Method for obtaining mesenchyma stromal cells from connective tissue

Abstract

The invention discloses a method for obtaining a mesenchyma stromal cell from a connective tissue, comprising a separation method of the mesenchyma stromal cell of the umbilical cord connective tissue and a separation extraction method of adipose stromal cells. The method comprises the following steps: mild oxidizers such as oxidized glutathione, malonaldehyde, and the like are used, and the mild oxidizers exist in the human body; polysaccharide is degraded and broken through the oxidability to oxidize sugar molecule hydroxyl to generate unsaturated carbonyl or polymerize into a double polymer so as to destroy a polysaccharide structure; the substances are also act on a collagen, and react with the proximal amino acid to peroxide collagen peptide so that a polypeptied chain of the collagen peptide is broken to reduce the solution viscosity; and degradation of an elastic fiber is effectively improved. The method can obtain the cells with the number of 1*10<6> to 8*10<6>. The flow cytometry proves that the obtained cells have improved uniformity and multi-differentiation capacity.

Description

technical field [0001] The invention relates to a method for obtaining mesenchymal stromal cells from connective tissue. Background technique [0002] Mesenchymal stromal cells (MSCs) are a type of stem cells that originate from the mesoderm and mainly exist in the bone marrow. MSC has the common characteristics of stem cells, small size, large nucleoplasm, and does not express differentiation-related markers, nor does it express markers of hematopoietic stem cells. Using in vitro cell culture method, MSC can be directional induced to differentiate into osteogenic tissue, cartilage tissue, adipose tissue, muscle and other mesoderm tissues, which lays a good foundation for the application of MSC. [0003] Using MSC for tissue engineering research and application has many advantages; the tissue induced by it does not have the problems of tissue matching and immune rejection when transplanted. Just because MSC has its unique advantages in terms of source, isolation and tissue...

AI Technical Summary

Problems solved by technology

According to the experiment, simply using collagenase digestion (taking Wharton's jelly as an example), the highest cell yield can barely reach 10 6 order of magnitude (this number is quite different from the number of nucleated cells contained in umbilical cord tissue, and mesenchymal stromal cells usually only account for a small part of the nucleated cells), which is consistent with the literature report that each umbilical cord contains about 4,000,000 mesenchymal cells. The number of mesenchymal stromal cells varies greatly, which fully demonstrates the low efficiency of this method

[0007] At the same time, if the method of prolonging the digestion time is adopted, although the yield of cells can be partially increased, it will also cause a decrease in cell activity and a sharp drop in the adherence rate. Faced with the dilemma of a small number of cells, we had to adopt a large number of expanded cells in subsequent experiments to make up for the shortage of primary cells

However, this will also cause the undesired consequences that the original characteristics of mesenchymal stromal cells will gradually disappear with multiple expansion passages

[0008] The main reason for the low yield of cells after digestion is that there are gelatinous substances in the digestive juice after collagenase digestion, and these gelatinous substances adhere to cells in a large number. In some patents and literatures, it is difficult to effectively separate them under the centrifugation condition of 15min1000xg, while Long-term high-speed centrifugation also greatly damages the viability of cells

For the jelly, the analysis shows that the low concentration of collagenase opens up the three-dimensional structure of collagen but there is not enough quantity to completely destroy the chemical structure of collagen, causing collagen to continue to wrap around cells, but if the amount of collagenase is increased, not only The digestion efficiency has not been significantly improved, and the excessive use of collagenase has adverse effects on the cells, resulting in a decrease in the paste rate; on the other hand, connective tissue contains a large amount of macromolecular long-chain structures such as elastic fibers and polysaccharides, and these substances are not Can not be destroyed by collagenase, and is very soluble in water, can bind to cells through cell surface receptors, wrap around cells, and suspend in digestive juice

At the same time, the existence of the above substances also increases the viscosity of the solution and reduces the centrifugal efficiency.

[0009] The technology disclosed in patent 200510015492.2 is to introduce trypsin for further digestion after the collagen is digested, in order to use trypsin to digest the elastic fiber structure in the connective tissue and release the cells from the tissue. Enzyme does not significantly improve the extraction efficiency, and because trypsin has a significant effect on cell damage, it will produce irreversible damage after more than 10 minutes

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