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Method for dissolving microcarrier agglomerate state in microcarrier cell culture system

A cell culture and microcarrier technology, applied in the field of cell culture, can solve the problems of not being able to use the ball to transfer the ball, damage, and enter the aggregation state, etc., to achieve the effect of reducing the aggregation of microcarriers, improving the quality of cells, and prolonging the culture time

Active Publication Date: 2021-11-12
BEIJING CYTONICHE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, trypsin, which is commonly used for cell subculture, can damage the 3D TableTrix series microcarriers. The addition of trypsin will cause the microcarrier structure to be gradually destroyed and finally disintegrated, so it cannot be used for ball transfer.
However, the ball-to-ball technique without digestion will make the microcarriers stick together with cell proliferation and gradually enter a state of aggregation.
Aggregation of microcarriers will block the cells in the center of the aggregate from obtaining sufficient nutrients, thereby affecting the quality and quantity of the cells, and once the cells are aggregated, the aggregate cannot be opened by simply adjusting the cell seeding density and the stirring speed of the reactor, which will affect the subsequent continuous passage

Method used

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  • Method for dissolving microcarrier agglomerate state in microcarrier cell culture system
  • Method for dissolving microcarrier agglomerate state in microcarrier cell culture system
  • Method for dissolving microcarrier agglomerate state in microcarrier cell culture system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1, physical method can't dissolve microcarrier aggregate

[0044] 1. Cell 3D culture

[0045] Set up two treatment groups: blank and treatment. Repeat each set three times.

[0046] The umbilical cord mesenchymal stem cells were resuspended in the medium to obtain a cell suspension, and the cell concentration in the cell suspension was 2.5×10 6pieces / ml. The cell suspension was inoculated into a bioreactor equipped with microcarriers and 50ml of medium (the ratio of microcarriers to medium was 20mg / 15mL), and the ratio of cell suspension to microcarriers was 200μL / 20mg. The inoculated bioreactor was placed in a 37° C. carbon dioxide incubator for cultivation, and the stirring program of the reactor was 35 rpm / min. On the second day of inoculation, 25ml of medium was supplemented.

[0047] 2. Observation of microcarrier aggregation

[0048] Take pictures to observe the aggregation of the microcarriers on day 0-4 of the cultured cells in step 1 above. Fo...

Embodiment 2

[0053] Example 2, using cell digestion solution to depolymerize microcarrier aggregates and passage

[0054] 1. Cell 3D culture

[0055] Step 1 of Example 1 was used to culture cells, and four treatment groups were set up, namely control group, experimental group 1, experimental group 2 and experimental group 3. Repeat each set three times.

[0056] 2. Dissolution of carrier aggregation and passage

[0057] Take the experimental group and the control group cultured for 4 days in the above step 1 to carry out the following experiments.

[0058] The experimental group was processed as follows:

[0059] 1) Stop the stirring procedure of the bioreactor during the cultivation.

[0060] 2) After 20 min, remove 55 ml of the medium supernatant in the culture system.

[0061] 3) Use a microscope to take pictures to observe the saturation and aggregation of the microcarriers in the above treatment groups. Observe the cell saturation, and treat with digestive enzymes after the cell...

Embodiment 3

[0077] Embodiment 3, adopt trypsin to depolymerize microcarrier aggregation

[0078] Cells were cultured according to Step 1 of Example 1, and two treatment groups were set up, a control group and a trypsin group. Repeat each set three times. Take 10 mg of the culture system of the control group and the trypsin group cultured for 4 days to carry out the following experiments respectively.

[0079] Control group: Add 1ml of microcarrier lysate to the culture system of the control group, mix well, and let stand for 10 minutes.

[0080] Trypsin group: Add 1ml of trypsin digestion solution to the culture system of trypsin group, mix well, and let stand for 10 minutes.

[0081] The aggregation of microcarriers in the control group and the trypsin group was observed under a microscope. The cell viability was detected by live-dead fluorescent staining count, and the detection method was the same as step 2 of Example 2.

[0082] Microcarrier aggregation see Figure 8 , wherein, 1...

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Abstract

The invention discloses a method for dissolving a microcarrier agglomerate state in a microcarrier cell culture system. The method comprises the following steps: adding a cell digestion solution into a microcarrier cell culture system in a microcarrier agglomerate state, performing an enzymolysis reaction, adding a culture medium and a microcarrier after the reaction is finished, and removing the agglomerate state of the microcarrier, wherein the cell digestion solution is TrypLE<TM> Express or Trypzyme® recombinant trypsin. The method dissolves the microcarrier agglomeration, the microcarrier agglomeration phenomenon after cell passage is obviously reduced, and the cell growth speed and the cell activity are not obviously changed.

Description

technical field [0001] The invention relates to the field of cell culture, in particular to a method for dissolving the aggregation state of microcarriers in a microcarrier cell culture system. Background technique [0002] The large-scale industrial culture technology of animal cells is very important for biological industries such as biopharmaceuticals, vaccine production, and cell therapy. Traditional two-dimensional cell culture methods are difficult to meet the needs of industrialized cell production. Microcarrier cell culture technology makes the process of large-scale industrial production of animal cells possible. During the entire large-scale culture process, the transfer of cell spheroids is the key to scale-up culture. According to market demand, Beijing Huakan Biotechnology Co., Ltd. has developed a degradable three-dimensional microcarrier aggregate (CN201910079680.3, trade name 3D TableTrix microcarrier). At present, trypsin, which is commonly used for cell ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0668C12N2513/00C12N2531/00
Inventor 鄢晓君刘伟张元元
Owner BEIJING CYTONICHE BIOTECH CO LTD
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