Feeder cell processing method applied to conditional reprogramming technology

A feeder cell and treatment method technology, applied in the feeder cell treatment field, can solve the problems of difficult cells, affect the secretion of nutrients of feeder cells, etc., and achieve the effects of good effect, protection of biological characteristics, and simple and effective treatment method.

Inactive Publication Date: 2017-10-20
GUANGZHOU QIZHI BIOLOGICAL ENG EQUIP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in practical applications, although the method of radiation treatment can inhibit the differentiation and growth of feeder cells, it also affects the secretion of nutrients in feeder cells, which brings many unfavorable factors when applied to CR, such as long-term cell inhibition. It is difficult to obtain the required conditional reprogramming cells objectively and accurately

Method used

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  • Feeder cell processing method applied to conditional reprogramming technology
  • Feeder cell processing method applied to conditional reprogramming technology
  • Feeder cell processing method applied to conditional reprogramming technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Preparation of conditioned medium:

[0032] Under sterile conditions, take F12 basal medium, DMEM basal medium, hydrocortisone / EGF mixture, amphotericin B, botulinum toxin fibronectin and ROCK inhibitor Y-27632, and mix well, namely Obtain the conditioned medium described in the embodiments of the present invention;

[0033] The dosage of each component of the conditioned medium is as follows: F12 basal medium 25% (v / v), fetal bovine serum 5% (v / v), hydrocortisone / EGF mixture 30 μg / mL, amphotericin B 250 μg / mL mL, botulinum toxin fibronectin 5 μg / mL, ROCK inhibitor Y-276325nmol / L, and the balance is DMEM basal medium; wherein, hydrocortisone / EGF mixture, by mass ratio, hydrocortisone: EGF is 5:1.

Embodiment 2

[0035] (1) Treatment of 3T3-J2 feeder cells

[0036] S1: Take 3T3-J2 feeder cells and inoculate at a density of 1×10 6 Inoculate cells / mL into cell culture flasks, add conditioned medium to culture until the cell confluence reaches 50%, remove the medium, and wash the cells with PBS solution for 3 times;

[0037] S2: Add 2 μg / mL mitomycin C, treat at 37°C for 3 hours, remove the mitomycin C solution, wash the cells with PBS solution 3 times, add conditioned medium, at 37°C, 5%CO 2 cultured in an incubator.

[0038] (2) Acquisition of primary lung cancer cells

[0039] Take the surgically resected lung cancer tissue, cut it into 2-3mm tissue pieces, rinse to remove blood and other impurities, add 1% collagenase to digest overnight at 4°C, add conditioned medium, repeatedly blow and beat the tissue piece to obtain lung cancer single cells The suspension was filtered to obtain the lung cancer primary cell suspension.

[0040] (3) CRCs culture of lung cancer primary cells

...

Embodiment 3

[0044] (1) Treatment of 3T3-J2 feeder cells

[0045] S1: Take 3T3-J2 feeder cells and inoculate at a density of 1×10 6 Inoculate cells / mL into cell culture flasks, add conditioned medium to culture until the cell confluence reaches 70%, remove the medium, and wash the cells with PBS solution for 3 times;

[0046] S2: Add 3 μg / mL mitomycin C, treat at 37°C for 2 hours, remove the mitomycin C solution, wash the cells with PBS solution 3 times, add conditioned medium, at 37°C, 5%CO 2 cultured in an incubator.

[0047] (2) Acquisition of primary lung cancer cells

[0048] Take the surgically resected lung cancer tissue, cut it into 2-3mm tissue pieces, rinse to remove blood and other impurities, add 1% collagenase to digest overnight at 4°C, add conditioned medium, repeatedly blow and beat the tissue piece to obtain lung cancer single cells The suspension was filtered to obtain the lung cancer primary cell suspension.

[0049] (3) CRCs culture of lung cancer primary cells

...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a feeder cell processing method applied to conditional reprogramming technology, the feeder cell processing method comprises the following steps: S1, taking 3T3-J2 feeder cells, inoculating the 3T3-J2 feeder cells into a cell culture flask, adding a conditioned culture medium for culture until cell fusion degree is 50-90%, removing the culture medium, using a PBS solution for washing the cells for 2 to 3 times; and S2, adding 2-4 mu g / mL of mitomycin C for processing for 1.5-3h, removing the mitomycin C, using the PBS solution for washing the cells for 2 to 3 times, adding the conditioned culture medium for culture at 37 DEG C in a 5% CO2 incubator. The processing method provided by the invention is simple and effective, and can protect the biological characteristics of the feeder cells, makes secretion of cytokines and nutrients of the feeder cells sustainable, and can eliminate the damage to conditionally reprogrammed cells caused by the continuous radiation of rays.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for treating feeder cells applied to conditional reprogramming technology. Background technique [0002] For a long time, culturing tissue cells in vitro and constructing preclinical models have been important research methods in the field of biomedicine. Cell models not only help researchers better understand the molecular mechanisms of disease and its causes, but also assist in drug discovery and the development of new treatments. However, cells cultured in vitro cannot maintain their proliferative ability for a long time in the natural state. After reaching a certain number of divisions, the cells will stop dividing and gradually decline. There are existing methods that allow cells cultured in vitro to regain unlimited proliferation, such as induced pluripotent stem cells (iPSC), using viral oncogenes to modify the genome of host cells, changing the cell cycl...

Claims

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Application Information

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IPC IPC(8): C12N5/073C12N5/09
CPCC12N5/0693C12N2501/11C12N2501/39C12N2501/727C12N2501/998C12N2501/999C12N2502/02
Inventor 任政华尹顺义伍活镰杨晓红
Owner GUANGZHOU QIZHI BIOLOGICAL ENG EQUIP
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