Cell scale culture method, purification method and cell messenger

A culture method and purification method technology, applied in the field of large-scale cell culture, cell messenger and purification, can solve the problems of harsh operating conditions, complicated process, and low cell survival rate

Inactive Publication Date: 2019-08-06
张永国
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The method for culturing cells with microcarriers in the prior art has complex processes, harsh operating conditions, low cell survival rate, low inoculation density, and high requirements for microcarriers, so special microcarriers are needed to ensure normal cell culture. Indirectly increase the operating cost and limit the application of microcarriers

Method used

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  • Cell scale culture method, purification method and cell messenger
  • Cell scale culture method, purification method and cell messenger
  • Cell scale culture method, purification method and cell messenger

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] The cell-scale culture method and the subsequent purification method are carried out according to the following steps:

[0092] 1) After digesting the adipocytes and mesenchymal cells attached to the wall, inoculate them in a biological incubator 20 and co-culture with GE microcarriers. The inoculation density of the cells is 8*10 3 cell / ml, the final concentration of the culture solution after inoculation is 0.2wt%;

[0093] 2) culture fluid is housed in the sterile culture bag 10, and a joint 50 is installed on the side wall of the sterile culture bag, which is connected with the inlet of the biological incubator 20 through the joint, so as to transport nutrients through the adjustable variable speed pump 30, and the biological culture The outlet of the device 20 is provided with a joint 50, and the culture solution in the biological incubator 20 is discharged through the variable speed pump 30;

[0094] 3) After stirring and culturing at a slow speed of 10-15rpm for...

Embodiment 2

[0099] The cell-scale culture method and the subsequent purification method are carried out according to the following steps:

[0100] 1) Treat the microcarriers: prepare 4L of PBS 0.01M pH7.4, autoclave at 121°C for 15min, soak the Cytodex 1GE microcarriers with the above PBS overnight, wash 3 times, the beads settle to the bottom, discard the broken sterilized supernatant After that, the medium was used to equilibrate overnight at room temperature, and the medium was changed once in the middle. Aliquot 10ml / tube, store at 4°C, take 1ml and add 100ml medium when used;

[0101] 2) After digesting the adherent adipocytes and mesenchymal cells, inoculate them in a biological culture device 20 to co-culture with microcarriers, and the seeding density of the cells is 9.0*10 3 cell / ml, the final concentration of the culture solution after inoculation is 0.8wt%;

[0102] 3) culture fluid is housed in the sterile culture bag 10, and the side wall of the sterile culture bag is equip...

Embodiment 3

[0108] The large-scale culture method of adipocytes and the subsequent purification method are carried out according to the following steps:

[0109] 1) Treat the microcarriers: prepare 4L of PBS 0.01M pH7.4, autoclave at 121°C for 15min, soak the Cytodex 1GE microcarriers with the above PBS overnight, wash 3 times, the beads settle to the bottom, discard the broken sterilized supernatant After that, the medium was used to equilibrate overnight at room temperature, and the medium was changed once in the middle. Aliquot 10ml / tube, store at 4°C, take 1ml and add 100ml medium when used;

[0110] 2) After digesting the adherent adipocytes, inoculate them in a biological culture device 20 to co-culture with microcarriers, and the inoculation density of adipocytes is 8.6*10 3 cell / ml, the final concentration of the culture solution after inoculation is 0.1wt%;

[0111] 3) culture fluid is housed in the sterile culture bag 10, and the side wall of the sterile culture bag is equippe...

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Abstract

The invention provides a cell scale culture method, which comprises the following steps: inoculating cells to-be-cultured and a sterilized GE microcarrier into a serum-containing medium, culturing thematerials to a high density in a biological incubator, then switching to a serum-free medium, continuously culturing the material for 4-7 days in serum-free medium, and collecting a supernatant. Theculture method is simple in operation, mild in operating conditions, and high in cell survival rate, provides a reference method for subsequent cell-scale culture, and is worthy of widespread application.

Description

technical field [0001] The invention relates to the field of cell culture, in particular to a large-scale cell culture method, a cell messenger and a purification method. Background technique [0002] Every adult body contains about 30 billion white fats, whose function is to store energy in the form of fat cells. Each fat cell contains triglycerides, commonly known as fat globules. When the amount of fat globules becomes larger, the volume of fat cells will expand, resulting in obesity; on the contrary, when triglycerides are burned, the cells will shrink and the body will lose weight. Under normal circumstances, the number of fat cells does not increase after puberty. The distribution of body fat is partly determined by genetic and hormonal influences. For example, women's subcutaneous fat mostly accumulates in the lower abdomen, buttocks and thighs, while men accumulate it in the upper abdomen and waist. [0003] Umbilical cord mesenchymal cells (Mesenchymal Stem Cells...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12N5/077
CPCC12N5/0653C12N5/0668C12N2500/36C12N2531/00
Inventor 张永国何君
Owner 张永国
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