CN102787094B - Substratum, cell cultures test kit and cell culture processes - Google Patents

Substratum, cell cultures test kit and cell culture processes Download PDF

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CN102787094B
CN102787094B CN201110448806.3A CN201110448806A CN102787094B CN 102787094 B CN102787094 B CN 102787094B CN 201110448806 A CN201110448806 A CN 201110448806A CN 102787094 B CN102787094 B CN 102787094B
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epithelial
substratum
epithelium
feeder
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CN102787094A (en
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李晖
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Shenzhen Yongtai Biotechnology 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
    • C12N2500/14—Calcium; Ca chelators; Calcitonin
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    • C12N2502/00—Coculture with; Conditioned medium produced by

Abstract

The present invention relates to substratum, cell cultures test kit and cell culture processes, especially for substratum, cell cultures test kit and cell culture processes that epithelial cell is cultivated.Described substratum comprises serum, calcium component and ROCK inhibitor.

Description

Substratum, cell cultures test kit and cell culture processes
The application is application number is 201110127667.4, and denomination of invention is substratum, the divisional application of the Chinese invention patent application (applying date is on May 17th, 2011) of cell cultures test kit and cell culture processes.
Technical field
The present invention relates to substratum, cell cultures test kit and cell culture processes, especially for substratum, cell cultures test kit and cell culture processes that epithelial cell is cultivated.
Background technology
The epithelial cell of human body vitals as lung, kidney, liver, pancreas and skin are all broken up by organ specificity is its moiety.The epithelial cell of these specific differentiation is directly related with the specific function of Different Organs, as the gaseous interchange function of lung, the filtering function of kidney, the removing toxic substances of liver and in and function, pancreatic cell produce Regular Insulin, skin has the injury of protection body from external environment.And these vitals are once pathology occur or degenerates to threaten the health of the mankind, because these vitals are difficult to be replaced, the specific cell of Different Organs can not replace the cell of other organ mutually.
The cell of specific differentiation, if the insulin-producing cells of renal epithelial cell, pancreas, the hair follicle of corium and gland cell are all be difficult to regeneration, let alone and carries out cultivation in vitro and bred.Laboratory animal (comprising various transgenosis and cloned animal) is the essential tool for disease pathogenesis research and new drug development.But, separation is remained very difficult from the vitro culture of people and mammiferous primary epithelial cells.Apply current serum free medium, original cuiture (the survive for days that can only carry out short-term had, as the epithelial cell such as lung and pancreas), what have can only carry out limited Secondary Culture (1-3 generation, as tracheae/epithelial cell such as segmental bronchus and prostate gland), what have even at all can not vitro culture (as the epithelial cell such as liver and colon), and the superficial cell only coming from human body skin can about Secondary Culture 10 generation.And the epithelial cell output obtained from each animal or biopsy sample is still very low, it is all very low for comprising the quantity of cell, the directly cell purity of separation or Short-term Culture, and these are primary and the epithelial rate of propagation that goes down to posterity is also very limited.
In order to carry out epithelial cultivation in vitro, abroad attempt by genetic manipulation at present, as proceeded to virus (SV40T or HPV16E6E7) or cellular oncogene, can the algebraically of the outer cell survival of extension body.But the disadvantage of genetic manipulation is genetic background and the phenotype that can change these cells, so that these normal epithelium cells are allowed to lose its normal physiological function, as p53 and pRB signal path is usually suppressed.And these genetically modified cells can not be transplanted into body again again.But owing to lacking the epithelial technology of effective vitro culture at present, these cells above-mentioned still enjoy favor in the medical science and life science of the world today.At non-cancer research field, they just represent tissue or the organ of " function " property in initial source.At cancer research field, contrast that they are also usually used as " normal cell ".And their market value is also very expensive.
The first step of antitumor drug research is exactly that medicine is tested the specific toxicities of cancer cells, how JEG-3 (as HeLa and A539 etc.) used at present goes down to posterity several years even many decades in vitro, they can not react the characteristic of human malignant tumor of the same type to a great extent, and some laboratories also exist the crossed contamination of a large amount of JEG-3.And for different individualities, even if the tumour of same type also may be caused by different reasons or mechanism, therefore for each tumour, all need and can represent its specific pathogenetic cell strain to carry out new drug development.More outstanding example, the research field as prostate cancer does not have the clone of the mankind's former prostate cancer at all, only clone (LnCAP, PC-3, DU-145) be all from transfer after tumor tissues.However, the research of the tumor research in the whole world more than 90% and nearly all PTS is also continuing to use may not have representational JEG-3 at all.Therefore, the key issue that current cancer research and new drug development will solve is, how to obtain reaction all kinds human tumor characteristic, cancer cells that same tumor represents Different Individual characteristic or different onset mechanism.Certainly, a ultimate real difficult problem lacks effective epithelial primary and Secondary Culture technology.In addition, a lot of toxic side effect that antitumor drug exists are because new drug research lacks normal cell controls to a great extent.Come from the cancer cells of the same tissue of same individuality and normal cell (the above-mentioned HPVE6E7 of Asterand company of the U.S. transforms the prostatic cell of so-called " normally " and the cancer obtained if can obtain, although price is up to 1,2000 U.S. dollars/right, but very in great demand), the first step of new drug development will return science, and the history of PTS research will rewrite.
Regenerative medicine is a kind of brand-new Therapeutic mode of current clinical medicine, namely with the modern cellular replacement therapy art regenerating, reproduce, replace and new life is base therapy principle, utilize stem cell (embryonic stem cell and adult stem cell) to have and change ability to various cytodifferentiation, treatment is sex change that is numerous, that there is no treatment way at present, gangrenosum acne and injury disease clinically, has remarkable and unique medical effect.But the predicament faced at present is, although embryonic stem cell has differentiation capability, reproducibility and plasticity-are strong, may tumorigenesis and face dispute of ethic, how to induce its directed differentiation and increase the surviving rate of transplanting to remain a difficult problem of not separating; And adult stem cell source is rare, is difficult to identify, be separated and purifying, therefore hinders its clinical application; Study hotspot emerging at present---inductive pluripotent stem cells, because it relates to the change that genetic manipulation causes cytogenetics background, and the foreign gene imported and virogene have the potential of carcinogenic or teratogenesis and can not be used for clinical treatment.
Individuality medicine is modern medicine developing direction from now on.According to the disease of clinical patient, relevant genetic background and correlative factor occur/develop, diagnose exactly within the shortest time to disease, monitor, formulating real individualized treatment scheme is the key of dealing with problems.Such as, World Health Organization's investigation finds that safety of medicine sex chromosome mosaicism is one of lethal most important reason of inpatient.Untoward reaction caused by drug reaction individual difference has become the important public hygiene problem of harm humans health.And inherited genetic factors causes the major cause of drug reaction individual difference.In the U.S., had more than 70 to plant medicine and sticked genetic tags through FDA approval, the clinical patients being used to indicate different genotype when drug application to the predicting function of curative effect and toxicity, and genetic background clearly new drug development will become the trend of future development.How to utilize that the biopsy of extremely trace (as cell etc. inhaled by pin) carries out Rapid&Early diagnosis to diseases such as malignant tumours, In vitro chemo-drug sensitive test, curative effect monitoring are key to tumour patient individualized treatment.All these will depend on primary cell culture technology fast and effectively and could really realize.
Biobank (Times is referred to as one of ten large ideas changing the world) just rises in worldwide.The researchist of NIH is just making great efforts to create the first preservation in the whole America mankind biological organization sample, tumour cell, DNA, and the national biobank of blood.English, add, Norway and Sweden set up this kind of nationwide mechanism.Collect and store abundant, from cancer, brain disorder to the biological organization sample of disease of metabolism, for setting up individuality medicine and new drug development lays the foundation, allow screening and treatment patient more targeted.But just comprise frozen tissue, fixing tissue, DNA, RNA, protein, blood, blood plasma, serum and urine etc. at present stored by biobank, these extremely valuable resources with namely go, cannot regenerate.Effectively primary and passage cell culture technique will inject real " work " power for biobank.This appearance with the biobank of " regeneration " ability will have milestone significance to modern medicine study, Individual Diagnosis and treatment, new drug development.
In sum, current urgent problem is, how to carry out propagation and these epithelial Secondary Culture of the primary epithelial cells in histoorgan source quickly and efficiently, and effectively can extend epithelial cultivation algebraically, the genetic background of cell can not be changed again simultaneously.
Summary of the invention
The present inventor conducts in-depth research for the problems referred to above, propose new substratum and test kit, by using this substratum or test kit in-vitro multiplication primary epithelial cells and Secondary Culture epithelial cell, when not changing cytogenetics background, can effectively extend epithelial cultivation algebraically.
That is, the invention provides following substratum (also referred to as substratum of the present invention):
1. a substratum, it comprises
Serum,
Calcium component, and
ROCK inhibitor.
2. the substratum according to item 1, wherein, the content of described serum is 1.0 ~ 35v/v%.
3. the substratum according to item 1, wherein, described ROCK inhibitor be selected from fasudil, H-1152, Y-27632, HA1100, HA1077 and GSK429286 one or more.
4. the substratum according to item 1, it is for cultivating epithelial cell.
5. the substratum according to item 4, wherein, described epithelial cell is non-Eponychium cell.
6. the substratum according to item 4, wherein, described epithelial cell is primary cell.
7. the substratum according to item 4, wherein, described epithelial cell is passage cell.
8. the substratum according to item 4, wherein, described epithelial cell is tumour cell.
9. the substratum according to item 5, wherein, described non-keratinocyte epithelial cell is squamous cell, columnar epithelial cell, gland epithelial cells or transitional type epithelial cell.
10. the substratum according to item 5, wherein, described non-keratinocyte epithelial cell is oral cavity, nasal mucosa, tracheae, bronchial, lung epithelial, epithelium of esophagus, gastric epithelial, large intestine epithelium, small intestine epithelium, hepatocellular, epithelial duct, gall-bladder, pancreas, beta Cell of islet, kidney, bladder, urothelial, testis epithelium, ovarian epithelium, thyroid, follicular epithelial cell, adrenal, thymus gland, prostatic, mammary gland, tonsilar, the cell of hypophysis, corneal epithelial cell, retinal pigment epithelium.
11. substratum according to item 1, wherein, the content of described calcium component counts 0.1 μM ~ 30mM with calcium ion.
12. substratum according to item 1, wherein, this substratum also comprises feeder cell.
13. substratum according to item 1, wherein, comprise conditioned medium in this substratum.
In addition, the present invention also provides following test kit (also referred to as test kit of the present invention):
14. 1 kinds of test kits, it comprises substratum, serum and ROCK inhibitor.
15. test kits according to item 14, this test kit is for cultivating epithelial cell.
16. test kits according to item 15, wherein, described epithelial cell is non-Eponychium cell.
17. test kits according to item 15, wherein, described epithelial cell is primary cell.
18. test kits according to item 15, wherein, described epithelial cell is passage cell.
19. test kits according to item 15, wherein, described epithelial cell is tumour cell.
20. test kits according to item 16, wherein, described non-keratinocyte epithelial cell is squamous cell, columnar epithelial cell, gland epithelial cells or transitional type epithelial cell.
21. test kits according to item 16, wherein, described non-keratinocyte epithelial cell is oral cavity, nasal mucosa, tracheae, bronchial, lung epithelial, epithelium of esophagus, gastric epithelial, large intestine epithelium, small intestine epithelium, hepatocellular, epithelial duct, gall-bladder, pancreas, beta Cell of islet, kidney, bladder, urothelial, testis epithelium, ovarian epithelium, thyroid, follicular epithelial cell, adrenal, thymus gland, prostatic, mammary gland, tonsilar, the cell of hypophysis, corneal epithelial cell, retinal pigment epithelium.
22. test kits according to item 14, it also comprises calcium component.
23. test kits according to item 14, it also comprises feeder cell.
24. test kits according to item 23, wherein, described feeder cell are cryopreservations.
25. test kits according to item 23, wherein, described feeder cell are low-temperature storage.
26. test kits according to item 14, wherein, it also comprises the container for holding separately described feeder cell when carrying out cell cultures, and this container has permeability membrane structure.
27. test kits according to item 23, wherein, described feeder cell are inoblasts of mouse or people.
28. test kits according to item 14, wherein, described substratum comprises conditioned medium.
29. test kits according to item 14, wherein, described ROCK inhibitor be selected from fasudil, H-1152, Y-27632, HA1100, HA1077 and GSK429286 one or more.
30. 1 kinds of test kits, it comprises substratum according to any one of item 1 ~ 13 and feeder cell.
31. test kits according to item 30, wherein, described feeder cell are cryopreservations.
32. test kits according to item 30, wherein, described feeder cell are low-temperature storage.
33. test kits according to item 30, wherein, it also comprises the container for holding separately described feeder cell when carrying out cell cultures, and this container has permeability membrane structure.
In addition, the present invention also provides the following epithelial method of cultivation (also referred to as cultural method of the present invention):
Cultivate epithelial method for 34. 1 kinds, the method comprises
Steps A: the substratum of use according to any one of item 1 ~ 13 is by epithelial cell and feeder cell Dual culture.
35. epithelial methods of cultivation according to item 34, wherein, described epithelial cell is primary cell.
36. epithelial methods of cultivation according to item 34, wherein, described epithelial cell is passage cell.
37. epithelial methods of cultivation according to item 34, wherein, described epithelial cell is tumour cell.
38. epithelial methods of cultivation according to item 34, wherein, described epithelial cell is non-Eponychium cell.
39. epithelial methods of cultivation according to item 38, wherein, described non-keratinocyte epithelial cell is squamous cell, columnar epithelial cell, gland epithelial cells or transitional type epithelial cell.
40. epithelial methods of cultivation according to item 38, wherein, described non-keratinocyte epithelial cell is oral cavity, nasal mucosa, tracheae, bronchial, lung epithelial, epithelium of esophagus, gastric epithelial, large intestine epithelium, small intestine epithelium, hepatocellular, epithelial duct, gall-bladder, pancreas, beta Cell of islet, kidney, bladder, urothelial, testis epithelium, ovarian epithelium, thyroid, follicular epithelial cell, adrenal, thymus gland, prostatic, mammary gland, tonsilar, the cell of hypophysis, corneal epithelial cell, retinal pigment epithelium.
41. epithelial methods of cultivation according to item 34, wherein, the epithelial method of this cultivation is epithelial cell Secondary Culture method.
42. epithelial methods of cultivation according to item 34, wherein, the epithelial method of this cultivation is primary epithelial cells multiplication culture method.
43. epithelial methods of cultivation according to item 34, wherein, are placed in same culture vessel by epithelial cell and feeder cell.
Feeder cell wherein, are placed in separately the container with permeability membrane structure by 44. epithelial methods of cultivation according to item 34.
45. epithelial methods of cultivation according to item 34, wherein, described feeder cell are inoblasts of mouse or people.
Cultivate epithelial method for 46. 1 kinds, the method comprises
Step B: use the culture medium culturing epithelial cell described in item 12 or 13.
47. epithelial methods of cultivation according to item 46, wherein, described epithelial cell is primary cell.
48. epithelial methods of cultivation according to item 46, wherein, described epithelial cell is passage cell.
49. epithelial methods of cultivation according to item 46, wherein, described epithelial cell is tumour cell.
50. epithelial methods of cultivation according to item 46, wherein, described epithelial cell is non-Eponychium cell.
51. epithelial methods of cultivation according to item 50, wherein, described non-keratinocyte epithelial cell is squamous cell, columnar epithelial cell, gland epithelial cells or transitional type epithelial cell.
52. epithelial methods of cultivation according to item 50, wherein, described non-keratinocyte epithelial cell is oral cavity, nasal mucosa, tracheae, bronchial, lung epithelial, epithelium of esophagus, gastric epithelial, large intestine epithelium, small intestine epithelium, hepatocellular, epithelial duct, gall-bladder, pancreas, beta Cell of islet, kidney, bladder, urothelial, testis epithelium, ovarian epithelium, thyroid, follicular epithelial cell, adrenal, thymus gland, prostatic, mammary gland, tonsilar, the cell of hypophysis, corneal epithelial cell, retinal pigment epithelium.
53. epithelial methods of cultivation according to item 46, wherein, the epithelial method of this cultivation is epithelial cell Secondary Culture method.
54. epithelial methods of cultivation according to item 46, wherein, the epithelial method of this cultivation is primary epithelial cells multiplication culture method.
The substratum of the application of the invention or test kit cultivate epithelial cell, can when not changing cytogenetics background, and Effective multiplication cultivates primary epithelial cells, and effectively extends epithelial cultivation algebraically.
Accompanying drawing explanation
Fig. 1 is cultural method schematic diagram of the present invention, and wherein, 1., 2., 3. scheme is three kinds of culture systems that substratum of the present invention and feeder cell combine.
Fig. 2 is the workflow diagram adopting conditioned medium of the present invention to carry out epithelial cell cultivation.
Fig. 3 is the workflow diagram adopting indirect (Transwell) co-culture system of feeder cell of the present invention to carry out epithelial cell cultivation.
Fig. 4 is the workflow diagram adopting feeder cell of the present invention and epithelial cell Co-culture system.
Fig. 5 is that the segmental bronchus primary epithelial cells of normal people compares at serum free medium (left side) with containing the growth conditions cultivated in blood serum medium (right side).Show in figure, bronchial epithelial cell can not be bred in containing the substratum of serum.The primary bronchial epithelial cell of preparation is separated by the method for embodiment 5, finally by the cell precipitation Eddy diffusion that obtains, in DMEM substratum completely, [composition is DMEM, add 10% foetal calf serum (FBS), 100U/ml penicillin (penicillin) and 100 μ g/ml Streptomycin sulphates (streptomycin)] in, or the epithelial cell substratum SFM (GIBCO#10744-019) (see embodiment 10) of serum-free.After one week, observe under conventional inverted microscope and take a picture.Although culture effect is not good, serum free medium is still widely used in the primary and Secondary Culture of conventional epithelial cell.
Fig. 6 is that the normal human bronchial's primary epithelial cells growth conditions cultivated compares.Show in figure, for bronchial epithelial cell, adopt three kinds of culture systems of the present invention to cultivate, the growing multiplication of cell is obviously better than using conventional serum free medium.The primary bronchial epithelial cell of preparation is separated by the method for embodiment 5, by the cell precipitation Eddy diffusion (method is shown in Fig. 2) in the conditioned medium of embodiment 8 obtained, or re-suspended cell in HL substratum of the present invention (see embodiment 2, HL substratum 6) in, by the feeder cell Indirect co-culture illustrated by the method for Fig. 3 and embodiment 9, or carry out original cuiture by Fig. 4 and embodiment 6.After one week, observe under conventional inverted microscope and take a picture.Feeder cell required in this illustrated research carry out going down to posterity and conservation according to embodiment 1.HL substratum required in this illustrated research is prepared by embodiment 2.Feeder cell required in this illustrated research obtain by embodiment 3 and 4.
Fig. 7 is the comparison that normal human bronchial's epithelial cell subculture in vitro separately is cultivated.X-coordinate represents cultivated days, and ordinate zou represents the multiple of cell proliferation.Show in figure, for bronchial epithelial cell, adopt three kinds of culture systems of the present invention to cultivate, cell can go down to posterity about at least 10 generations in vitro, is obviously better than the cultivation adopting serum free medium.The primary bronchial epithelial cell of preparation is separated by the method for embodiment 5, by the cell precipitation Eddy diffusion (method is shown in Fig. 2) in the conditioned medium of embodiment 8 obtained, or re-suspended cell in HL substratum of the present invention (see embodiment 2, HL substratum 6) in, by the feeder cell Indirect co-culture illustrated by the method for Fig. 3 and embodiment 9, or carry out Secondary Culture by the explanation of Fig. 4 and embodiment 6 and 7.Feeder cell required in this illustrated research carry out going down to posterity and conservation according to embodiment 1.HL substratum required in this illustrated research is prepared by embodiment 2.Feeder cell required in this illustrated research obtain by embodiment 3 and 4.
Fig. 8 summarizes the epithelial germline and type that the inventive method successfully cultivated.These cells come from people, Mouse and rat respectively.The organization type of these cells has, but is not limited to: oral cavity, trachea-bronchial epithelial cell, lung epithelial, stomach, colon, prostate gland, mammary gland, liver, pancreas, bladder, ovary and tonsil.Concrete implementation step is separated by the method for embodiment 5 prepares primary epithelial cells, the cell precipitation Eddy diffusion of acquisition is carried out in the conditioned medium of embodiment 8 cultivate (method is shown in Fig. 2), or re-suspended cell in HL substratum of the present invention, (see embodiment 2, wherein cultivate for the epithelial cell of mammary gland, prostate gland, stomach, bladder-derived by HL substratum 1; The epithelial cell that HL substratum 2 is originated for liver is cultivated; HL substratum 3 is cultivated for the epithelial cell in colon, tonsil, mammary gland, stomach source; HL substratum 4 is cultivated for the epithelial cell in ovary, tonsil source; HL substratum 5 is cultivated for the epithelial cell in pancreas, tonsil source; HL substratum 6 is cultivated for the epithelial cell in oral cavity, trachea-bronchial epithelial cell, lung source), by the feeder cell Indirect co-culture illustrated by Fig. 3 and embodiment 9, or carry out subculture by the explanation of Fig. 4 and embodiment 6 and 7.Feeder cell required in this illustrated research carry out going down to posterity and conservation according to embodiment 1.HL substratum required in this illustrated research is prepared by embodiment 2.Feeder cell required in this illustrated research obtain by embodiment 3 and 4.
Figure 9 shows that and derive from people, the epithelial cultivation example of rat and mouse.Left figure is the prostate epithelial cell deriving from people, be separated by the method for embodiment 5 and prepare primary epithelial cells, the cell precipitation of acquisition is resuspended in the HL conditioned medium (namely by HL substratum 1 and feeder cell Dual culture, obtaining by diagram 2 method) of embodiment 8 and cultivates.Middle figure and right figure derives from the mammary epithelial cell of rat and the pulmonary epithelial cells of mouse, be separated by the method for embodiment 5 and prepare primary epithelial cells, the cell precipitation of acquisition is resuspended in the HL substratum 1 in embodiment 2 or HL substratum 6, carries out Secondary Culture by the method for Fig. 4 and embodiment 6 and 7.Feeder cell required in this illustrated research carry out going down to posterity and conservation according to embodiment 1.HL substratum required in this illustrated research is prepared by embodiment 2.Feeder cell required in this illustrated research are prepared by embodiment 3 and 4.
The more different foetal calf serum concentration of Figure 10 is on the impact of epithelial cell growth.Prepare HL substratum by embodiment 2, add foetal calf serum and make its final concentration be respectively 1%, 2%, 5%, 10%.15％，20％。By normal human bronchial's epithelial cell of 5000 s-generations (from Fig. 7 research, the method of embodiment 9) be suspended in respectively 3 milliliters above-mentioned containing in the HL substratum of different foetal calf serum concentration, then the Tissue Culture Plate in 6 holes is placed in, again 500000 feeder cell (preparing by embodiment 3) are placed in transwell basket (Corning Products), according to the method for Fig. 3, at 5%CO2, cultivate 6 days in the incubator of 37 DEG C.Discard the feeder cell of transwell basket and wherein splendid attire, discard remaining substratum in culture plate, to wash once without the phosphoric acid buffer of calcium magnesium, add 1ml0.05%EDTA/ trysinization liquid, hatch about 2-3 minute for 37 DEG C, after the completely de-wall of cell, add the DMEM substratum of 2ml containing 10% foetal calf serum, abundant mixing cell, takes out 50ul cell suspension and is mixed in dyeing in the trypan blue (trypan blue) of 4% of 50ul, carry out cell counting according to a conventional method.
The chemical structure of the ROCK inhibitor that Figure 11 substratum of the present invention adds.
Figure 12 shows the impact of concentration on epithelial cell growth of ROCK inhibitor.Prepare HL substratum 6 by embodiment 2, add different ROCK inhibitor and make its final concentration be respectively 1uM, 5uM, 10uM, 20uM, 50uM.By the normal bronchial epithelial cell of 5000 s-generations (from Fig. 7 research, the method of embodiment 9) be suspended in respectively 3 milliliters above-mentioned containing in the HL substratum 6 of different concns ROCK inhibitor, then the Tissue Culture Plate in 6 holes is placed in, again 500000 feeder cell (preparing by embodiment 3) are placed in transwell basket (Corning Products), according to the method for Fig. 3, at 5%CO2, cultivate 8 days in the incubator of 37 DEG C.Discard the feeder cell of transwell basket and wherein splendid attire, discard remaining substratum in culture hole, to wash once without the phosphoric acid buffer of calcium magnesium, add 1ml 0.05%EDTA/ trysinization liquid, hatch about 2-3 minute for 37 DEG C, after the completely de-wall of cell, add the DMEM nutrient solution of 2ml containing 10% foetal calf serum, abundant mixing cell, takes out 50ul cell suspension and is mixed in dyeing in the trypan blue (trypan blue) of 4% of 50ul, carry out cell counting according to a conventional method.
the embodiment of invention
substratum of the present invention
A first aspect of the present invention relates to a kind of substratum (also referred to as substratum of the present invention, i.e. HL substratum), and it comprises serum, calcium component and ROCK inhibitor.
In this specification sheets, substratum refers to the matrix for cultivating biomaterial.Being not particularly limited for biomaterial, can be virus, cell (such as microorganism cells, zooblast, vegetable cell), tissue (such as animal tissues, plant tissue) etc.Substratum of the present invention is preferably the substratum for cell cultures, i.e. cell culture medium.Substratum of the present invention is more preferably the substratum cultivated for epithelial cell.Epithelial cell in this specification sheets can be such as non-keratinocyte epithelial cell, such as, derive from body of gland, comprises the non-keratinocyte epithelial cell of mammary gland, prostate gland, liver and gut epithelium.Non-keratinocyte epithelial cell is divided into the active cells with absorption or secreting function, and non-keratinocyte epithelial cell is not the squamous cell of height keratinization structure.The non-keratinocyte epithelial cell of available culture medium culturing of the present invention can derive from the histoorgan of any kind.The non-keratinocyte epithelial cell that can cultivate comprises but is not defined as Types Below: oral cavity, nasal mucosa, tracheae, bronchial, lung epithelial, epithelium of esophagus, gastric epithelial, large intestine epithelium, small intestine epithelium, hepatocellular, epithelial duct, gall-bladder, pancreas (comprising beta Cell of islet), kidney, bladder, urothelial, testis epithelium, ovarian epithelium, thyroid, follicular epithelial cell, adrenal, thymus gland, prostatic, mammary gland, tonsilar, the cell of hypophysis, corneal epithelial cell, retinal pigment epithelium.In addition, substratum of the present invention can be solid powdery substratum, also can be liquid nutrient medium, preferably liquid nutrient medium.When substratum of the present invention is solid powdery substratum, preferably be modulated into liquid nutrient medium in use to carry out cell cultures, the various compositions such as serum, calcium component and ROCK inhibitor can be added in modulation process, make it meet important document of the present invention.
In this specification sheets, when object is cell, " cultivation " or " cell cultures " refers to and manually maintains in vitro, and cell is bred.Cell cultures is aseptically carried out usually, can be to cultivate individual cells or tissue.
Serum should be contained in substratum of the present invention.As serum, serum conventional in cell cultures can be used, such as calf serum or foetal calf serum, preferred foetal calf serum.As serum, also comprise various serum substitute, product (the such as Knockout of Invitrogen company that serum substitute is bought including but not limited to commercialization tMserum substitute, the Ultroser of Pall Corporation company tM), also comprise milk and milk constituents (the skimmed milk dry powder such as filtered).Amount for the serum in substratum is not particularly limited, can be identical with the amount being added into the serum in substratum in the cell cultures of routine, and those skilled in the art can suitably adjust as required.Preferably, with volume basis (v/v%), in substratum the lower limit of serum content can be 1.0%, more preferably 2.0%, more preferably 3.0%, more preferably 4.0%, more preferably 4.5%, more preferably 5.0%; In substratum the upper limit of serum content can be 35%, more preferably 30%, more preferably 25%, more preferably 20%, more preferably 17%, more preferably 15%, more preferably 13%, more preferably 11%, more preferably 10%.
In this specification sheets, ROCK refers to Rho kinases 1 (ROCK 1) and/or Rho kinases 2 (ROCK 2).ROCK is suppressed to refer to the activity of at least one enzyme reduced in ROCK1 or ROCK2, expression or function.Compared with undressed cell, ROCK activity, expression or function can be totally constrained and namely reach non-activity, nothing expression or nonfunctional, also can be activity, expression or functional level reduction.In this specification sheets, ROCK inhibitor refers to: the material with the effect suppressing ROCK.For ROCK inhibitor, be not particularly limited, can use ROCK inhibitor well known in the art, such as fasudil (Fasudil), H-1152, Y-27632, HA1100, HA1077 and GSK429286 etc., these are all commercial common agents.In addition, ROCK inhibitor is also included in PCT application (WO 03/059913, WO 03/064397, WO 05/003101, WO 04/112719, WO 03/062225 and WO03/062227) disclosed in or at US granted patent (7,217,722 and 7,199,147) and small molecules ROCK inhibitor disclosed in U.S. Patent (2003/0220357,2006/0241127,2005/0182040 and 2005/0197328).The content of ROCK inhibitor in substratum of the present invention is not particularly limited, as long as significant quantity.Described significant quantity refers to the amount that can suppress ROCK, the method defining effective amount well known to a person skilled in the art, those skilled in the art can define effective amount aptly according to conditions such as the kind of institute's cultured cells, the kinds of ROCK inhibitor that uses.Such as, although may be different because of the kind of the kind of cell, ROCK inhibitor, cell culture condition etc., generally speaking, ROCK inhibitor content can between 0.1 μM ~ 1mM.
Feeder cell can also be comprised in substratum of the present invention.In this specification sheets, feeder cell refer to: with the cell of wishing cultured cells co-cultivation, wherein, describedly wish that cultured cells is preferably non-keratinocyte epithelial cell.Feeder cell are generally through gamma radiation exposure and/or mitomycin process, and its mitotic division is suppressed, but still can maintenance metabolism active.Therefore, feeder cell have metabolic capacity but the non-proliferative cell that can not divide.Process and block cell mitogen and the method maintaining cell metabolic activity can be the ordinary method of cytobiology.Feeder cell can derive from any Mammals, but its source is different from wishing the kind that cultured cells (preferred non-keratinocyte epithelial cell) is originated.Feeder cell can be but be not limited to following source, as the feeder cell of mouse, rat, dog, cat, ox, horse, pig, primates and the mankind.Typical feeder cell type comprises splenocyte, hugely bites thymocyte and/or inoblast, and they become non-proliferative cell after treatment.In addition, J2 also can be utilized in the present invention as feeder cell, and J2 cell is the subclone building the mouse fibroblast cell Swiss 3T3cells being, through gamma radiation exposure and/or mitomycin process.Amount for the feeder cell comprised in substratum of the present invention is not particularly limited, and those skilled in the art can be suitable for determining as required, usual feeder cell with wish that the epithelial ratio of cultivating can be 1: 9 ~ 9: 1, preferably 2: 8 ~ 8: 2.
In addition, conditioned medium can be comprised in substratum of the present invention.In this specification sheets, conditioned medium refers to the substratum comprising feeder cell secretory product and/or extract.Typical conditioned medium is with after culture medium culturing feeder cell for some time, then the substratum removing feeder cell and obtain.Herein, the substratum for cultivating feeder cell preferably comprises the substratum of serum, calcium component and ROCK inhibitor, such as substratum of the present invention.For the time of cultivating feeder cell, be not particularly limited, those skilled in the art can be suitable for determining as required, can be 1 ~ 300 hour usually, preferably 5 ~ 200 hours, more preferably 10 ~ 150 hours, more preferably 10 ~ 150 hours, more preferably 20 ~ 100 hours, more preferably 24 ~ 72 hours, more preferably 35 ~ 60 hours, more preferably 40 ~ 50 hours.Generally speaking, preparation condition substratum such as comprises cell cultures in a kind of substratum (as F substratum), collects these substratum after several days.Conditioned medium can be, the conditioned medium after diluting by a certain percentage with fresh culture, also can be the conditioned medium after not diluted collection.Fresh culture: the Dilution ratio of conditioned medium can be 1: 99 ~ 99: 1, determines according to specific experiment condition." conditioned medium " described in the present invention comprises through any dilution proportion or undiluted conditioned medium.Preferably, substratum of the present invention can be the conditioned medium of the not diluted containing serum, calcium component and ROCK inhibitor.
Substratum of the present invention can use the conventional medium for cell cultures to prepare.Conventional medium refers to the substratum for cell cultures (being preferred for epithelial cell to cultivate) well-known in the art, include but not limited to: DMEM (Dulbecco ' s Modified Essential Medium), Ham ' s F12 substratum, Ham ' s F-10 substratum, RPMI 1640, Eagle ' s Basal Medium (EBM), Eagle ' sMinimum Essential Medium (MEM), HEPES, Medium 199 and correlation type substratum.Conventional medium also can be the combination of two or more substratum, the mixed culture medium of such as DMEM and F12.Generally do not comprise serum in the formula of conventional medium, therefore, when using conventional medium to prepare substratum of the present invention, a certain amount of serum can be added separately, making the serum content in substratum in the scope of above-mentioned restriction.The operation of the serum increased or reduce in substratum is the routine operation of the art.
Calcium component should be contained in substratum of the present invention.Alleged herein " calcium component " refers to and can provide calcium ion (Ca for substratum of the present invention 2+) composition, include but not limited to the compound of calcic.Usually, calcium component derives from serum or serum substitute, or derives from the salt of the calcic added in substratum.Described calcium component such as can be added in substratum with the form of calcium salt (such as calcium chloride, calcium sulfate).The content range (in calcium ion) of calcium component can be identical with for the calcium component content in the conventional medium of cell cultures, and those skilled in the art can suitably adjust as required.Such as, with Ca 2+meter, the lower limit of calcium component content can be 0.1 μM, preferably 10 μMs, more preferably 100 μMs, more preferably 200 μMs, more preferably 400 μMs, more preferably 500 μMs, more preferably 800 μMs, more preferably 1mM, more preferably 1.2mM, more preferably 1.3mM, more preferably 1.5mM; The upper limit of calcium component content can be 30mM, more preferably 25mM, more preferably 20mM, more preferably 15mM, more preferably 12mM, more preferably 10mM, more preferably 8mM, more preferably 7mM, more preferably 6mM, more preferably 5mM, more preferably 4mM, more preferably 3mM, more preferably 2.8mM, more preferably 2.5mM.
When using conventional medium to prepare substratum of the present invention, preferably carrying out adjusting and make calcium component content in the scope of above-mentioned restriction.If comprise calcium in the classics formula of conventional medium and calcium component content in the scope of above-mentioned restriction, then with regard to calcium component content, this conventional medium is without the need to adjusting; If comprise calcium in the classics formula of conventional medium but calcium component content not in the scope of above-mentioned restriction, then should correspondingly increase or reduce formula in calcium, make the calcium component content of substratum in the scope of above-mentioned restriction; If do not comprise calcium in the classics formula of conventional medium, then should increase calcium in formula, make the calcium component content of substratum in the scope of above-mentioned restriction.The operation of the calcium component content increased or reduce in substratum is the routine operation of the art.
Other conventional ingredient can be added in substratum of the present invention, including but not limited to: amino acids, vitamins, inorganic salts, VITAMIN B4, thanomin (ethanolamine), D-Glucose, heparin (heparin), N-[2-hydroxyethyl (hydroxyethylpiperazine)-N '-[2-ethanesulfonic acid (ethanesulfonic acid)] (HEPES), hydrocortisone (hydrocortisone), insulin (Regular Insulin), Urogastron (EGF), suprarenin (epinephrine), Thioctic Acid (lipoic acid), phenol red, phosphorylethanolamine (phosphoethanolamine), putrescine (putrescine), Toxins,exo-, cholera (choleratoxin), Sodium.alpha.-ketopropionate (sodium pyruvate), triiodothyronine (triiodothyronine, T3), thymus pyrimidine and transferrin (transferrin).Wherein, heparin and transferrin can use ironic citrate (ferric citrate) or chelating ferric sulfate (ferrous sulfate chelates) to substitute.Above-mentioned added ingredients all can be bought in commercialization.
Amino acids in substratum of the present invention is including but not limited to ALANINE, L-arginine, altheine (L-asparagine), L-Aspartic acid, Cys, Pidolidone, L-glutaminate, glycine, L-Histidine, ILE, L-Leu, 1B, METHIONINE, L-Phe, L-PROLINE, Serine, L-threonine, L-Trp, TYR and Valine.
Vitamins in substratum of the present invention is including but not limited to vitamin H, choline chloride 60 (choline chloride), D-Ca + 2-pantothenic acid (pantothenate), folic acid (folic acid), i-inositol (i-inositol), niacinamide (niacinamide), many alcohol (pyridoxine), riboflavin (riboflavin), VITMAIN B1 (thiamine) and vitamin B12.
Inorganic salts composition including but not limited to: calcium salt is (as CaCl 2), CuSO 4, FeSO 4, KCl, magnesium salts is (as MgCl 2), manganese salt is (as MnCl 2), sodium-acetate, NaCl, NaHCO 3, Na 2hPO 4, Na 2sO 4and other element of trace is as selenium (selenium), silicon, molybdenum (molybdenum), vanadium (vanadium), nickel, tin, zinc, these trace elementss can occur in a variety of forms, but the form of preferably salt, as Na 2seO 3, Na 2siO 3, (NH 4) 6Mo 7o 24, NH 4vO 3, NiSO 4, SnCl and ZnSO 4.
Other added ingredients of substratum of the present invention is including but not limited to heparin, Urogastron (epidermal growth factor, EGF), at least one can increase intracellular cyclic adenosine one phosphoric acid (cyclic adenosine monophosphate, cAMP) factor, at least one fibroblast growth factor (fibroblast growth factor, FGF) of level.Above-mentioned added ingredients can directly join in basic medium, also can become mixed solution with DPBS (Dulbecco ' s Phosphate Buffered Saline) buffer, and freezen protective is until row interpolation again when preparing substratum.Heparin in substratum of the present invention can be bought in commercialization.The Main Function of heparin is the activity of the stable growth factor, as FGF.The interpolation concentration of heparin in basic medium is 1-500U.S.P. units/L.EGF also can buy in commercialization, and the interpolation concentration of EGF in basic medium is 0.00001-10mg/L.
The factor that can increase intracellular cAMP levels of multiple kind can be comprised in substratum of the present invention, can be directly increase cAMP level [as two butyryl (dibutyryl) cAMP], also can be cause intracellular cAMP levels to raise [as Toxins,exo-, cholera and forskolin (forskolin)] by interacting with cell G-protein, or increase intracellular cAMP levels as the antagonist (as Racemic isoproterenol) of receptor,β (β-adrenergic receptors), or make intracellular cAMP levels increase [as isobutylmethylxanthine (IBMX) and theophylline (theophylline)] by suppressing the activity of cAMP phosphodiesterase.These compounds above-mentioned can be bought in commercialization.
test kit of the present invention
A second aspect of the present invention relates to a kind of test kit (test kit of the present invention), and it comprises substratum, serum, calcium component and ROCK inhibitor.
Test kit of the present invention is preferred for culturing cell.Described cell is preferably epithelial cell, is more preferably non-keratinocyte epithelial cell.
Described substratum can be any substratum for culturing cell, conventional medium as escribed above, can be preferably above-mentioned conditioned medium.Described serum, calcium component and ROCK inhibitor are with above-mentioned.Test kit of the present invention may be used for the substratum preparing the invention described above.In test kit of the present invention, the ratio of substratum, serum and ROCK inhibitor three preferably meet will three mix after can form the substratum of the invention described above.
Preferably, test kit of the present invention also comprises calcium component, and now, the ratio of substratum, serum, calcium component and ROCK inhibitor preferably meets the substratum by forming the invention described above after four mixing.
Preferably, test kit of the present invention also comprises feeder cell.Described feeder cell are with above-mentioned.Described feeder cell can be conventional adherent, 3-5 days of can surviving under normal temperature; Described feeder cell also can be cryopreservations, and cryopreservation refers to standing storage below-80 DEG C, generally can store several days to several years.Described feeder cell can also be low-temperature storage, and low-temperature storage refers to storage at 4 DEG C, generally can store 1 ~ 7 day.
Preferably, test kit of the present invention also comprises the container for holding separately described feeder cell when carrying out cell cultures, and this container has permeability membrane structure.Particularly, when test kit of the present invention comprises feeder cell, preferably it also comprises described container.The effect of described container is, when carrying out cell cultures, by feeder cell with need cultured cells to keep apart to cultivate, and make the secretory product of feeder cell be entered into the substratum cultivated and need cultured cells by above-mentioned permeability membrane structure.For the shape, material, specification etc. of described container all without particular restriction, those skilled in the art can be suitable for selecting as required.Such as, Transwell basket (Corning Products) can be used.
Preferably, test kit of the present invention comprises substratum of the present invention and feeder cell, more preferably also comprises described container.
Other composition for adding in substratum can also being comprised in test kit of the present invention, including but not limited to the added ingredients described in aforesaid " substratum of the present invention " saves.
Other composition for cell cultures or assembly can also be comprised, such as indicator, pancreatin, culturing bottle, culture dish, working instructions etc. in test kit of the present invention.
In test kit of the present invention, preferred described each composition is separated, is more preferably placed in independently container respectively.
cultural method of the present invention
A third aspect of the present invention relates to the epithelial method of a kind of cultivation (also claiming cultural method of the present invention), and the method comprises
Steps A: use the substratum of the invention described above by epithelial cell and feeder cell Dual culture; Or
Step B: use the above-mentioned culture medium culturing epithelial cell (see Fig. 2) of the present invention comprising feeder cell or conditioned medium.
Cultural method of the present invention can be epithelial cell Secondary Culture method, also can be primary epithelial cells multiplication culture method.
Here, described epithelial cell and feeder cell are all with aforementioned.
Can by epithelial cell and feeder cell Co-culture in steps A, also can by epithelial cell and feeder cell Indirect co-culture.Described Co-culture refers to and epithelial cell and feeder cell is placed in same culture vessel, carries out cultivating (see Fig. 4); Described Indirect co-culture refers to and feeder cell is placed in separately the above-mentioned container (such as Transwell basket) with permeability membrane structure, then, the container that such as again this can be equipped with feeder cell is placed in cultivates epithelial substratum, carries out cultivating (see Fig. 3).
Other step in cultural method of the present invention can be carried out according to the ordinary method of the art.
The inoculum density of cell suitably can adjust according to concrete experiment condition.In conventional disposable plastic culture vessel, general inoculating cell number is 1x10 4~ 10x10 5cell/cm 2, i.e. 75cm 2culturing bottle inoculating cell numerical example is as being 1 x 10 6.Go down to posterity each time and all will require to adjust cell density according to specific experiment.
Mammalian cell is incubated at 37 DEG C, suitable CO usually 2in the incubator of concentration (3 ~ 10%), certain humidity, temperature, CO 2concentration and humidity can adjust according to specific experiment condition.The scope of the pH of substratum can adjust as required, is generally pH7.1 ~ 7.6, is preferably pH7.1 ~ 7.3
Within the usual 1-2 of cell culture medium days, change once, also can adjust according to concrete cultured cells type.As long as cell grows to full abundance in culture vessel, just can carry out having gone down to posterity.In this specification sheets, passage refers to and divides kind of a part in new culture vessel in cell.
Embodiment
Below, by embodiment, more specific description is carried out to the present invention, but the present invention is not by the restriction of these embodiments.In addition, without the reagent of special mark in embodiment, all purchased from GIBCO company.
The subculture of embodiment 1Swiss 3T3 cell
Swiss 3T3 cell is taught laboratory from the Howard Green of Harvard University and is obtained.
Required reagent:
DMEM substratum: DMEM (GIBCO#11965-092) completely, 10% foetal calf serum (FBS), 100U/ml penicillin (penicillin) and 100 μ g/ml Streptomycin sulphate (streptomycin) trysinization liquid: 0.05% pancreatin/EDTA (GIBCO#25300-062)
Without calcium magnesium phosphoric acid buffer: Ca 2+, Mg 2+free PBS
Secondary Culture operation steps:
Inoculation 1x10 5swiss 3T3 cell, in T75 Tissue Culture Dish, adds the complete DMEM substratum that 10ml is above-mentioned, is placed in CO 2incubator is at 5%CO 2, cultivate 2-5 days for 37 DEG C.When cell monolayer abundance is 80-90%, remove the substratum in culture dish, with 10ml without calcium magnesium phosphoric acid buffer washed cell individual layer 2 times, add 2ml trysinization liquid, hatch 1 minute at 37 DEG C.Pat culture dish fully to discharge adherent cell, with in 10ml completely DMEM substratum and digestion reaction.4 DEG C of centrifugal 5 minutes (1000 revs/min) sedimentation cells, remove supernatant, and re-suspended cell is deposited in 10ml and cultivates in DMEM substratum completely.As required can 1: 10,1: 20 or 1: 50 ratio goes down to posterity in the culture dish of new T75 (10-15ml is DMEM substratum completely) or T175 (25-30ml is DMEM substratum completely), changes weekly the fresh substratum of DMEM completely 2-3 time as required.If desired can by 1x10 6cell is resuspended in the cells frozen storing liquid (60%DMEM, 30% foetal calf serum and 10%DMSO) of 1-2ml, is stored in liquid nitrogen for subsequent use.
Embodiment 2HL substratum
HL substratum 1 is the mixed culture medium of DMEM (GIBCO#11965-092) and Ham ' s F-12NUTRIENTMIX (GIBCO#11765-054), volume proportion is 3: 1, add the foetal calf serum of 5% simultaneously, and 0.4 μ g/ml hydrocortisone (hydrocortisone), 5 μ g/ml Regular Insulin (insulin), 8.4ng/ml Toxins,exo-, cholera (cholera toxin), 10ng/ml Urogastron (epithelial growthfactor (EGF)), 24 μ g/ml VITAMIN B4 (adenine), 100U/ml penicillin (penicillin) and 100 μ g/ml Streptomycin sulphates (streptomycin), 0.25 μ g/ml amphotericin B (Fungizone, ) 30 μMs of fasudils (Fasudil) (or 0.5 μM of H-1152 or 5 μM Y-27632, 30 μMs of HA1100, 10uM GSK429286), above-mentioned substratum need through 0.22 μ aperture membrane filtration.
HL substratum 2 is at DMEM (Low Glucose, No Glutamine, GIBCO#11054-020) foetal calf serum of 10% is added in, and 0.4 μ g/ml hydrocortisone (hydrocortisone), 5 μ g/ml Regular Insulin (insulin), 8.4ng/ml Toxins,exo-, cholera (cholera toxin), 10ng/ml Urogastron (epithelial growth factor (EGF)), 24 μ g/ml VITAMIN B4 (adenine), 100U/ml penicillin (penicillin) and 100 μ g/ml Streptomycin sulphates (streptomycin), 0.25 μ g/ml amphotericin B (Fungizone, ) 30 μMs of fasudils (Fasudil) (or 0.5 μM of H-1152 or 5 μM Y-27632, 30 μMs of HA1100, 10 μMs of GSK429286), above-mentioned substratum need through 0.22 μ aperture membrane filtration.
HL substratum 3 is that RPMI substratum 1640 (GIBCO#22400-121) is containing 5mMHEPES, add the foetal calf serum of 8%, and 0.4 μ g/ml hydrocortisone (hydrocortisone), 5 μ g/ml Regular Insulin (insulin), 8.4ng/ml Toxins,exo-, cholera (cholera toxin), 10ng/ml Urogastron (epithelial growth factor (EGF)), 24 μ g/ml VITAMIN B4 (adenine), 100U/ml penicillin (penicillin) and 100 μ g/ml Streptomycin sulphates (streptomycin), 0.25 μ g/ml amphotericin B (Fungizone, ) 30 μMs of fasudils (Fasudil) (or 0.5 μM of H-1152 or 5 μM Y-27632, 30 μMs of HA1100, 10 μMs of GSK429286), above-mentioned substratum need through 0.22 μ aperture membrane filtration.
HL substratum 4 is at L-15 (Leibovitz) substratum (L-glutamine, No HEPES, GIBCO#11415064) foetal calf serum of 5% is added in, and 0.4 μ g/ml hydrocortisone (hydrocortisone), 5 μ g/ml Regular Insulin (insulin), 8.4ng/ml Toxins,exo-, cholera (choleratoxin), 10ng/ml Urogastron (epithelial growth factor (EGF)), 24 μ g/ml VITAMIN B4 (adenine), 100U/ml penicillin (penicillin) and 100 μ g/ml Streptomycin sulphates (streptomycin), 0.25 μ g/ml amphotericin B (Fungizone, ) 30 μMs of fasudils (Fasudil) (or 0.5 μM of H-1152 or 5 μM Y-27632, 30 μMs of HA1100, 10 μMs of GSK429286), above-mentioned substratum need through 0.22 μ aperture membrane filtration.
HL substratum 5 is at substratum 199 (with Earle ' s salts but no L-glutamine, nosodium bicarbonate, GIBCO#11825015) foetal calf serum of 10% is added in, and 0.4 μ g/ml hydrocortisone (hydrocortisone), 5 μ g/ml Regular Insulin (insulin), 8.4ng/ml Toxins,exo-, cholera (cholera toxin), 10ng/ml Urogastron (epithelial growth factor (EGF)), 24 μ g/ml VITAMIN B4 (adenine), 100U/ml penicillin (penicillin) and 100 μ g/ml Streptomycin sulphates (streptomycin), 0.25 μ g/ml amphotericin B (Fungizone, ) 30 μMs of fasudils (Fasudil) (or 0.5 μM of H-1152 or 5 μM Y-27632, 30 μMs of HA1100, 10 μMs of GSK429286), above-mentioned substratum need through 0.22 μ aperture membrane filtration.
HL substratum 6 is DMEM (GIBCO#11965-092) and the serum free medium SFM (mixed culture medium of GIBCO#10744-019, volume proportion is 1: 3, add the foetal calf serum of 5% simultaneously, and 0.4 μ g/ml hydrocortisone (hydrocortisone), 5 μ g/ml Regular Insulin (insulin), 8.4ng/ml Toxins,exo-, cholera (cholera toxin), 10ng/ml Urogastron (epithelial growthfactor (EGF)), 24 μ g/ml VITAMIN B4 (adenine), 100U/ml penicillin (penicillin) and 100 μ g/ml Streptomycin sulphates (streptomycin), 0.25 μ g/ml amphotericin B (Fungizone, ) 30 μMs of fasudils (Fasudil) (or 0.5 μM of H-1152 or 5 μM Y-27632, 30 μMs of HA1100, 10 μMs of GSK429286), above-mentioned substratum need through 0.22 μ aperture membrane filtration.
The preparation (Mitomycin C process swiss 3T3 cell) of embodiment 3 feeder cell (losing the Swiss 3T3 cell of splitting ability)
1) as the swiss 3T3 cell of feeder cell, the cell that Secondary Culture is early stage should be adopted.When swiss 3T3 Growth of Cells is to when expiring abundance, add the Mitomycin C (water-soluble, storage liquid concentration is 0.5mg/mL) that final concentration is 10 μ g/mL in substratum, 37 DEG C process 2 hours;
2) substratum (DMEM) of the 1xPBS or serum-free that add temperature bath again washes 3 times, abandons washing lotion;
3) add 0.05% pancreatin/EDTA predigestion cell 30-40 to discard after second, again add 0.05% pancreatin/EDTA and digest 30 seconds, pat culture dish and make cell dispersal, then add perfect medium (DMEM containing 10% foetal calf serum) neutralization reaction;
4) low-speed centrifugal (1000rpm) removes supernatant, obtains cell precipitation;
5) cell precipitated directly can be used as feeder cell, or stores for subsequent use;
A) be directly used as the situation of feeder cell, operation steps is as follows:
-add the warm 1xPBS bathed to wash cell precipitation 1 time, recentrifuge
-cell is suspended from HL substratum
-in the proper ratio (as 1: 3) epithelial cell of feeder cell and some amount is suspended in HL substratum, the suitableeest inoculum density of epithelial cell is 2.5x10 5the culture dish of individual/100mm;
B) feeder cell are second day situations for subsequent use, and operation steps is as follows:
-add the warm 1xPBS bathed to wash cell precipitation 1 time, recentrifuge
-cell is suspended from complete DMEM substratum (DMEM containing 10% foetal calf serum)
-ratio with 1: 3 is inoculated in culture dish, overnight incubation
Within-the second day, wash cell 2 times with the 1xPBS of temperature bath, change HL substratum into
-being inoculated in epithelial cell containing feeder cell culture dish again, the suitableeest inoculum density of epithelial cell is 2.5x10 5the culture dish of individual/100mm;
C) feeder cell store situation for subsequent use, and operation steps is as follows:
-add the warm 1xPBS bathed to wash cell precipitation 1 time
-feeder cell are resuspended in the frozen storing liquid (90% foetal calf serum and 10%DMSO) of 6ml, divide in 3 cryopreservation tubes
-frozen under liquid nitrogen or-150 DEG C of conditions
-face the used time to get 1 recovery, add complete DMEM substratum (DMEM containing 10% foetal calf serum) and be inoculated in 100mm culture dish
Remarks: be used as the 3T3 cell of feeder cell after Mitomycin C process, need use in 3-5 days or cryopreservation for subsequent use.
The preparation (radioactive rays process swiss 3T3 cell) of embodiment 4. feeder cell (losing the Swiss 3T3 cell of splitting ability)
As the swiss 3T3 cell of feeder cell, should adopt the cell that Secondary Culture is early stage, these cell cultures, in DMEM substratum, grow to nearly completely abundance, with not containing Ca 2+/ Mg 2+1xPBS wash cell 1 time, add 1ml 0.05% trypsin digestion cell 20-40 second, then add in the complete DMEM substratum of 9ml (DMEM containing 10% foetal calf serum) and digestion reaction, in 4 DEG C of low-speed centrifugal 1000rmp collecting cell precipitations, add 10ml DMEM re-suspended cell, radiation exposure cell suspension, namely Cesium-137 alpha cellulose a gage (JL Shepherd Mark) processes 3000Rad (30Grey).These are through the swiss 3T3 cell of radioactive rays process, are inoculated in HL substratum and are directly used as feeder cell; Or be incubated at complete DMEM substratum or be placed in 4 DEG C and store and used within 1-7 days; Or Long-term Cryopreservation is for subsequent use in-80 DEG C or liquid nitrogen.
Separation and Culture primary epithelial cells in embodiment 5. flesh tissue
When patient or patient care people informed consent, the normal or neoplasmic tissue sample of collector.Concrete operation step is as follows:
1. the preparation of Digestive system: containing the HL substratum (i.e. the mixed culture medium of DMEM: F12 volume ratio 3: 1) of collagenase/Unidasa (final concentration is the mixed solution of 1x), adds 5%FBS.
Determine the consumption of Digestive system according to the size of flesh tissue, usually need 10 times to the consumption of the Digestive system of sample volume, such as 2-3 mouse prostate needs 2-5ml Digestive system;
2. wash the flesh tissue 1 time of separation with the ethanol of 95-100%, then wash 2 times with PBS, then by organizing the sterile petri dish put into containing precooling PBS, under dissecting microscope, with dissecting tweezers and fat residual in tissue removed by scissors.
3. tissue sample is put into 14ml or the 50ml centrifuge tube containing above-mentioned Digestive system, 37 DEG C of digestion 1-3 hour.
4. organize low-speed centrifugal (1000rpm) 5 minutes by postdigestive, remove supernatant.
5. cell precipitation is resuspended in the 0.25% pancreatin/EDTA of 2-5mL, is placed in 1 hour or room temperature 10 minutes on ice.
6. then add the DMEM substratum of 10ml containing 10%FBS, centrifugal 5 minutes of low speed 1000rmp.
7. supernatant is removed clean as far as possible.
8. add the 5mg/mL Dispase of 2ml temperature bath and the 1mg/mL DNase I of 200 μ L, repeatedly blow and beat sample 1 minute with aseptic P1000 disposable plastic rifle head.
9. add the DMEM of 10ml containing 10%FBS, with the metre filter cell suspension in 40-70 μm of aperture, collect the cell suspension after filtering, centrifugal 5 minutes of low speed 1000rmp, remove supernatant.
10. re-suspended cell is deposited in HL substratum, is inoculated in the culturing bottle of T25 or T75 and cultivates by either a program of the present invention (see Fig. 1-4).
Embodiment 6. feeder cell/epithelial Co-culture (see Fig. 4 and Fig. 6)
The feeder cell (losing the Swiss3T3 cell of splitting ability) of above-mentioned Mitomycin C process or radiation exposure are undertaken by following three kinds of modes as feeder layer and epithelial Dual culture:
1. culturing directly cell/epithelial cell Dual culture.The 1xPBS adding temperature bath washes feeder cell and precipitates 1 time, recentrifuge, and be suspended from by cell in HL substratum, be suspended in HL substratum with 1: 3 ratio by the epithelial cell of feeder cell and some amount, the suitableeest inoculum density of epithelial cell is 2.5x10 5the culture dish of individual/100mm, is placed in 37 DEG C, 5%CO by culturing bottle 2cultivate under condition.
2. feeder cell are adherent in advance, then add epithelial cell Dual culture.The feeder cell of above-mentioned Mitomycin C process or radiation exposure are suspended from complete DMEM substratum (DMEM containing 10% foetal calf serum), ratio with 1: 3 is inoculated in culture dish, cultivate 2-3 hour or spend the night for 37 DEG C, cell is washed 2 times with the 1xPBS of temperature bath, change HL substratum into, be inoculated in by epithelial cell in the culture dish containing feeder cell, the suitableeest inoculum density of epithelial cell is 2.5x10 again 5the culture dish of individual/100mm, adherent feeder cell can use within 3-5 days any times.Culturing bottle is placed in 37 DEG C, 5%CO 2cultivate under condition.
3. feeder cell 4 DEG C or liquid nitrogen storage for subsequent use.The feeder cell of above-mentioned Mitomycin C process or radiation exposure are suspended from complete DMEM substratum (DMEM containing 10% foetal calf serum) and are stored in 4 DEG C (1-7 days) or standing storage in liquid nitrogen, 2 to carry out and epithelial Dual culture (in HL substratum) as stated above after recovery.The suitableeest inoculum density of epithelial cell is 2.5x10 5the culture dish of individual/100mm.Culturing bottle is placed in 37 DEG C, cultivates under 5%CO2 condition.
No matter with the Dual culture of which kind of mode above-mentioned, fresh HL substratum within second day, should be changed.When the feeder cell more than 50% come off, fresh above-mentioned Mitomycin C process or the feeder cell of radiation exposure should be supplemented, until when epithelial cell growth to 70-90% abundance needs to go down to posterity.
Embodiment 7. feeder cell/epithelial separation is gone down to posterity
1. epithelial cell growth is to 70-90% abundance, sucking-off substratum, washes 1 time with the PBS of temperature bath,
2. sucking-off PBS, adds the PBS washed cell containing 0.02%EDTA (0.68mM) of 10-12mL temperature bath, to remove feeder cell Swiss 3T3; Pay special attention to, washed cell need be tried one's best fast, first EDTA solution is added to the cell of culture dish periphery, rotates wave and culture ware, and then EDTA solution is circled round to the cell of culture dish inner circumferential, repeats convolution washed cell 10 times.
3. sucking-off EDTA washing lotion, adds the cell inside 0.02%EDTA to culture dish again, rotates wave and culture ware, the cell then making EDTA solution circle round to culture dish, repeats convolution washed cell 30 times.The Eponychium cell of usual people needs 40 EDTA washings, and other cell is different according to adherent tightness degree, suitably can adjust washing times.
4. sucking-off EDTA washing lotion, the PBS adding temperature bath washes 3 times, remains that culture dish tilts.Before last washings sucking-off, examine under a microscope, confirm that all feeder cell Swiss 3T3 are washed off completely, otherwise, need again increase EDTA washing times.
5. adopt conventional pancreatin method digestive epithelium cell, for Eponychium cell, pancreatin normal temperature digests 2 minutes, sucking-off Digestive system, and again in 37 DEG C of trysinizations 5 minutes, pat culture dish and make cell dispersal, re-suspended cell is in HL substratum.Then inoculate epithelial cell on the feeder cell of ametycin or radioactive rays process, optimal cell inoculum density is 2.5x10 5the culture dish of individual/100mm;
7. if desired can by 1x10 6epithelial cell is resuspended in the cells frozen storing liquid (90% foetal calf serum and 10%DMSO) of 1-2ml, is stored in liquid nitrogen for subsequent use.
The preparation of embodiment 8. feeder cell conditioned medium and epithelial subculture (see Fig. 2 and Fig. 6)
According to Fig. 2, the feeder cell of above-mentioned Mitomycin C process or radiation exposure are incubated at (T75 culturing bottle: 2x10 in HL substratum 6cell, 15ml HL substratum; T175: 5x10 6cell, 35mlHL substratum) at 37 DEG C, 5%CO 2incubator in cultivate 48 hours.Collect substratum, centrifugal 15 minutes of 2000rpm, through 0.22 μ membrane filtration, mix with freshly prepared HL substratum in 1: 5 ratio (can optimize by 1: 99-99: 1) and namely obtain HL conditioned medium.The primary cell suspension or passage cell that are separated preparation are directly cultivated in HL conditioned medium, culture condition is 37 DEG C, 5%CO 2.When cell proliferation is to 70-90% abundance, 1xPBS washed cell twice, 0.05% pancreatin/EDTA digests monolayer cell 2-5 minute, and 10ml is completely in DMEM and digestion reaction, centrifugal 5 minutes of 1000rmp, remove supernatant, with certain proportion (1: 2,1: 3,1: 4,1: 5, as long as cell attachment and normal growth can be maintained) re-suspended cell is deposited in inoculation culture in 10ml HL conditioned medium.If desired can by 1x10 6epithelial cell is resuspended in the cells frozen storing liquid (90% foetal calf serum and 10%DMSO) of 1-2ml, is stored in liquid nitrogen for subsequent use.
Embodiment 9. epithelial cells/feeder cell Indirect co-culture (Transwell culture systems) and go down to posterity (see Fig. 3 and Fig. 6)
According to Fig. 3, the feeder cell of above-mentioned Mitomycin C process or radiation exposure are placed in Transwell basket (Transwell insert, Corning company) in, the primary cell suspension or the passage cell that are separated preparation can directly be cultivated in HL substratum, and culture condition is 37 DEG C, 5%CO 2.When cell proliferation is to 70-90% abundance, first remove Transwell basket, 1xPBS washs epithelial cell twice, and 0.05% pancreatin/EDTA digests monolayer cell 2-5 minute, 10ml is completely in DMEM and digestion reaction, centrifugal 5 minutes of 1000rpm, removes supernatant, with certain proportion (1: 2,1: 3,1: 4,1: 5, as long as cell attachment and normal growth can be maintained) re-suspended cell is deposited in 10ml HL inoculation of medium and cultivates.If desired can by 1x10 6epithelial cell is resuspended in the cells frozen storing liquid (90% foetal calf serum and 10%DMSO) of 1-2ml, is stored in liquid nitrogen for subsequent use.
Embodiment 10. is used for epithelial cultivation (see Fig. 5) containing blood serum medium and serum free medium
DMEM substratum: DMEM completely, 10% foetal calf serum (FBS), 100U/ml penicillin (penicillin) and 100 μ g/ml Streptomycin sulphates (streptomycin)
Serum free medium: the epithelial cell substratum SFM (GIBCO#10744-019) of serum-free, gentamicin (10 μ g/m1)
Trysinization liquid: 0.05% pancreatin/EDTA
Without calcium magnesium phosphoric acid buffer: Ca 2+, Mg 2+free PBS
The primary cell suspension or the passage cell that are separated preparation are directly cultivated in above-mentioned complete DMEM substratum or serum free medium, and culture condition is 37 DEG C, 5%CO 2.When cell proliferation is to 70-90% abundance, 1xPBS washed cell twice, 0.05% pancreatin/EDTA digests monolayer cell 2-5 minute, 10ml is completely in DMEM substratum and digestion reaction, centrifugal 5 minutes of 1000rmp, remove supernatant, be deposited in the complete DMEM of 10ml or serum free medium with certain proportion (normally 1: 2 or 1: 3) re-suspended cell and cultivate.If desired can by 1x10 6cell is resuspended in the cells frozen storing liquid (90% foetal calf serum and 10%DMSO) of 1-2ml, is stored in liquid nitrogen for subsequent use.
Also it should be noted that, can implement and not obvious run counter to the prerequisite of purport of the present invention under, in this manual as a certain technical scheme component part described by arbitrary technical characteristic or the combination of technical characteristic equally also go for other technical scheme; Further, can implement and not obvious run counter to the prerequisite of purport of the present invention under, as different technologies scheme component part described by technical characteristic between also can combine in any way, form other technical scheme.The technical scheme that the present invention obtains by combining under being also contained in above-mentioned situation, and these technical schemes are equivalent to record in this manual.
Below describe the present invention through the specific embodiment and the embodiment, but it will be understood by those skilled in the art that these are not intended to limit scope of the present invention, scope of the present invention should be determined by claims.
Industrial applicibility
Substratum of the present invention, cell cultures test kit, epithelial cell cultural method and the every field such as basal cell biological study, tumor research, aging research, new drug development, gene therapy, external transgenosis and gene knockout research, tissue regeneration, clinical regenerative medicine, clinical personalized treatment, molecule and genetic epidemiology research can be widely used in by the epithelial cell that the method obtains.

Claims (33)

1. substratum is for cultivating an epithelial purposes, and wherein, described substratum comprises:
Serum,
Calcium component,
Feeder cell or comprise the conditioned medium of feeder cell secretory product, described feeder cell are the Swiss 3T3 cell losing splitting ability, and
ROCK inhibitor, wherein when described ROCK inhibitor is fasudil, Y-27632, HA1100 or GSK429286, its content is in the medium 1-50 μM; When described ROCK inhibitor is H-1152, its content is in the medium 0.5-50 μM;
Described epithelial cell is tumour cell.
2. purposes according to claim 1, wherein, in described substratum, the content of serum is 1.0 ~ 35 v/v%.
3. purposes according to claim 1, wherein, described ROCK inhibitor be selected from fasudil, H-1152, Y-27632, HA1100 and GSK429286 one or more.
4. purposes according to claim 1, wherein, described epithelial cell is squamous cell, columnar epithelial cell, gland epithelial cells or transitional type epithelial cell.
5. purposes according to claim 1, wherein, described epithelial cell is oral cavity, nasal mucosa, tracheae, bronchial, lung epithelial, epithelium of esophagus, gastric epithelial, large intestine epithelium, small intestine epithelium, hepatocellular, epithelial duct, gall-bladder, pancreas, kidney, bladder, urothelial, testis epithelium, ovarian epithelium, thyroid, follicular epithelial cell, adrenal, thymus gland, prostatic, mammary gland, tonsilar, the cell of hypophysis, corneal epithelial cell, retinal pigment epithelium.
6. purposes according to claim 1, wherein, in described substratum, the content of calcium component counts 0.1 μM ~ 30 mM with calcium ion.
7. test kit is for cultivating an epithelial purposes, wherein, described test kit comprise substratum, serum, ROCK inhibitor and
Feeder cell or comprise the conditioned medium of feeder cell secretory product, described feeder cell are the Swiss 3T3 cell losing splitting ability;
Wherein when described ROCK inhibitor is fasudil, Y-27632, HA1100 or GSK429286, its content is in the medium 1-50 μM; When described ROCK inhibitor is H-1152, its content is in the medium 0.5-50 μM,
Wherein said epithelial cell is tumour cell.
8. purposes according to claim 7, wherein, described epithelial cell is squamous cell, columnar epithelial cell, gland epithelial cells or transitional type epithelial cell.
9. purposes according to claim 7, wherein, described epithelial cell is oral cavity, nasal mucosa, tracheae, bronchial, lung epithelial, epithelium of esophagus, gastric epithelial, large intestine epithelium, small intestine epithelium, hepatocellular, epithelial duct, gall-bladder, pancreas, kidney, bladder, urothelial, testis epithelium, ovarian epithelium, thyroid, follicular epithelial cell, adrenal, thymus gland, prostatic, mammary gland, tonsilar, the cell of hypophysis, corneal epithelial cell, retinal pigment epithelium.
10. purposes according to claim 7, wherein, described test kit also comprises calcium component.
11. purposes according to claim 7, wherein, described feeder cell are cryopreservations.
12. purposes according to claim 7, wherein, described feeder cell are low-temperature storage.
13. purposes according to claim 7, wherein, described test kit also comprises the container for holding separately described feeder cell when carrying out cell cultures, and this container has permeability membrane structure.
14. purposes according to claim 7, wherein, described ROCK inhibitor be selected from fasudil, H-1152, Y-27632, HA1100 and GSK429286 one or more.
Cultivate epithelial method for 15. 1 kinds, the method comprises
Steps A: use the substratum comprising serum, calcium component and ROCK inhibitor by epithelial cell and feeder cell Dual culture;
Wherein, described epithelial cell is tumour cell, described feeder cell are the Swiss 3T3 cell losing splitting ability, and wherein when described ROCK inhibitor is fasudil, Y-27632, HA1100 or GSK429286, its content is in the medium 1-50 μM; When described ROCK inhibitor is H-1152, its content is in the medium 0.5-50 μM.
The epithelial method of 16. cultivation according to claim 15, wherein, in described substratum, the content of serum is 1.0 ~ 35 v/v%.
17. methods according to claim 15, wherein, described ROCK inhibitor be selected from fasudil, H-1152, Y-27632, HA1100 and GSK429286 one or more.
The epithelial method of 18. cultivation according to claim 15, wherein, described epithelial cell is squamous cell, columnar epithelial cell, gland epithelial cells or transitional type epithelial cell.
The epithelial method of 19. cultivation according to claim 15, wherein, described epithelial cell is oral cavity, nasal mucosa, tracheae, bronchial, lung epithelial, epithelium of esophagus, gastric epithelial, large intestine epithelium, small intestine epithelium, hepatocellular, epithelial duct, gall-bladder, pancreas, kidney, bladder, urothelial, testis epithelium, ovarian epithelium, thyroid, follicular epithelial cell, adrenal, thymus gland, prostatic, mammary gland, tonsilar, the cell of hypophysis, corneal epithelial cell, retinal pigment epithelium.
The epithelial method of 20. cultivation according to claim 15, wherein, in described substratum, the content of calcium component counts 0.1 μM ~ 30 mM with calcium ion.
The epithelial method of 21. cultivation according to claim 15, wherein, comprises the conditioned medium containing feeder cell secretory product in described substratum.
The epithelial method of 22. cultivation according to claim 15, wherein, the epithelial method of this cultivation is epithelial cell Secondary Culture method.
The epithelial method of 23. cultivation according to claim 15, wherein, the epithelial method of this cultivation is primary epithelial cells multiplication culture method.
The epithelial method of 24. cultivation according to claim 15, wherein, is placed in same culture vessel by epithelial cell and feeder cell.
Feeder cell wherein, are placed in separately the container with permeability membrane structure by the epithelial method of 25. cultivation according to claim 15.
Cultivate epithelial method for 26. 1 kinds, the method comprises
Step B: use the culture medium culturing epithelial cell comprising serum, calcium component and ROCK inhibitor; Wherein said substratum also comprises feeder cell or comprises the conditioned medium of feeder cell secretory product, described epithelial cell is tumour cell, described feeder cell are the Swiss 3T3 cell losing splitting ability, wherein when described ROCK inhibitor is fasudil, Y-27632, HA1100 or GSK429286, its content is in the medium 1-50 μM; When described ROCK inhibitor is H-1152, its content is in the medium 0.5-50 μM.
The epithelial method of 27. cultivation according to claim 26, wherein, in described substratum, the content of serum is 1.0 ~ 35 v/v%.
The epithelial method of 28. cultivation according to claim 26, wherein, described ROCK inhibitor be selected from fasudil, H-1152, Y-27632, HA1100 and GSK429286 one or more.
The epithelial method of 29. cultivation according to claim 26, wherein, described epithelial cell is squamous cell, columnar epithelial cell, gland epithelial cells or transitional type epithelial cell.
The epithelial method of 30. cultivation according to claim 26, wherein, described epithelial cell is oral cavity, nasal mucosa, tracheae, bronchial, lung epithelial, epithelium of esophagus, gastric epithelial, large intestine epithelium, small intestine epithelium, hepatocellular, epithelial duct, gall-bladder, pancreas, kidney, bladder, urothelial, testis epithelium, ovarian epithelium, thyroid, follicular epithelial cell, adrenal, thymus gland, prostatic, mammary gland, tonsilar, the cell of hypophysis, corneal epithelial cell, retinal pigment epithelium.
The epithelial method of 31. cultivation according to claim 26, wherein, in described substratum, the content of calcium component counts 0.1 μM ~ 30 mM with calcium ion.
The epithelial method of 32. cultivation according to claim 26, wherein, the epithelial method of this cultivation is epithelial cell Secondary Culture method.
The epithelial method of 33. cultivation according to claim 26, wherein, the epithelial method of this cultivation is primary epithelial cells multiplication culture method.
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