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Preparation method of MDCK suspension cell

A technique of suspending cells and cells, which is applied in the field of cell culture, can solve the problems of difficult cell sampling and counting, small amplification ratio, and large site area, etc., and achieve the effect of fast cell recovery, excellent performance, and simple method

Inactive Publication Date: 2018-04-03
吉林冠界生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Disadvantages: limited by the small specific surface area of ​​square bottles and roller bottles, to achieve higher cell density, more attachment area must be provided, which takes up a lot of space; cell passage needs to be digested, and it is difficult to expand and cultivate cells; cell sampling and counting tougher
Disadvantages: There are also certain limitations, such as: cells are difficult to digest and pass; during the process of microcarrier suspension amplification, the amplification ratio is small; microcarriers are expensive; the process of recovering microcarriers is complicated

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] A preparation method of MDCK suspension cells, the steps are as follows:

[0044] Adherent cultured MDCK cells were introduced from ATCC by the Gansu Animal Cell Engineering Technology Research Center (GsACC) of Northwest University for Nationalities. The introduction time: February 2011, ATCC number: CCL-34, generation: P56, preservation number: 58860056 . A cell bank was established after GsACC expansion and culture, and the number of the cell bank is: GsACC2B0000090.

[0045] The MDCK cells were revived with DMEM / F12 medium containing 10% FBS, grown densely and then passaged.

[0046] Select well-grown cells and gradually domesticate and cultivate them, as follows:

[0047] Subcultured with DMEM / F12 medium containing 8% fetal bovine serum for 3 times;

[0048] Subcultured 5 times with DMEM / F12 medium containing 5% fetal bovine serum;

[0049] Subcultured 5 times with DMEM / F12 medium containing 2% fetal bovine serum;

[0050] DMEM / F12 medium and low-serum medium we...

Embodiment 2

[0063] A preparation method of MDCK suspension cells, the steps are as follows:

[0064] Adherent cultured MDCK cells were introduced from ATCC by the Gansu Animal Cell Engineering Technology Research Center (GsACC) of Northwest University for Nationalities. The introduction time: February 2011, ATCC number: CCL-34, generation: P56, preservation number: 58860056 . A cell bank was established after GsACC expansion and culture, and the number of the cell bank is: GsACC2B0000090.

[0065] The MDCK cells were revived with DMEM / F12 medium containing 10% FBS, grown densely and then passaged.

[0066] Select well-grown cells and gradually domesticate and cultivate them, as follows:

[0067] Subcultured with DMEM / F12 medium containing 8% fetal bovine serum for 3 times;

[0068] Subcultured 5 times with DMEM / F12 medium containing 5% fetal bovine serum;

[0069] Subcultured 5 times with DMEM / F12 medium containing 2% fetal bovine serum;

[0070] DMEM / F12 medium and low-serum medium ...

Embodiment 3

[0083] A preparation method of MDCK suspension cells, the steps are as follows:

[0084] Adherent cultured MDCK cells were introduced from ATCC by the Gansu Animal Cell Engineering Technology Research Center (GsACC) of Northwest University for Nationalities. The introduction time: February 2011, ATCC number: CCL-34, generation: P56, preservation number: 58860056 . A cell bank was established after GsACC expansion and culture, and the number of the cell bank is: GsACC2B0000090.

[0085] The MDCK cells were revived with DMEM / F12 medium containing 10% FBS, grown densely and then passaged.

[0086] Select well-grown cells and gradually domesticate and cultivate them, as follows:

[0087] Subcultured with DMEM / F12 medium containing 8% fetal bovine serum for 3 times;

[0088] Subcultured 5 times with DMEM / F12 medium containing 5% fetal bovine serum;

[0089] Subcultured 5 times with DMEM / F12 medium containing 2% fetal bovine serum;

[0090] DMEM / F12 medium and low-serum medium ...

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Abstract

The invention relates to the field of cell culture, and particularly discloses a preparation method of MDCK suspension cells, wherein the preparation method comprises the following steps: subculturingthe MDCK cells by sequentially using DMEM / F12 culture medium containing 8%, 5% and 2% of fetal calf serum; sequentially culturing the mixed culture liquid of the DMEM / F12 culture medium and low-serumculture medium according to the volume ratio of 1:1, 1:5 and 0:1; performing digestion and centrifugation; performing shaking culture on the obtained cells by using the low-serum medium at a rotate speed of 20-30r / min; gradually increasing the rotate speed of the shaking table until the cells completely losing the ability of adhering to the flask wall are obtained, and the cells are cell strainsobtained through low-serum suspension culture; collecting the cells, and gradually increasing the content of serum-free culture medium, after stable growth, the MDCK cells obtained through serum-freesuspension culture are obtained. The method, sought gradually through a large number of experiments, is simple and convenient and easy in factory production.

Description

technical field [0001] The invention relates to the field of cell culture, in particular to a method for preparing MDCK suspension cells. Background technique [0002] At present, the mature culture methods mainly include single-layer static adherent culture and adsorption culture. [0003] The monolayer static adherent culture belongs to the static culture process. Under this method, MDCK cells are statically attached to the surface of containers such as square bottles or roller bottles, and then the cells are expanded and grown until a monolayer is formed. Advantages: Since the cells are attached to the surface of the object, the separation of the cells and the medium can be achieved without centrifugation; the cells are easy to clean; and the cell morphology is easy to observe. Disadvantages: limited by the small specific surface area of ​​square bottles and roller bottles, to achieve higher cell density, more attachment area must be provided, which takes up a lot of sp...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/02A01N1/02
CPCA01N1/0221C12N5/0686
Inventor 李莉赵海源陈宏王玉红冯玉强蒋晓梅张天舒朱长动杨柳张丽娜杜鑫唐东雪赵丹刘金伟曾晓敏
Owner 吉林冠界生物技术有限公司
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