CN107142237A - Culture medium, cell culture kit and cell culture processes - Google Patents

Culture medium, cell culture kit and cell culture processes Download PDF

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CN107142237A
CN107142237A CN201710271857.0A CN201710271857A CN107142237A CN 107142237 A CN107142237 A CN 107142237A CN 201710271857 A CN201710271857 A CN 201710271857A CN 107142237 A CN107142237 A CN 107142237A
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李晖
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Shenzhen Yongtai Biotechnology Co., Ltd
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Abstract

The present invention relates to culture medium, cell culture kit and cell culture processes, culture medium, cell culture kit and cell culture processes particularly for epithelial cell culture.The culture medium includes serum, calcium component and ROCK inhibitor.

Description

Culture medium, cell culture kit and cell culture processes
The application is Application No. 201110127667.4, entitled culture medium, cell culture kit and thin The divisional application of the Chinese invention patent application (applying date is on May 17th, 2011) of born of the same parents' cultural method.
Technical field
The present invention relates to culture medium, cell culture kit and cell culture processes, particularly trained for epithelial cell Foster culture medium, cell culture kit and cell culture processes.
Background technology
The human body vitals epithelial cell that for example lung, kidney, liver, pancreas and skin are all broken up by organ specificity constitutes for it Composition.The epithelial cell of these specific differentiations is directly related with the specific function of Different Organs, such as the gas exchange work of lung Energy, the filtering function of kidney, the removing toxic substances of liver and neutralization function, pancreatic cell generation insulin, skin, which have, protects body from outer The injury of boundary's environment.And these vitals will threaten the health of the mankind once occurring lesion or degeneration, because these are important Organ is difficult to be replaced, and the specific cell of Different Organs can not mutually replace the cell of other organs.
The cell of specific differentiation, such as renal epithelial cell, the insulin-producing cells of pancreas, the hair follicle of corium and body of gland are thin It is difficult regeneration that born of the same parents, which are, and culture is let alone carried out in vitro and is bred.Experimental animal (including various transgenosis and clone move Thing) it is that the essential tool with new drug development is studied for disease pathogenesis.But, the original for being isolated from people and mammal In vitro culture for epithelial cell is still highly difficult.Using current serum free medium, can only carrying out for having is short-term Original cuiture (survival a couple of days, such as lung and pancreas epithelial cell), what is had can only carry out limited Secondary Culture (1-3 generations, such as gas The epithelial cell such as pipe/bronchus and prostate), what is had is even unable in vitro culture (such as liver and colon epithelial cell) at all, The superficial cell for only coming from human body skin can be with or so generation of Secondary Culture 10.And from each animal or biological tissue The epithelial cell yield obtained in sample is still very low, including cell quantity, be directly separated or Short-term Culture cell purity All it is very low, these primary and passage epithelial cell growth rates are also extremely limited.
It is external at present to attempt, by genetic manipulation, to be such as transferred to virus in order to carry out the culture of epithelial cell in vitro (SV40T or HPV16E6E7) or cellular oncogene, can extend the algebraically of external cell survival.But the maximum of genetic manipulation Have the disadvantage the genetic background and phenotype that can change these cells, so that allowing these normal epithelium cells to lose its normal physiological work( Can, such as p53 and pRB signal paths are usually suppressed.Moreover, these genetically modified cells be impossible transplant again again into Body.But it is due to the technology for lacking effective in vitro culture epithelial cell at present, these above-mentioned cells are in the world today Still favor is enjoyed in medical science and life science.In non-cancer research field, they just represent the " work(initially originated The tissue or organ of energy " property.In cancer research field, they are also frequently used to compare as " normal cell ".And they The market price it is also very expensive.
The first step of antineoplastic research is exactly that medicine is tested to the specific toxicities of cancer cell, and presently used cancer is thin Several years even many decades are passed in born of the same parents' strain (such as HeLa and A539) in vitro more, and they can not largely react similar Also there is the cross pollution of a large amount of JEG-3s in the characteristic of type human malignant tumor, some laboratories.And for different Body, even if same type of tumour may also be by the reason for different or caused by mechanism, therefore for each tumour, is required for The cell line of its specific pathogenesis can be represented to carry out new drug development by having.The research of more prominent example, such as prostate cancer Cell line of the field at all without the primary prostate cancer of the mankind, only cell line (LnCAP, PC-3, DU-145) is from Tumor tissues after transfer.Nevertheless, the research of the tumor research in the whole world more than 90% and nearly all PTS is also It is being continuing with may not having representational JEG-3 at all.Therefore, current cancer research and new drug development to be solved It is a key issue that how to obtain reaction all kinds human tumor characteristic, same tumor represents Different Individual characteristic or difference The cancer cell of pathogenesis.Certainly, real problem after all is a lack of the primary and Secondary Culture of effective epithelial cell Technology.In addition, many toxic side effects that antineoplastic is present, are largely because new drug research and lack normal cell Control.If obtaining coming from the cancer cell and normal cell of same individual same tissue, (Asterand companies of the U.S. are with above-mentioned So-called " normal " and the prostatic cell of cancer that HPVE6E7 conversions are obtained, although price up to 1,2000 U.S. dollars/right, also It is very in great demand), the first step of new drug development will return science, and the history of PTS research will be rewritten.
Regenerative medicine is a kind of brand-new Therapeutic mode of current clinical medicine, i.e., using regenerate, reconstruction, instead of and new life as The modern cellular replacement therapy art of base therapy principle, has to each using stem cell (embryonic stem cell and adult stem cell) Cell differentiation transformation ability is planted, denaturation, gangrenosum acne and damaging disease clinically numerous, that there is no treatment method at present is treated Disease, with notable and unique medical effect.But the predicament faced at present is, although embryonic stem cell has differentiation capability, Reproducibility and plasticity are strong, but tumorigenesis and may face dispute of ethic, how to induce surviving for its directed differentiation and increase transplanting Rate is still the problem not solved；And adult stem cell source is rare, it is difficult to recognize, separate and purify, therefore it is clinical to hinder it Using；Study hotspot --- inductive pluripotent stem cells emerging at present, cause cytogenetics to be carried on the back because it is related to genetic manipulation The change of scape, and import foreign gene and viral gene have carcinogenic or teratogenesis potential and cannot be used for clinical treatment.
Individuality medicine is the developing direction of modern medicine from now on.It is relevant according to the disease of clinical patient generation/development Genetic background and correlative factor, are diagnosed to disease, are monitored exactly within the shortest time, formulate real individualized treatment Scheme is the key solved the problems, such as.For example, World Health Organization's investigation finds that safety of medicine sex chromosome mosaicism is that inpatient is lethal most One of important the reason for.The important public health that adverse reaction caused by drug response individual difference has turned into harm human health is asked Topic.And inherent cause is to cause the main cause of drug response individual difference.In the U.S., there are more than 70 kind medicines through FDA batches Standard has sticked genetic tags, for indicating that prediction of the clinical patients of different genotype in drug application to curative effect and toxicity is made With, and clearly new drug development will turn into the trend of future development to genetic background.How extremely micro biopsy is utilized It is to tumour that (such as pin inhales cell) carries out Rapid＆Early diagnosis, In vitro chemo-drug sensitive test, curative effect monitoring to diseases such as malignant tumours The key of individual's treatment.It is all these all will could be really real dependent on fast and effectively primitive cell culture technology It is existing.
Biobank (Times is referred to as to change one of the ten big ideas in the world) just rises in worldwide.The U.S. The researcher of NIH just makes great efforts to create the first preservation mankind biological organization sample in the whole America, tumour cell, DNA, And the national biobank of blood.English plus, Norway and Sweden set up this kind of nationwide mechanism.Collect and store Enough, from cancer, the biological organization sample of brain disorder to disease of metabolism, ground to set up individuality medicine and new drug Hair lays the foundation, and makes screening and treatment patient more targeted.But simply include frost group stored by biobank at present Knit, fixed tissue, DNA, RNA, protein, blood, blood plasma, serum and urine etc., these extremely valuable resources are with it Go, it is impossible to regenerate.Effective primary and passage cell culture technique will be real " work " power of biobank injection.This tool The appearance for having the biobank of " regeneration " ability will have inner to modern medicine study, Individual Diagnosis and treatment, new drug development Journey upright stone tablet meaning.
In summary, current urgent problem be how quickly and efficiently to carry out histoorgan source it is primary on The Secondary Culture of the propagation of chrotoplast and these epithelial cells, and can effectively extend the culture algebraically of epithelial cell, together When can not change the genetic background of cell again.
The content of the invention
The present inventor has made intensive studies regarding to the issue above, it is proposed that new culture medium and kit, by using The culture medium or kit in-vitro multiplication primary epithelial cells and Secondary Culture epithelial cell, can not change the cytogenetics back of the body In the case of scape, effectively extend the culture algebraically of epithelial cell.
That is, the present invention provides following culture mediums (the also referred to as culture medium of the present invention)：
1. a kind of culture medium, it is included
Serum,
Calcium component, and
ROCK inhibitor.
2. the culture medium according to item 1, wherein, the content of the serum is 1.0~35v/v%.
3. the culture medium according to item 1, wherein, the ROCK inhibitor is to be selected from Fasudil, H-1152, Y- 27632nd, more than one or both of HA1100, HA1077 and GSK429286.
4. the culture medium according to item 1, it is used to cultivate epithelial cell.
5. the culture medium according to item 4, wherein, the epithelial cell is non-Eponychium cell.
6. the culture medium according to item 4, wherein, the epithelial cell is primary cell.
7. the culture medium according to item 4, wherein, the epithelial cell is passage cell.
8. the culture medium according to item 4, wherein, the epithelial cell is tumour cell.
9. the culture medium according to item 5, wherein, the non-keratinocyte epithelial cell is squamous cell, columnar epithelium Cell, gland epithelial cells or transitional type epithelial cell.
10. the culture medium according to item 5, wherein, the non-keratinocyte epithelial cell is oral cavity, schneiderian membrane, tracheae , it is bronchial, lung epithelial, epithelium of esophagus, gastric epithelial, large intestine epithelium, small intestine epithelium, liver cell, Epithelial duct, gall-bladder, pancreas, beta Cell of islet, kidney, bladder, urothelial, testis epithelium, ovarian epithelium , thyroid, follicular epithelial cell, adrenal, thymus gland, prostate, mammary gland, tonsilar, hypophysis Cell, corneal epithelial cell, retinal pigment epithelium.
11. the culture medium according to item 1, wherein, the content of the calcium component is calculated as 0.1 μM~30mM with calcium ion.
12. the culture medium according to item 1, wherein, the culture medium also includes feeder cells.
13. the culture medium according to item 1, wherein, conditioned medium is included in the culture medium.
In addition, the present invention also provides following kits (the also referred to as kit of the present invention)：
14. a kind of kit, it includes culture medium, serum and ROCK inhibitor.
15. the kit according to item 14, the kit is used to cultivate epithelial cell.
16. the kit according to item 15, wherein, the epithelial cell is non-Eponychium cell.
17. the kit according to item 15, wherein, the epithelial cell is primary cell.
18. the kit according to item 15, wherein, the epithelial cell is passage cell.
19. the kit according to item 15, wherein, the epithelial cell is tumour cell.
20. the kit according to item 16, wherein, the non-keratinocyte epithelial cell is squamous cell, in column Chrotoplast, gland epithelial cells or transitional type epithelial cell.
21. the kit according to item 16, wherein, the non-keratinocyte epithelial cell is oral cavity, schneiderian membrane, tracheae , it is bronchial, lung epithelial, epithelium of esophagus, gastric epithelial, large intestine epithelium, small intestine epithelium, liver cell, Epithelial duct, gall-bladder, pancreas, beta Cell of islet, kidney, bladder, urothelial, testis epithelium, ovarian epithelium , thyroid, follicular epithelial cell, adrenal, thymus gland, prostate, mammary gland, tonsilar, hypophysis Cell, corneal epithelial cell, retinal pigment epithelium.
22. the kit according to item 14, it also includes calcium component.
23. the kit according to item 14, it also includes feeder cells.
24. the kit according to item 23, wherein, the feeder cells are cryopreservations.
25. the kit according to item 23, wherein, the feeder cells are low-temperature storages.
26. the kit according to item 14, wherein, it also includes described for individually being held when carrying out cell culture The container of feeder cells, the container has permeability membrane structure.
27. the kit according to item 23, wherein, the feeder cells are mouse or the fibroblast of people.
28. the kit according to item 14, wherein, the culture medium includes conditioned medium.
29. the kit according to item 14, wherein, the ROCK inhibitor is to be selected from Fasudil, H-1152, Y- 27632nd, more than one or both of HA1100, HA1077 and GSK429286.
30. a kind of kit, it includes the culture medium and feeder cells any one of item 1~13.
31. the kit according to item 30, wherein, the feeder cells are cryopreservations.
32. the kit according to item 30, wherein, the feeder cells are low-temperature storages.
33. the kit according to item 30, wherein, it also includes described for individually being held when carrying out cell culture The container of feeder cells, the container has permeability membrane structure.
In addition, the present invention also provides the method (the also referred to as cultural method of the present invention) of following culture epithelial cells：
34. a kind of method for cultivating epithelial cell, this method includes
Step A：Epithelial cell and feeder cells are co-cultured using the culture medium any one of item 1~13.
35. the method for the culture epithelial cell according to item 34, wherein, the epithelial cell is primary cell.
36. the method for the culture epithelial cell according to item 34, wherein, the epithelial cell is passage cell.
37. the method for the culture epithelial cell according to item 34, wherein, the epithelial cell is tumour cell.
38. the method for the culture epithelial cell according to item 34, wherein, the epithelial cell is that non-Eponychium is thin Born of the same parents.
39. the method for the culture epithelial cell according to item 38, wherein, the non-keratinocyte epithelial cell is scaly epithelium Cell, columnar epithelial cell, gland epithelial cells or transitional type epithelial cell.
40. according to item 38 culture epithelial cell method, wherein, the non-keratinocyte epithelial cell be oral cavity, Schneiderian membrane, it is tracheae, bronchial, lung epithelial, epithelium of esophagus, gastric epithelial, large intestine epithelium, small intestine epithelium , it is liver cell, epithelial duct, gall-bladder, pancreas, beta Cell of islet, kidney, bladder, urothelial, on testis Skin, ovarian epithelium, thyroid, follicular epithelial cell, it is adrenal, thymus gland, prostate, mammary gland, It is tonsilar, the cell of hypophysis, corneal epithelial cell, retinal pigment epithelium.
41. the method for the culture epithelial cell according to item 34, wherein, the method for the culture epithelial cell is that epithelium is thin Born of the same parents' Secondary Culture method.
42. the method for the culture epithelial cell according to item 34, wherein, the method for the culture epithelial cell is on primary Epithelial cell proliferation cultural method.
43. the method for the culture epithelial cell according to item 34, wherein, epithelial cell and feeder cells are placed in same In culture vessel.
44. the method for the culture epithelial cell according to item 34, wherein, feeder cells are individually placed in can be penetrating In the container of property membrane structure.
45. the method for the culture epithelial cell according to item 34, wherein, the feeder cells are mouse or people into fibre Tie up cell.
46. a kind of method for cultivating epithelial cell, this method includes
Step B：Use the medium culture epithelial cell described in item 12 or 13.
47. the method for the culture epithelial cell according to item 46, wherein, the epithelial cell is primary cell.
48. the method for the culture epithelial cell according to item 46, wherein, the epithelial cell is passage cell.
49. the method for the culture epithelial cell according to item 46, wherein, the epithelial cell is tumour cell.
50. the method for the culture epithelial cell according to item 46, wherein, the epithelial cell is that non-Eponychium is thin Born of the same parents.
51. the method for the culture epithelial cell according to item 50, wherein, the non-keratinocyte epithelial cell is scaly epithelium Cell, columnar epithelial cell, gland epithelial cells or transitional type epithelial cell.
52. according to item 50 culture epithelial cell method, wherein, the non-keratinocyte epithelial cell be oral cavity, Schneiderian membrane, it is tracheae, bronchial, lung epithelial, epithelium of esophagus, gastric epithelial, large intestine epithelium, small intestine epithelium , it is liver cell, epithelial duct, gall-bladder, pancreas, beta Cell of islet, kidney, bladder, urothelial, on testis Skin, ovarian epithelium, thyroid, follicular epithelial cell, it is adrenal, thymus gland, prostate, mammary gland, It is tonsilar, the cell of hypophysis, corneal epithelial cell, retinal pigment epithelium.
53. the method for the culture epithelial cell according to item 46, wherein, the method for the culture epithelial cell is that epithelium is thin Born of the same parents' Secondary Culture method.
54. the method for the culture epithelial cell according to item 46, wherein, the method for the culture epithelial cell is on primary Epithelial cell proliferation cultural method.
By using the culture medium or kit culture epithelial cell of the present invention, cytogenetics background can not changed In the case of, Effective multiplication culture primary epithelial cells, and effectively extend the culture algebraically of epithelial cell.
Brief description of the drawings
Fig. 1 is cultural method schematic diagram of the present invention, wherein, 1., 2., 3. scheme is the culture medium and feeder cells of the present invention Three kinds of culture systems of combination.
Fig. 2 is the workflow diagram that epithelial cell culture is carried out using the conditioned medium of the present invention.
Fig. 3 is feeder cells (Transwell) co-culture system progress epithelial cell culture indirectly using the present invention Workflow diagram.
Fig. 4 is the feeder cells using the present invention and the workflow diagram of epithelial cell Co-culture system.
Fig. 5 is the bronchus primary epithelial cells of normal person in serum free medium (left side) and serum-containing media (right side) The growth conditions of culture compare.Shown in figure, bronchial epithelial cell can not breed in the culture medium containing serum.By embodiment 5 method separation prepares primary bronchial epithelial cell, and the cell precipitation of acquisition finally is resuspended in into complete DMEM trains Supporting base, [composition is DMEM, adds 10% hyclone (FBS), 100U/ml penicillin (penicillin) and 100 μ g/ml strepto-s Plain (streptomycin)] in, or serum-free epithelial cell culture medium SFM (GIBCO#10744-019) (see embodiment 10).After one week, observe and take a picture under conventional inverted microscope.Although culture effect is not good, serum free medium is still wide It is general to be used for that conventional epithelial cell to be primary and Secondary Culture.
Fig. 6 is that normal human bronchial's primary epithelial cells growth conditions of culture compare.Shown in figure, on bronchus Chrotoplast, is cultivated using three kinds of culture systems of the present invention, and the growing multiplication of cell is substantially better than using conventional without blood Clear culture medium.Separation prepares primary bronchial epithelial cell as described in Example 5, and the cell precipitation of acquisition is resuspended in In the conditioned medium of embodiment 8 (method is shown in Fig. 2), or HL culture medium of the cell in the present invention is resuspended (see embodiment 2, HL Culture medium 6) in, the feeder cells Indirect co-culture as illustrated by Fig. 3 method and embodiment 9, or enter by Fig. 4 and embodiment 6 Row original cuiture.After one week, observe and take a picture under conventional inverted microscope.Feeder cells required in the research of this diagram Passed on according to embodiment 1 and conservation.HL culture mediums required in the research of this diagram are prepared by embodiment 2.This diagram Research in required for feeder cells by embodiment 3 and 4 acquisition.
Fig. 7 is the comparison of normal human bronchial's epithelial cell subculture in vitro separately culture.Abscissa represents cultivated days, ordinate Represent the multiple of cell propagation.It has been shown that, for bronchial epithelial cell, trained using three kinds of culture systems of the present invention in figure Support, cell can pass at least or so 10 generations in vitro, hence it is evident that better than the culture using serum free medium.By embodiment 5 Method separation prepares primary bronchial epithelial cell, and the cell precipitation of acquisition is resuspended in the conditioned medium of embodiment 8 In (method is shown in Fig. 2), or be resuspended cell in the present invention HL culture mediums (see embodiment 2, HL culture mediums 6) in, by Fig. 3 side Feeder cells Indirect co-culture illustrated by method and embodiment 9, or carry out Secondary Culture by the explanation of Fig. 4 and embodiment 6 and 7. Feeder cells required in the research of this diagram are passed on and conservation according to embodiment 1.Required in the research of this diagram HL culture mediums prepared by embodiment 2.Feeder cells required in the research of this diagram are obtained by embodiment 3 and 4.
Fig. 8 summarize the inventive method succeeded culture epithelial cell germline and type.These cells come respectively From in people, mouse and rat.The organization type of these cells has, but is not limited to：Oral cavity, trachea-bronchial epithelial cell, lung epithelial, stomach, Colon, prostate, mammary gland, liver, pancreas, bladder, ovary and tonsil.Specific implementation step separates system as described in Example 5 Standby primary epithelial cells, the cell precipitation of acquisition are resuspended in being cultivated (method is shown in the conditioned medium of embodiment 8 Fig. 2), or be resuspended cell in the present invention HL culture mediums (see embodiment 2, wherein HL culture mediums 1 be used for mammary gland, prostate, The epithelial cell culture of stomach, bladder-derived；HL culture mediums 2 are used for the epithelial cell culture that liver is originated；HL culture mediums 3 are used to tie Intestines, tonsil, mammary gland, the epithelial cell culture in stomach source；HL culture mediums 4 are used for the epithelial cell training that ovary, tonsil are originated Support；HL culture mediums 5 are used for the epithelial cell culture that pancreas, tonsil are originated；HL culture mediums 6 be used for oral cavity, trachea-bronchial epithelial cell, The epithelial cell culture in lung source), the feeder cells Indirect co-culture as illustrated by Fig. 3 and embodiment 9, or by Fig. 4 and implementation The explanation of example 6 and 7 carries out subculture.In the research of this diagram required for feeder cells according to embodiment 1 carry out passage and Conservation.HL culture mediums required in the research of this diagram are prepared by embodiment 2.Raising required in the research of this diagram is thin Born of the same parents are obtained by embodiment 3 and 4.
Fig. 9 show the culture example from people, rat and mouse epithelial cells.Left figure is derived from the prostate of people Epithelial cell, as described in Example 5 separation prepares primary epithelial cells, and the cell precipitation of acquisition is resuspended in into embodiment 8 Cultivated in HL conditioned mediums (being co-cultured by HL culture mediums 1 and feeder cells, obtained by 2 methods of diagram).Middle figure and right figure The galactophore epithelial cell of rat and the pulmonary epithelial cells of mouse are derived from respectively, and separation as described in Example 5 prepares primary Epithelial cell, the cell precipitation of acquisition is resuspended in HL culture mediums 1 or HL culture mediums 6 in embodiment 2, by Fig. 4 and embodiment 6 Method with 7 carries out Secondary Culture.Feeder cells required in the research of this diagram are passed on and protected according to embodiment 1 Kind.HL culture mediums required in the research of this diagram are prepared by embodiment 2.Feeder cells required in the research of this diagram Prepared by embodiment 3 and 4.
Influence of the more different hyclone concentration of Figure 10 to epithelial cell growth.HL culture mediums are prepared by embodiment 2, plus Entering hyclone makes its final concentration be respectively 1%, 2%, 5%, 10%.15%, 20%.By normal person's branch of 5000 second generations Tracheal epithelial cell (in Fig. 7 researchs, the method for embodiment 9) is suspended in 3 milliliters and above-mentioned contains different hyclones respectively In the HL culture mediums of concentration, in the Tissue Culture Plate for being subsequently placed in 6 holes, then 500000 feeder cells (are made by embodiment 3 It is standby) it is placed in transwell baskets (Corning Products), according to Fig. 3 method, trained in 5%CO2,37 DEG C of incubator Support 6 days.Transwell baskets and the feeder cells wherein contained are discarded, remaining culture medium in culture plate are discarded, with without calcium and magnesium Phosphate buffer wash once, add 1ml 0.05%EDTA/ pancreatin digestive juices, 37 DEG C be incubated about 2-3 minutes, treat that cell is complete Entirely after de- wall, the DMEM culture mediums that 2ml contains 10% hyclone are added, cell is fully mixed, take out the mixing of 50ul cell suspensions Dyed in 50ul 4% trypan blue (trypan blue), cell count is carried out according to a conventional method.
The chemical constitution for the ROCK inhibitor that the culture medium of Figure 11 present invention is added.
Figure 12 shows influence of the concentration of ROCK inhibitor to epithelial cell growth.HL culture mediums 6 are prepared by embodiment 2, Adding different ROCK inhibitors makes its final concentration respectively 1uM, 5uM, 10uM, 20uM, 50uM.By 5000 second generations just Normal bronchial epithelial cell (from Fig. 7 researchs, the method for embodiment 9) is suspended in 3 milliliters and above-mentioned contains various concentrations respectively In the HL culture mediums 6 of ROCK inhibitor, in the Tissue Culture Plate for being subsequently placed in 6 holes, then by 500000 feeder cells (by implementation It is prepared by example 3) it is placed in transwell baskets (Corning Products), according to Fig. 3 method, in 5%CO2,37 DEG C of culture Cultivated 8 days in case.Transwell baskets and the feeder cells wherein contained are discarded, remaining culture medium in culture hole is discarded, with Phosphate buffer without calcium and magnesium is washed once, adds 1ml 0.05%EDTA/ pancreatin digestive juices, and 37 DEG C are incubated about 2-3 minutes, treat Cell is taken off after wall completely, is added the DMEM nutrient solutions that 2ml contains 10% hyclone, is fully mixed cell, is taken out 50ul cells and is hanged Liquid is mixed in 50ul 4% trypan blue (trypan blue) and dyed, and cell count is carried out according to a conventional method.
The embodiment of invention
The culture medium of the present invention
The first aspect of the present invention is related to a kind of culture medium (the also referred to as culture medium of the present invention, i.e. HL culture mediums), and it is wrapped Containing serum, calcium component and ROCK inhibitor.
In this specification, culture medium refers to the matrix for cultivating biomaterial.It is not particularly limited for biomaterial, Can be virus, cell (such as microbial cell, zooblast, plant cell), tissue (such as animal tissue, plant tissue) Deng.The culture medium of the present invention is preferably used to the culture medium of cell culture, i.e. cell culture medium.The culture medium of the present invention is more preferably It is the culture medium for epithelial cell culture.Epithelial cell in this specification can be such as non-keratinocyte epithelial cell, for example From body of gland, including mammary gland, prostate, the non-keratinocyte epithelial cell of liver and gut epithelium.Non-keratinocyte epithelial cell is divided into Competent cell with absorption or secreting function, non-keratinocyte epithelial cell is not that the scaly epithelium of height keratinization structure is thin Born of the same parents.Any kind of histoorgan can be derived from the non-keratinocyte epithelial cell of the medium culture of the present invention.It can train Foster non-keratinocyte epithelial cell is included, but are not limited to such as Types Below：It is oral cavity, schneiderian membrane, on tracheae, bronchial, lung Skin, epithelium of esophagus, gastric epithelial, large intestine epithelium, small intestine epithelium, liver cell, epithelial duct, gall-bladder , pancreas (including beta Cell of islet), kidney, bladder, urothelial, testis epithelium, ovarian epithelium, it is thyroid, It is follicular epithelial cell, adrenal, thymus gland, prostate, mammary gland, tonsilar, on the cell of hypophysis, cornea Chrotoplast, retinal pigment epithelium.In addition, the present invention culture medium can be solid powdery culture medium or Fluid nutrient medium, preferably fluid nutrient medium.When the culture medium of the present invention is solid powdery culture medium, preferably when in use It is modulated into fluid nutrient medium to carry out cell culture, serum, calcium component and ROCK can be added in modulated process and is suppressed The various compositions such as agent, the important document for making it meet the present invention.
In this specification, when object is cell, " culture " or " cell culture " refers to manually be maintained in vitro, makes Cell is bred.Cell culture is generally aseptically carried out, and can be culture individual cells or tissue.
It should contain serum in the culture medium of the present invention.As serum, the serum commonly used in cell culture, example can be used Such as calf serum or hyclone, preferably hyclone.It is used as serum, in addition to various serum substitutes, serum substitute bag Include but be not limited to product (such as Knockout of Invitrogen companies of commercialization purchaseTMSerum substitute, Pall The Ultroser of Corporation companiesTM), in addition to milk and milk constituents (the skim milk dry powder of such as filtering).For The amount of serum in culture medium is not particularly limited, can be with the amount in conventional cell culture added to the serum in culture medium It is identical, and those skilled in the art can suitably be adjusted as needed.Preferably, with volume basis (v/v%), culture In base 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%th, more preferably 5.0%；In culture medium the upper limit of serum content can be 35%, more preferably 30%, more preferably 25%, it is more excellent Select 20%, more preferably 17%, more preferably 15%, more preferably 13%, more preferably 11%, more preferably 10%.
In this specification, ROCK refers to Rho kinases 1 (ROCK 1) and/or Rho kinases 2 (ROCK 2).Suppress ROCK to refer to Reduce activity, expression or the function of at least one of ROCK1 or ROCK2 enzyme.Compared with undressed cell, ROCK lives Property, expression or function can be totally constrained reach it is inactive, without expression or nonfunctional or activity, expression or work( Can level reduction.In this specification, ROCK inhibitor refers to：Material with the effect for suppressing ROCK.For ROCK inhibitor, It is not particularly limited, ROCK inhibitor well known in the art, such as Fasudil (Fasudil), H-1152, Y- can be used 27632, HA1100, HA1077 and GSK429286 etc., these are all the common agents of commercialization.In addition, ROCK inhibitor is also wrapped Include in PCT application (WO 03/059913, WO 03/064397, WO 05/003101, WO 04/112719, WO 03/062225 With WO 03/062227) disclosed in or it is special in US granted patent (7,217,722 and 7,199,147) and U. S. application Small molecule ROCK suppressions disclosed in sharp (2003/0220357,2006/0241127,2005/0182040 and 2005/0197328) Preparation.Content for ROCK inhibitor in the culture medium of the present invention is not particularly limited, as long as effective dose.It is described to have Effect amount is the amount for referring to suppress ROCK, and the method for determining effective dose is that well known to a person skilled in the art people in the art Member can suitably determine effective dose according to conditions such as species, the species of used ROCK inhibitor of the cell cultivated. For example, although may be different because of the species of cell, the species of ROCK inhibitor, cell culture condition etc., but in general, ROCK Inhibitor content can be between 0.1 μM~1mM.
Feeder cells can also be included in the culture medium of the present invention.In this specification, feeder cells refer to：With wishing to cultivate Cell co-culture cell, wherein, it is described wish culture cell be preferably non-keratinocyte epithelial cell.Feeder cells are general It is that, by gamma radiation exposures and/or mitomycin processing, its mitosis is suppressed, but still can maintenance metabolism activity. Therefore, feeder cells are with metabolic capability but the non-proliferative cell that can not divide.Processing and blocking cell mitogen are simultaneously tieed up The method for holding cell metabolic activity can be the conventional method of cell biology.Feeder cells can be moved from any lactation Thing, but its source is different from the kind in cell (the preferably non-keratinocyte epithelial cell) source for wishing culture.Feeder cells can be with It is but is not limited to the feeder cells in following source, such as mouse, rat, dog, cat, ox, horse, pig, primate and the mankind.Typical case Feeder cells type include splenocyte, macrophage thymocyte and/or fibroblast, they after processing turn into non-proliferative it is thin Born of the same parents.In addition, can also be by the use of J2 as feeder cells in the present invention, J2 cells are to build the mouse fibroblast cell Swiss 3T3 for being The subclone of cell line, is handled through gamma radiation exposures and/or mitomycin.Included in culture medium for the present invention The amount of feeder cells is not particularly limited, and those skilled in the art can suitably determine as needed, and usual feeder cells are with wishing The ratio for hoping the epithelial cell of culture can be 1：9~9：1, preferably 2：8~8：2.
In addition, conditioned medium can be included in the culture medium of the present invention.In this specification, conditioned medium refers to include The culture medium of feeder cells secretion and/or extract.Typical conditioned medium is to use one section of medium culture feeder cells After time, then remove culture medium obtained from feeder cells.Herein, blood is preferably included for cultivating the culture medium of feeder cells Clearly, calcium component and the culture medium of ROCK inhibitor, culture medium of the invention.Time for cultivating feeder cells, do not have Specifically limited, those skilled in the art can suitably determine as needed, generally can be 1~300 hour, preferably 5~200 is small When, more preferably 10~150 hours, more preferably 10~150 hours, more preferably 20~100 hours, more preferably 24~72 hours, more It is preferred that 35~60 hours, more preferably 40~50 hours.In general, preparation condition culture medium for example including by cell culture in one Plant in culture medium (such as F culture mediums), these culture mediums are collected after a few days.Conditioned medium can press one with fresh culture Conditioned medium after certainty ratio dilution or the conditioned medium after not diluted collection.Fresh culture：Condition The dilution ratio of culture medium can be 1:99~99:1, depending on specific experiment condition.Heretofore described " CMC model Base " is included through any dilution proportion or undiluted conditioned medium.Preferably, culture medium of the invention can be containing blood Clearly, calcium component and the not diluted conditioned medium of ROCK inhibitor.
The culture medium of the present invention can use the conventional medium for cell culture to prepare.Conventional medium refers to this Technical field is known to be used for the culture medium of cell culture (being preferred for epithelial cell culture), includes but is not limited to：DMEM (Dulbecco ' s Modified Essential Medium), Ham ' s F12 culture mediums, Ham ' s F-10 culture mediums, RPMI 1640,Eagle’s Basal Medium(EBM),Eagle’s Minimum Essential Medium(MEM),HEPES, Medium 199 and correlation type culture medium.Conventional medium can also be the combination of two or more culture mediums, such as DMEM With F12 mixed culture medium.Do not include serum in the formula of conventional medium typically, therefore, made when using conventional medium During the culture medium of the standby present invention, a certain amount of serum can be separately added so that the serum content in culture medium is in above-mentioned restriction In the range of.The operation for increasing or decreasing the serum in culture medium is the routine operation of the art.
It should contain calcium component in the culture medium of the present invention." calcium component " referred to herein is to refer to the training for the present invention Support base and calcium ion (Ca is provided2+) composition, including but not limited to calcic compound.Generally, calcium component derives from serum or blood Clear substitute, or the calcic added in culture medium salt.The calcium component for example can be with calcium salt (such as chlorination Calcium, calcium sulfate) form be added to culture medium in.The content range (in terms of calcium ion) of calcium component can with for cell culture Conventional medium in calcium component content it is identical, and those skilled in the art can suitably be adjusted as needed.Example Such as, with Ca2+Meter, the lower limit of calcium component content can be 0.1 μM, preferably 10 μM, more preferably 100 μM, more preferably 200 μM, more excellent Select 400 μM, more preferably 500 μM, more preferably 800 μM, 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 preparing the culture medium of the present invention using conventional medium, preferably it is adjusted so that calcium component content is upper In the range of stating restriction.If in the classical formula of conventional medium comprising calcium and calcium component content above-mentioned restriction scope Interior, then for calcium component content, the conventional medium need not be adjusted；If included in the classical formula of conventional medium Calcium but calcium component content is not in the range of above-mentioned restriction, then should correspondingly increase or decrease the calcium in formula so that culture The calcium component content of base is in the range of above-mentioned restriction；, should be if not including calcium in the classical formula of conventional medium Increase calcium in formula so that the calcium component content of culture medium is in the range of above-mentioned restriction.Increase or decrease the calcium in culture medium The operation of component content is the routine operation of the art.
Other conventional ingredients can be added in the culture medium of the present invention, including but not limited to：Amino acids, vitamin Class, inorganic salts, adenine, monoethanolamine (ethanolamine), D-Glucose, heparin (heparin), N- [2- ethoxys (hydroxyethylpiperazine)-N'- [2-ethanesulfonic acid (ethanesulfonic acid)] (HEPES), cortisol (hydrocortisone), insulin (insulin), EGF (EGF), adrenaline (epinephrine), sulphur are pungent Sour (lipoic acid), phenol red, phosphoethanolamine (phosphoethanolamine), putrescine (putrescine), cholera poison Plain (cholera toxin), Sodium Pyruvate (sodium pyruvate), triiodo thryonine (triiodothyronine, T3), thymidine and transferrin (transferrin).Wherein, heparin and transferrin can use ironic citrate (ferric Citrate) or chelating ferric sulfate (ferrous sulfate chelates) substitute.Above-mentioned adding ingredient can be purchased with commercialization Buy.
Amino acids in the culture medium of the present invention includes but is not limited to：ALANINE, L-arginine, altheine (L-asparagine), L-Aspartic acid, Cys, Pidolidone, Glu, glycine, L-Histidine, L- are different Leucine, L-Leu, 1B, METHIONINE, L-phenylalanine, L-PROLINE, Serine, L-threonine, L- Tryptophan, TYR and Valine.
Vitamins in the culture medium of the present invention includes but is not limited to：Biotin, Choline Chloride (choline chloride)、D-Ca+2- pantothenic acid (pantothenate), folic acid (folic acid), i- inositols (i-inositol), niacinamide (niacinamide), polyol (pyridoxine), riboflavin (riboflavin), vitamin B1 (thiamine) and vitamin B12。
Inorganic salts composition includes but is not limited to：Calcium salt (such as CaCl2)、CuSO4、FeSO4, KCl, magnesium salts (such as MgCl2), manganese salt (such as MnCl2), sodium acetate, NaCl, NaHCO3、Na2HPO4、Na2SO4, and trace other elements such as selenium (selenium), silicon, molybdenum (molybdenum), vanadium (vanadium), nickel, tin, zinc, these trace elements can be in a variety of forms Occur, it is preferred that the form of salt, such as Na2SeO3,Na2SiO3,(NH4)6Mo7O24,NH4VO3,NiSO4, SnCl and ZnSO4.
Other adding ingredients of the culture medium of the present invention include but is not limited to：Heparin, EGF (epidermal growth factor, EGF), at least one can increase the phosphoric acid (cyclic of intracellular cyclic adenosine one Adenosine monophosphate, cAMP) factor of level, at least one fibroblast growth factor (fibroblast Growth factor, FGF).Above-mentioned adding ingredient can be added directly into basal medium, can also use DPBS (Dulbecco's Phosphate Buffered Saline) buffer is cultivated into mixed liquor, freezen protective until preparing Row addition again during base.Heparin in the culture medium of the present invention can be commercialized purchase.The main function of heparin be stable growth because The activity of son, such as FGF.Addition concentration of the heparin in basal medium is 1-500U.S.P.units/L.EGF can also business Change purchase, addition concentration of the EGF in basal medium is 0.00001-10mg/L.
It can increase the factor of intracellular cAMP levels comprising a variety of species in the culture medium of the present invention, can be straight Meet [such as two butyryl (dibutyryl) cAMP] of increase cAMP levels or by causing with the interaction of cell G-protein Intracellular cAMP levels raise [such as cholera toxin and forskolin (forskolin)], or as receptor,β (β- Adrenergic receptors) antagonist (such as isoprel) increase intracellular cAMP levels or pass through suppress CAMP phosphodiesterases activity and make intracellular cAMP levels increase [such as isobutylmethylxanthine (IBMX) and tea Alkali (theophylline)].These above-mentioned compounds can be commercialized purchase.
The kit of the present invention
The second aspect of the present invention is related to a kind of kit (kit of the invention), its comprising culture medium, serum, calcium into Divide and ROCK inhibitor.
The kit of the present invention is preferred for cultivating cell.The cell is preferably epithelial cell, more preferably non-keratinocyte Epithelial cell.
The culture medium can be any culture medium for being used to cultivate cell, and conventional medium as escribed above preferably may be used To be above-mentioned conditioned medium.The serum, calcium component and ROCK inhibitor are with above-mentioned.The kit of the present invention can be used for Prepare the culture medium of the invention described above.In the kit of the present invention, the ratio of culture medium, serum and ROCK inhibitor three is preferred Satisfaction can form the culture medium of the invention described above after three is mixed.
Preferably, kit of the invention also includes calcium component, now, culture medium, serum, calcium component and ROCK inhibitor Preferred meet of four ratio will can form the culture medium of the invention described above after four mixing.
Preferably, kit of the invention also includes feeder cells.The feeder cells are with above-mentioned.The feeder cells can To be conventional adherent, it can be survived 3-5 days under normal temperature；The feeder cells can also be cryopreservation, and cryopreservation refers to The long term storage below -80 DEG C, can typically be stored several days to several years.The feeder cells can also be low-temperature storage, low Temperature storage refers to store at 4 DEG C, can typically store 1~7 day.
Preferably, kit of the invention, which is also included, is used to individually hold the feeder cells when carrying out cell culture Container, the container has permeability membrane structure.Particularly, it is excellent in the case where the kit of the present invention includes feeder cells It is selected also to include the container.The effect of the container is, when carrying out cell culture, by feeder cells with needing the thin of culture Born of the same parents, which keep apart, to be cultivated, and causes the secretion of feeder cells to enter culture needs by above-mentioned permeability membrane structure In the culture medium of the cell of culture.Shape, material, specification for the container etc. are without specifically limited, people in the art Member can suitably select as needed.It is, for example, possible to use Transwell baskets (Corning Products).
Preferably, kit of the invention includes the culture medium and feeder cells of the present invention, more preferably also holds comprising described Device.
The other compositions for being used for being added in culture medium can also be included in the kit of the present invention, are included but is not limited to Adding ingredient described in the foregoing section of " culture medium of the invention " one.
The other compositions or component for cell culture, such as indicator, pancreas can also be included in the kit of the present invention Enzyme, blake bottle, culture dish, operation instructions etc..
In the kit of the present invention, preferably described each composition is separated, is more preferably respectively placed in independent container.
The cultural method of the present invention
The third aspect of the present invention is related to a kind of method (the also referred to as cultural method of the present invention) for cultivating epithelial cell, the party Method includes
Step A：Epithelial cell and feeder cells are co-cultured using the culture medium of the invention described above；Or
Step B：Use the above-mentioned medium culture epithelial cell of the invention comprising feeder cells or conditioned medium (see Fig. 2).
The cultural method of the present invention can be epithelial cell Secondary Culture method or primary epithelial cells propagation training The method of supporting.
Here, the epithelial cell and feeder cells are with foregoing.
Can be by epithelial cell and feeder cells Co-culture in step A, can also be by epithelial cell and feeder cells Indirect co-culture.The Co-culture refers to epithelial cell and feeder cells being placed in same culture vessel, is cultivated (see Fig. 4)；The Indirect co-culture refers to being individually placed in feeder cells into the above-mentioned container (example with permeability membrane structure Such as Transwell baskets) in, it is then possible to which the container equipped with feeder cells to be for example placed in the culture of culture epithelial cell again In base, cultivated (see Fig. 3).
Other steps in the cultural method of the present invention can be carried out according to the conventional method of the art.
The inoculum density of cell can suitably be adjusted according to specific experiment condition.Conventional disposable plastic culture In vessel, general inoculating cell number is 1x104~10x105Cell/cm2, i.e. 75cm2Blake bottle inoculating cell number is, for example, 1x106.Passage each time will be adjusted according to specific experiment requirement to cell density.
Mammalian cell is generally incubated in 37 DEG C, appropriate CO2In concentration (3~10%), the incubator of certain humidity, temperature Degree, CO2Concentration and humidity can be adjusted according to specific experiment condition.The pH of culture medium scope can be adjusted as needed It is whole, usually pH7.1~7.6, preferably pH7.1~7.3
Cell culture medium is changed once for usual 1-2 days, can be also adjusted according to the cell type specifically cultivated.As long as thin Born of the same parents grow to full abundance in culture vessel, it is possible to which progress is passed on.In this specification, passage refers to cell point kind A part is into new culture vessel.
Embodiment
Hereinafter, more specific description is carried out to the present invention by embodiment, but the present invention is not restricted by the embodiments. In addition, without the reagent especially marked in embodiment, being purchased from GIBCO companies.
The subculture of the Swiss 3T3 cells of embodiment 1
Swiss 3T3 cells are obtained from Harvard University Howard Green professors laboratory.Required examination Agent：
Complete DMEM culture mediums：DMEM (GIBCO#11965-092), 10% hyclone (FBS), 100U/ml moulds Plain (penicillin) and 100 μ g/ml streptomysins (streptomycin)
Pancreatin digestive juice:0.05% pancreatin/EDTA (GIBCO#25300-062)
Without calcium and magnesium phosphate buffer：Ca2+,Mg2+free PBS
Secondary Culture operating procedure：
It is inoculated with 1x105Swiss 3T3 cells add the above-mentioned complete DMEM culture mediums of 10ml, put in T75 Tissue Culture Dish In CO2Incubator is in 5%CO2, 37 DEG C are cultivated 2-5 days.When cell monolayer abundance is 80-90%, the training in culture dish is removed Base is supported, cell monolayer is washed 2 times without calcium and magnesium phosphate buffer with 10ml, 2ml pancreatin digestive juices is added, is incubated 1 minute at 37 DEG C. Culture dish is patted fully to discharge adherent cell, with the complete DMEM culture mediums of 10ml and digestion reaction.4 DEG C centrifuge 5 points Clock (1000 revs/min) sedimentation cell, removes supernatant, and cell precipitation is resuspended and is cultivated in the complete DMEM culture mediums of 10ml.Root Can be with 1 according to needs：10,1：20 or 1：50 ratios are passed in new T75 (the complete DMEM culture mediums of 10-15ml) or T175 In the culture dish of (the complete DMEM culture mediums of 25-30ml), fresh complete DMEM culture mediums 2-3 is changed weekly as needed It is secondary.If necessary can be by 1x106Cell is resuspended in 1-2ml cells frozen storing liquid (60%DMEM, 30% hyclone and 10% DMSO in), it is stored in standby in liquid nitrogen.
The HL culture mediums of embodiment 2
HL culture mediums 1 are DMEM (GIBCO#11965-092) and Ham ' s F-12 NUTRIENT MIX (GIBCO# Mixed culture medium 11765-054), volume proportion is 3:1, while the hyclone of addition 5%, and 0.4 μ g/ml cortisols (hydrocortisone), 5 μ g/ml insulin (insulin), 8.4ng/ml cholera toxins (cholera toxin), 10ng/ Ml EGFs (epithelial growth factor (EGF)), 24 μ g/ml adenines (adenine), 100U/ml Penicillin (penicillin) and 100 μ g/ml streptomysins (streptomycin), 0.25 μ g/ml amphotericin Bs (Fungizone) 30 μM of Fasudils (Fasudil) (or 0.5 μM of H-1152 or 5 μM of Y-27632,30 μM of HA1100, 10uM GSK429286), above-mentioned culture medium need to be through 0.22 μ apertures membrane filtration.
HL culture mediums 2 are the addition 10% in DMEM (Low Glucose, No Glutamine, GIBCO#11054-020) Hyclone, and 0.4 μ g/ml cortisols (hydrocortisone), 5 μ g/ml insulin (insulin), 8.4ng/ml Cholera toxin (cholera toxin), 10ng/ml EGFs (epithelial growth factor (EGF)), 24 μ g/ml adenines (adenine), 100U/ml penicillin (penicillin) and 100 μ g/ml streptomysins (streptomycin), 0.25 30 μM of μ g/ml amphotericin Bs (Fungizone) Fasudil (Fasudil) (or 0.5 μM of H-1152 or 5 μM of Y- 27632,30 μM of HA1100,10 μM of GSK429286), above-mentioned culture medium need to be through 0.22 μ apertures membrane filtration.
HL culture mediums 3 are RPMI culture mediums 1640 (GIBCO#22400-121) HEPES containing 5mM, the tire ox blood of addition 8% Clearly, and 0.4 μ g/ml cortisols (hydrocortisone), 5 μ g/ml insulin (insulin), 8.4ng/ml cholera toxins (cholera toxin), 10ng/ml EGFs (epithelial growth factor (EGF)), 24 μ g/ml glands Purine (adenine), 100U/ml penicillin (penicillin) and 100 μ g/ml streptomysins (streptomycin), 0.25 μ g/ 30 μM of Fasudils (Fasudil) of ml amphotericin Bs (Fungizone) (or 0.5 μM H-1152 or 5 μM Y-27632,30 μM HA1100,10 μM of GSK429286), above-mentioned culture medium need to be through 0.22 μ apertures membrane filtration.
HL culture mediums 4 are in L-15 (Leibovitz) culture medium (L-glutamine, No HEPES, GIBCO# 11415064) hyclone of addition 5% in, and 0.4 μ g/ml cortisols (hydrocortisone), 5 μ g/ml insulin (insulin), 8.4ng/ml cholera toxins (cholera toxin), 10ng/ml EGFs (epithelial Growth factor (EGF)), 24 μ g/ml adenines (adenine), 100U/ml penicillin (penicillin) and 100 μ g/ Ml streptomysins (streptomycin), 0.25 30 μM of μ g/ml amphotericin Bs (Fungizone) Fasudil (Fasudil) (or 0.5 μM of H-1152 or 5 μM Y-27632,30 μM HA1100,10 μM of GSK429286), above-mentioned culture medium need to be through 0.22 μ apertures Membrane filtration.
HL culture mediums 5 are in culture medium 199 (with Earle's salts but no L-glutamine, no Sodium bicarbonate, GIBCO#11825015) in addition 10% hyclone, and 0.4 μ g/ml cortisols (hydrocortisone), 5 μ g/ml insulin (insulin), 8.4ng/ml cholera toxins (cholera toxin), 10ng/ Ml EGFs (epithelial growth factor (EGF)), 24 μ g/ml adenines (adenine), 100U/ml Penicillin (penicillin) and 100 μ g/ml streptomysins (streptomycin), 0.25 μ g/ml amphotericin Bs (Fungizone) 30 μM of Fasudils (Fasudil) (or 0.5 μM H-1152 or 5 μM Y-27632,30 μM of HA1100,10 μ M GSK429286), above-mentioned culture medium need to be through 0.22 μ apertures membrane filtration.
HL culture mediums 6 are that (GIBCO#10744-019's is mixed by DMEM (GIBCO#11965-092) and serum free medium SFM Culture medium is closed, volume proportion is 1:3, while the hyclone of addition 5%, and 0.4 μ g/ml cortisols (hydrocortisone), 5 μ g/ml insulin (insulin), 8.4ng/ml cholera toxins (cholera toxin), 10ng/ Ml EGFs (epithelial growth factor (EGF)), 24 μ g/ml adenines (adenine), 100U/ml Penicillin (penicillin) and 100 μ g/ml streptomysins (streptomycin), 0.25 μ g/ml amphotericin Bs (Fungizone) 30 μM of Fasudils (Fasudil) (or 0.5 μM H-1152 or 5 μM Y-27632,30 μM of HA1100,10 μ M GSK429286), above-mentioned culture medium need to be through 0.22 μ apertures membrane filtration.
Preparation (the Mitomycin C processing of the feeder cells of embodiment 3 (the Swiss 3T3 cells for losing splitting ability) Swiss 3T3 cells)
1) as the swiss 3T3 cells of feeder cells, preferably using the cell of Secondary Culture early stage.When swiss 3T3 are thin During intracellular growth to full abundance, the Mitomycin C that final concentration of 10 μ g/mL are added in culture medium (is dissolved in water, storing liquid concentration is 0.5mg/mL), 37 DEG C are handled 2 hours；
2) 1xPBS or serum-free that add warm bath culture medium (DMEM) are washed 3 times, abandon washing lotion；
3) 0.05% pancreatin/EDTA predigestion cells are added to discard after 30-40 seconds, 0.05% pancreatin/EDTA is added again Digestion 30 seconds, patting culture dish disperses cell, then adds complete medium (DMEM for containing 10% hyclone) neutralization anti- Should；
4) low-speed centrifugal (1000rpm) removes supernatant, obtains cell precipitation；
5) cell precipitated can be directly used as feeder cells, or store standby；
A) situation of feeder cells is directly used as, operating procedure is as follows:
The 1xPBS of-addition warm bath washes cell precipitation 1 time, centrifuges again
- cell is suspended from HL culture mediums
- in the proper ratio (such as 1：3) feeder cells and a number of epithelial cell are suspended in HL culture mediums, on The most suitable inoculum density of chrotoplast is 2.5x105Individual/100mm culture dish；
B) feeder cells are second day standby situations, and operating procedure is as follows:
The 1xPBS of-addition warm bath washes cell precipitation 1 time, centrifuges again
- cell is suspended from complete DMEM culture mediums (DMEM for containing 10% hyclone)
- with 1:3 ratio is inoculated in culture dish, overnight incubation
Cell is washed with the 1xPBS of warm bath 2 times within-the second day, change HL culture mediums into
- epithelial cell is inoculated in the culture dish containing feeder cells again, the most suitable inoculum density of epithelial cell is 2.5x105 Individual/100mm culture dish；
C) feeder cells are the standby situations of storage, and operating procedure is as follows:
The 1xPBS of-addition warm bath washes cell precipitation 1 time
- feeder cells are resuspended in 6ml frozen stock solution (90% hyclone and 10%DMSO), divide in 3 cryopreservation tubes In
- freeze under the conditions of liquid nitrogen or -150 DEG C
- face the used time and take 1 recovery, add complete DMEM culture mediums (DMEM for containing 10% hyclone) and be inoculated in 100mm Culture dish
Remarks：It is used as after the 3T3 cells of feeder cells handle through Mitomycin C, need to be used or low temperature in 3-5 days Freeze standby.
Preparation (the radioactive ray process swiss of the feeder cells of embodiment 4. (the Swiss 3T3 cells for losing splitting ability) 3T3 cells)
As the swiss 3T3 cells of feeder cells, preferably using the cell of Secondary Culture early stage, these cell culture in In DMEM culture mediums, growing to closely expires abundance, with without Ca2+/Mg2+1xPBS wash cell 1 time, add the pancreatin of 1ml 0.05% Vitellophag 20-40 seconds, then adds the complete DMEM culture mediums of 9ml (DMEM for containing 10% hyclone) and neutralizes digestion reaction, Cell precipitations are collected in 4 DEG C of low-speed centrifugal 1000rmp, 10ml DMEM is added and is resuspended cell, radioactive ray irradiating cell suspension, i.e., Cesium-137 alpha cellulose a gage (JL Shepherd Mark) processing 3000Rad (30Grey).These swiss through radioactive ray process 3T3 cells, are inoculated in HL culture mediums and are directly used as feeder cells；Or be incubated at complete DMEM culture mediums or be placed in 4 DEG C of storages Deposit and used within 1-7 days；Or Long-term Cryopreservation is standby in -80 DEG C or liquid nitrogen.
Primary epithelial cells are separately cultured in the flesh tissue of embodiment 5.
In the case of patient or patient care people's informed consent, the normal or neoplasmic tissue sample of collector.
Concrete operation step is as follows:
1. the preparation of digestive juice：HL culture mediums containing clostridiopetidase A/hyaluronidase (final concentration of 1x mixed liquor) are (i.e. DMEM:F12 volume ratios 3：1 mixed culture medium), add 5%FBS.The consumption of digestive juice is determined according to the size of flesh tissue, It is generally necessary to which the consumption of the digestive juice of 10 times of sample volumes, such as 2-3 mouse prostate needs 2-5ml digestive juices；
2. washing the flesh tissue 1 time of separation with 95-100% ethanol, then washed 2 times, tissue is put into containing pre- with PBS then In cold PBS sterile petri dish, under disecting microscope, the fat remained in tissue is removed with dissection tweezers and scissors.
3. tissue sample is put into 14ml the or 50ml centrifuge tubes containing above-mentioned digestive juice, 37 DEG C digest 1-3 hours.
4. organizing low-speed centrifugal (1000rpm) 5 minutes by postdigestive, supernatant is removed.
5. cell precipitation is resuspended in 2-5mL 0.25% pancreatin/EDTA, 1 hour or room temperature 10 minutes on ice are placed in.
6. and then add DMEM culture mediums of the 10ml containing 10%FBS, low speed 1000rmp centrifugations 5 minutes.
7. supernatant is removed as far as possible clean.
8. the 5mg/mL dispases of 2ml warm bath and 200 μ L 1mg/mL DNase I are added, with sterile P1000 once Property plastics pipette tips blow and beat sample 1 minute repeatedly.
9. adding DMEMs of the 10ml containing 10%FBS, with the filter filtration cell suspension in 40-70 μm of aperture, filtering is collected Cell suspension afterwards, low speed 1000rmp is centrifuged 5 minutes, removes supernatant.
10. cell precipitation is resuspended in HL culture mediums, it is inoculated in T25 or T75 blake bottle by either one of the invention Case (see Fig. 1-4) is cultivated.
The Co-culture of 6. feeder cells of embodiment/epithelial cell (see Fig. 4 and Fig. 6)
The feeder cells (the Swiss3T3 cells for losing splitting ability) of above-mentioned Mitomycin C processing or radioactive ray irradiation are made Carried out for the co-cultivation of feeder layer and epithelial cell by following three kinds of modes：
1. direct feeder cells/epithelial cell is co-cultured.Add warm bath 1xPBS wash feeder cells precipitate 1 time, again from The heart, cell is suspended from HL culture mediums, with 1:Feeder cells and a number of epithelial cell are suspended in HL culture mediums by 3 ratios In, the most suitable inoculum density of epithelial cell is 2.5x105Individual/100mm culture dish, 37 DEG C, 5%CO are placed in by blake bottle2Under the conditions of Culture.
2. feeder cells are adherent in advance, epithelial cell co-cultivation is added.Above-mentioned Mitomycin C is handled or radioactive ray shine The feeder cells penetrated are suspended from complete DMEM culture mediums (DMEM for containing 10% hyclone), with 1:3 ratio is inoculated in culture dish In, 37 DEG C are cultivated 2-3 hours or overnight, cell are washed with the 1xPBS of warm bath 2 times, change HL culture mediums into, then epithelial cell is connect Plant in the culture dish containing feeder cells, the most suitable inoculum density of epithelial cell is 2.5x105Individual/100mm culture dish, it is adherent Feeder cells can be used within 3-5 days any times.Blake bottle is placed in 37 DEG C, 5%CO2Under the conditions of cultivate.
3. feeder cells are standby in 4 DEG C or liquid nitrogen storage.Above-mentioned Mitomycin C is handled or the raising of radioactive ray irradiation is thin Born of the same parents are suspended from complete DMEM culture mediums (DMEM for containing 10% hyclone) and are stored in 4 DEG C (1-7 days) or long term storage in liquid nitrogen, multiple 2 progress and the co-cultivation of epithelial cell as stated above after Soviet Union (in HL culture mediums).The most suitable inoculum density of epithelial cell is 2.5x105Individual/100mm culture dish.Blake bottle is placed in 37 DEG C, cultivated under the conditions of 5%CO2.
No matter with the co-cultivation of which kind of above-mentioned mode, fresh HL culture mediums should be changed within second day.When the feeding more than 50% When supporting cell detachment, the feeder cells that fresh above-mentioned Mitomycin C is handled or radioactive ray irradiate should be supplemented, until epithelial cell Grow to 70-90% abundance need passage when.
The separation passage of 7. feeder cells of embodiment/epithelial cell
1. epithelial cell growth is to 70-90% abundance, culture medium is suctioned out, is washed with the PBS of warm bath 1 time,
2. suctioning out PBS, the washing cells of the PBS containing 0.02%EDTA (0.68mM) of 10-12mL warm bath are added, to remove feeding Support cell Swiss 3T3；Pay special attention to, washing cell need to be as far as possible quick, and EDTA solution is first added to the cell of culture dish periphery, Culture dish is shaken in rotation, EDTA solution is circled round to the cell of culture dish inner circumferential again, repeats convolution washing cell 10 times.
3. suctioning out EDTA washing lotions, culture dish is shaken in the cell added again on the inside of 0.02%EDTA to culture dish, rotation, so EDTA solution is circled round the cell to culture dish afterwards, repeat convolution washing cell 30 times.The Eponychium cell of usual people 40 EDTA washings are needed, other cells are different according to adherent tightness degree, can suitably adjust washing times.
4. suctioning out EDTA washing lotions, the PBS for adding warm bath is washed 3 times, remains that culture dish is tilted.Last time cleaning solution is inhaled Before going out, observe under the microscope, confirm that all feeder cells Swiss 3T3 are washed off completely, otherwise, need to increase again EDTA washing times.
5. using conventional pancreatin method digestive epithelium cell, for Eponychium cell, pancreatin normal temperature digests 2 points Clock, suctions out digestive juice, is digested 5 minutes in 37 DEG C of pancreatin again, and patting culture dish disperses cell, and cell is resuspended in HL culture mediums In.Then inoculation epithelial cell is on the feeder cells that mitomycin C or radioactive ray process are crossed, and optimal cell inoculum density is 2.5x105Individual/100mm culture dish；
7. if necessary can be by 1x106Epithelial cell is resuspended in 1-2ml cells frozen storing liquid (90% hyclone and 10% DMSO in), it is stored in standby in liquid nitrogen.
The preparation of the feeder cells conditioned medium of embodiment 8. and the subculture of epithelial cell (see Fig. 2 and Fig. 6)
According to Fig. 2, above-mentioned Mitomycin C is handled or the feeder cells of radioactive ray irradiation are incubated at (T75 in HL culture mediums Blake bottle：2x106Cell, 15ml HL culture mediums；T175:5x106Cell, 35ml HL culture mediums) in 37 DEG C, 5%CO2Training Support in case and cultivate 48 hours.Culture medium is collected, 2000rpm is centrifuged 15 minutes, through 0.22 μ membrane filtrations, by 1：5 ratios (can be pressed 1：99-99：1 optimization) mixed with the HL culture mediums of Fresh and obtain HL conditioned mediums.The primary thin of preparation will be separated Born of the same parents' suspension or passage cell are directly cultivated in HL conditioned mediums, and condition of culture is 37 DEG C, 5%CO2.When cell increases When growing to 70-90% abundance, twice, 0.05% pancreatin/EDTA digests cell monolayer 2-5 minutes to 1xPBS washings cell, 10ml In complete DMEM and digestion reaction, 1000rmp is centrifuged 5 minutes, supernatant is removed, with certain proportion (1:2,1:3,1:4,1:5 is equal Can, as long as cell attachment and normal growth can be maintained) cell precipitation inoculated and cultured in 10ml HL conditioned mediums is resuspended. If necessary can be by 1x106Epithelial cell is resuspended in 1-2ml cells frozen storing liquid (90% hyclone and 10%DMSO), storage It is stored in standby in liquid nitrogen.
9. epithelial cells of embodiment/feeder cells Indirect co-culture (Transwell culture systems) and passage (see Fig. 3 and Fig. 6)
According to Fig. 3, above-mentioned Mitomycin C is handled or the feeder cells of radioactive ray irradiation are placed in Transwell baskets In (Transwell insert, Corning companies), separate the primary cell suspension prepared or passage cell can be directly in HL Cultivated in culture medium, condition of culture is 37 DEG C, 5%CO2.When cell is bred to 70-90% abundance, first remove Transwell baskets, twice, 0.05% pancreatin/EDTA digests cell monolayer 2-5 minutes to 1xPBS washings epithelial cell, and 10ml is complete In full DMEM and digestion reaction, 1000rpm is centrifuged 5 minutes, supernatant is removed, with certain proportion (1:2,1:3,1:4,1:5, As long as cell attachment and normal growth can be maintained) cell precipitation is resuspended in 10ml HL inoculation of medium cultures.If necessary may be used By 1x106Epithelial cell is resuspended in 1-2ml cells frozen storing liquid (90% hyclone and 10%DMSO), is stored in liquid nitrogen In it is standby.
The serum-containing media of embodiment 10. and serum free medium are used for the culture of epithelial cell (see Fig. 5)
Complete DMEM culture mediums：DMEM, 10% hyclone (FBS), 100U/ml penicillin (penicillin) and 100 μ g/ml streptomysins (streptomycin)
Serum free medium：The epithelial cell culture medium SFM (GIBCO#10744-019) of serum-free, gentamicin (10 μ g/ml)
Pancreatin digestive juice:0.05% pancreatin/EDTA
Without calcium and magnesium phosphate buffer：Ca2+,Mg2+free PBS
The primary cell suspension prepared or passage cell is separated directly to train in above-mentioned complete DMEM culture mediums or serum-free Support and cultivated in base, condition of culture is 37 DEG C, 5%CO2.When cell is bred to 70-90% abundance, 1xPBS washing cells Twice, in 0.05% pancreatin/EDTA digestion cell monolayer 2-5 minutes, the complete DMEM culture mediums of 10ml and digestion reaction, 1000rmp is centrifuged 5 minutes, removes supernatant, (is typically 1 with certain proportion:2 or 1:3) cell precipitation is resuspended in the complete DMEM of 10ml Or cultivated in serum free medium.If necessary can be by 1x106Cell is resuspended in 1-2ml cells frozen storing liquid (90% hyclone And 10%DMSO) in, it is stored in standby in liquid nitrogen.
It should also be noted that, on the premise of it can implement and substantially not run counter to the purport of the present invention, in this manual It can also be equally applicable as the combination of any technical characteristic or technical characteristic described by the composition part of a certain technical scheme In other technical schemes；Also, on the premise of it can implement and substantially not run counter to the purport of the present invention, it is used as different technologies scheme Composition part described by technical characteristic between can also be combined in any way, to constitute other technical schemes.This Invention be also contained in it is above-mentioned in the case of by technical scheme obtained from combination, and these technical schemes are equivalent to being documented in this In specification.
Above by embodiment and embodiment, the present invention is described, but those skilled in the art should manage Solution, these are not intended to be defined the scope of the present invention, and the scope of the present invention should be determined by claims.
Industrial applicibility
The culture medium of the present invention, cell culture are obtained with kit, epithelial cell cultural method and by this method Epithelial cell can be widely applied to basal cell biological study, tumor research, aging research, new drug development, gene therapy, External transgenosis and gene knockout research, regeneration, clinical regenerative medicine, clinical personalized treatment, molecule and heredity are popular Disease learns the every field such as research.

Claims (9)

1. a kind of culture medium is used for the purposes for cultivating epithelial cell, wherein, the culture medium is included：
Serum,
Calcium component, and
ROCK inhibitor；
The content of calcium component is calculated as 0.1 μM~10mM with calcium ion in the culture medium；
The content of serum is 2.0~20v/v% in the culture medium；
Content of the ROCK inhibitor in the culture medium is 0.1 μM~1mM；
The epithelial cell is non-Eponychium cell, the non-keratinocyte epithelial cell be oral cavity, tracheae, it is bronchial, Lung epithelial, gastric epithelial, colon, liver cell, pancreas, bladder, ovarian epithelium, thyroid, prostatitis Gland, mammary gland, it is tonsilar；
The epithelial cell is primary cell.
2. purposes according to claim 1, wherein, the ROCK inhibitor is to be selected from Fasudil, H-1152, Y- 27632nd, more than one or both of HA1100 and GSK429286.
3. purposes according to claim 1, wherein, the culture medium also includes feeder cells.
4. purposes according to claim 1, wherein, conditioned medium, the conditioned medium bag are included in the culture medium Secretion containing feeder cells and/or extract.
5. purposes according to claim 1, wherein, the lower limit of the content of calcium component is calculated as with calcium ion in the culture medium 10 μM, more preferably 100 μM, more preferably 200 μM, more preferably 400 μM, more preferably 500 μM, more preferably 800 μM, more preferably 1mM, more It is preferred that 1.2mM, more preferably 1.3mM, more preferably 1.5mM；The upper limit of the content of calcium component is calculated as with calcium ion in the culture medium 8mM, more preferably 7mM, more preferably 6mM, more preferably 5mM, more preferably 4mM, more preferably 3mM, more preferably 2.8mM, more preferably 2.5mM。
6. purposes according to claim 1, wherein, the lower limit of the content of serum is 1.0v/v%, more in the culture medium It is preferred that 2.0v/v%, more preferably 3.0v/v%, more preferably 4.0v/v%, more preferably 4.5v/v%, more preferably 5.0v/v%；It is described The upper limit of serum content is 35v/v%, more preferably 30v/v%, more preferably 25v/v%, more preferably 20v/v%, more in culture medium It is preferred that 17v/v%, more preferably 15v/v%, more preferably 13v/v%, more preferably 11v/v%, more preferably 10v/v%.
7. a kind of kit is used for the purposes for cultivating epithelial cell, wherein, the kit includes culture medium, serum, calcium component And ROCK inhibitor,
The epithelial cell be non-keratinocyte epithelial cell, the non-keratinocyte epithelial cell be colon, ovary, oral cavity, gas It is pipe, bronchial, lung epithelial, gastric epithelial, liver cell, pancreas, bladder, prostate, mammary gland or It is tonsilar,
The epithelial cell is primary cell；
The kit is used to prepare in the culture medium for including serum, calcium component and ROCK inhibitor, the culture medium, calcium component Content is calculated as 0.1 μM~10mM with calcium ion, and the content of serum is 2.0~20v/v%, and the content of ROCK inhibitor is 0.1 μM ~1mM.
8. a kind of method for cultivating epithelial cell, this method includes
Step A：Using comprising serum, the culture medium of calcium component and ROCK inhibitor co-cultures epithelial cell and feeder cells； Wherein,
The epithelial cell be non-keratinocyte epithelial cell, the non-keratinocyte epithelial cell be colon, ovary, oral cavity, gas It is pipe, bronchial, lung epithelial, gastric epithelial, liver cell, pancreas, bladder, prostate, mammary gland or It is tonsilar,
The epithelial cell is primary cell；
The content of calcium component is calculated as 0.1 μM~10mM with calcium ion in the culture medium；
The content of serum is 2.0~20v/v% in the culture medium；
Content of the ROCK inhibitor in the culture medium is 0.1 μM~1mM.
9. a kind of method for cultivating epithelial cell, this method includes
Step B：Use the medium culture epithelial cell comprising serum, calcium component and ROCK inhibitor；Wherein described culture medium Feeder cells or conditioned medium are also included, the epithelial cell is non-Eponychium cell,
The non-keratinocyte epithelial cell be colon, ovary, oral cavity, tracheae, bronchial, lung epithelial, gastric mucosa It is epithelium, liver cell, pancreas, bladder, prostate, mammary gland or tonsilar,
The epithelial cell is primary cell；
The content of calcium component is calculated as 0.1 μM~10mM with calcium ion in the culture medium；
The content of serum is 2.0~20v/v% in the culture medium；
Content of the ROCK inhibitor in the culture medium is 0.1 μM~1mM；
The conditioned medium includes feeder cells secretion and/or extract.
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