Subcloned cell strain TF-1-A2, preparation method thereof and application

A technology of TF-1-A2 and TF-1, which is applied in the field of determination of the biological activity of TF-1 subclone stable cell line TF-1-A2 and NGF products, which can solve the problem of difficult to ensure the repeatability and stability of the results , Large deviation of results, low signal-to-noise ratio, etc., to achieve the effect of easy statistical analysis of specific activity, convenient operation, and high signal-to-noise ratio

Active Publication Date: 2012-09-12
北京福睿君安科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the application, we found that due to the strong effect of GM-CSF on the growth stimulation of GM-CSF-dependent TF-1 cells, when using GM-CSF-dependent TF-1 cells to measure the biological activity of NGF, it is necessary to repeatedly Adjust the concentration of GM-CSF, only in this way can NGF promote the growth of TF-1 cells at an appropriate GM-CSF concentration, but there are problems such as complicated operation, low signal-to-noise ratio and large deviation of results
More importantly, it is often difficult to guarantee the repeatability and stability of the results due to the strong activity of GM-CSF that conceals the effect of promoting the growth of NGF

Method used

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  • Subcloned cell strain TF-1-A2, preparation method thereof and application
  • Subcloned cell strain TF-1-A2, preparation method thereof and application
  • Subcloned cell strain TF-1-A2, preparation method thereof and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Domestication of GM-CSF-dependent TF-1 cells

[0040] 1. Routine culture of GM-CSF-dependent TF-1 cells

[0041] GM-CSF-dependent TF-1 cells were purchased from the Basic Medical Cell Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, maintained in RPMI 1640 complete medium containing 10% fetal bovine serum, and added rhGM-CSF at a final concentration of 8ng / ml nourish. The cell concentration was maintained at 3 × 10 4 -5×10 5 cells / ml, at 37°C, 5% CO 2 Under the conditions of cultivation, the medium was changed once every 2-3 days.

[0042] 2. GM-CSF-dependent domestication of TF-1 cells

[0043] In the presence of a certain concentration of NGF, the GM-CSF-dependent TF-1 cells were domesticated into NGF-dependent TF-1 cells by gradually reducing the concentration of GM-CSF by serial dilution and serial passage. The specific domestication method is as follows: with the RPMI 1640 complete medium containing 10% fetal ...

Embodiment 2

[0055] Example 2: Cloning of NGF-dependent TF-1 cells

[0056] Use methylcellulose semi-solid medium to clone the domesticated mNGF-dependent TF-1 cells, the specific method is as follows:

[0057] 1) The methylcellulose semi-solid medium used is METHOCULT TM H4100 (Stem cell company, Catalog #04100) methylcellulose concentrate (2.6% concentration, stored at -20°C for future use), fully melted at 4°C before use. Add RPMI 1640 complete culture solution containing 10% fetal bovine serum and mNGF and mix it thoroughly for 2.5-fold dilution, so that the final concentration of mNGF is 25ng / ml, and the final concentration of methylcellulose is 1.0%;

[0058] 2) Take mNGF-dependent TF-1 cells in the logarithmic growth phase, collect the cells by centrifugation at 1000 rpm for 10 min, resuspend in RPMI 1640 complete culture medium containing 10% fetal bovine serum, and adjust the cell concentration to 2×10 4 cells / ml of cell suspension;

[0059]3) Take 1ml of diluted cell suspensi...

Embodiment 3

[0063] Example 3: Screening of NGF-dependent TF-1 monoclonal strains

[0064] According to the method described in Example 1, the dose-effect-response curves of the 15 monoclonal cell strains obtained by the cloning of the methylcellulose semi-solid medium were plotted respectively, and the maximum proliferation effect (Maximal effect, Emax) of the cells was calculated. The interval and signal-to-noise ratio are shown in Table 2. According to the data in Table 2 and the dose-effect-response curve, select a cell line with a good curve, carry out expanded culture and cryopreserve to establish a cell bank.

[0065] Screening results of table 215 TF-1 monoclonal cell lines

[0066]

[0067] The formula for calculating the signal-to-noise ratio is:

[0068] Signal-to-noise ratio (S / N value) = NGF maximum effect OD value / negative control OD value;

[0069] The formula for calculating the maximum effect interval of a cell is:

[0070] Emax interval = NGF maximum effect OD valu...

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Abstract

The invention relates to 'a subcloned cell strain TF-1-A2, a preparation method thereof and application' and belongs to the technical field of biology. The preparation method of the stable subcloned cell strain TF-1-A2 includes the steps: domesticating GM-CSF (granulocyte-macrophage colony-stimulating factor) dependent TF-1 cells into NGF (nerve growth factor) dependent strains; cloning the induced NGF dependent TF-1 cells by the aid of methylcellulose semisolid media; picking monoclonal cells from the semisolid media and enlarging and cultivating the monoclonal cells; and preparing a dose-effect curve of each monoclonal cell strain to the NGF and performing screening to obtain the subcloned cell strain TF-1-A2. The subcloned cell strain TF-1-A2 obtained by the preparation method is highly sensitive to the NGF and has fine reactivity and continuous inheritance stability. A TF-1 cell growth method established by the aid of the subcloned cell strain TF-1-A2 is used for externally and quantitatively measuring biological activity of the nerve growth factor, and is high in signal-to-noise ratio, fine in repeatability and superior to an existing method.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a TF-1 subclone stable cell line TF-1-A2 for quantitatively detecting the biological activity of nerve growth factor, its preparation method and its use in the determination of the biological activity of NGF products the use of. Background technique [0002] With the development of genetic engineering technology, a large number of genetically engineered drugs continue to enter clinical and production. Since genetically engineered drugs are different from general chemicals, most of them are peptides and proteins, derived from living organisms, with complex molecular structures, and are easily affected by various physical and chemical conditions during the production process. Therefore, for Such products are subject to comprehensive and strict quality control in all aspects. For cytokine biological products, since their chemical nature is a polypeptide or protein, their activity is d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/078C12Q1/02
Inventor 刘荷中乐伟霍立红史权威李曼
Owner 北京福睿君安科技有限公司
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