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Method for preparing human chorionic stem cell culture solution and application thereof

A technology of stem cell culture and chorion, applied in the application field of stem cell technology, can solve problems such as lack of research on photoaging repair potential, and achieve the effects of promoting paracrine function, high ratio, and low cost

Inactive Publication Date: 2019-01-18
深圳市乔水生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, factors secreted by stem cells derived from fat and bone marrow have been used to treat skin wrinkles, but there is still a lack of research on the repair potential of stem cells derived from human placenta for photoaging

Method used

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  • Method for preparing human chorionic stem cell culture solution and application thereof
  • Method for preparing human chorionic stem cell culture solution and application thereof
  • Method for preparing human chorionic stem cell culture solution and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] 1.1 Preparation of human chorionic stem cell culture medium (CDSC-CNM)

[0030] Preparation of serum-free medium

[0031] Transfer 2μg EGF, 2μg bFGF, 2ml B27, 400μg BSA, 400μg insulin, 1ml penicillin and streptomycin mixture into 100ml sterilized glass serum bottle, and make DMEM medium to 100ml to prepare stem cell culture medium , mix well, package and store in a 4°C refrigerator for later use.

[0032] cell counting method

[0033] Prepare a clean cell counting plate, and place the coverslip on the counting tank. After the cells to be tested are made into a single-cell suspension and mixed by blowing, suck out 5-10 μL and drip slowly along the edge of the cover glass. The cell suspension siphons into the counting tank under the cover glass by capillary action and fills the cover glass. and counting board. Make sure that there are no air bubbles under the coverslip, and avoid letting the suspension flow into the small groove next to it. Put the counter under the ...

Embodiment 2

[0053] This example is set to be compared with Example 1 to demonstrate the promotion effect of the technical solution of Example 2 on the paracrine function of CDSCs.

[0054] 2.1 cobalt 60 Effects of γ-ray irradiation on paracrine function of CDSCs

[0055] In this part, it is set to receive 2, 4, and 8 Gy doses of cobalt in the process of subculture CDSCs in the same example 1. 60 γ-ray irradiation (the corresponding experimental groups are respectively marked as 2Gy group, 4Gy group, and 8Gy group), and the irradiation dose rate is 165.44cGy / min. 60 Except for gamma ray irradiation, other processing steps of each experimental group were consistent with that of Example 1. Embodiment 1 is set as the control group and compares, adopts ELISA to measure the EGF, TGF-β content of control group and 2Gy group, 4Gy group, 8Gy group, wherein 60 The EGF content of each group before γ-ray was 5.11±0.36ng / mL, and the TGF-β content was 2.65±0.04ng / mL. The test results are shown in th...

Embodiment 3

[0075]This example is set to verify the repair and regeneration effect of CDSC-CNM, and an emulsion with the function of repairing photoaging skin is prepared using CDSC-CNM as an active substance.

[0076] 3.1 Obtaining photoaged human immortalized epidermal cells

[0077] Human immortalized epidermal cells (HaCaT) were purchased from the American Type Culture Collection (ATCC) Cell Line Service Company. Growth medium (growth medium, GM) was composed of DMEM, 10% FBS and 1% penicillin and streptomycin (0.1 mg / mL penicillin and 100 U / mL streptomycin), normal HaCaT cells were cultured with GM, and placed at 37°C , 5% CO 2 incubator. When the cell fusion rate reaches more than 90%, the cells are subcultured to 6-well plates, 24-well plates and 96-well plates according to the needs of subsequent experimental analysis. HaCaT cells induced by photoaging were washed with PBS, added a small amount of PBS to keep the cells moist, and fully exposed to UVB ultraviolet lamps, the UVB ...

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Abstract

The invention discloses a method for preparing human chorionic stem cell culture solution and application thereof. The method comprises the following steps of: using trypsin to digest JEG in logarithmic growth phase; 3. Human chorionic cells; Enrichment of human chorionic stem cells in cell suspension by serum-free suspension culture after termination of digestion to obtain JEG-3 suspension of cell ball; After digestion with trypsin and EDTA, the cells were cultured in stem cell culture medium. During the culture, the cells were irradiated with 2 - 4 Gy cobalt 60 [gamma] rays at a dose rate of165.44 cGy / min, and stromal cell derived factor was added to the culture medium. 1-alpha., obtain a human chorionic stem cell culture; Centrifuge, filter and remove human chorionic stem cells to obtain filtrate which is that culture medium of human chorionic stem cells. Human chorionic stem cells can be enriched effectively by this method, and the culture solution containing various high-contentparacrine components can be obtained by improving the passage culture conditions, which can be used as an active substance to be added into skin care cosmetics, so that the skin care cosmetics can have anti-aging effect and the like.

Description

technical field [0001] The invention relates to the application of stem cell technology, in particular to a method for preparing human chorionic stem cell culture fluid and its application. Background technique [0002] In the 1970s, it was discovered that a type of adherent cell derived from mesoderm and having fibroblast-like features, namely mesenchymal stem cells, has rapidly become a Research hotspots at home and abroad. At present, there have been a number of mesenchymal stem cell-related application preparations applied to clinical research abroad, and more clinical trials are in progress or applied for. At present, mesenchymal stem cells have been isolated and extracted from bone marrow, fat, dental pulp, amniotic membrane, chorion, decidua, umbilical cord, umbilical cord blood, amniotic fluid, testis, tonsil and other tissues. [0003] The mechanism of mesenchymal stem cells promoting tissue regeneration includes two aspects of differentiation and paracrine. The d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775A61K8/99A61K8/9794A61Q19/00
CPCA61K8/99A61K2800/10A61Q19/00A61K8/9794C12N5/0668C12N2500/90C12N2509/00
Inventor 魏色东
Owner 深圳市乔水生物科技有限公司
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