CN103966168B - Tumor living tissue in-vitro culture system and culture method - Google Patents
Tumor living tissue in-vitro culture system and culture method Download PDFInfo
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- CN103966168B CN103966168B CN201410212174.4A CN201410212174A CN103966168B CN 103966168 B CN103966168 B CN 103966168B CN 201410212174 A CN201410212174 A CN 201410212174A CN 103966168 B CN103966168 B CN 103966168B
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Abstract
The invention relates to a tumor living tissue in-vitro culture system and a corresponding tumor living tissue in-vitro culture method. The tumor living tissue in-vitro culture system comprises a support system for simulating an in-vivo basilemma structure and a blood supply system of tumor, a tumor tissue specimen treatment liquid which is a RPMI1640 culture liquid containing 2mu g/ml of amphotericin, 1000U/ml of penicillin and 1000mu g/ml of streptomycin, and a special culture medium for tumor tissue culture, wherein the culture medium contains 20mu g/L of bFGF (Basic Fibroblast Growth Factor), 20mu g/L of EGF (Epidermal Growth Factor), 10mu g/L of PDGF (Platelet Derived Growth Factor), 10mu g/L of HGF (Hepatocyte Growth Factor), 10mu g/L of IGF (Insulin-like Growth Factor), 10mu g/L of NRG1, 10mu g/L of VEGF (Vascular Endothelial Growth Factor), 5% of fetal calf serum as well as 500U/ml of penicillin and 500mu g/ml of streptomycin on the basis of a mixture solution containing 50 percent of DMEM (Dulbecco Modified Eagle Medium) and 50 percent of HamF 12. The invention provides a systematic solution scheme and an appropriate culture medium for the in-vitro culture of tumor tissue cells.
Description
Technical field
The present invention relates to a kind of vitro culture system of tumor biopsies, more particularly, to the special external of tumor biopsies
Culture fluid, is directed to the extracorporeal culturing method of corresponding tumor biopsies.
Background technology
Research shows that different types of tumor has different biological characteristicses；Same type of tumor, different are individual
Body, its biological characteristics are also differed.In order to study the biological characteristicses of the tumor tissue cell of each individuality, such as tumor tissues
Sensitivity of the cell to Radiotherapy chemotherapy, the diagnosis and treatment for tumour patient provide foundation, it is necessary to carry out the training of Primary Tumor living tissue cells
Support.
The internal microenvironment of simulation
First, the tumor cell of human body is left due to changing the absorption mode and the microenvironment of existence of nutrition, often occur
Anoikis (anoikis).Therefore, during the culture of Vitro Tumor histiocyte, consider first to simulate internal microenvironment, this
It is the key of In vitro culture tumor tissue cell existence.The tumor cell of epithelial origin is relied on and is attached at ability on certain holder
Growth, i.e., so-called anchorage-dependent cell.In culture vessel pan coating growth substrate, such as collagen or other extracellular matrixs
Composition etc., simulates internal based film structure, is not only particularly conducive to the attaching and growth of epithelial cell, is also beneficial to cultured cells
Differentiation, make cell polarity show become apparent from.Though tumor cell has stronger clonal growth power, but still have certain groupment or
With other cell phase dependences.First, the mutual dependence for existence of tumor cell and tumor cell；Second, tumor cell and substrate into
Fibrocellular dependence.Also these dependences can be eliminated or weaken simultaneously after external dispersion culture and exclusion fibroblast,
The activity of cancerous cell existence and growth may be affected.
The special composition of In vitro culture
Due to different tumor cells, which drives gene different, and advantage signal Signal Transduction Pathways are also differed.It is swollen after in vitro
Tumor tissue is diluted due to correlation factor of surviving in autocrine system, does not reach tumor existence or the demand for growing, therefore, in training
Respective components be must be added in foster base.
The selection of minimal medium
Different culture medium, the culture to tumor tissues have certain difference.Such as M199 and RPMI1640 culture medium only
Short term culture suitable for epithelial tissue and its existence is maintained, the long-term cultivation and proliferation to epithelial tissue simultaneously fail to understand
It is aobvious.DMEM and HamFl2 culture medium is used to having its special role during epithelial tissue culture.The attaching of epithelial cell can be promoted；Maintain
The differentiation of epithelial cell cultivation conditions in vitro.The culture of the particularly suitable primary epithelial cells of particularity of HamFl2 culture medium,
Add the inorganic ionss of trace element in medium component, be more favorable for the growth and metabolism of cell.In actual culture, will
DMEM and HamFl2 is mixed for the culture of the tumor tissue cell of epithelial origin by a certain percentage.Normal cell culture is being not added with
Serum or low serum are difficult to grow or survive, and tumor cell can be also grown in low blood serum medium.
Take precautions against microorganism pollution
The tumor of Different Organs, may contaminated degree difference.In body surface or lining patch digestive tract, respiratory tract etc.
The tumor tissues at place, directly expose or contact with external environment, what tissue was polluted per se with various pathogenic microorganisms or by which
Chance is more.During In vitro culture it may first have to carry out pretreatment to tumor tissue.Need treatment fluid and culture medium.
Remove non-tumor cell
Epithelial tissue is connected with connective tissue by means of thin layer basement membrane.Draw materials separation cell when can bring into a small amount of connective tissue into
Point, often result in fibroblast and growth is mixed with epithelial cell, therefore, it is necessary to take cancer nests tissue as far as possible.
As the In vitro culture of tumor tissue cell is affected by above-mentioned many factors, system is had no at present both at home and abroad
Solution tumor tissue cell culture scheme and suitable culture medium, culture medium also without the special tumor tissues culture that can be sold and
Culture technique, it is therefore necessary to develop effective tumor biopsies vitro culture system and corresponding cultural method.
The content of the invention
It is an object of the invention to provide a kind of tumor biopsies vitro culture system, including simulate internal based film structure
And the support system and tumor tissues sample disposal liquid and special culture media of the blood supply system of tumor, it is the body of tumor tissue cell
The solution and suitable culture medium of outer culture offer system.
A kind of tumor biopsies vitro culture system of the present invention, including：The internal based film structure of simulation and tumor
Blood supply system support system；Tumor tissues sample disposal liquid, be containing amphotericin 2ug/ml, penicillin 1000U/ml,
The RPMI1640 culture fluid of streptomycin 1000ug/ml；And tumor tissues culture special culture media, it is with DMEM and HamF l2
Each 50% mixed liquor basis, contains：20ug/L bFGF、20ug/L EGF、10ug/L PDGF、10ug/L HGF、10ug/L
The hyclone of IGF, 10ug/L NRG1,10ug/L VEGF and 5%, and 500U/ml penicillins, 500ug/ml streptomycins.
According to the further feature of tumor biopsies vitro culture system of the present invention, the support system is to prop up
The filter paper of the coated high-hydroscopicity of collagen that frame is supported.
It is a further object of the present invention to provide a kind of tumor biopsies extracorporeal culturing method.
Tumor biopsies extracorporeal culturing method of the present invention, comprises the following steps：
A. the culture pre-treatment of tumor tissues：Tumor tissues 15-20 minutes are fully soaked with tumor tissues treatment fluid；Institute
Tumor tissues sample disposal liquid is stated, is containing amphotericin 2ug/ml, penicillin 1000U/ml, streptomycin 1000ug/ml
RPMI1640 culture fluid；
B. the tumor tissues for soaking are taken out, under mirror, is cut into 0.5-1mm3Fritter, be placed in support system using swollen
Tumor tissue culture special culture media is cultivated；The support system is the blood supply system of the internal based film structure of simulation and tumor
Support system；The tumor tissues culture special culture media, is, with each 50% mixed liquor bases of DMEM and HamF l2, to contain：
20ug/L bFGF、20ug/L EGF、10ug/L PDGF、10ug/L HGF、10ug/L IGF、10ug/L NRG1、10ug/L
The hyclone of VEGF and 5%, and 500U/ml penicillins, 500ug/ml streptomycins.
According to the further feature of cultural method of the present invention, the tumor tissues are derived from body surface or lining patch disappears
Change road, the tumor tissues in respiratory tract etc., directly exposure or the tumor tissues contacted with external environment.
Preferably, the support system is the filter paper of the coated high-hydroscopicity of collagen supported with support.
Tumor biopsies vitro culture system of the present invention, and corresponding cultural method, with following beneficial effect
Really：
(1) treatment effect of preventing microbial contamination：Tumor tissues especially derive from body surface or lining patch digestive tract, respiratory tract
Interior etc. tumor tissues, the tumor tissues for directly exposing or contacting with external environment, at the treatment fluid developed by the Jing present invention
Reason, puts special culture media culture, and its pollution rate is about 1.26%, and it is 24.1% that untreated specimen carries out cultivating pollution rate.
(2) the invention provides the filter paper of the coated high-hydroscopicity of collagen supported using support carries out group as support system
Block culture is knitted, test result indicate that：All shown with the existence of the tumor tissues of support system culture of the present invention and growth conditions
Write better than in immersion cultivation conditions.
(3) under support system of the present invention, respectively with the present invention develop special tumor tissues culture medium with
RPMI1640 culture medium is cultivated to tumor tissues, and culture was detected after 5 days, was as a result shown：Special tumor tissues culture
The tumor tissues existence of base is significantly better than the cultivation conditions under RPMI1640 culture medium conditions with growth conditions.
Description of the drawings
Fig. 1 be using tumor biopsies vitro culture system of the present invention with using conventional immersion culture and
Culture experiment result of the RPMI1640 culture medium to tumor tissues.
Specific embodiment
Embodiment one：Tumor biopsies vitro culture system of the present invention
Tumor biopsies vitro culture system of the present invention is using the internal based film structure of simulation and the blood supply of tumor
The support system of system, preferably adopts the filter paper of the coated high-hydroscopicity of collagen supported with support.
The present invention has prepared two kinds of culture fluid, and the first is tumor tissues sample disposal liquid, for the culture of tumor tissues
Pre-treatment；Second is tumor tissues culture special culture media, for being used together with reference to the support system, is different from routine
Immersion culture.
Tumor tissues sample disposal liquid of the present invention be based on conventional RPMI1640 culture fluid, add it is following into
Point：Amphotericin 2ug/ml, penicillin 1000U/ml, streptomycin 1000ug/ml.
Tumor tissues culture special culture media of the present invention be with each 50% mixed liquor basis of DMEM and HamF l2, plus
Enter following component：20ug/L bFGF、20ug/L EGF、10ug/L PDGF、10ug/L HGF、10ug/L IGF、10ug/L
The hyclone of NRG1,10ug/L VEGF and 5%, and 500U/ml penicillins, 500ug/ml streptomycins.
The composition adopted in above-mentioned treatment fluid and culture medium, is commercially available reagent.
The tumor tissues sample disposal liquid configured by embodiment one and tumor tissues culture special culture media are with later
Embodiment.These tumor tissues sample disposal liquid and tumor tissues culture special culture media and support system can be used to owning
The In vitro culture of the tumor biopsies of type, such as from body surface or lining patch digestive tract, the tumor tissues in respiratory tract etc.,
The tumor tissues for directly exposing or contacting with external environment, and it is not limited to the gastrointestinal tumor illustrated by following examples
Tissue.
Embodiment two：The decontamination of tumor tissues sample disposal liquid of the present invention, preventing polluting effect
Micro-assembly robot block culture pre-treatment is carried out with gastrointestinal tumor, fresh tumor tissue is taken, one group with conventional containing dual anti-
RPMI1640 culture fluid process, another group with tumor tissues sample disposal liquid of the present invention process.
As a result show, with the conventional culture fluid treatment group containing dual anti-RPMI1640,6 pollutions in 32 gastric cancer, 47
In intestinal cancer, 13 are contaminated.And with the specimen of tumor tissues sample disposal liquid of the present invention process, only 1 intestinal cancer is dirty
Dye.
Conclusion：Tumor tissues sample disposal liquid of the present invention processes decontamination, anti-fouling effect and is significantly better than routine containing dual anti-
RPMI1640 culture fluid process (P<0.05).It is shown in Table 1.
Table 1：The decontamination of tumor tissues sample disposal liquid, preventing polluting effect
| It is pollution-free | It is contaminated | Pollution rate | |
| Dedicated specimen treatment fluid | 78 | 1 | 1.26% |
| Containing dual anti-RPMI1640 | 60 | 19 | 24.1% |
Embodiment three：Support system culture of the present invention is grown to tumor micro-assembly robot block and is survived with conventional immersion culture
Impact
On the basis of embodiment two, by the tumor tissue after process, 0.5-1mm is cut into3Tumor micro-assembly robot block, one
Group carries out tumor tissues culture with support system of the present invention, and another group is soaked culture in conventional manner.
Culture is detected after 5 days, is as a result shown：Tumor tissues existence and the growth of support system culture of the present invention
State is significantly better than immersion culture (P<0.01) (see Fig. 1).
Example IV：Tumor tissues culture special culture media of the present invention is with routine RPMI1640 culture medium to tumor group
Knit the impact of existence and growth
On the basis of embodiment two, by the tumor tissue after process, 0.5-1mm is cut into3Tumor micro-assembly robot block, one
Group carries out tumor tissues culture with tumor tissues culture special culture media of the present invention, and another group is trained with conventional RPMI1640
Foster base is cultivated.
Culture is detected after 5 days, is as a result shown：It is swollen after tumor tissues culture special culture media culture of the present invention
Tumor tissue to be survived and be significantly better than the culture (P under RPMI1640 culture medium conditions with growth conditions<0.01) (see Fig. 1).
Claims (1)
1. a kind of tumor biopsies vitro culture system, it is characterised in that the system includes：
The support system of the blood supply system of the internal based film structure of simulation and tumor, the support system are the glue supported with support
The filter paper of the high-hydroscopicity of primordial covering；
Tumor tissues sample disposal liquid, is to add amphotericin 2ug/ml, penicillin 1000U/ml, streptomycin 1000ug/ml
RPMI1640 culture fluid；And
Tumor tissues culture special culture media, is, with each 50% mixed liquor bases of DMEM and HamF l2, to add：20ug/L
BFGF, 20ug/L EGF, 10ug/L PDGF, 10ug/L HGF, 10ug/L IGF, 10ug/LNRG1,10ug/L VEGF and 5%
Hyclone, and 500U/ml penicillins, 500ug/ml streptomycins；
The tumor tissues are derived from the tumor tissues that body surface or lining are pasted in digestive tract, respiratory tract, directly exposure or same outer shroud
The tumor tissues that border contacts.
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| AU2019440405A1 (en) * | 2019-04-11 | 2021-12-09 | Genex Health Co., Ltd | Method for culturing primary cells of gastric cancer and gallbladder and bile duct cancer, and supporting reagents |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2453011A1 (en) * | 2010-11-12 | 2012-05-16 | Commissariat à l'Énergie Atomique et aux Énergies Alternatives | Glioma stem cells and methods for obtaining them |
| CN102465113A (en) * | 2010-11-12 | 2012-05-23 | 复旦大学附属中山医院 | Human hepatoma carcinoma cell line and application thereof |
| CN103756904A (en) * | 2014-01-10 | 2014-04-30 | 赵海涛 | Three-dimensional culture system for tumor cell culture |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| EP2453011A1 (en) * | 2010-11-12 | 2012-05-16 | Commissariat à l'Énergie Atomique et aux Énergies Alternatives | Glioma stem cells and methods for obtaining them |
| CN102465113A (en) * | 2010-11-12 | 2012-05-23 | 复旦大学附属中山医院 | Human hepatoma carcinoma cell line and application thereof |
| CN103756904A (en) * | 2014-01-10 | 2014-04-30 | 赵海涛 | Three-dimensional culture system for tumor cell culture |
Non-Patent Citations (2)
| Title |
|---|
| 人脑胶质瘤中肿瘤干细胞的体外培养及生物学特性;潘志刚等;《中国组织工程研究与临床康复》;20081216;第12卷(第51期);第10055-10058页 * |
| 肝肿瘤细胞HepG2培养条件的摸索及代谢能力的研究;李欣等;《广东医学》;20070831;第28卷(第8期);第1220-1222页 * |
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