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A method for three-dimensional microcarrier cell adsorption culture

A technology of cell adsorption and microcarriers, which is applied in general culture methods, tissue culture, biochemical equipment and methods, etc., can solve the problem of repair and regeneration function decline, and the two-dimensional microenvironment cannot simulate and reproduce the three-dimensional microenvironment well , stem cell survival rate and function reduction, to achieve the effect of promoting function, easy cultivation, and avoiding bacterial infection

Active Publication Date: 2022-02-11
BEIJING CYTONICHE BIOTECH CO LTD
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Problems solved by technology

Therefore, this simplified two-dimensional microenvironment cannot simulate and reproduce the three-dimensional microenvironment in the body well.
However, depending on the stem cells cultured in the traditional two-dimensional environment, during the stem cell transplantation treatment, due to the drastic changes in the living environment in vivo and in vitro, the survival rate and function of stem cells are reduced, and the repair and regeneration functions are reduced, which restricts the use of stem cell therapy. The effect of clinical application

Method used

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  • A method for three-dimensional microcarrier cell adsorption culture
  • A method for three-dimensional microcarrier cell adsorption culture
  • A method for three-dimensional microcarrier cell adsorption culture

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1, the method that adopts stirrer rotation adsorption method to carry out three-dimensional microcarrier cell adsorption

[0030] 1. Microcarrier preparation: Weigh 200 mg of microcarrier powder, carry out ultraviolet sterilization, and pour it into a sterile built-in impeller cell culture bottle (such as figure 1 shown); prepare three groups;

[0031] 2. Cell preparation: Prepare adipose-derived mesenchymal stem cell suspension in advance, 5×10 6 Cells were resuspended in 60mL complete medium for use; prepare three groups;

[0032] 3. Cell inoculation: mix the above cell suspension into the cell culture flask with built-in impeller, and mix with 200mg microcarrier;

[0033] 4. Cell adsorption: Place the built-in impeller cell culture flask on a low-speed stirrer and put it in a 37°C, 5% carbon dioxide incubator for 80rpm stirring, so that the cells adhere to the microcarrier by rotating;

[0034] 5. In situ counting to obtain the adsorption rate:

[0035...

Embodiment 2

[0038] Embodiment 2, the method for the three-dimensional microcarrier cell adsorption of small volume high density swelling adsorption method

[0039] 1. Microcarrier preparation: Weigh 20 mg of microcarrier powder for ultraviolet sterilization and set aside;

[0040] 2. Cell preparation: Prepare adipose-derived mesenchymal stem cell suspension in advance, with a density of 2.5×10 6 cells / mL, prepare 200 μL for every 20 mg of three-dimensional microcarriers;

[0041] 3. Cell inoculation: pipette 200 μL of cell suspension and add dropwise to 20 mg of microcarrier to fully mix the cell suspension and microcarrier;

[0042] 4. Cell adsorption: put the microcarrier of the mixed cell suspension into a 37°C, 5% carbon dioxide incubator and incubate for 2 hours to make the cells adhere to the microcarrier;

[0043] 5. Cell culture: After the cells adhere, add 3mL of complete medium, put it in a 37°C, 5% carbon dioxide incubator and culture it for 24 hours, then observe the cells; ...

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Abstract

The invention discloses a three-dimensional microcarrier cell adsorption culture method. It comprises the following steps: 1) inoculating cells: mixing the cell suspension with a dry three-dimensional microcarrier to obtain a microcarrier of the mixed cell suspension; 2) cell adsorption: mixing the mixed cell suspension obtained in step 1) The microcarriers are incubated by a rotational dynamic adsorption method, so that the cells adhere to the three-dimensional microcarriers; 3) Cell culture: after the cells are adhered in step 2), complete medium is added and cultured. The invention provides more growth area for cells, simplifies the operation of inoculating cells on microcarriers, and avoids the risk of bacterial contamination; it is suitable for the cultivation of large-scale bioreactors; the unique imbibition of three-dimensional microcarriers can be a large amount The cells enter the inside of the connected pores of the carrier, better form a bionic three-dimensional growth pattern, and promote the function of the in vitro cell culture process.

Description

technical field [0001] The invention relates to a three-dimensional microcarrier cell adsorption culture method, which belongs to the technical field of cell adsorption culture. Background technique [0002] With the rapid development of interdisciplinary subjects such as biomedicine, materials science, mechanics, and engineering, more and more people are paying attention to the difference between the microenvironment of cells cultured in vitro and the microenvironment in vivo. The traditional two-dimensional cell culture (based on commercial culture dishes or multi-well plates) technology has been developed for more than 100 years, and it has been widely used in basic life science research, medical research and other fields. However, with the rapid development of microscopic imaging technology, researchers have gradually discovered the living state of cells in a two-dimensional culture environment, and the cell morphology is far from the culture conditions in the real envir...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/00
CPCC12N5/0075
Inventor 鄢晓君刘伟
Owner BEIJING CYTONICHE BIOTECH CO LTD
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