Method for extracting umbilical cord mesenchymal stem cells by combining grinding with mixed enzyme

Abstract

The invention discloses a method for extracting umbilical cord mesenchymal stem cells by combining grinding with mixed enzyme. The method comprises the steps of 1, removing both ends of the umbilicalcord, and washing arteriovenous blood stains of the umbilical cord; 2, removing umbilical arteries and umbilical veins, cutting the umbilical cord into small pieces, peeling off an adhesive layer, cutting it into smaller pieces; 3, splitting and charging the tissue pieces into a centrifuging tube, and grinding the tissue into meat paste with a tissue grinder; 4, mixing collagenase and trypsin witha meat paste suspension and shaking the mixture in a shaker; 5, after neutralizing with a culture medium, centrifuging supernatant, mixing with a culture solution is executed, and adding the mixtureinto a cell culture flask; 6, standing and culture, digesting with enzyme, and then executing subculture; 7, continuing to culture for a period of time, subculturing P1 generation and cryopreserving the P2 generation. Based on the method of combining tissue grinding with digestion, the yield of primary cells can be greatly improved, and cell damage can be decreased.

Description

technical field [0001] The invention relates to an extraction method used in the field of mammalian cell genetic engineering, in particular to a method for extracting umbilical cord mesenchymal stem cells which can be effectively ground and combined with mixed enzymes. Background technique [0002] Umbilical cord mesenchymal stem cells are a kind of mesenchymal stem cells present in Wharton's jelly and perivascular tissue of neonatal umbilical cord. The mesenchymal stem cells derived from the umbilical cord are easy to obtain and have no moral and ethical disputes. The number of cells that can be obtained is large, the proliferation ability is strong, the immune regulation is strong, and the total amount of cell growth factors secreted is also very high, which is beneficial to skin tissue damage. Repair and skin allergies are solved, the research and development of cosmeceutical products based on this is constantly deepening, and the stem cell industry is gradually becoming ...

AI Technical Summary

Problems solved by technology

[0003] Specifically, most umbilical cord mesenchymal stem cells are cultured using the adherence method. The umbilical cord is cut into small pieces and pasted on the surface of a petri dish. After about a week of adherence, the generation cells climb out of the overgrowth and then digest and passage. This method takes a long time. It usually takes about a month to carry out the first subculture operation. The most common problem is that the tissue is not firmly adhered to the wall, it is easy to fall off, and the cells crawl out less. If the primary culture time is too long, the cells will age and the state will deteriorate. Use The effect becomes worse, etc.; the amount of work of the wall-attachment method is huge. For example, the number of cells in an umbilical cord reaches 1 billion in the P1 generation. Due to the incomplete wall-attachment, the difficulty of operation is greatly increased. In the later stage, the cell texture is not uniform, which affects the quality control and use of the cells; the production cost caused by the adherent method is that the primary culture takes a long time, consumes more culture medium and serum, and greatly increases the cost

[0004] The usual enzymatic digestion method usually uses collagenase and trypsin to digest the Wharton's jelly tissue cells of the umbilical cord. Because the surface of the umbilical cord tissue is slippery, it is difficult to cut it up. Visible small pieces of tissue, and trypsin separates cells by dissociating intercellular glycoproteins and mucins. Excessive digestion can easily cause damage to cells, damage cell membranes, and destroy cytoskeleton to affect subsequent cell proliferation and differentiation. Enzymes act on tissues It often leads to overdigestion of surface tissue cells and the internal cells of the tissue are still not in contact with the digestive juice, resulting in low yield of mesenchymal stem cells extracted by digestion method, poor cell activity, and difficulty in proliferating to the ideal number

[0005] When it is necessary to prepare a large number of umbilical cord mesenchymal stem cells, digestion is often used to extract and prepare primary cells. Due to individual differences, it is difficult to control the infiltration and digestion rate of digestive enzymes on tissues. Excessive digestion on the tissue surface will cause cell A large amount of damage, the amount of cells obtained from the primary generation is small, which increases the difficulty of cell expansion, the state and activity of the cells after expansion become worse, destroys the cell wall structure, and affects cell adhesion

[0006] To sum up, the large-scale culture of umbilical cord mesenchymal stem cells uses the adherent method for the primary generation, the adherence is not firm, the culture time is long, the cell state becomes old, the state of the cells climbing out is not uniform, and the use effect becomes poor; The wall method requires a huge amount of engineering, which consumes a lot of manpower and material resources, and the production cost is high; a large number of umbilical cord mesenchymal stem cells are cultivated by conventional digestion method, and enzymes act on the surface of the tissue block, which often leads to excessive digestion of surface tissue cells and tissue internal cells. Still not in contact with the digestive juice, resulting in low yield of mesenchymal stem cells extracted by digestion method, poor cell activity, damaged cell wall, low degree of adherence, poor cell state, difficult to proliferate to the ideal number, and seriously affect the later expansion and induction or genetic modification work

Images