Preparation method of Mesenchymal Stem Cell (MSC) medium for cosmetics

A technology of mesenchymal stem cells and culture medium, which is applied in the field of mesenchymal stem cell culture medium and cosmetics preparation, can solve the problems of difficult preservation and transportation of tumorigenicity, low utilization rate of mesenchymal stem cells, and increase of preparation cost, etc. Preservation and transportation, no immune rejection, the effect of reducing the cost of preparation

Inactive Publication Date: 2020-07-28
中科晟和干细胞技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The mesenchymal stem cell culture medium in the prior art is used in the preparation method of cosmetics, not only the use effect is not good, but the mesenchymal stem cell conditioned medium contains live cells, which have immune rejection and potential pathogenicity when using the cells. Tumorous, difficult to store and transport, etc., and the utilization rate of mesenchymal stem cells is low, which increases the preparation cost of mesenchymal stem cell conditioned medium

Method used

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  • Preparation method of Mesenchymal Stem Cell (MSC) medium for cosmetics
  • Preparation method of Mesenchymal Stem Cell (MSC) medium for cosmetics
  • Preparation method of Mesenchymal Stem Cell (MSC) medium for cosmetics

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Example 1 Identification of surface markers of mesenchymal stem cells (MSC)

[0023] Take 3x10 MSCs cultured in vitro for the fourth passage (P4) before cryopreservation 6 After washing with DPBS, centrifuge at 800g for 5 minutes, resuspend in 300ul of DPBS containing 1% bovine serum albumin (BSA), and divide into three parts on average, one of which is added with the isotype control antibody for flow cytometry detection, and the other two Two groups of flow detection antibodies were added to each part: FITC-HLA-DR, APC-CD90, PE-CD105 and Percp-CD34 were added to one sample, and PE-CD73 and Percp-CD45RA were added to the other sample, stained at 4°C After 30 minutes, wash once with DPBS containing 1% BSA, centrifuge at 800g for 5 minutes, and test on the machine. The model of the flow meter is BD-FacsCalibur. The results of flow cytometry were analyzed by Flowjo software, the cell part was selected in the FSCvsSSC graph, and the expression of MSC surface markers was de...

Embodiment 2

[0024] Detection (ELISA) of important biologically active substances in embodiment 2MSC-CM

[0025] Taking SDF-1α, the most common factor in MSC culture, as the object, the content of SDF-1α factor in MSC-CM was determined by quantitative ELISA method. According to the instructions provided by the kit manufacturer, the main steps are briefly described as follows:

[0026]①Preparation of test samples: Take one piece of freeze-dried MSC-CM prepared by the two methods, dissolve them in 0.5ml sterile, pyrogen-free deionized water, take 100ul of dissolved MSC-CM, and add 900ul of the kit to provide dilute the sample 10 times, then take 200ul of the 10 times diluted sample, continue to dilute 2 times with the sample diluent provided by the kit, and set aside;

[0027] ②Preparation of SDF-1α standard substance: according to the instructions, dilute the SDF-1α standard substance (100ug / ml) provided by the kit with the sample diluent to obtain concentrations of 10000pg / ml, 5000pg / ml, ...

Embodiment 3

[0039] Example 3 Detection of the Effect of MSC-CM on the Growth of Epithelial Cells

[0040] The function of biologically active molecules in MSC-CM was determined by detecting the effect of MSC-CM on the growth of human dermal fibroblasts. Human dermal fibroblasts were determined using HDF (Human dermal fiborblast, human dermal fibroblasts, purchased from Cell Applications, USA (Cat#: 106K-05a), isolated from foreskin cells of a 16-year-old human). MTT kit was purchased from Biyuntian Biotechnology Co., Ltd., catalog number: C0009.

[0041] ①Preparation of test sample culture medium: first prepare 500ml of DMEM / F12 medium containing 10% fetal bovine serum for use (this medium is also a growth medium for HDF cells), and take 2 different ways of preparing MSC-CM freeze-dried products , add 1ml of the above-mentioned medium to each freeze-dried product to fully dissolve it (this medium is the stock solution of MSC-CM medium), and then dilute MSC-CM1 and MSC with the above-ment...

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Abstract

The invention discloses a preparation method of a Mesenchymal Stem Cell (MSC) medium for cosmetics. The preparation method comprises the following steps of: (1) preparation of a No.1 medium; (2) preparation of lithium chloride mother solution; (3) preparation of a No.2 medium; (4) separation and culture of MSCs; and (5) preparation of a Mesenchymal Stem Cell Conditional Medium (MSC-CM), i.e., taking cells subjected to passage in the fourth time, respectively inoculating the cells into the No.1 medium and the No.2 medium with a density of 1*105/ml, discarding culture supernatant, adding a proper volume of 1X HBSS solution to wash the cells for twice, completely sucking the residual HBSS solution dry, adding a serum free high-glucose DMEM medium, removing cell debris, adding a proper volumeof 20% sterile sugar solution into each supernatant, subpackaging the obtained liquid into 10ml per bottle, transferring into a vacuum freeze-drying bottle, and after freeze-drying in a vacuum freezerdryer, preserving the obtained product in a refrigerator with a temperature of minus 80 DEG C for later use. Compared to the prior art, the preparation method has the advantages of good use effect, high utilization rate and low cost.

Description

technical field [0001] The invention relates to the technical field of mesenchymal stem cells, in particular to a method for preparing a mesenchymal stem cell culture medium for cosmetics. Background technique [0002] Mesenchymal stem cells (MSCs) are a type of heterogeneous cells that are ubiquitous in various tissues and are closely related to the repair of corresponding tissue damage. They are one of the main sources of raw materials for repairing tissues and organs; MSCs in vivo During the growth process, it will secrete some cytokines to inhibit the development of inflammation, regulate the formation of new blood vessels, promote the formation of blood vessels, promote the growth of wound tissue cells, participate in the repair of wound damage, and finally promote the formation of wound epithelium and granulation tissue to achieve accelerated The process of wound healing; these biologically active molecules secreted by MSCs cultured in vitro, which have the function of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775A61K8/99A61Q19/00A61Q19/08A61Q17/02
CPCC12N5/0668A61K8/99A61Q19/00A61Q19/08A61Q17/02C12N2500/32C12N2500/30C12N2500/12C12N2509/10C12N2509/00C12N2501/727C12N2500/44C12N2501/11
Inventor 杨洁韩丽娜王浠李东梅
Owner 中科晟和干细胞技术有限公司
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