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Method for establishing human umbilical cord mesenchymal stem cell bank by adopting blood serum substituent

A serum substitute and quality stem cell technology, applied in the field of cell cryopreservation system, can solve the problems of long time in the establishment of cell bank and the small number of obtained cells, and achieve the effect of avoiding individual differences, solving the difficulty of sample collection, and strong repeatability

Inactive Publication Date: 2016-09-28
天晴干细胞股份有限公司
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Problems solved by technology

[0003] The present invention uses the animal free culture system to cultivate and freeze the umbilical cord mesenchymal stem cells, and adopts the secondary slide method that makes full use of the umbilical cord tissue when establishing the cell bank, so as to solve the problems of long time in the establishment process of the cell bank and the small number of obtained cells, etc. , and the cell quality was tested, the results showed that its cell quality and biological ability were superior to traditional culture methods

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  • Method for establishing human umbilical cord mesenchymal stem cell bank by adopting blood serum substituent
  • Method for establishing human umbilical cord mesenchymal stem cell bank by adopting blood serum substituent
  • Method for establishing human umbilical cord mesenchymal stem cell bank by adopting blood serum substituent

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specific Embodiment approach 1

[0024] Embodiment 1: A method of using serum substitutes to establish a human umbilical cord mesenchymal stem cell bank in this embodiment is carried out in accordance with the following steps:

[0025] 1. Separation of umbilical cord Wharton's jelly: cut the umbilical cord into 1-3 cm, peel off the blood vessels and epidermis, and separate Wharton's jelly;

[0026] 2. Tissue block culture and cell passage: cut the umbilical cord Huatong jelly separated in step 1 into small pieces, add it to serum-free medium for culture, replace the medium every 5 days, and suspend the tissue block when the cell density reaches 70%. And collect, then centrifuge, discard the supernatant, add trypsin substitute, digest for 3min, then add normal saline, collect the cell suspension, centrifuge, and resuspend the cells;

[0027] 3. Centrifuge the resuspended cells in step 2, discard the supernatant, resuspend with serum-free medium for secondary culture, replace the medium every 5 days, and suspen...

specific Embodiment approach 2

[0030] Specific embodiment two: the difference between this embodiment and specific embodiment one is: the specific operation of separating umbilical cord Huatong jelly is as follows:

[0031] Disinfect the umbilical cord tissue, cut it into 2cm pieces, peel off the blood vessels and skin, separate the Wharton's jelly tissue, cut the tissue into 2-3mm pieces, resuspend each tissue piece in 10mL serum-free medium, That is to complete the operation of separating the umbilical cord Huatong jelly. Others are the same as in the first embodiment.

specific Embodiment approach 3

[0032] Specific embodiment 3: The difference between this embodiment and specific embodiment 1 is that the components of the serum-free medium are: α-MEM basal medium with a volume percentage content of 95% and UltraGRO serum with a volume percentage content of 5% substitute, and 2U / mL sodium heparin. Others are the same as in the first embodiment.

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Abstract

The invention provides a method for establishing a human umbilical cord mesenchymal stem cell bank by adopting a serum substituent and relates to a method for establishing a stem cell bank. The method comprises the following steps: separating umbilical cord Wharton jelly and adding into a serum-free culture medium for culturing; replacing the culture medium for one time every 5 days; after cells grow to 70 percent, reproducing; collecting tissue blocks again and adding into the serum-free culture medium for secondary culture; replacing the culture medium for one time every 5 days; after cells grow to 70 percent, reproducing; after subculture, cryopreserving to establish the umbilical cord mesenchymal stem cell bank. The invention adopts serum-free culture and a cryopreserving system to establish the cell bank and a tissue block secondary climbing method. The method has the advantages that all tissues are effectively utilized and the cells can climb out as much as possible; the quantity of obtained cells is twice of that of a traditional method. A cell culture system adopts the serum substituent to replace bovine serum and adopts a pancreatin substituent to replace porcine pancreatin, so that pollution to human by animal sources is effectively avoided, and a quality control link of detecting cattle and pigs is saved.

Description

technical field [0001] The invention relates to a new method for establishing an umbilical cord mesenchymal stem cell bank and a cell cryopreservation system. Background technique [0002] Mesenchymal stem cells are pluripotent stem cells, which have the characteristics of multi-directional differentiation potential, hematopoietic support and promotion of stem cell implantation, immune regulation and self-replication, so they have attracted increasing attention. Major breakthroughs have been made in the clinical application of mesenchymal stem cells and in solving various blood system diseases, cardiovascular and cerebrovascular diseases, nervous system diseases, bone and joint diseases, and autoimmune diseases. With the continuous development of clinical applications, the quality of mesenchymal stem cells has also attracted more and more attention. The traditional methods of culturing mesenchymal stem cells use serum (bovine source) and trypsin (pig source) system. This an...

Claims

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Application Information

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IPC IPC(8): C40B50/06C12N5/0775
CPCC40B50/06C12N5/0665C12N2500/12C12N2501/91C12N2509/00
Inventor 芦慧颖张怡孟庆雪刘春香
Owner 天晴干细胞股份有限公司
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