CN107267446A - A kind of processing method for the feeder cells that technology is reprogrammed applied to conditionity - Google Patents

A kind of processing method for the feeder cells that technology is reprogrammed applied to conditionity Download PDF

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CN107267446A
CN107267446A CN201710620966.9A CN201710620966A CN107267446A CN 107267446 A CN107267446 A CN 107267446A CN 201710620966 A CN201710620966 A CN 201710620966A CN 107267446 A CN107267446 A CN 107267446A
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feeder cells
conditionity
processing method
cell
reprogrammed
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任政华
尹顺义
伍活镰
杨晓红
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GUANGZHOU QIZHI BIOLOGY ENGINEERING EQUIPMENT Co Ltd
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GUANGZHOU QIZHI BIOLOGY ENGINEERING EQUIPMENT Co Ltd
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/02—Coculture with; Conditioned medium produced by embryonic cells

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Abstract

The invention belongs to biological technical field, and in particular to a kind of processing method for the feeder cells that technology is reprogrammed applied to conditionity, the treating method comprises following steps：S1：3T3 J2 feeder cells are taken, are seeded in Tissue Culture Flask, when addition Conditioned immunolresponse culture to cell fusion degree is 50~90%, culture medium are removed, cell is rinsed 2~3 times with PBS solution；S2：2~4 μ g/mL of addition mitomycin C, handles 1.5~3h, removes mitomycin C, and cell is rinsed 2~3 times with PBS solution, adds Conditioned immunolresponse, in culture in 37 DEG C, 5%CO2 incubators.The processing method that the present invention is provided is easy and effective, can both protect the biological characteristics of feeder cells, feeder cells can be excluded injury of the continuation effect of irradiation of ray to conditionity reprogrammed cell again with the secrete cytokines and nutriment of continuation.

Description

A kind of processing method for the feeder cells that technology is reprogrammed applied to conditionity
Technical field
The invention belongs to biological technical field, and in particular to a kind of feeder cells that technology is reprogrammed applied to conditionity Processing method.
Background technology
For a long time, in vitro cultured tissue cell and build before clinical pattern, be always that biomedicine field is important Research method.Cell model can not only help researcher to more fully understand the molecule mechanism of disease, producing cause, can be with Assist the exploitation of drug discovery and new treatment.But the cell of in vitro culture, it is impossible to long-term in its natural state to keep propagation Ability, after certain division number of times is reached, cell can stop division, taper off.The existing cell weight that can allow in vitro culture The new method for obtaining unlimited multiplication capacity, such as induced multi-potent stem cell (iPSC), utilizes viral oncogenes engineered host cell Genome, the cell cycle that change has p53 to regulate and control is that cell obtains unlimited multiplication capacity etc., it is necessary to change cell in itself Genotype and phenotype, so as to influence the feature of cell in itself, or even produce the side effect for being difficult to predict, and the effect of these methods Rate is relatively low, and technical difficulty is larger, it is difficult to wide popularization and application.
Conditionity reprogramming technology (Conditioanl Reprogramming, CR) is that only one can in the world at present So that the technology of infinite multiplication is carried out to cancer cell and normal cell simultaneously.CR is made carefully by using media environment " condition " Born of the same parents obtain unlimited multiplication capacity, with growth controllability, can stop cell growth and differentiation by changing media environment Only or continue.CR need not change the genotype and phenotype of cell in itself, and cell can be grown and nothing in its natural state Limit propagation, will not produce side effect for the development of cell and tissue.
Although the concrete principle that CR makes cell obtain unlimited multiplication capacity is not revealed completely now, existing research Show, addition Rho kinase inhibitors and feeder cells are CR two key conditions in the medium.By Rho kinase inhibitors Be added to feeder cells can be changed into cell with inducing cell can infinite multiplication stem cell-like cell；When condition removal, Cell can lose its stem cell-like properties, the cell type before can reverting to.This stem cell-like cell is referred to as conditionity Reprogrammed cell (CRCs).
The method that many reports use ray radiation treatment applied to CR feeder cells before at present, such as Li Zhangyan Etc. in the paper " the primary lung carcinoma cell longterm culture in vitro of people and the research of individuation susceptibility " delivered, feeder cells is pass through The 3T3 fibroblasts of 35gray X-ray radiations.But in actual applications, although using the method for ray radiation treatment can be with Suppress the differentiation and growth of feeder cells, but also influence the secretion of the nutritional ingredient of feeder cells, brought when applied to CR Radioactive ray continuous action in many unfavorable factors, such as prolonged Carbazole alkaloid and the secretion of prolonged nutritional ingredient, very The difficult objective and accurate conditionity reprogrammed cell for obtaining needs.
The content of the invention
It is an object of the invention to overcome existing technological deficiency, there is provided a kind of feeding applied to conditionity reprogramming technology Support the processing method of cell.The processing method that the present invention is provided is easy and effective, can both protect the biological characteristics of feeder cells, make Feeder cells can exclude the continuation effect of irradiation of ray to condition again with the secrete cytokines and nutriment of continuation The injury of property reprogrammed cell.
Technical scheme is as follows：
A kind of processing method for the feeder cells that technology is reprogrammed applied to conditionity, comprises the following steps：
S1：3T3-J2 feeder cells are taken, are seeded in Tissue Culture Flask, Conditioned immunolresponse culture are added to cell fusion Spend for 50~90% when, remove culture medium, cell 2~3 times rinsed with PBS solution；
S2：2~4 μ g/mL mitomycin C is added, 1.5~3h is handled, mitomycin c solution is removed, is rushed with PBS solution Wash cell 2~3 times, Conditioned immunolresponse is added, in 37 DEG C, 5%CO2Cultivated in incubator.
Preferably, in step S1, S2, the Conditioned immunolresponse is composed of the following components：F12 basal mediums 24.5~ 25.5% (v/v), hyclone 4.5~5% (v/v), the μ g/mL of hydrocortisone/EGF mixtures 28~32, amphotericin B 245~255 μ g/mL, the μ g/mL of botulin toxin fibronectin 4~6, ROCK inhibitor Y-276324~6nmol/L, surplus For DMEM basal mediums.
Preferably, the Conditioned immunolresponse is composed of the following components：F12 basal mediums 250mL/L, hydrogenation can The μ g/mL of pine/EGF mixtures 30, the μ g/mL of amphotericin B 250, the μ g/mL of botulin toxin fibronectin 5, ROCK inhibitor Y-276325nmol/L, surplus is DMEM basal mediums.
Wherein, the hydrocortisone/EGF mixtures, are counted, hydrocortisone in mass ratio：EGF is 5:1.The EGF (Epidermal Growth Factor) is epithelical cell growth factor, can promote the Proliferation, Differentiation of cell, so that with new life Cell replace aging and dead cell.
The Rho kinase inhibitors Y-27632 is a kind of related FZ formation serine-Soviet Union's ammonia of efficient Rho The small molecule specific inhibitor of pka acid (ROCK) family.
Preferably, in step S1, the inoculum density of the 3T3-J2 feeder cells is 1 × 106cells/mL。
Preferably, in step S1, the cell fusion degree is 70%.
Preferably, in step S2, the concentration of the mitomycin C is 4 μ g/mL.
Preferably, in step S2, the time of the mitomycin C processing is 2h.
Preferably, in step S2, the temperature of the mitomycin C processing is 37 DEG C.
Feeder cells after the processing method processing of the feeder cells provided through the present invention can be applied to conditionity reprogramming Tumour cell and its normal tissue cell and the stem cells such as technology culture breast cancer, lung cancer, oophoroma, the carcinoma of the rectum, colon cancer, obtain To corresponding conditionity reprogrammed cell (CRCs), but this is not limited only to, other cell culture for using feeder cells also can use The method processing feeder cells that the present invention is provided.
Compared with prior art, the beneficial effects of the invention are as follows：
(1) processing method that the present invention is provided is easy and effective, can both protect the biological characteristics of feeder cells, make raising thin Born of the same parents can be with the secrete cytokines and nutriment of continuation, and the continuation effect of irradiation that ray can be excluded again is rearranged to conditionity The injury of journey cell；
(2) the processing method cost that the present invention is provided is low, special irradiation apparatus and the special installation of offer without buying Environment, and mitomycin C is cheap；
(3) processing method that provides of the present invention coordinates the Conditioned immunolresponse that the present invention is provided to feeder cells and primary Cell is effectively cultivated, and conditionity reprogrammed cell culture success ratio is high, and effect is good；In addition, the processing side that the present invention is provided Method is simple to operate, enables to need the cell culture of feeder cells more rapidly, more convenient.
Brief description of the drawings
Fig. 1 is co-cultured through concentration for the 3T3-J2 feeder cells after 2 μ g/mL mitomycin C processing with lung carcinoma cell The lung cancer cell conditionity reprogrammed cell arrived.
Fig. 2 is co-cultured through concentration for the 3T3-J2 feeder cells after 3 μ g/mL mitomycin C processing with lung carcinoma cell The lung cancer cell conditionity reprogrammed cell arrived.
Fig. 3 is co-cultured through concentration for the 3T3-J2 feeder cells after 4 μ g/mL mitomycin C processing with lung carcinoma cell The lung cancer cell conditionity reprogrammed cell arrived.
Embodiment
The present invention is further described below by way of embodiment, but the present invention is not limited only to following examples. In the scope of the present invention or not departing from present disclosure, in spirit and scope, to pharmaceutical composition of the present invention Be suitably modified, replace effect identical component, it will become apparent to those skilled in the art that they all by regarding To be included within the scope of the present invention.
The present invention is raw materials used, is conventional commercial raw material unless otherwise specified.
Embodiment 1
The preparation of Conditioned immunolresponse：
Under sterile conditions, F12 basal mediums, DMEM basal mediums, hydrocortisone/EGF mixtures, two are taken Property mycin B, botulin toxin fibronectin and ROCK inhibitor Y-27632, mix, produce bar described in the embodiment of the present invention Part culture medium；
Conditioned immunolresponse each component consumption is as follows：F12 basal mediums 25% (v/v), hyclone 5% (v/v), hydrogen Change the μ g/mL of cortisone/EGF mixtures 30, the μ g/mL of amphotericin B 250, botulin toxin fibronectin 5 μ g/mL, ROCK Inhibitor Y-276325nmol/L, surplus is DMEM basal mediums；Wherein, hydrocortisone/EGF mixtures, in mass ratio Meter, hydrocortisone：EGF is 5:1.
Embodiment 2
(1) processing of 3T3-J2 feeder cells
S1：3T3-J2 feeder cells are taken, by inoculum density 1 × 106Cells/mL is seeded in Tissue Culture Flask, adds bar When part medium culture to cell fusion degree is 50%, culture medium is removed, cell is rinsed 3 times with PBS solution；
S2：2 μ g/mL mitomycin C is added, 3h is handled under conditions of temperature is 37 DEG C, removes mitomycin C molten Liquid, cell is rinsed 3 times with PBS solution, Conditioned immunolresponse is added, in 37 DEG C, 5%CO2Cultivated in incubator.
(2) lung cancer cell is obtained
The cancerous lung tissue of surgery excision is taken, 2-3mm tissue block is shredded into, blood stains and other impurities are washed off, added 1% clostridiopetidase A is put 4 DEG C of digestion and stayed overnight, and adds Conditioned immunolresponse, and tissue block is blown and beaten repeatedly and obtains lung cancer single cell suspension, warp Filtering, obtains lung cancer cell suspension.
(3) lung cancer cell CRCs is cultivated
Take lung cancer cell suspension to be added in the blake bottle for the 3T3-J2 feeder cells handled well, add conditionity training Support after base, in 37 DEG C, 5%CO2Cultivated in incubator.
Cultivation results are as shown in figure 1, the lung cancer cell CRCs iuntercellulars that culture is obtained arrange close, and cellular morphology is complete Whole, majority is in irregular shape, and minority is in oval, consistent with the cellular morphology of lung cancer cell.
Embodiment 3
(1) processing of 3T3-J2 feeder cells
S1：3T3-J2 feeder cells are taken, by inoculum density 1 × 106Cells/mL is seeded in Tissue Culture Flask, adds bar When part medium culture to cell fusion degree is 70%, culture medium is removed, cell is rinsed 3 times with PBS solution；
S2：3 μ g/mL mitomycin C is added, 2h is handled under conditions of temperature is 37 DEG C, removes mitomycin C molten Liquid, cell is rinsed 3 times with PBS solution, Conditioned immunolresponse is added, in 37 DEG C, 5%CO2Cultivated in incubator.
(2) lung cancer cell is obtained
The cancerous lung tissue of surgery excision is taken, 2-3mm tissue block is shredded into, blood stains and other impurities are washed off, added 1% clostridiopetidase A is put 4 DEG C of digestion and stayed overnight, and adds Conditioned immunolresponse, and tissue block is blown and beaten repeatedly and obtains lung cancer single cell suspension, warp Filtering, obtains lung cancer cell suspension.
(3) lung cancer cell CRCs is cultivated
Take lung cancer cell suspension to be added in the blake bottle for the 3T3-J2 feeder cells handled well, add conditionity training Support after base, in 37 DEG C, 5%CO2Cultivated in incubator.
Cultivation results are as shown in Fig. 2 the lung cancer cell CRCs iuntercellulars that culture is obtained arrange close, and cellular morphology is complete Whole, majority is in irregular shape, and minority is in oval, consistent with the cellular morphology of lung cancer cell.
Embodiment 4
(1) processing of 3T3-J2 feeder cells
S1：3T3-J2 feeder cells are taken, by inoculum density 1 × 106Cells/mL is seeded in Tissue Culture Flask, adds bar When part medium culture to cell fusion degree is 70%, culture medium is removed, cell is rinsed 3 times with PBS solution；
S2：4 μ g/mL mitomycin C is added, 2h is handled under conditions of temperature is 37 DEG C, removes mitomycin C molten Liquid, cell is rinsed 3 times with PBS solution, Conditioned immunolresponse is added, in 37 DEG C, 5%CO2Cultivated in incubator.
(2) lung cancer cell is obtained
The cancerous lung tissue of surgery excision is taken, 2-3mm tissue block is shredded into, blood stains and other impurities are washed off, added 1% clostridiopetidase A is put 4 DEG C of digestion and stayed overnight, and adds Conditioned immunolresponse, and tissue block is blown and beaten repeatedly and obtains lung cancer single cell suspension, warp Filtering, obtains lung cancer cell suspension.
(3) lung cancer cell CRCs is cultivated
Take lung cancer cell suspension to be added in the blake bottle for the 3T3-J2 feeder cells handled well, add conditionity training Support after base, in 37 DEG C, 5%CO2Cultivated in incubator.
Cultivation results are as shown in figure 3, the lung cancer cell CRCs iuntercellulars that culture is obtained arrange close, and cellular morphology is complete Whole, majority is in irregular shape, and minority is in oval, consistent with the cellular morphology of lung cancer cell.
Above content is to combine specific preferred embodiment further description made for the present invention, it is impossible to assert The specific implementation of the present invention is confined to these explanations.For general technical staff of the technical field of the invention, On the premise of not departing from present inventive concept, some simple deduction or replace can also be made, should all be considered as belonging to the present invention's Protection domain.

Claims (9)

1. a kind of processing method for the feeder cells that technology is reprogrammed applied to conditionity, it is characterised in that the processing method Comprise the following steps：
S1：3T3-J2 feeder cells are taken, are seeded in Tissue Culture Flask, adding Conditioned immunolresponse culture to cell fusion degree is When 50~90%, culture medium is removed, cell is rinsed 2~3 times with PBS solution；
S2：2~4 μ g/mL mitomycin C is added, 1.5~3h is handled, mitomycin c solution is removed, rinses thin with PBS solution Born of the same parents 2~3 times, add Conditioned immunolresponse, in 37 DEG C, 5%CO2Cultivated in incubator.
2. the processing method of the feeder cells according to claim 1 that technology is reprogrammed applied to conditionity, its feature exists In in step S1, S2, the Conditioned immunolresponse is composed of the following components：F12 basal mediums 24.5~25.5% (v/v), Hyclone 4.5~5% (v/v), hydrocortisone/μ g/mL of EGF mixtures 28~32, the μ g/ of amphotericin B 245~255 ML, the μ g/mL of botulin toxin fibronectin 4~6, ROCK inhibitor Y-276324~6nmol/L, surplus are the training of DMEM bases Support base.
3. the processing method of the feeder cells according to claim 2 that technology is reprogrammed applied to conditionity, its feature exists In the Conditioned immunolresponse is composed of the following components：F12 basal mediums 250mL/L, hydrocortisone/EGF mixtures 30 μ g/mL, the μ g/mL of amphotericin B 250, μ g/mL, Rho kinase inhibitors Y- of botulin toxin fibronectin 5 276325nmol/L, surplus is DMEM basal mediums.
4. it is applied to the processing method that conditionity reprograms the feeder cells of technology, its feature according to Claims 2 or 3 It is that the hydrocortisone/EGF mixtures is counted, hydrocortisone in mass ratio：EGF is 5:1.
5. the processing method of the feeder cells according to claim 1 that technology is reprogrammed applied to conditionity, its feature exists In in step S1, the inoculum density of the 3T3-J2 feeder cells is 1 × 106cells/mL。
6. the processing method of the feeder cells according to claim 1 that technology is reprogrammed applied to conditionity, its feature exists In in step S1, the cell fusion degree is 70%.
7. the processing method of the feeder cells according to claim 1 that technology is reprogrammed applied to conditionity, its feature exists In in step S2, the concentration of the mitomycin C is 4 μ g/mL.
8. the processing method of the feeder cells according to claim 1 that technology is reprogrammed applied to conditionity, its feature exists In in step S2, the time of the mitomycin C processing is 2h.
9. the processing method of the feeder cells according to claim 1 that technology is reprogrammed applied to conditionity, its feature exists In in step S2, the temperature of the mitomycin C processing is 37 DEG C.
CN201710620966.9A 2017-07-27 2017-07-27 A kind of processing method for the feeder cells that technology is reprogrammed applied to conditionity Pending CN107267446A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115029316A (en) * 2022-06-20 2022-09-09 华中科技大学同济医学院附属协和医院 Primary cervical cancer cell line with radiotherapy sensitivity and radiotherapy tolerance characteristics and construction method and application thereof

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Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2012065067A2 (en) * 2010-11-12 2012-05-18 Georgetown University Immortalization of epithelial cells and methods of use

Non-Patent Citations (3)

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Title
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ROBERT E. HYNDS ET AL.: "Expansion of Human Airway Basal Stem Cells and Their Differentiation as 3D Tracheospheres", 《METHODS IN MOLECULAR BIOLOGY》 *
SARA LLAMES ET AL.: "Feeder Layer Cell Actions and Applications", 《TISSUE ENGINEERING: PART B》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115029316A (en) * 2022-06-20 2022-09-09 华中科技大学同济医学院附属协和医院 Primary cervical cancer cell line with radiotherapy sensitivity and radiotherapy tolerance characteristics and construction method and application thereof
CN115029316B (en) * 2022-06-20 2024-04-30 华中科技大学同济医学院附属协和医院 Primary cervical cancer cell line with radiotherapy sensitivity and radiotherapy tolerance characteristics, and construction method and application thereof

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