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Culture medium composition for culturing amnion-derived mesenchymal stem cell, and method for culturing amnion-derived mesenchymal stem cell by using same

A technology of mesenchymal stem cells and culture medium, applied in biochemical equipment and methods, embryonic cells, animal cells, etc., can solve the problem of long proliferation of stem cells

Inactive Publication Date: 2013-06-12
RNL BIO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the disadvantage of the conventional method of culturing amnion-derived stem cells is that the stem cells proliferate for a long time

Method used

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  • Culture medium composition for culturing amnion-derived mesenchymal stem cell, and method for culturing amnion-derived mesenchymal stem cell by using same
  • Culture medium composition for culturing amnion-derived mesenchymal stem cell, and method for culturing amnion-derived mesenchymal stem cell by using same
  • Culture medium composition for culturing amnion-derived mesenchymal stem cell, and method for culturing amnion-derived mesenchymal stem cell by using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Isolation of mesenchymal stem cells from amnion tissue

[0040]The amnion tissue used in the examples of the present invention was isolated from the placenta, and the isolated tissue was placed in saline containing antibiotics or DMEM (Dulbecco modified Eagle Medium) medium, and then transferred to the laboratory.

[0041] On a clean bench, wash 5 g of amnion tissue with sterile saline, and cut the washed amnion tissue as small as possible with surgical scissors in a petri dish. Add collagenase solution and mix well with the trimmed tissue. Put the mixture into a flask, and then place the flask at 37 °C, 5% CO 2 Incubate for 90 minutes in an incubator to lyse the tissue. Lysed tissue was filtered through a cell strainer in a 50 mL tube and centrifuged. After centrifugation, the supernatant was removed, and the cell pellet was suspended in DMEM-P and inoculated into square flasks, and then incubated at 37 °C, 5% CO 2 cultured in an incubator for 3-4 days...

Embodiment 2

[0042] Example 2: Cultivate isolated amniotic mesenchyme in DMEM-P and KSFM-P mixed medium Stem cells

[0043] The amnion-derived mesenchymal stem cells obtained in Example 1 were mixed with 1.0×10 6 The density of the cells was dispersed in T175 flasks, and cultured in a mixed medium consisting of DMEM-P medium (Table 1) and KSFM-P medium (Table 3) mixed at the ratio shown in Table 4, while each The medium was changed every 2 days. The pictures of the cells on the 3rd and 4th day of culture are as follows figure 1 and 2 shown.

[0044] Table 1: Components of DMEM-P medium

[0045] components

commercial source

concentration

DMEM-HG

WelGENE, Inc.

L-Ascorbic acid 2-phosphate

Sigma-aldrich

0.2mM

fetal bovine serum

Invitrogen

10%

b-FGF

Invitrogen

10ng / ml

NEAA

Invitrogen

10μl / ml

[0046] Table 2: Components of NEAA

[0047] NEAA component

Concentration (m...

Embodiment 3

[0058] Example 3: Immunological characteristics of amnion-derived mesenchymal stem cells cultured in mixed medium Flow cytometric analysis of surface antigen expression

[0059] The amnion-derived mesenchymal stem cells cultured in the mixed medium of Example 2 were characterized by CD series antigen markers. FACS analysis was performed using CD29 (monocyte marker), CD31 (endothelial and stem cell marker), CD34 (hematopoietic stem cell marker), CD44, CD45 (PTPR, ASV, leukocyte marker) and CD73.

[0060] The amnion-derived mesenchymal stem cells obtained in Example 1 were washed with PBS and treated with trypsin, after which the cells were collected and centrifuged at 1500 rpm for 5 minutes. The supernatant was removed, and the cells were incubated in blocking buffer solution (5% serum (normal goat serum + normal horse serum)) for 60 minutes at 4°C, followed by centrifugation at 1500 rpm for 5 minutes. After removing the supernatant, the cells were suspended in PBS and the...

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Abstract

The present invention relates to a culture medium for culturing a mesenchymal stem cell, and more specifically, to a culture medium composition for culturing a mesenchymal stem cell, comprising a basic culture medium, L-ascorbic acid 2-phosphate, fetal bovine serum, basic fibroblast growth factor (b-FGF), non-essential amino acids (NEAA), insulin, N-acetyl-L-cysteine, calcium chloride and hydrocortisone,and a method for culturing a mesenchymal stem cell by using the same. According to the present invention, it is possible to obtain the number of mesenchymal stem cells necessary for stem cell treatment within a short time and the differentiation of mesenchymal stem cells is improved, and is useful for the treatment of cells using a stem cell.

Description

technical field [0001] The present invention relates to a medium for culturing mesenchymal stem cells, more particularly to a medium composition for culturing mesenchymal stem cells and a method for cultivating mesenchymal stem cells using said medium composition, wherein The medium composition contains basal medium, L-ascorbic acid 2-phosphate, fetal bovine serum, basic fibroblast growth factor (b-FGF), non-essential amino acid (NEAA), insulin, N-acetyl- L-cysteine, calcium chloride and hydrocortisone. Background technique [0002] Stem cells refer to cells that not only have self-replication ability but also have the ability to differentiate into at least two types of cells, and can be divided into totipotent stem cells, pluripotent stem cells and multipotent stem cells ). [0003] Adult stem cells are obtained by obtaining cells from various human organs and developing the cells into stem cells, which are characterized in that they differentiate only into specific tissu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12N5/02
CPCC12N5/0605C12N5/0668C12N2500/14C12N2500/32C12N2501/115C12N2501/33C12N2501/39
Inventor 罗廷灿姜成根徐宙延金孝银
Owner RNL BIO
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