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Large-scale culture method and device for fibroblasts

A technology for fibroblasts and a culturing method is applied in the field of a large-scale culturing method of fibroblasts and a culturing device, and can solve the problems of increasing operating costs, harsh operating conditions and high requirements

Inactive Publication Date: 2019-08-16
张永国
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] 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 the microcarrier itself. It is necessary to use special microcarriers to ensure normal cell culture. Indirectly increase the operating cost and limit the application of microcarriers

Method used

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  • Large-scale culture method and device for fibroblasts
  • Large-scale culture method and device for fibroblasts
  • Large-scale culture method and device for fibroblasts

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] The large-scale culture method of fibroblasts is carried out according to the following steps:

[0076] 1) Get the attached fibroblasts and digest them, inoculate them in a biological culture device 20 and co-culture with GE microcarriers, and the inoculation density of fibroblasts is 8*10 3 cell / ml, the final concentration after inoculation is 0.2wt% (total concentration of GE microcarrier);

[0077] 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;

[0078] 3) After stirring and culturing at a slow speed of 10-15rpm for 24 hours, then rapidly c...

Embodiment 2

[0081] The large-scale culture method of fibroblasts is carried out according to the following steps:

[0082] 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;

[0083] 2) After digesting the fibroblasts attached to the wall, inoculate them in a biological culture device 20 and co-culture with microcarriers. The inoculation density of the fibroblasts is 10*10 3 cell / ml, the final concentration after inoculation is 0.8wt% (total concentration of GE microcarrier);

[0084] 3) culture fluid is housed in the sterile culture bag 10, and the side wall of the sterile culture bag is equipped with a...

Embodiment 3

[0088] The large-scale culture method of fibroblasts is carried out according to the following steps:

[0089] 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;

[0090] 2) After digesting the adherent fibroblasts, inoculate them in a biological culture device 20 to co-culture with microcarriers, and the inoculation density of fibroblasts is 9*10 3 cell / ml, the final concentration after inoculation is 0.1wt% (total concentration of GE microcarrier);

[0091] 3) culture fluid is housed in the sterile culture bag 10, and the side wall of the sterile culture bag is equipped with a connector 50,...

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Abstract

The invention provides a large-scale culture method and device for fibroblasts. The culture method comprises the following steps: inoculating cells to be cultured and sterilized GE microcarriers intoa culture medium containing serum, wherein the total concentration of the inoculated GE microcarriers is 0.1-1wt%; and stirring and culturing for 24 h or above. The inoculating density of the cells tobe cultured is (8-10)* 103 cells / ml. The culture method is simple to operate, mild in operating conditions and high in cell survival rate. The large-scale culture method for the fibroblasts is provided, and is worthy of wide popularization and application.

Description

technical field [0001] The invention relates to the field of fibroblast culture, in particular to a large-scale culture method of fibroblasts and a culture device thereof. Background technique [0002] Fibroblasts, also known as fibroblasts, are the main cellular component of loose connective tissue and are terminally differentiated cells that make proteins such as collagen. The number of fibroblasts is the largest, the cell body is large, and it is multi-projected spindle-shaped or star-shaped flat cells. The nucleus is regular oval, and the cell outline is unclear and has protrusions. Its shape can still change according to the functional changes of the cells and the physical properties of the attachment. [0003] Microcarriers refer to microbeads with a diameter of 60-250 μm that are suitable for the growth of adherent cells. It is generally composed of natural dextran or various synthetic polymers. [0004] The principle of microcarrier application is to add the harml...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077C12M3/00C12M1/00
CPCC12N5/0656C12M23/14C12M23/08C12N2531/00C12N2533/70
Inventor 张永国何君
Owner 张永国
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