] Arginine 25%;

[0083] Transforming Growth ...

Embodiment 3

[0087] Take the basal culture medium DMEM / F12, first add serum substitutes and non-essential amino acids to it, mix well and wait until the solution is stable, then add the essential amino acids L-glutamine, dithiothreitol, growth factors and system stability factors in sequence Mix evenly; Expressed by volume fraction and substance molar volume ratio, the contents of various components in the prepared culture solution are:

[0088] Serum Replacement 20%

[0089] Non-essential amino acid reagent 1%;

[0090] Essential amino acid L-glutamine 2mmol / L;

[0091] Dithiothreitol 0.05mmol / L;

[0092] Basic fibroblast growth factor 8ng / ml;

[0093] Epidermal growth factor 10ng / ml;

[0094] Hepatocyte growth factor 20ng / ml;

[0095] Keratinocyte growth factor-2 10ng / ml;

[0096] The system stability factor is 1.2%.

[0097] Among them, the distribution ratio of each component of the system stability factor is:

[0098] β-taurine 10%;

[0099] Arginine 20%;

[0100] Transformi...

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Abstract

The invention discloses culture fluid for maintaining the pluripotency of human amniotic epithelial stem cells. The culture fluid comprises base culture fluid, 15%-25% of serum substitutes, 0.1%-2% of non-essential amino acid, 1 mmol / L-5 mmol / L of essential amino acid, 0.01 mmol / L-1 mmol / L of dithiothreitol, 30 ng / ml-120 ng / ml of growth factors and 0.1%-2% of system stabilizing factors. The culture fluid has the advantages that in-vivo survival environments of the stem cells can be simulated by the culture fluid, accordingly, the pluripotency of the stem cells can be maintained when the stem cells are subjected to in-vitro culture, and stem cells with high pluripotency still can be obtained after repeated pass culture is carried out.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a culture medium for maintaining the pluripotency of human amniotic membrane epithelial stem cells. Background technique [0002] The amniotic membrane is located inside the chorion of the embryo, which is a transparent film without blood vessels, nerves, lymph, and muscles, and is closely connected with the developing fetus. Cells derived from human amnion mainly consist of two types of cells: human amnion epithelial cells (hAECs) and human amnion mesenchyme cells (hAMCs). [0003] Human amniotic epithelial cells (hAEC) have the characteristics of expressing multiple embryonic stem cell markers and have a relatively comprehensive multi-lineage differentiation potential. Studies by Miki et al. have confirmed the potential of hAECs to differentiate into three germ layers. In addition to expressing embryonic stem cell surface antigens: SSEA-3 and SSEA-4, tumor resistance gene...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/073
CPCC12N5/0605C12N2500/32C12N2500/33C12N2500/44C12N2501/11C12N2501/115C12N2501/117C12N2501/12C12N2501/148C12N2501/22C12N2501/998
Inventor 申重阳陈洁王仲陈勇军
Owner COBAXER BIOTECH
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