Human umbilical cord mesenchymal stem cell separation culture method
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A technology for the separation and cultivation of stem cells, applied in the field of cell separation and cultivation of biological products, can solve the problems of long time, low cell yield, cumbersome operation, etc., and achieve the effect of easy separation, high purity and yield of stem cells
Inactive Publication Date: 2018-02-02
南京三生生物技术股份有限公司
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However, the enzymatic digestion method requires a variety of enzymes, which is cumbersome and takes a long time; at the same time, it has the disadvantages that the digestion time is difficult to control and the cell yield is low.
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Embodiment 1
[0019] A method for isolating and culturing human umbilical cord mesenchymal stem cells, comprising the following steps:
[0020] (1) Take the isolated umbilical cord of a full-term cesarean section fetus, put it in a sterile tissue preservation solution, and store it at 4°C for 12 hours;
[0021] (2) Take out the umbilical cord from the sterile tissue preservation solution under aseptic conditions, rinse it with normal saline several times, and remove the residual blood;
[0022] (3) Cut the umbilical cord cleaned in step (2) into small pieces of 2 cm with sterile scissors, cut the small pieces longitudinally, remove the two umbilical veins and two umbilical arteries of the tissue block with toothed forceps, and peel off Put Huatong glue into normal saline, and cut Huatong glue completely with sterile scissors;
[0023] (4) Transfer the shredded Huatong jelly in step (3) to medium containing DMEM / F12, oscillate and centrifuge at a speed of 1500 rpm, centrifuge for 5 minutes ...
Embodiment 2
[0028] A method for isolating and culturing human umbilical cord mesenchymal stem cells, comprising the following steps:
[0029] (1) Take the isolated umbilical cord of a full-term cesarean section fetus, put it in a sterile tissue preservation solution, and store it at 4°C for 24 hours;
[0030] (2) Take out the umbilical cord from the sterile tissue preservation solution under aseptic conditions, rinse it with normal saline several times, and remove the residual blood;
[0031] (3) Use sterile scissors to cut the umbilical cord cleaned in step (2) into small pieces of 3 cm, cut the small pieces longitudinally, remove the two umbilical veins and two umbilical arteries of the tissue block with toothed forceps, and peel off Put Huatong glue into normal saline, and cut Huatong glue completely with sterile scissors;
[0032] (4) Transfer the shredded Huatong jelly in step (3) to medium containing DMEM / F12, shake and centrifuge at a speed of 2000 rpm, centrifuge for 10 minutes a...
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Abstract
The invention belongs to the technical field of biological product separation culture, and particularly relates to a human umbilical cord mesenchymal stem cell separation culture method. The method comprises the following steps that an in-vitro umbilical cord of a term fetus born by caesarean delivery is taken and put into a sterile tissue preserving fluid for storage; flushing is conducted multiple times with normal saline, and remained bloodstains are removed; two umbilical veins and two umbilical arteries of tissue mass are removed with toothed tweezers, and Wharton's jelly is completely cut into pieces with sterile scissors; the Wharton's jelly which is cut into pieces is transferred into a culture medium, and vibration and centrifugation are conducted in sequence; the centrifugal sedimentation part is transferred into a cell culture flask; after culture is conducted for 5-6 days, it can be seen that part of cells climb out from the periphery of small pieces of the tissue, then a culture substrate is replaced once every three days, and culture continuous to be conducted; on the fourteenth day or so, the degree of cell fusion reaches 80% or above, and spiral growth is achieved;afterwards, each transmission of the next generation takes three days, and adequate mesenchymal stem cells can be obtained. Compared with a traditional tissue method, the method is simpler, and the purity and yield of stem cells are higher.
Description
technical field [0001] The invention belongs to the technical field of cell separation and culture of biological products, and in particular relates to a method for separation and culture of human umbilical cord mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (MSCs) are a type of pluripotent stem cells derived from mesoderm and ectoderm, capable of self-renewal and multi-directional differentiation, as well as hematopoietic support and immune regulation functions. It mainly exists in adult bone marrow and various tissues. At present, mesenchymal stem cells in adult bone marrow have become the main source of research, but the number is small, and the proliferation and differentiation potential will decrease with age, the virus infection rate is high, and the immunogenicity is strong. In addition, bone marrow aspiration is required. collection, so its research and application are limited; in recent years, a large number of studies have shown that ...
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