CN103756904A - Three-dimensional culture system for tumor cell culture - Google Patents

Three-dimensional culture system for tumor cell culture Download PDF

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CN103756904A
CN103756904A CN201410012583.XA CN201410012583A CN103756904A CN 103756904 A CN103756904 A CN 103756904A CN 201410012583 A CN201410012583 A CN 201410012583A CN 103756904 A CN103756904 A CN 103756904A
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culture system
tumor
gel
dimensional culture
dimensional
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赵海涛
李光兵
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/12—Well or multiwell plates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/20—Material Coatings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2513/00—3D culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin

Abstract

The invention relates to a three-dimensional culture system for tumor cell culture. Furthermore, the invention relates to a three-dimensional culture system containing gel serving as a tumor tissue growth scaffold, a culture medium, a coagulant and a tumor tissue.

Description

For the dimensional culture system of tumor cell culture
Technical field
The present invention relates to the dimensional culture system for tumor cell culture.Further, the present invention relates to the screening of described dimensional culture system for tumor chemotherapeutic drug.
Background technology
The first-selected excision primary lesion for the treatment of malignant tumor scheme at present, but some malignant tumor patient when medical in advanced tumor, cannot row operative treatment, and after excision primary tumor, tumor recurrence has all affected oncotherapy effect, has reduced malignant tumor patient lifetime.Chemotherapeutic treatment is to improve oncotherapy effect, improves patient's prognosis and extends one of important means of survival of patients phase.The chemotherapeutic treatment of malignant tumour is mainly to take whole body treatment with chemotherapy medicine at present, before treatment with chemotherapy medicine, by vitro culture tumor tissues primary cell, thereby filters out susceptibility chemotherapeutics, instructs clinical formulation chemotherapy regimen.
Cell in vitro culture technique can not perfect simulation tumor tissues internal milieu, and destroyed the microenvironment of tumor tissues, institute's treatment with chemotherapy medicine is only for tumor epithelial cell cell, and can not act on mesenchyma stroma of tumors cell simultaneously.In cell cultures, cell is grown at two dimensional surface, and is to grow in three dimension system at the tumour cell of body, and existing cell culture technology can not be simulated changing at bulk-growth environment and biodynamics of tumor tissues completely.
In order to overcome above-mentioned defect of the prior art, the invention provides a kind of dimensional culture system for culture of tumor cell, utilize the gel that contains substratum to construct the support of tumor tissue growth.Tumor tissue is fixed in gel stent, tumor tissue self formed with body on all four tumor tissue growth microenvironment.And gel stent is simulated and the condition of tumor tissues at bulk-growth is provided.In dimensional culture system, tumor cell proliferation can moving, has got rid of the contact inhibition effect in two-dimentional culture systems, the growth of tumour cell in this system, complete reaction the reaction of tumour cell to intervening measure.
According to dimensional culture system provided by the invention and prior art shape ratio, have the following advantages:
1, dimensional culture system is established as cell and tissue culture a kind of culture systems of 3 D stereo is provided, more meet the biological behaviour of Growth of Cells, can promote the gathering of cell, maintain histiocytic spheroid-like, and support the normal growth of tumour cell.
2, this technology is utilized three-dimensional histoculture technology, intact simulation live body environment.Compared with conventional cell culture technique, three-dimensional histoculture technology, can simulate the microenvironment of tumor tissues, and the more perfect tumor tissues that presents is to chemotherapy drug susceptibility.
3, native system gathers the tumor specimen of excision, and for each individuality, the susceptibility of every kind of tumour is screened.Accomplish individuation, at utmost increased chemosensitivity, alleviated toxic and side.
4, native system can be used in stem cell cultivation and breaks up acute and chronic toxicity test; The screening of medicine prediction curative effect; Vasculogenesis test etc.
Summary of the invention
The invention provides a kind of dimensional culture system for tumor cell culture, it is characterized in that, described dimensional culture system contains the gel as tumor tissue growth's support.
Further, according to the dimensional culture system for tumor cell culture of the present invention, it is characterized in that, described dimensional culture system also contains substratum, setting accelerator.
Further, according to the dimensional culture system for tumor cell culture of the present invention, it is characterized in that, described dimensional culture system further contains tumor tissues.
Further, according to the dimensional culture system for tumor cell culture of the present invention, it is characterized in that, described tumor tissues is fixed in gel stent.
Alternatively, according to the dimensional culture system for tumor cell culture of the present invention, it is characterized in that, described tumor tissues is liver cancer tissue.
On the other hand, the invention provides the method for a kind of foundation for the dimensional culture system of tumor cell culture, it comprises step:
A) mixture of obtaining liq gel and setting accelerator;
B) gel mixture configuring is added in culture plate, wait for that liquid gel forms translucent;
C) tumor tissue is positioned in translucent gel, is placed in center, hole, standing, treat that tissue culture gel formation is opaque gelatin.
On the other hand, the invention provides the purposes of the dimensional culture system for tumor cell culture according to the present invention in chemotherapeutics screening.
Accompanying drawing explanation
Fig. 1: liver cancer tissue dimensional culture, Microscopic observation different treatment group, liver cancer cell growth and cell migration situation, cell migration is apart from having reacted liver cancer cell to chemotherapy drug susceptibility.Chemotherapy medicament sensitive, cell migration distance is short.A is control group, and B-D is different chemotherapeutics treatment group.
Fig. 2: utilize dimensional culture system, filter out liver cancer tissue susceptibility chemotherapeutics.
Should understand, appended accompanying drawing has not shown the technique of painting of slightly simplifying of the exemplifying various features of ultimate principle of the present invention pari passu.Specific design feature of the present invention disclosed herein for example comprises that concrete size, direction, position and profile will partly will be applied and the environment of use is determined by concrete.
In these figures, run through several figures of accompanying drawing, Reference numeral is quoted part equally or that be equal to of the present invention.
Embodiment
Dimensional culture system provided by the invention is to utilize the gel that contains substratum to construct the support of tumor tissue growth.Tumor tissue is fixed in gel stent, tumor tissue self formed with body on all four tumor tissue growth microenvironment.And gel stent is simulated and the condition of tumor tissues at bulk-growth is provided.In dimensional culture system, tumor cell proliferation can moving, has got rid of the contact inhibition effect in two-dimentional culture systems, the growth of tumour cell in this system, complete reaction the reaction (Fig. 1) of tumour cell to intervening measure.
The foundation of embodiment 1, dimensional culture system
First prepare tissue culture gel: containing the setting accelerator (Adittive1) of Urogastron, add 12mL tissue culture with in matrigel 1.5mL, described matrigel is adhered albumen, collagen iv containing layer, fibroblast growth factor, tissue fibers proenzyme activation factor, slowly add, limit edged fully mixes, and mixed solution is faint yellow limpid.The indicator (Adittive2) that 1.5mL is contained to phenolphthalein adds in mixed solution, and limit slowly adds, and limit mixes.Just added fashionablely, visible topical solutions be pink, becomes faint yellow, limpidly after mixing, and after each component mixes, is configured to 15mL tissue culture gel.
The gel configuring is added in 48 well culture plates, and every hole adds 250uL gel.Be positioned in super clean bench, wait for that liquid gel forms translucent.
The tumor tissues sample that extracts excision, is positioned over tissue in PBS solution, is separated into 0.5cm 3size tissue block.
Tissue block is positioned in translucent gel, is placed in center, hole, standing, treat that tissue culture gel formation is opaque gelatin.
In 12mL substratum, add 3mL foetal calf serum.In each tissue culture hole, add 250uL to contain the substratum of foetal calf serum.
The detection of chemotherapy drug susceptibility in embodiment 2, dimensional culture system
Utilize substratum or PBS to prepare chemotherapeutics to be measured, according to working concentration shown in medicine specification sheets, keep culture volume and chemotherapeutics volume ratio to be greater than 100.
Tumor tissue is cultivated in culture systems to 24h, 48h, 72h, 96h and 120h, every day, observation of cell growing state, and measured cell migration distance.
Collect related data.The impact of statistical study different pharmaceutical on growth of tumour cell and migration.
Utilize above-mentioned three-dimensional histoculture technology, we study (Fig. 2) to Hepatocellular Carcinoma to chemotherapy drug susceptibility, PRELIMINARY RESULTS demonstration, and this culture systems can be reacted the susceptibility of liver cancer tissue to different chemotherapeutics.

Claims (8)

1. for a dimensional culture system for tumor cell culture, it is characterized in that, described dimensional culture system contains the gel as tumor tissue growth's support.
2. according to claim 1 for the dimensional culture system of tumor cell culture, it is characterized in that, described dimensional culture system also contains substratum, setting accelerator.
3. according to the dimensional culture system for tumor cell culture described in claim 1 or 2 any one, it is characterized in that, described dimensional culture system further contains tumor tissues.
4. according to the dimensional culture system described in claim 1-3 any one, it is characterized in that, described gel contains layer and is adhered albumen, collagen iv, fibroblast growth factor, tissue fibers proenzyme activation factor.
5. according to the dimensional culture system for tumor cell culture described in claim 1-4 any one, it is characterized in that, described tumor tissues is fixed in gel stent.
6. according to the dimensional culture system for tumor cell culture described in claim 1-5 any one, it is characterized in that, described tumor tissues is liver cancer tissue.
7. foundation is for a method for the dimensional culture system of tumor cell culture, and it comprises step:
A) mixture of obtaining liq gel and setting accelerator;
B) gel mixture configuring is added in culture plate, wait for that liquid gel forms translucent;
C) tumor tissue is positioned in translucent gel, is placed in center, hole, standing, treat that tissue culture gel formation is opaque gelatin.
8. the dimensional culture system of preparing according to the dimensional culture system for tumor cell culture described in claim 1-6 any one or method according to claim 7 is in the purposes of chemotherapeutics screening.
CN201410012583.XA 2014-01-10 2014-01-10 Three-dimensional culture system for tumor cell culture Pending CN103756904A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103966168A (en) * 2014-05-19 2014-08-06 广州恒迪生物科技有限公司 Tumor living tissue in-vitro culture system and culture method
CN104531618A (en) * 2014-12-21 2015-04-22 朱熹 Method for obtaining lots of three-dimensional growing tumour cells at low cost by utilizing graphene nano material
CN106337078A (en) * 2016-10-13 2017-01-18 成都无界精准生物科技有限公司 Novel method for detecting sensitivity of tumor medicine
CN107058456A (en) * 2017-06-02 2017-08-18 四川精准医学检验有限责任公司 A kind of circulating tumor cell 3D susceptibility test methods

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102952279A (en) * 2012-05-10 2013-03-06 东南大学 Hydrogel for three-dimensional culturing of tumor cells and applications thereof
CN103477222A (en) * 2010-09-29 2013-12-25 麻省理工学院 Device for high throughput investigations of cellular interactions

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CN102952279A (en) * 2012-05-10 2013-03-06 东南大学 Hydrogel for three-dimensional culturing of tumor cells and applications thereof

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103966168A (en) * 2014-05-19 2014-08-06 广州恒迪生物科技有限公司 Tumor living tissue in-vitro culture system and culture method
CN103966168B (en) * 2014-05-19 2017-05-03 广州恒迪生物科技有限公司 Tumor living tissue in-vitro culture system and culture method
CN104531618A (en) * 2014-12-21 2015-04-22 朱熹 Method for obtaining lots of three-dimensional growing tumour cells at low cost by utilizing graphene nano material
CN104531618B (en) * 2014-12-21 2018-06-26 朱熹 The method that three dimensional growth tumour cell is obtained using graphene nano material
CN106337078A (en) * 2016-10-13 2017-01-18 成都无界精准生物科技有限公司 Novel method for detecting sensitivity of tumor medicine
CN107058456A (en) * 2017-06-02 2017-08-18 四川精准医学检验有限责任公司 A kind of circulating tumor cell 3D susceptibility test methods

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Application publication date: 20140430