Methods for freezing and thawing whole cell of umbilical cord and separating and augmenting stem cell
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A technology of whole cells and stem cells, applied in the field of cryopreservation and resuscitation of umbilical cord whole cells and the isolation and expansion of stem cells, which can solve the problems of consumption and incomplete maturity.
Active Publication Date: 2012-09-12
BOYALIFE
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[0005] However, the method and technology for isolating stem cells from umbilical cord tissue such as umbilical cord whole cells are not yet fully mature, and the processing and isolation of each umbilical cord whole cell requires a certain amount of time and labor.
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Embodiment 1
[0102] Embodiment 1, umbilical cord whole cell cryopreservation, resuscitation and method of separation and expansion of stem cells after resuscitation Law
[0103] The method of cryopreservation of umbilical cord tissue includes the following steps:
[0104] (1) Disinfection and cleaning: In a biological safety cabinet, disinfect the surface of the umbilical cord tissue with alcohol, cut the umbilical cord from the middle, spread it on a sterile 10cm cell culture plate, and clean the tissue with PBS to reduce the size of the tissue surface. red blood cells;
[0105] (2) Digestion treatment: Add 0.1 g of type I collagenase to 100 ml of DMEM-F12, then filter it with a 20 μm filter to obtain the digestion solution, transfer the umbilical cord tissue obtained in step (1) to another 10 cm cell culture dish, Cut the umbilical cord tissue into 1cm 3 Tissue pieces of different sizes, put the tissue pieces into the prepared digestive solution, digest them in a constant temperatur...
Embodiment 2
[0119] Embodiment 2, umbilical cord whole cell cryopreservation, resuscitation and method of separation and expansion of stem cells after resuscitation Law
[0120] Carried out with reference to the method of Example 1. The thawed umbilical cord whole cells began to have adherent cells on the 14th day of culture, and the cell fusion rate reached 80% by the 24th day of culture. After 3 passages, the cell purity was greater than 90%.
Embodiment 3
[0121] Embodiment 3, umbilical cord whole cell cryopreservation, resuscitation and method of separation and expansion of stem cells after resuscitation Law
[0122] Carried out with reference to the method of Example 1. The thawed umbilical cord whole cells began to have adherent cells on the 13th day of culture, and the cell fusion rate reached 80% by the 24th day of culture. After 3 passages, the cell purity was greater than 85%.
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Abstract
The invention relates to methods for separating, freezing and thawing a whole cell of an umbilical cord and separating and augmenting a thawed stem cell. The method for separating and freezing the whole cell of the umbilical cord comprises the following steps of: sterilizing and cleaning an umbilical cord tissue; shearing the tissue into a block shape and carrying out digestion treatment for 1.5 hours; preparing a umbilical cord tissue frozen solution for later use; and adding the whole cell and frozen solution which are obtained by sterilizing treatment into a freezing tube, refrigerating the freezing tube for 0.5 hour at low temperature of 4DEG C, freezing the freezing tube for one day under the temperature condition of -80DEG C and then freezing the freezing tube in liquid nitrogen for later use. The method for thawing the whole cell of the umbilical cord comprises the following steps of: when the whole cell of the umbilical cord is required, extracting the whole cell of the umbilical cord from the liquid nitrogen, thawing the whole cell of the umbilical cord in a constant-temperature water bath; cleaning the whole cell of the umbilical cord by using a mesenchymal stem cell culture medium and a drop method; and augmenting a mesenchymal stem cell by using the thawed whole cell of the umbilical cord through cell culture and cell passage. According to the methods disclosed by the invention, the frozen umbilical cord tissue can be effectively protected and is convenient for thawing; and the method is especially suitable for separating and augmenting the mesenchymal stem cell after the frozen umbilical cord tissue is thawed.
Description
technical field [0001] The invention relates to a method for processing whole umbilical cord cells, in particular to a method for freezing and resuscitating whole umbilical cord cells, and a method for separating and amplifying stem cells from the whole umbilical cord cells after resuscitation, in particular to freezing and storing whole umbilical cord cells, A method of resuscitating, then isolating and expanding mesenchymal stem cells therefrom. Background technique [0002] Mesenchymal stem cells (MSCs) originate from mesoderm and ectoderm in the early stages of development, and have the characteristics of multilineage differentiation potential, immune regulation and self-replication, and have attracted increasing attention. Under specific induction conditions in vivo or in vitro, mesenchymal stem cells can differentiate into fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelial and other tissue cells. It has multi-directional differen...
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