Serum-free medium of human amniotic epithelial stem cell and culture method thereof

A serum-free culture medium and serum-free culture technology, applied in cell dissociation methods, cell culture active agents, embryonic cells, etc., can solve the problems of non-immunogenicity and achieve strong clinical applicability, strong expansion ability, Strong immunogenic effect

Active Publication Date: 2017-06-13
沈阳艾米奥生物工程技术研发中心有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention aims at the problems existing in the above-mentioned prior art and provides a serum-free medium for human amniotic membrane epithelial stem cells and a culture method thereof. Also addresses allogeneic, broad source, unethical and non-immunogenic issues

Method used

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  • Serum-free medium of human amniotic epithelial stem cell and culture method thereof
  • Serum-free medium of human amniotic epithelial stem cell and culture method thereof
  • Serum-free medium of human amniotic epithelial stem cell and culture method thereof

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Experimental program
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Effect test

Embodiment 1

[0058] Such as figure 1 Shown is a schematic diagram of an inverted microscope (× 40) for primary cultured hAESCs. The primary amnion epithelial cells extracted from human amnion are uniform in shape and arranged like cobblestones.

[0059] Such as figure 2 As shown, the cell immunofluorescence method was used to detect and identify the surface markers of the extracted hAECs, and the expression of the epithelial marker epithelial keratin CK19 and the mesenchymal cell marker vimentin (× 100) in the P0 hAESCs extracted from the primary culture. The expression of CK19 was strongly positive and the expression of vimentin was weakly positive when hybridized with red fluorescent protein-labeled donkey anti-mouse fluorescent secondary antibody.

[0060] Such as image 3 As shown, the serum-free medium cultured the P1 generation hAESCs for 96 hours in an inverted microscope image (×40). Under the microscope, the cells were uniform in shape, and the cells were arranged like cobbl...

Embodiment 2

[0070] The human amniotic membrane epithelial stem cell serum-free medium of the present invention comprises:

[0071] Dulbecco's Modified Eagle's Medium / F12 (referred to as DMEM / F12) mixed by volume 1:1, 15.0~15.6g / L

[0072] Epidermal Growth Factor 0.005mg / L

[0073] Human transferrin 1.0 mg / L

[0074] Human insulin 5.5 mg / L

[0075] Sodium selenite 5.0×10 3 mg / L

[0076] L-alanyl-L-glutamine dipeptide 300 mg / L

[0077] L-alanine 10mg / L

[0078] L-Asparagine 4.9 mg / L

[0079] L-Aspartic Acid 9.3 mg / L

[0080] L-L-glutamic acid 10.7 mg / L

[0081] Glycine 5.5 mg / L

[0082] L-proline 7.5 mg / L

[0083] L-serine 6.5 mg / L

[0084] made into an aqueous solution.

Embodiment 3

[0086] The human amniotic membrane epithelial stem cell serum-free medium of the present invention comprises:

[0087] Dulbecco's Modified Eagle's Medium / F12 (referred to as DMEM / F12) mixed by volume 1:1, 15.3 g / L

[0088] Epidermal growth factor 0.01 mg / L

[0089] Human transferrin 3.5mg / L

[0090] Human insulin 10mg / L

[0091] Sodium selenite 6.0×10 3 mg / L

[0092] L-alanyl-L-glutamine dipeptide 400mg / L

[0093] L-alanine 17mg / L

[0094] L-Asparagine 7mg / L

[0095] L-Aspartic Acid 13mg / L

[0096] L-L-glutamic acid 14mg / L

[0097] Glycine 7.0mg / L

[0098] L-proline 10mg / L

[0099] L-serine 8mg / L

[0100] made into an aqueous solution.

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Abstract

The invention relates to a serum-free medium of a human amniotic epithelial stem cell and a culture method thereof. The serum-free medium is formed by adding a human epidermal growth factor, human transferring, human insulin, sodium selenite, alanyl-L-glutamine dipeptide, alanine, asparaginate, aspartic acid, glutamic acid, glycine, proline and serine to a DMEM/F12 basic medium. The culture method comprises the steps of digesting 2.5g/L of trypsin for a human amniotic membrane, obtaining the human amniotic epithelial stem cell and filtering to prepare a single cell suspension; putting the human amniotic epithelial stem cell into a CO2 incubator of which the saturated humidity and the volume fraction are 5% at 37 DEG C through the serum-free medium under a serum-free condition and achieving growth and amplification of the amniotic epithelial stem cell through liquid exchange and subculture under the serum-free culture condition, the serum-free medium has the characteristics of stem cells, and is free of other animal sources, wide in source and free of ethical restriction.

Description

technical field [0001] The invention belongs to an epithelial cell culture medium and a culture method thereof in the field of biotechnology, and specifically relates to a human amniotic membrane epithelial stem cell serum-free culture medium and a culture method thereof. Background technique [0002] Human amnion is easy to obtain, does not cause ethical controversy, contains rich amnion stem cells (amnion epithelial stem cells and amnion mesenchymal stem cells), and has low immunogenicity. It can become an important source of seed cells for clinical application of regenerative medicine. [0003] Human amniotic epithelial stem cells (AESCs) are one of the stem cells isolated from amniotic membrane. hAESC is differentiated from the original two-germ blastoderm, which is developed from ectoderm cells on the 8th day after fertilization, earlier than the formation of triple-germ embryos. This unique source of tissue embryology endows hAESC with the potential and uniqueness of m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735
CPCC12N5/0606C12N2500/05C12N2500/25C12N2500/32C12N2500/90C12N2501/11C12N2501/998C12N2509/00
Inventor 庞希宁施萍赵峰郎宏鑫
Owner 沈阳艾米奥生物工程技术研发中心有限公司
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