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Preparation method and recovery method of decidua parietalis mesenchymal stem cells

A technique for stem cells and parietal decidua, which is applied in the field of preparation of parietal decidua mesenchymal stem cells, can solve the problems of inability to effectively maintain cell activity and biological function, difficult cells, and low cell concentration, and achieves the ability to maintain viability. and biological functions, improving purity, and accelerating growth

Active Publication Date: 2021-07-20
GUANGDONG VITALIFE BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the existing preparation method of PMSCs, it is difficult for cells to climb out of the tissue and grow on the wall, and the existing medium cannot effectively promote the growth of PMSCs, so that the concentration of cells is low and the quality is poor, thus affecting the inhibitory effect of PMSCs on tumor cells
In addition, the existing cryopreservation and recovery methods of PMSCs cannot effectively maintain cell viability and biological function, and cannot guarantee that each batch of recovered cells has similar cell viability

Method used

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  • Preparation method and recovery method of decidua parietalis mesenchymal stem cells
  • Preparation method and recovery method of decidua parietalis mesenchymal stem cells
  • Preparation method and recovery method of decidua parietalis mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] A method for preparing parietal decidual mesenchymal stem cells, comprising the following steps:

[0052] S11. Separation of parietal decidua tissue: use tissue cleaning solution to clean the blood and blood clots on the placental tissue of healthy full-term newborns, use surgical stripping instruments to separate parietal decidua tissue, and then use surgical scissors to cut it into 1-4mm 3 and cleaned with tissue cleaning solution; the tissue cleaning solution is prepared from the following volume percentage raw materials: 1% penicillin and streptomycin mixture, 51.1% erythrocyte lysate, 47% normal saline, and the normal saline is the mass fraction 0.9%;

[0053] S12. Digestive wall decidua tissue block: After adding tissue digestive juice to the chopped tissue block, shake and digest at a constant temperature for 2 hours at 37°C, with a rotation speed of 200rpm / min; the tissue digestive juice contains a volume concentration of 50% Tryple- EDTA enzyme and 10mg / ml typ...

Embodiment 2

[0061] The recovery method of the parietal decidual mesenchymal stem cell culture medium, including:

[0062] S21. Dissolving the frozen decidual mesenchymal stem cells cryopreserved in Example 1 in a water bath at 37°C;

[0063] S22. Use the selection medium to resuspend the epithelial decidual mesenchymal stem cells of step S21, centrifuge at 1300rpm for 6min, wash the cell surface twice with PBS buffer, centrifuge at 1300rpm for 6min, add the selection medium to resuspend the pellet for subculture;

[0064] S23. Collect the PMSCs of the P5 generation, add selection medium to resuspend the cells into a low concentration group, a medium concentration group and a high concentration group, wherein the concentration of PMSCs in the low concentration group is 1×10 5 per well, the concentration of PMSCs in the middle concentration group was 2×10 5 per well, the concentration of PMSCs in the high concentration group was 4×10 5 cells / well, each group was plated in the upper chambe...

Embodiment 3

[0066] Co-cultivation experiment of parietal decidual mesenchymal stem cell medium and cervical cancer cells, including:

[0067] S31. Dissolve the cervical cancer cell line Hela rapidly in a 37°C water bath, add complete medium to resuspend the cells, centrifuge, wash twice with PBS buffer, centrifuge at 1000rpm for 3min, add fresh complete medium to resuspend the cells, transfer Cultivate in a T25 culture bottle, which is designated as the P1 generation; the complete medium is a serum-free DMEM medium containing 10% fetal bovine serum and 1% anti-penicillin and streptomycin;

[0068] S32, Hela cell passage: observe the cell morphology and medium changes after recovery, replace the complete medium every 2 days, and collect the cell suspension when the cell growth and confluence is greater than 80%, count and calculate the survival rate, microbiological detection, and passage ;

[0069] S33. Cultivate Hela cells in the lower chamber of the Transwell chamber: Discard the origi...

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Abstract

The invention discloses a preparation method and a resuscitation method of decidua parietalis mesenchymal stem cells, and the preparation method comprises the following steps that tissue blocks are digested by using a high-glucose DMEM culture medium containing 40-60% by volume of Tryple-EDTA enzyme and 8-12 mg / ml of type II collagenase as a tissue digestion solution, so that the decidua parietalis mesenchymal stem cells can climb out of tissues for adherent growth; a DMEM (dulbecco's modified eagle medium) serum-free culture medium containing a serum substitute with the volume concentration of 8-12%, L-glutamine with the volume concentration of 0.5-1 mol / ml, a basic fibroblast growth factor with the volume concentration of 18-25 ng / ml, an epidermal growth factor with the volume concentration of 16-22 ng / ml and a stem cell growth factor with the volume concentration of 6-12 ng / ml is adopted as a selective culture medium to terminate digestion, and the decidua parietalis mesenchymal stem cells are resuspended, so that the purity of the decidua parietalis mesenchymal stem cells is improved, the growth of the decidua parietalis mesenchymal stem cells is accelerated, and the in-vitro rapid amplification of the decidua parietalis mesenchymal stem cells is realized.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a preparation method and recovery method of parietal decidual mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (mesenchymal stem cells, MSCs) are pluripotent progenitor cells with the ability to chemoattract tumors, and the research on gynecological tumors has made significant progress. However, there are different reports on the influence of uterine-derived mesenchymal stem cells on the biological behavior of gynecological malignant tumor cells. The study of the adaptability and selectivity of parietal decidua MSCs (Pariduval Mesenchymal stem cells, PMSCs) and gynecological tumors has become an important topic in the clinical treatment of gynecological tumors. new method. [0003] Pariduval Mesenchymal stem cells (PMSCs) have the characteristics of simple in vitro proliferation and culture, no moral ethics involved, good migration ability, homing to tumo...

Claims

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Application Information

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IPC IPC(8): C12N5/0775A01N1/02
CPCC12N5/0668A01N1/0221C12N2509/00C12N2509/10C12N2500/32C12N2501/115C12N2501/11C12N2501/125C12N2500/90A01N1/0226C12N2500/76C12N2502/30C12N2501/734C12N2500/34C12N2500/10
Inventor 刘小翠许峻荣唐淑艳邓燕莲蒙燕瑶杨景利王进辉
Owner GUANGDONG VITALIFE BIOTECHNOLOGY CO LTD
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