Culture method of umbilical cord mesenchymal stem cells

A technology of quality stem cells and culture methods, applied in cell dissociation methods, animal cells, tissue culture, etc., can solve the problems of storage contamination, increased risk, leakage of tube caps, etc., to save storage space, ensure safety, The effect of avoiding pollution

Inactive Publication Date: 2019-03-19
陕西森凯组织工程与再生医学科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] 1. During normal delivery, because the umbilical cord is delivered through a sterile vagina, many umbilical cords of normal delivery cannot be well aseptically treated, so that these umbilical cords cannot be used for the preparation of umbilical cord mesenchymal stem cells
[0015] 2. Because some umbilical cords are contaminated by pathogens, pathogenic microorganisms such as bacteria and molds reproduce during the cultivation of umbilical cords, causing pollution to other cells and other individual storage
[0016] 3. What is more serious is that in the current general storage process, immersion in liquid nitrogen is often used for storage, which will increase the risk of cross-contamination between the stored cells through the liquid nitrogen liquid phase
[0017] 4. When using non-screw-capped plastic storage tubes with caps, leakage from the cap opening also greatly increases the chance of contamination of other specimens
[0018] The pollution of the normal delivery umbilical cord itself greatly limits the availability of these umbilical cords. What is more serious is that the bacteria, mold, liquid phase cross-contamination and cross-contamination caused by the leakage of the nozzle will cause serious damage to future clinical research applications. huge problem
Make these umbilical cord mesenchymal stem cells lose their clinical application value

Method used

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  • Culture method of umbilical cord mesenchymal stem cells
  • Culture method of umbilical cord mesenchymal stem cells
  • Culture method of umbilical cord mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Turn on the ultraviolet lamp in the clean area to irradiate for more than 30 minutes, turn on the fan in the clean area; take the umbilical cord storage and transportation bottle to the mesenchymal stem cell preparation room, and check whether the storage and transportation bottle has liquid leakage, whether the seal is tight, whether there are cracks, and whether the label is complete , Accurate, whether the umbilical cord information is complete and meets the preparation requirements.

[0057] Then wipe it with alcohol cotton balls and put the storage bottle into a normal operating biological safety cabinet; disinfect surgical instrument boxes, 50ml centrifuge tubes, 75% medical alcohol, normal saline, petri dishes and other items with 75% medical alcohol after disinfection Put it in a biological safety cabinet; open the cap of the umbilical cord storage and transportation bottle, draw 1ml of umbilical cord preservation solution into a 2ml EP tube for detection of bact...

Embodiment 2

[0060] Routine collection and storage of primary (P0) and first-generation (P1) and second-generation (P2) umbilical cord mesenchymal stem cells after culture. Dulbecco's modified eagle's medium (DMEM, Gibco, USA), 1% glutamine (Gibco, USA) and serum-free medium were used for culture. The umbilical cord mesenchymal stem cells were collected in a CryoFlex 2ml EP cryopreservation tube in a sterile manner. . Storage tubes containing umbilical cord mesenchymal stem cells with caps. The capped cell storage tube was further wrapped with a double-layer polyester film. Use a dedicated storage rack to store in the gas phase of the Cry Extra high-efficiency gas phase liquid nitrogen storage tank. All treated cells were stored in the gas phase at -196°C in Thermo Scientific Cry Extra liquid nitrogen storage tanks.

[0061] The umbilical cord mesenchymal stem cells processed by the above method, and the umbilical cord mesenchymal stem cell bank and storage method can avoid cross-contam...

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Abstract

The invention relates to the field of medical treatment, and particularly relates to a culture method of umbilical cord mesenchymal stem cells. The method disclosed by the invention comprises the following steps: 1, umbilical cord disinfecting treatment: (a) treating the surface of the umbilical cord with 75% alcohol; (b) removing umbilical cord adventitia with hands; and (c) performing asepsis treatment on umbilical cord tissues; 2, separation of the umbilical cord tissues; 3, preparation and culture of umbilical cord mesenchymal stem cells: (a) performing manual asepsis treatment on the tissues; (b) cutting up the tissues to be particles with size of 1 cubic millimeter in an asepsis manner; (c) treating the tissues prepared in 3b with a digestive enzyme; (d) cleaning, centrifugally separating and collecting cells; and (e) performing primary cell culture; 4, safe storage of the umbilical cord mesenchymal stem cells: (a) collecting the cells in a CryoFlex 2ml freezing storage tube in an asepsis manner; (b) covering the storage tube with an inward-turning seal cover; and (c) storing the cells in the gaseous phase of a Cry Extra high-efficiency gaseous-phase liquid nitrogen storage tank.

Description

technical field [0001] The present invention relates to the medical field, in particular to a method for culturing umbilical cord mesenchymal stem cells. Background technique [0002] Mesenchymal Stem Cell (MSC) is a type of stem cell, named for its ability to differentiate into bone, cartilage, fat, muscle and other mesenchymal tissues in a specific environment in vivo and in vitro. Widely present in various tissues, such as umbilical cord, placenta, fat, lung, heart, brain, small intestine, bone marrow, etc. [0003] Characteristics of mesenchymal stem cells include: [0004] 1) It grows adherently in a plastic petri dish and proliferates highly in a medium containing serum; [0005] 2) Express cell surface markers CD105, CD73, CD90, but not CD45, CD34, CD14 or CD11b, CD79a or CD19, HLA-DR; [0006] 3) It can be differentiated into osteoblasts, adipocytes and chondrocytes in vitro. [0007] The differentiation of mesenchymal stem cells is related to its microenvironmen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775A01N1/02A61K35/28
CPCA01N1/0221A61K35/28C12N5/0665C12N2509/00
Inventor 念沛沛扈晖陈寅嘉澍任敬村
Owner 陕西森凯组织工程与再生医学科技有限公司
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