Large-scale cell culture method and used microcarrier

A culture method and microcarrier technology, applied in general culture methods, microcarriers, cell culture supports/coatings, etc., can solve problems such as increased product production costs, increased process flow, and difficulties in large-scale cell culture, achieving Facilitates adhesion and reduces contact area

Pending Publication Date: 2021-07-02
ZHUJIANG HOSPITAL SOUTHERN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, as a cell product for clinical implantation, the introduction of various proteins and enzymes must be avoided during the preparation process, which brings difficulties to the large-scale cultivation of cells
Especially for the large-scale culture of anchorage-dependent cells, trypsin digestion is still required to collect the cultured cells. The introduction of trypsin increases the subsequent purification process of cell products and increases the production cost of the product

Method used

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  • Large-scale cell culture method and used microcarrier
  • Large-scale cell culture method and used microcarrier

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Effect test

preparation example Construction

[0040] The preparation method of the microcarrier of the present application is as follows:

[0041] Prepare gel molecule solution and exogenous matrix molecule solution respectively;

[0042] mixing calcium carbonate powder with the gel molecule solution, and then mixing with the exogenous matrix molecule solution to form a premix;

[0043] Promoting the gelation reaction of the premix solution through fluid granulation technology to generate hydrogel microcarriers;

[0044] The hydrogel microcarrier is dried and sterilized to obtain dry powder of the microcarrier.

[0045] Further, the concentration of the gel molecule solution and the exogenous matrix solution affects the size of the hydrogel microcarrier, and those skilled in the art can determine the specific value according to the experimental results, but preferably, the gel in the premix solution The concentration of the molecular solution should be kept above 10g / L to ensure the smooth progress of the gelation react...

Embodiment 1

[0048] refer to figure 1 , the preparation method of the microcarrier is as follows:

[0049] A, solution preparation: concentration 10-50g / L sodium alginate solution; concentration 1-10g / L chitosan solution;

[0050] B. Collagen solution, chitosan solution, matrigel solution, chondroitin sulfate solution or fibronectin solution with a concentration of 2-50g / L are prepared;

[0051] C. Mix the sodium alginate solution prepared in step A with calcium carbonate powder to form a uniform suspension, and then mix it with the collagen solution, chitosan solution, matrigel solution, chondroitin sulfate solution or fibronectin solution prepared in step B One or more of them are uniformly mixed, and the concentration of sodium alginate in the mixture is kept above 10g / L;

[0052] D. The mixed solution prepared in step C is formed into uniform droplets through fluid granulation technology, cross-linked by calcium ions, and combined with temperature changes, a gelation reaction occurs ...

Embodiment 2

[0057] 【1】Prepare sodium alginate solution with a concentration of 30g / L;

[0058] [2] Prepare a collagen solution with a concentration of 5g / L and adjust it to neutral with NaOH;

[0059] 【3】Prepare the gel bath solution, containing calcium chloride (concentration 20g / L) in the gel bath;

[0060] [4] Mix the sodium alginate solution prepared in step [1] with the collagen solution prepared in step [2] in a volume ratio of 2:1;

[0061] [5] The mixture of sodium alginate and collagen prepared in step [4] forms a stable jet under a high-voltage electrostatic field, sprays it into the gel bath prepared in step [3], and keeps the gel bath at 37°C After 30 minutes of gelation reaction, sodium alginate-collagen blended hydrogel microspheres were prepared;

[0062] [6] After the sodium alginate-collagen blended hydrogel microspheres prepared in step [5] are autoclaved, they are dried by a fluidized bed dryer into a microcarrier dry powder for later use.

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Abstract

The invention discloses a microcarrier which comprises gel molecules and outer matrix molecules, the gel molecules and the outer matrix molecules are mixed and then induced to perform gelation reaction, and the hydrogel microcarrier is generated, wherein the gel molecules are sodium alginate or calcium alginate, and the external matrix molecules are one or a combination of more than one of collagen, matrigel protein, chondroitin sulfate, fibronectin and chitosan. The main component of the microcarrier contains a calcium alginate composite material. The material has the greatest advantage that the material is easy to swell and deform in the presence of a calcium ion chelating agent, so that the contact area between the material and cells is reduced, the cells can fall off under slight physical shearing force, and the separation of the cells and the microcarrier is realized. The whole process does not need pancreatin, and cell separation can be realized.

Description

technical field [0001] The present application relates to the technical field of cell culture, in particular to a method for large-scale cell culture and the microcarrier used. Background technique [0002] With the development of stem cell technology, cell therapy has provided the possibility for the radical cure of many major diseases such as tumors, liver and kidney failure, and neuro-endocrine system diseases such as Parkinson's and diabetes. However, the introduction of various proteins and enzymes should be avoided in the preparation process of cell products for clinical implantation, which brings difficulties to the large-scale cultivation of cells. Especially for the large-scale culture of anchorage-dependent cells, trypsinization is still required to collect the cultured cells. The introduction of trypsin increases the subsequent purification process of cell products and increases the production cost of the product. Contents of the invention [0003] The purpose ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/00
CPCC12N5/0075C12N2500/12C12N2500/30C12N2531/00C12N2533/72
Inventor 于炜婷郑国爽高毅彭青李阳
Owner ZHUJIANG HOSPITAL SOUTHERN MEDICAL UNIV
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