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CN102787094A - Culture medium, kit used for culturing cells and method for culturing cells - Google Patents

Culture medium, kit used for culturing cells and method for culturing cells Download PDF

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CN102787094A
CN102787094A CN2011104488063A CN201110448806A CN102787094A CN 102787094 A CN102787094 A CN 102787094A CN 2011104488063 A CN2011104488063 A CN 2011104488063A CN 201110448806 A CN201110448806 A CN 201110448806A CN 102787094 A CN102787094 A CN 102787094A
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substratum
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serum
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CN102787094B (en
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李晖
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Shenzhen Yongtai Biotechnology Co Ltd
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Abstract

The invention relates to a culture medium, a kit used for culturing cells and a method for culturing cells, and is especially relates to the culture medium used for culturing the epithelial cells, the kit used for culturing the cells and the method for culturing the cells. The culture medium comprises serum, a calcium component and a ROCK inhibitor.

Description

Substratum, cell cultures are with test kit and cell culture processes
The application is that application number is 201110127667.4, denomination of invention the dividing an application with Chinese invention patent application (applying date is on May 17th, 2011) of test kit and cell culture processes that be substratum, cell cultures.
Technical field
The present invention relates to substratum, cell cultures with test kit and cell culture processes, the substratum of cultivating especially for epithelial cell, cell cultures are with test kit and cell culture processes.
Background technology
Human body vitals such as lung, kidney, liver, pancreas and skin are its moity by the epithelial cell of organ specificity differentiation all.The epithelial cell of these specificitys differentiation is directly related with the specific function of Different Organs, as the detoxifcation of the filtering function of the gaseous interchange function of lung, kidney, liver and in and function, pancreatic cell produce Regular Insulin, skin and have and protect body to avoid the injury of external environment.And in a single day pathology takes place or degenerates to threaten human beings'health in these vitals, because these vitals are difficult to be replaced, the specific cell of Different Organs can not replace the cell of other organ each other.
The cell of specificity differentiation all is to be difficult to regenerated like the insulin-producing cells of renal epithelial cell, pancreas, the hair follicle and the gland cell of corium, lets alone external and has carried out multiplication by culture.Laboratory animal (comprising various transgenics and cloned animal) is the essential tool that is used for disease pathogenesis research and new drug development.But, for separate from the people and mammiferous former generation epithelial vitro culture remain difficulty very.Use present serum free medium; Former foster (survival a couple of days of being commissioned to train that can only carry out short-term that has; Like epithelial cells such as lung and pancreas), what have can only carry out the limited cultivation of going down to posterity (1-3 generation, like epithelial cells such as tracheae/segmental bronchus and prostate glands); That have even at all can not vitro culture (like epithelial cells such as liver and colons), the superficial cell that only comes from human body skin can go down to posterity and cultivate about 10 generations.And the epithelial cell output that from each animal or biological tissue sample, obtains is still very low, and the cell purity that comprises quantity, direct separation or the Short-term Culture of cell all is very low, and these former generations are also very limited with the epithelial rate of propagation that goes down to posterity.
In order to carry out epithelial cultivation external, external at present trial the, as change virus (SV40T or HPV16E6E7) or cellular oncogene over to, algebraically that can the outer cell survival of extension body through genetic manipulation.Yet the disadvantage of genetic manipulation is to change the genetic background and the phenotype of these cells, so that lets these normal epithelium cells lose its normal physiological function, usually is suppressed like p53 and pRB signal path.And these genetically modified cells can not be transplanted into body again again.But owing to lack at present the epithelial technology of effective vitro culture, above-mentioned these cells still enjoy favor in the medical science of the world today and life science.In non-cancer research field, they are just representing the tissue or the organ of " function " property in initial source.In the cancer research field, they also usually are used as " normal cell " contrast.And their market value is also very expensive.
The first step of antitumor drug research is exactly the specificity toxicity test of medicine to cancer cells; At present used JEG-3 (like HeLa and A539 etc.) is many at subculture in vitro separately several years even many decades; They can not react the characteristic of human malignant tumor of the same type to a great extent, and also there is the crossed contamination of a large amount of JEG-3s in some laboratories.And,,, all need and to represent its specific pathogenetic cell strain to carry out new drug development therefore for each tumour even the tumour of same type also possibly caused by different reasons or mechanism for different individualities.More outstanding example does not have the clone of human former prostate cancer like the research field of prostate cancer at all, and (LnCAP, PC-3 are from the tumor tissues after shifting DU-145) to only clone.However, the research of tumor research and the nearly all PTS of the whole world more than 90% also possibly not have representational JEG-3 continuing using at all.Therefore, cancer research at present with the key issue that new drug development will solve is, how to obtain to react all kinds human tumor characteristic, tumour of the same race is represented Different Individual characteristic or the machine-processed cancer cells of different onset.Certainly, a ultimate real difficult problem is to lack effectively epithelial former generation and go down to posterity culture technique.In addition, a lot of toxic side effect that antitumor drug exists are because new drug research lacks normal cell contrast to a great extent.If can obtain coming from cancer cells and normal cell (so-called " normally " and the prostatic cell of cancer that U.S. Asterand company obtains with above-mentioned HPVE6E7 conversion of the same tissue of same individuality; Though 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 treatment pattern of current clinical medicine; Promptly to regenerate, to reproduce, to replace and new life is the modern cellular replacement therapy art of base therapy principle; Utilize stem cell (embryonic stem cell and adult stem cell) to have and change ability to various cytodifferentiation; Treat sex change, gangrenosum acne and injury disease numerous clinically, that still do not have the treatment way at present, have remarkable and unique medical effect.Yet the predicament that faces at present is, though embryonic stem cell has differentiation capability, reproducibility and plasticity-are strong, maybe tumorigenesis with face the ethics dispute, how to induce its directed differentiation and increase the surviving rate of transplanting and remain a difficult problem of not separating; It is rare to form soma cell source, is difficult to identification, separation and purifying, has therefore hindered its clinical application; Present emerging research focus---inductive pluripotent stem cells, because it relates to the change that genetic manipulation causes the cytogenetics background, and the foreign gene and the virogene that import have the potential of carcinogenic or teratogenesis and can not be used for clinical treatment.
Individuality medicine is a modern medicine developing direction from now on.Genetic background and the correlative factor relevant according to the disease generation/development of clinical patient were diagnosed, are monitored disease exactly in the shortest time, and formulating real individualized treatment scheme is the key of dealing with problems.For example, World Health Organization's investigation finds that the drug safety problem is one of deadly most important reason of inpatient.Untoward reaction has become the healthy important public hygiene problem of harm humans due to the drug reaction difference between individuals.And inherited genetic factors is to cause the major cause of drug reaction difference between individuals.In the U.S., had 70 surplus kind of medicine sticked hereditary label through FDA approval, the clinical patients that is used to indicate different genotype when drug application to curative effect and toxic predicting function, and genetic background clearly new drug development will become developing tendency in future.It is the key to the tumour patient individualized treatment that the biopsy (inhaling cell etc. like pin) that how to utilize extremely trace carries out early stage quick diagnosis, external drug sensitive experiment, curative effect monitoring to diseases such as malignant tumours.All these will depend on fast and effectively, and the primary cell culture technology could really realize.
Biobank (Times is referred to as one of ten big ideas that change in the world) just rises in worldwide.The researchist of NIH is just making great efforts to create the human biological organization sample of the first preservation in the whole America, tumour cell, DNA, and the national biobank of blood.English, add, Norway and Sweden set up the nationwide mechanism of this type.Collect and store abundant, the biological organization sample from cancer, brain disorder to disease of metabolism for setting up individuality medicine with new drug development lays the foundation, lets screening and to treat the patient more targeted.Just comprise frozen tissue, fixed tissue, DNA, RNA, protein, blood, blood plasma, serum and urine etc. but at present biobank is stored, what these extremely valuable resources were used promptly goes, and can't regenerate.Effective former generation and passage cell culture technique will be injected real " work " power for biobank.This appearance with biobank of " regeneration " ability will have milestone significance to modern medicine study, individuation diagnosis and treatment, new drug development.
In sum; Urgent problem is at present; How to carry out the epithelial propagation and these epithelial cultivation of going down to posterity of former generation in histoorgan source quickly and efficiently, and can prolong epithelial cultivation algebraically effectively, the while can not change the genetic background of cell again.
Summary of the invention
The inventor furthers investigate to the problems referred to above; New substratum and test kit have been proposed; Through using this substratum or test kit in-vitro multiplication epithelial cell of former generation and going down to posterity the cultivation epithelial cell; Can under the situation that does not change the cytogenetics background, prolong epithelial cultivation algebraically effectively.
That is, the present invention provides following substratum (being also referred to as substratum of the present invention):
1. substratum, it comprises
Serum,
Calcium component and
The ROCK suppressor factor.
2. according to item 1 described substratum, wherein, the content of said serum is 1.0～35v/v%.
3. according to item 1 described substratum, wherein, said ROCK suppressor factor is to be selected among fasudil, H-1152, Y-27632, HA1100, HA1077 and the GSK429286 one or more.
4. according to item 1 described substratum, it is used to cultivate epithelial cell.
5. according to item 4 described substratum, wherein, said epithelial cell is non-Eponychium cell.
6. according to item 4 described substratum, wherein, said epithelial cell is a primary cell.
7. according to item 4 described substratum, wherein, said epithelial cell is a passage cell.
8. according to item 4 described substratum, wherein, said epithelial cell is a tumour cell.
9. according to item 5 described substratum, wherein, said non-keratinocyte epithelial cell is squamous cell, columnar epithelial cell, body of gland epithelial cell or transitional type epithelial cell.
10. according to item 5 described substratum, wherein, said non-keratinocyte epithelial cell is oral cavity, nasal mucosa; Tracheae, bronchial, lung epithelial, epithelium of esophagus; Gastric epithelial, big enteric epithelium, small intestine epithelium, hepatocellular, epithelial duct, gall-bladder, pancreas, beta Cell of islet; Kidney, bladder, urothelial, the 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. according to item 1 described substratum, wherein, the content of said calcium component is counted 0.1 μ M～30mM with calcium ion.
12. according to item 1 described substratum, wherein, this substratum also comprises feeder cell.
13., wherein, comprise conditioned medium in this substratum according to item 1 described substratum.
In addition, the present invention also provides following test kit (being also referred to as test kit of the present invention):
14. a test kit, it comprises substratum, serum and ROCK suppressor factor.
15. according to item 14 described test kits, this test kit is used to cultivate epithelial cell.
16. according to item 15 described test kits, wherein, said epithelial cell is non-Eponychium cell.
17. according to item 15 described test kits, wherein, said epithelial cell is a primary cell.
18. according to item 15 described test kits, wherein, said epithelial cell is a passage cell.
19. according to item 15 described test kits, wherein, said epithelial cell is a tumour cell.
20. according to item 16 described test kits, wherein, said non-keratinocyte epithelial cell is squamous cell, columnar epithelial cell, body of gland epithelial cell or transitional type epithelial cell.
21. according to item 16 described test kits, wherein, said non-keratinocyte epithelial cell is oral cavity, nasal mucosa; Tracheae, bronchial, lung epithelial, epithelium of esophagus; Gastric epithelial, big enteric epithelium, small intestine epithelium, hepatocellular, epithelial duct, gall-bladder, pancreas, beta Cell of islet; Kidney, bladder, urothelial, the 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. according to item 14 described test kits, it also comprises calcium component.
23. according to item 14 described test kits, it also comprises feeder cell.
24. according to item 23 described test kits, wherein, said feeder cell are that low temperature is frozen.
25. according to item 23 described test kits, wherein, said feeder cell are low-temperature storage.
26. according to item 14 described test kits, wherein, it also comprises the container that is used for when carrying out cell cultures, holding separately said feeder cell, this container has the permeability membrane structure.
27. according to item 23 described test kits, wherein, said feeder cell are inoblasts of mouse or people.
28. according to item 14 described test kits, wherein, said substratum comprises conditioned medium.
29. according to item 14 described test kits, wherein, said ROCK suppressor factor is to be selected among fasudil, H-1152, Y-27632, HA1100, HA1077 and the GSK429286 one or more.
30. a test kit, it comprises each described substratum and feeder cell in the item 1～13.
31. according to item 30 described test kits, wherein, said feeder cell are that low temperature is frozen.
32. according to item 30 described test kits, wherein, said feeder cell are low-temperature storage.
33. according to item 30 described test kits, wherein, it also comprises the container that is used for when carrying out cell cultures, holding separately said feeder cell, this container has the permeability membrane structure.
In addition, the present invention also provides the following epithelial method of cultivation (being also referred to as cultural method of the present invention):
34. cultivate epithelial method for one kind, this method comprises
Steps A: each described substratum is cultivated epithelial cell and feeder cell altogether in the use item 1～13.
35. according to the epithelial method of item 34 described cultivations, wherein, said epithelial cell is a primary cell.
36. according to the epithelial method of item 34 described cultivations, wherein, said epithelial cell is a passage cell.
37. according to the epithelial method of item 34 described cultivations, wherein, said epithelial cell is a tumour cell.
38. according to the epithelial method of item 34 described cultivations, wherein, said epithelial cell is non-Eponychium cell.
39. according to the epithelial method of item 38 described cultivations, wherein, said non-keratinocyte epithelial cell is squamous cell, columnar epithelial cell, body of gland epithelial cell or transitional type epithelial cell.
40. according to the epithelial method of item 38 described cultivations, wherein, said non-keratinocyte epithelial cell is oral cavity, nasal mucosa; Tracheae, bronchial, lung epithelial, epithelium of esophagus; Gastric epithelial, big enteric epithelium, small intestine epithelium, hepatocellular, epithelial duct, gall-bladder, pancreas, beta Cell of islet; Kidney, bladder, urothelial, the 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. according to the epithelial method of item 34 described cultivations, wherein, this cultivates epithelial method is the epithelial cell cultural method that goes down to posterity.
42. according to the epithelial method of item 34 described cultivations, wherein, this cultivates epithelial method is former generation epithelial cell proliferation cultural method.
43., wherein, epithelial cell and feeder cell are placed same cultivation vessel according to the epithelial method of item 34 described cultivations.
44., wherein, feeder cell are placed container separately with permeability membrane structure according to the epithelial method of item 34 described cultivations.
45. according to the epithelial method of item 34 described cultivations, wherein, said feeder cell are inoblasts of mouse or people.
46. cultivate epithelial method for one kind, this method comprises
Step B: use 12 or 13 a described culture medium culturing epithelial cell.
47. according to the epithelial method of item 46 described cultivations, wherein, said epithelial cell is a primary cell.
48. according to the epithelial method of item 46 described cultivations, wherein, said epithelial cell is a passage cell.
49. according to the epithelial method of item 46 described cultivations, wherein, said epithelial cell is a tumour cell.
50. according to the epithelial method of item 46 described cultivations, wherein, said epithelial cell is non-Eponychium cell.
51. according to the epithelial method of item 50 described cultivations, wherein, said non-keratinocyte epithelial cell is squamous cell, columnar epithelial cell, body of gland epithelial cell or transitional type epithelial cell.
52. according to the epithelial method of item 50 described cultivations, wherein, said non-keratinocyte epithelial cell is oral cavity, nasal mucosa; Tracheae, bronchial, lung epithelial, epithelium of esophagus; Gastric epithelial, big enteric epithelium, small intestine epithelium, hepatocellular, epithelial duct, gall-bladder, pancreas, beta Cell of islet; Kidney, bladder, urothelial, the 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. according to the epithelial method of item 46 described cultivations, wherein, this cultivates epithelial method is the epithelial cell cultural method that goes down to posterity.
54. according to the epithelial method of item 46 described cultivations, wherein, this cultivates epithelial method is former generation epithelial cell proliferation cultural method.
Substratum of the application of the invention or test kit are cultivated epithelial cell, can be under the situation that does not change the cytogenetics background, and effective multiplication culture epithelial cell of former generation, and prolong epithelial cultivation algebraically effectively.
Description of drawings
Fig. 1 is a cultural method synoptic diagram of the present invention, and wherein, 1., 2., 3. scheme is three kinds of culture systems of substratum of the present invention and feeder cell combination.
Fig. 2 adopts conditioned medium of the present invention to carry out the workflow diagram that epithelial cell is cultivated.
Fig. 3 adopts indirect (Transwell) co-culture system of feeder cell of the present invention to carry out the workflow diagram that epithelial cell is cultivated.
Fig. 4 is the workflow diagram that adopts feeder cell of the present invention and the direct co-culture system of epithelial cell.
Fig. 5 is normal people's segmental bronchus epithelial cell of former generation at serum free medium (left side) and contains the growth conditions cultivated in the blood serum medium (right side) relatively.Show among the figure that bronchial epithelial cell can not be bred in containing the substratum of serum.Separate the former tracheal epithelial cell of paying out of preparation by the method for embodiment 5; [composition is DMEM at last the cell precipitation that obtains to be suspended in completely the DMEM substratum again; Add 10% foetal calf serum (FBS); 100U/ml penicillium mould (penicillin) and 100 μ g/ml Streptomycin sulphates (streptomycin)] in, or the epithelial cell substratum SFM (GIBCO#10744-019) (seeing embodiment 10) of serum-free.After one week, under conventional inverted microscope, observe and photograph.Though culture effect is not good, serum free medium still is widely used in the conventional former generation of epithelial cell and the cultivation of going down to posterity.
Fig. 6 is that normal people's segmental bronchus epithelial cell growth state of former generation of cultivating compares.Show among the figure, for bronchial epithelial cell, adopt three kinds of culture systems of the present invention to cultivate, the growing multiplication of cell obviously is superior to using conventional serum free medium.Separate the former tracheal epithelial cell of paying out of preparation by the method for embodiment 5; The cell precipitation that obtains is suspended in again in the conditioned medium of embodiment 8 (method is seen Fig. 2); Perhaps re-suspended cell (is seen embodiment 2 in HL substratum of the present invention; HL substratum 6) in, press method and the embodiment 9 illustrated feeder cell of Fig. 3 and cultivate altogether indirectly, or by Fig. 4 and embodiment 6 carry out former be commissioned to train foster.After one week, under conventional inverted microscope, observe and photograph.Needed feeder cell go down to posterity according to embodiment 1 and protect kind in this illustrated research.Needed HL substratum is pressed embodiment 2 preparations in this illustrated research.Needed feeder cell are pressed embodiment 3 and 4 acquisitions in this illustrated research.
Fig. 7 is the comparison that normal people's bronchial epithelial cell subculture in vitro separately is cultivated.X-coordinate representes to cultivate fate, and ordinate zou is represented the multiple of cell proliferation.Show among the figure that for bronchial epithelial cell, adopt three kinds of culture systems of the present invention to cultivate, cell can about at least 10 generations, obviously be superior to adopting the cultivation of serum free medium at subculture in vitro separately.Separate the former tracheal epithelial cell of paying out of preparation by the method for embodiment 5; The cell precipitation that obtains is suspended in again in the conditioned medium of embodiment 8 (method is seen Fig. 2); Perhaps re-suspended cell (is seen embodiment 2 in HL substratum of the present invention; HL substratum 6) in, press method and the embodiment 9 illustrated feeder cell of Fig. 3 and cultivate altogether indirectly, or by the explanation of Fig. 4 and embodiment 6 and 7 cultivation of going down to posterity.Needed feeder cell go down to posterity according to embodiment 1 and protect kind in this illustrated research.Needed HL substratum is pressed embodiment 2 preparations in this illustrated research.Needed feeder cell are pressed embodiment 3 and 4 acquisitions in this illustrated research.
Fig. 8 has summed up the inventive method epithelial kind system and the type of culture successful.These cells come from people, mouse and rat respectively.The types of organization of these cells has, but is not limited to: oral cavity, tracheae, segmental bronchus, lung epithelial, stomach, colon, prostate gland, mammary gland, liver, pancreas, bladder, ovary and tonsil.The practical implementation step is pressed the method for embodiment 5 and is separated preparation epithelial cell of former generation; The cell precipitation that obtains is suspended in again in the conditioned medium of embodiment 8 and cultivates (method is seen Fig. 2); Perhaps (see embodiment 2, wherein HL substratum 1 is used for the epithelial cell cultivation in mammary gland, prostate gland, stomach, bladder source to re-suspended cell in HL substratum of the present invention; HL substratum 2 is used for the epithelial cell in liver source and cultivates; HL substratum 3 is used for the epithelial cell in colon, tonsil, mammary gland, stomach source and cultivates; HL substratum 4 is used for the epithelial cell in ovary, tonsil source and cultivates; HL substratum 5 is used for the epithelial cell in pancreas, tonsil source and cultivates; HL substratum 6 is used for the epithelial cell in oral cavity, tracheae, segmental bronchus, lung source and cultivates), press the illustrated feeder cell cultivation altogether indirectly of Fig. 3 and embodiment 9, or cultivate by the explanation of Fig. 4 and embodiment 6 and 7 and to go down to posterity.Needed feeder cell go down to posterity according to embodiment 1 and protect kind in this illustrated research.Needed HL substratum is pressed embodiment 2 preparations in this illustrated research.Needed feeder cell are pressed embodiment 3 and 4 acquisitions in this illustrated research.
Shown in Figure 9 for deriving from the cultivation instance of people, rat and mouse epithelial cell.Left figure is the prostate epithelial cell that derives from the people; Press the method for embodiment 5 and separate preparation epithelial cell of former generation; The cell precipitation that obtains is resuspended in middle cultivation of HL conditioned medium (promptly cultivate altogether, obtain) of embodiment 8 by diagram 2 methods by HL substratum 1 and feeder cell.Middle figure derives from mammary epithelial cell of rat and the pulmonary epithelial cells of mouse with right figure; Press the method for embodiment 5 and separate preparation epithelial cell of former generation; The cell precipitation that obtains is resuspended in HL substratum 1 or HL substratum 6 among the embodiment 2, by the cultivation of going down to posterity of the method for Fig. 4 and embodiment 6 and 7.Needed feeder cell go down to posterity according to embodiment 1 and protect kind in this illustrated research.Needed HL substratum is pressed embodiment 2 preparations in this illustrated research.Needed feeder cell are pressed embodiment 3 and 4 preparations in this illustrated research.
The more different foetal calf serum concentration of Figure 10 are to the influence of epithelial cell growth.Press embodiment 2 preparation HL substratum, add foetal calf serum and make its final concentration be respectively 1%, 2%, 5%, 10%.15％，20％。With normal people's bronchial epithelial cell of 5000 s-generations (in Fig. 7 research; The method of embodiment 9) is suspended in respectively in 3 milliliters of above-mentioned HL substratum that contain different foetal calf serum concentration; Place the Tissue Culture Plate in 6 holes then, again 500000 feeder cell (by embodiment 3 preparations) are placed transwell basket (Corning Company products), according to the method for Fig. 3; At 5%CO2, cultivated 6 days in 37 ℃ the incubator.Discard the transwell basket and the feeder cell of splendid attire wherein, discard remaining substratum in the culture plate, wash once with the phosphoric acid buffer of no calcium magnesium; Add 1ml0.05%EDTA/ trysinization liquid, hatched about 2-3 minute for 37 ℃, treat that cell takes off wall fully after; Adding 2ml contains the DMEM substratum of 10% foetal calf serum; Abundant mixing cell takes out dyeing in 4% the trypan blue (trypan blue) that the 50ul cell suspension is mixed in 50ul, carries out cell counting by ordinary method.
The chemical structure of the ROCK suppressor factor that Figure 11 substratum of the present invention is added.
Figure 12 shows the influence of the concentration of ROCK suppressor factor to epithelial cell growth.Press embodiment 2 preparation HL substratum 6, add different ROCK suppressor factor and make its final concentration be respectively 1uM, 5uM, 10uM, 20uM, 50uM.With the normal bronchial epithelial cell of 5000 s-generations (from Fig. 7 research; The method of embodiment 9) is suspended in respectively in 3 milliliters of above-mentioned HL substratum 6 that contain different concns ROCK suppressor factor; Place the Tissue Culture Plate in 6 holes then, again 500000 feeder cell (by embodiment 3 preparations) are placed transwell basket (Corning Company products), according to the method for Fig. 3; At 5%CO2, cultivated 8 days in 37 ℃ the incubator.Discard the transwell basket and the feeder cell of splendid attire wherein, discard remaining substratum in the culture hole, wash once with the phosphoric acid buffer of no calcium magnesium; Add 1ml 0.05%EDTA/ trysinization liquid, hatched about 2-3 minute for 37 ℃, treat that cell takes off wall fully after; Adding 2ml contains the DMEM nutrient solution of 10% foetal calf serum; Abundant mixing cell takes out dyeing in 4% the trypan blue (trypan blue) that the 50ul cell suspension is mixed in 50ul, carries out cell counting by ordinary method.
The embodiment of invention
Substratum of the present invention
First aspect of the present invention relates to a kind of substratum (being also referred to as substratum of the present invention, i.e. the HL substratum), and it comprises serum, calcium component and ROCK suppressor factor.
In this specification sheets, substratum is meant the matrix that is used to cultivate biomaterial.Not having particular restriction for biomaterial, can be virus, cell (for example microorganism cells, zooblast, vegetable cell), tissue (for example animal tissues, plant tissue) etc.Substratum of the present invention preferably is used for the substratum of cell cultures, i.e. cell culture medium.Substratum of the present invention is more preferably and is used for the substratum that epithelial cell is cultivated.Epithelial cell in this specification sheets can for example derive from body of gland for for example non-keratinocyte epithelial cell, comprises the non-keratinocyte epithelial cell of mammary gland, prostate gland, liver and gut epithelium.The non-keratinocyte epithelial cell is divided into the active cells with absorption or secreting function, and the non-keratinocyte epithelial cell is not the squamous cell of high angle materialization structure.The non-keratinocyte epithelial cell of available culture medium culturing of the present invention can derive from the histoorgan of any kind of.The non-keratinocyte epithelial cell that can cultivate comprises but is not defined as following type: the oral cavity, nasal mucosa; Tracheae, bronchial, lung epithelial, epithelium of esophagus, gastric epithelial, big enteric epithelium, small intestine epithelium, hepatocellular, epithelial duct, gall-bladder, pancreas (comprising beta Cell of islet), kidney, bladder, urothelial, the 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 the solid powdery substratum, also can be liquid nutrient medium, preferably liquid nutrient medium.When substratum of the present invention is the solid powdery substratum; Preferably in use it is modulated into liquid nutrient medium and carries out cell cultures; In modulation process, can add various compositions such as serum, calcium component and ROCK suppressor factor, make it satisfy important document of the present invention.
In this specification sheets, when object was cell, " cultivation " or " cell cultures " was meant external and carries out manual work and maintain, make cell be able to propagation.Cell cultures is carried out under aseptic condition usually, can be to cultivate individual cells or tissue.
Should contain serum in the substratum of the present invention.As serum, can use serum commonly used in the cell cultures, for example calf serum or foetal calf serum, preferably foetal calf serum.As serum, also comprise various serum substitutes, serum substitute comprises but is not limited to the product that the commercialization buys (Knockout of Invitrogen company for example TMSerum substitute, the Ultroser of Pall Corporation company TM), also comprise milk and milk constituents (for example filtering skimmed milk dry powder).Amount for the serum in the substratum does not have particular restriction, can with the cell cultures of routine in to be added into the amount of the serum in the substratum identical, and those skilled in the art can suitably adjust as required.Preferably, in volume ratio (v/v%), 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 the 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 the substratum.
In this specification sheets, ROCK is meant Rho kinases 1 (ROCK 1) and/or Rho kinases 2 (ROCK 2).Suppress ROCK and be meant activity, expression or the function that reduces at least a enzyme among ROCK1 or the ROCK2.Compare with undressed cell, ROCK activity, expression or function can be suppressed promptly to reach non-activity, do not have expression or do not have function fully, also can be activity, expression or functional level reduction.In this specification sheets, the ROCK suppressor factor is meant: the material with the effect that suppresses ROCK.For the ROCK suppressor factor, there is not particular restriction, can use ROCK suppressor factor well known in the art, fasudil (Fasudil) for example, H-1152, Y-27632, HA1100, HA1077 and GSK429286 etc., these all are commercial common agents.In addition, the ROCK suppressor factor also is included in PCT and applies for that (WO 03/059913, and WO 03/064397; WO 05/003101, and WO 04/112719, WO 03/062225 and WO03/062227) in disclosed, or in U.S.'s granted patent (7; 217,722 and 7,199; 147) disclosed small molecules ROCK suppressor factor and in the U. S. application patent (2003/0220357,2006/0241127,2005/0182040 and 2005/0197328).Content for ROCK suppressor factor in the substratum of the present invention does not have particular restriction, so long as significant quantity gets final product.Said significant quantity is meant the amount that can suppress ROCK; The method of confirming significant quantity is well known to a person skilled in the art, those skilled in the art can confirm significant quantity aptly according to the conditions such as kind of the kind of institute's cultured cells, employed ROCK suppressor factor.For example, though maybe be different because of the kind of the kind of cell, ROCK suppressor factor, cell culture condition etc., generally speaking, the ROCK inhibitor content can be between 0.1 μ M～1mM.
Can also comprise feeder cell in the substratum of the present invention.In this specification sheets, feeder cell are meant: with the cell of hoping the cultured cells co-cultivation, wherein, saidly hope preferably non-keratinocyte epithelial cell of cultured cells.Feeder cell generally are to handle through gamma radiation exposure and/or MTC, and its mitotic division is suppressed, but still can keep metabolic activity.Therefore, feeder cell are to have metabolic capacity but can not the non-proliferative cell of splitted.The method of handling and blocking cell mitogen and keep cell metabolic activity can be the ordinary method of cytobiology.Feeder cell can derive from any Mammals, but its source is different with the kind of hoping cultured cells (preferred non-keratinocyte epithelial cell) source.Feeder cell can be but be not limited to following source, like mouse, rat, dog, cat, ox, horse, pig, primates and human feeder cell.Typical feeder cell type comprises splenocyte, hugely bites thymocyte and/or inoblast, and they become non-proliferative cell after treatment.In addition, also can utilize J2 as feeder cell among the present invention, the J2 cell is a subclone of building the mouse fibroblast cell Swiss 3T3cells that is, handles through gamma radiation exposure and/or MTC.Amount for the feeder cell that comprised in the substratum of the present invention does not have particular restriction, and those skilled in the art can suit to confirm as required, and feeder cell can be 1: 9～9: 1 with the epithelial ratio of hoping cultivation usually, preferred 2: 8～8: 2.
In addition, can comprise conditioned medium in the substratum of the present invention.In this specification sheets, conditioned medium is meant the substratum that comprises feeder cell secretory product and/or extract.Typical conditioned medium is with after culture medium culturing feeder cell for some time, removes feeder cell again and the substratum that obtains.The substratum that is used to cultivate feeder cell here, preferably comprises the substratum of serum, calcium component and ROCK suppressor factor, substratum for example of the present invention.Time for cultivating feeder cell does not have particular restriction, and those skilled in the art can suit to confirm as required; Usually can be 1～300 hour, preferred 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, the preparation condition substratum for example comprises cell cultures (like F substratum) in a kind of substratum, collects these substratum after several days.Conditioned medium can be that the conditioned medium after diluting by a certain percentage with fresh culture also can be the conditioned medium after the not diluted collection.Fresh culture: the Dilution ratio of conditioned medium can be 1: 99～99: 1, decides according to concrete 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 that contains the not diluted of serum, calcium component and ROCK suppressor factor.
Substratum of the present invention can use the conventional substratum that is used for cell cultures to prepare.Conventional substratum is meant the substratum that is used for cell cultures (being preferred for epithelial cell cultivates) well-known in the art, includes but not limited to: and 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 substratum also can be the combination of two or more substratum, the for example mixed culture medium of DMEM and F12.Generally do not comprise serum in the prescription of conventional substratum, therefore, when using conventional substratum to prepare substratum of the present invention, can add a certain amount of serum separately, make serum content in the substratum in above-mentioned restricted portion.The operation that increases or reduce the serum in the substratum is the routine operation in present technique field.
Should contain calcium component in the substratum of the present invention.Alleged here " calcium component " is meant can provide calcium ion (Ca for substratum of the present invention 2+) composition, include but not limited to calcareous compound.Usually, calcium component derives from serum or serum substitute, perhaps derives from the calcareous salt that adds in the substratum.Said calcium component for example can be added in the substratum with the form of calcium salt (for example calcium chloride, calcium sulfate).The content range of calcium component (in calcium ion) can be identical with the calcium component content of the conventional substratum that is used for cell cultures, and those skilled in the art can suitably adjust as required.For example, with Ca 2+Meter, the lower limit of calcium component content can be 0.1 μ M, preferred 10 μ M, more preferably 100 μ M, more preferably 200 μ M, more preferably 400 μ M, more preferably 500 μ M, more preferably 800 μ M, 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 substratum to prepare substratum of the present invention, preferably adjust and make calcium component content in above-mentioned restricted portion.If comprise calcium and calcium component content in the classics prescription of conventional substratum in above-mentioned restricted portion, then with regard to calcium component content, this routine substratum need not to adjust; If comprise calcium in the classics prescription of conventional substratum but calcium component content not in above-mentioned restricted portion, then should correspondingly increase or reduce the calcium in the prescription, make the calcium component content of substratum in above-mentioned restricted portion; If do not comprise calcium in the classics prescription of conventional substratum, then should in prescription, increase calcium, make the calcium component content of substratum in above-mentioned restricted portion.The operation that increases or reduce the calcium component content in the substratum is the routine operation in present technique field.
Can add other conventional ingredient in the substratum of the present invention; Comprise but be 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 (cholera toxin), 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 the substratum of the present invention comprises but is not limited to: L-L-Ala, L-l-arginine, altheine (L-asparagine), L-aspartic acid, L-halfcystine, L-L-glutamic acid, L-glutaminate, glycocoll, L-Histidine, L-Isoleucine, L-leucine, L-Methionin, L-methionine(Met), L-phenylalanine(Phe), L-proline(Pro), L-Serine, L-Threonine, L-tryptophane, L-tyrosine and L-Xie Ansuan.
Vitamins in the substratum of the present invention comprises but is not limited to: vitamin H, choline chloride 60 (choline chloride), D-Ca + 2-pantothenic acid (pantothenate), folic acid (folic acid), i-inositol (i-inositol), vitamin PP (niacinamide), many alcohol (pyridoxine), vitamin G (riboflavin), VITMAIN B1 (thiamine) and cobalamin.
The inorganic salts composition comprises but is not limited to: calcium salt is (like CaCl 2), CuSO 4, FeSO 4, KCl, magnesium salts be (like MgCl 2), manganese salt is (like MnCl 2), sodium-acetate, NaCl, NaHCO 3, Na 2HPO 4, Na 2SO 4, and other element of trace such 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 salt preferably, like 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 comprises but is not limited to: heparin, Urogastron (epidermal growth factor; EGF), at least aly can increase 3'5'-AMP (cyclic adenosine monophosphate in the cell; CAMP) factor of level, at least a fibroblast growth factor (fibroblast growth factor, FGF).Above-mentioned added ingredients can directly join in the basic medium, also can use DPBS (Dulbecco ' s Phosphate Buffered Saline) damping fluid to be mixed with mixed solution, and freezing preservation is row interpolation again when preparing culture medium.Heparin in the substratum of the present invention can be bought in commercialization.The main effect of heparin is the activity of the stable growth factor, like 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 cAMP level in the cell that can comprise multiple kind in the substratum of the present invention; Can be [like two butyryl (dibutyryl) cAMP] that directly increases the cAMP level; Also can be through causing in the cell cAMP level to raise [like Toxins,exo-, cholera and forskolin (forskolin)], perhaps increase cAMP level in the cell, or make cAMP level increase [like isobutylmethylxanthine (IBMX) and theophylline (theophylline)] in the cell through the activity that suppresses the cAMP phosphodiesterase as the antagonist (like Racemic isoproterenol) of receptor, (β-adrenergic receptors) with cell G protein-interacting.Above-mentioned these compounds can be bought in commercialization.
Test kit of the present invention
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 suppressor factor.
Test kit of the present invention is preferred for culturing cell.Said cell is epithelial cell preferably, is more preferably the non-keratinocyte epithelial cell.
Said substratum can be any substratum that is used for culturing cell, and for example above-mentioned conventional substratum preferably can be above-mentioned conditioned medium.Said serum, calcium component and ROCK suppressor factor are with above-mentioned.Test kit of the present invention can be used to prepare the substratum of the invention described above.In the test kit of the present invention, substratum, serum and ROCK suppressor factor three's ratio preferably satisfies the substratum that can form the invention described above after three's mixing.
Preferably, test kit of the present invention also comprises calcium component, and at this moment, the ratio of substratum, serum, calcium component and ROCK suppressor factor preferably satisfies the substratum that can form the invention described above after four mixing.
Preferably, test kit of the present invention also comprises feeder cell.Said feeder cell are with above-mentioned.Said feeder cell can be conventional adherent, can survive under the normal temperature 3-5 days; Said feeder cell also can be that low temperature is frozen, and low temperature is frozen to be meant in standing storage below-80 ℃, generally can store several days to several years.Said feeder cell can also be low-temperature storage, and low-temperature storage is meant at 4 ℃ and stores down, generally can store 1～7 day.
Preferably, test kit of the present invention also comprises the container that is used for when carrying out cell cultures, holding separately said feeder cell, and this container has the permeability membrane structure.Particularly, comprise at test kit of the present invention under the situation of feeder cell, preferably it also comprises said container.The effect of said container is, when carrying out cell cultures, with feeder cell with need cultured cells to keep apart to cultivate, and make the secretory product of feeder cell enter into the substratum that cultivation needs cultured cells through above-mentioned permeability membrane structure.All do not have particular restriction for the shape of said container, material, specification etc., those skilled in the art can suit to select as required.For example, can use Transwell basket (Corning Company products).
Preferably, test kit of the present invention comprises substratum of the present invention and feeder cell, more preferably also comprises said container.
Can also comprise other composition that is used for adding to substratum in the test kit of the present invention, include but not limited to the added ingredients of in aforesaid " substratum of the present invention " joint, describing.
Can also comprise other composition or the assembly that are used for cell cultures in the test kit of the present invention, for example indicator, pancreatin, culturing bottle, petridish, working instructions etc.
In the test kit of the present invention, preferred said each composition is placed apart, more preferably places independently container respectively.
Cultural method of the present invention
The third aspect of the invention relates to the epithelial method of a kind of cultivation (also claiming cultural method of the present invention), and this method comprises
Steps A: use the substratum of the invention described above that epithelial cell and feeder cell are cultivated altogether; Or
Step B: use the above-mentioned culture medium culturing epithelial cell (see figure 2) of the present invention that comprises feeder cell or conditioned medium.
Cultural method of the present invention can be the epithelial cell cultural method that goes down to posterity, and also can be former generation epithelial cell proliferation cultural method.
Here, said epithelial cell and feeder cell are all with aforementioned.
Can epithelial cell and feeder cell directly be cultivated altogether in the steps A, also can epithelial cell and feeder cell be cultivated indirectly altogether.Said direct cultivation altogether is meant epithelial cell and feeder cell is placed same cultivation vessel, cultivates (see figure 4); Said indirect cultivation altogether is meant feeder cell is placed the above-mentioned container with permeability membrane structure (for example Transwell basket) separately; Then; Can for example again this container that feeder cell are housed be placed and cultivate epithelial substratum, cultivate (see figure 3).
Other step in the cultural method of the present invention can be carried out according to the ordinary method in present technique field.
The inoculum density of cell can suitably be adjusted according to concrete experiment condition.Conventional disposable plastic is cultivated in the vessel, and 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 6Go down to posterity each time and all will adjust according to concrete requirement of experiment pair cell density.
Mammalian cell is incubated at 37 ℃, suitable CO usually 2In the incubator of concentration (3～10%), certain humidity, temperature, CO 2Concentration and humidity can be adjusted according to concrete experiment condition.The scope of the pH of substratum can be adjusted as required, is generally pH7.1～7.6, is preferably pH7.1～7.3
Changed once, and also can adjust in the common 1-2 of cell culture medium days according to concrete cultured cells type.As long as cell grows to full abundance in cultivating vessel, just can go down to posterity.In this specification sheets, passage is meant cell is divided in kind of a part to the new cultivation vessel.
Embodiment
Below, through embodiment the present invention is explained more specifically, but the present invention does not receive the restriction of these embodiment.In addition, among the embodiment without the reagent of special mark, all available from GIBCO company.
The cultivation of embodiment 1Swiss 3T3 cell is gone down to posterity
Swiss 3T3 cell obtains from the Howard Green professor laboratory of Harvard University.
Required reagent:
DMEM substratum: DMEM (GIBCO#11965-092) completely, 10% foetal calf serum (FBS), 100U/ml penicillium mould (penicillin) and 100 μ g/ml Streptomycin sulphate (streptomycin) trysinization liquid: 0.05% pancreatin/EDTA (GIBCO#25300-062)
No calcium magnesium phosphoric acid buffer: Ca 2+, Mg 2+Free PBS
The cultivation operation steps goes down to posterity:
Inoculation 1x10 5Swiss 3T3 cell adds the above-mentioned complete DMEM substratum of 10ml in the T75 Tissue Culture Dish, places CO 2Incubator is at 5%CO 2, cultivated 2-5 days for 37 ℃.When the cell monolayer abundance is 80-90%, remove the substratum in the petridish, there are not calcium magnesium phosphoric acid buffer washed cell individual layer 2 times with 10ml, add 2ml trysinization liquid, hatched 1 minute at 37 ℃.Pat petridish with the adherent cell of abundant release, with 10ml completely in the DMEM substratum and digestion reaction.4 ℃ of centrifugal 5 minutes (1000 rev/mins) sedimentation cells are removed supernatant, and re-suspended cell is deposited in 10ml and cultivates in the DMEM substratum completely.As required can 1: 10,1: 20 or 1: 50 ratio go down to posterity in the petridish of new T75 (10-15ml is the DMEM substratum completely) or T175 (25-30ml is the DMEM substratum completely), change the fresh substratum of DMEM completely 2-3 time as required weekly.In case of necessity can be with 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 the liquid nitrogen 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), and volume proportion is 3: 1, adds 5% foetal calf serum 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 growth factor (EGF)); 24 μ g/ml VITAMIN B4s (adenine), 100U/ml penicillium mould (penicillin) and 100 μ g/ml Streptomycin sulphates (streptomycin), 0.25 μ g/ml amphotericin B (Fungizone;) 30 μ M fasudils (Fasudil) (or 0.5 μ M H-1152 or 5 μ M Y-27632; 30 μ M HA1100,10uM GSK429286), above-mentioned substratum needs through 0.22 μ aperture membrane filtration.
HL substratum 2 is at DMEM (Low Glucose, No Glutamine, GIBCO#11054-020) middle 10% the foetal calf serum that adds; 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 B4s (adenine), 100U/ml penicillium mould (penicillin) and 100 μ g/ml Streptomycin sulphates (streptomycin), 0.25 μ g/ml amphotericin B (Fungizone;) 30 μ M fasudils (Fasudil) (or 0.5 μ M H-1152 or 5 μ M Y-27632; 30 μ M HA1100,10 μ M GSK429286), above-mentioned substratum needs through 0.22 μ aperture membrane filtration.
HL substratum 3 contains 5mMHEPES for RPMI substratum 1640 (GIBCO#22400-121), adds 8% foetal calf serum, 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 B4s (adenine); 100U/ml penicillium mould (penicillin) and 100 μ g/ml Streptomycin sulphates (streptomycin); 0.25 μ g/ml amphotericin B (Fungizone) 30 μ M fasudils (Fasudil) (or 0.5 μ M H-1152 or 5 μ MY-27632,30 μ M HA1100; 10 μ M GSK429286), above-mentioned substratum needs through 0.22 μ aperture membrane filtration.
HL substratum 4 is at L-15 (Leibovitz) substratum (L-glutamine, No HEPES, GIBCO#11415064) middle 5% the foetal calf serum that adds; 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 B4s (adenine), 100U/ml penicillium mould (penicillin) and 100 μ g/ml Streptomycin sulphates (streptomycin), 0.25 μ g/ml amphotericin B (Fungizone;) 30 μ M fasudils (Fasudil) (or 0.5 μ M H-1152 or 5 μ M Y-27632; 30 μ M HA1100,10 μ MGSK429286), above-mentioned substratum needs through 0.22 μ aperture membrane filtration.
HL substratum 5 is at substratum 199 (with Earle ' s salts but no L-glutamine, no sodium bicarbonate, GIBCO#11825015) middle 10% the foetal calf serum that adds; 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 B4s (adenine), 100U/ml penicillium mould (penicillin) and 100 μ g/ml Streptomycin sulphates (streptomycin), 0.25 μ g/ml amphotericin B (Fungizone;) 30 μ M fasudils (Fasudil) (or 0.5 μ M H-1152 or 5 μ M Y-27632; 30 μ M HA1100,10 μ MGSK429286), above-mentioned substratum needs through 0.22 μ aperture membrane filtration.
HL substratum 6 is that (mixed culture medium of GIBCO#10744-019, volume proportion are 1: 3 to DMEM (GIBCO#11965-092), add 5% foetal calf serum simultaneously with serum free medium SFM; 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 B4s (adenine), 100U/ml penicillium mould (penicillin) and 100 μ g/ml Streptomycin sulphates (streptomycin), 0.25 μ g/ml amphotericin B (Fungizone;) 30 μ M fasudils (Fasudil) (or 0.5 μ M H-1152 or 5 μ M Y-27632; 30 μ M HA1100,10 μ M GSK429286), above-mentioned substratum needs through 0.22 μ aperture membrane filtration.
The preparation of embodiment 3 feeder cell (losing the Swiss 3T3 cell of splitting ability) (silk splits the plain C of enzyme handles swiss 3T3 cell)
1) as the swiss 3T3 cell of feeder cell, should adopt goes down to posterity cultivates early stage cell.When swiss 3T3 cell grew to full abundance, the silk that adds final concentration in the substratum and be 10 μ g/mL split the plain C (water-soluble, storage liquid concentration is 0.5mg/mL) of enzyme, handles 2 hours for 37 ℃;
2) add the 1xPBS of temperature bath or the substratum (DMEM) of serum-free again and wash 3 times, abandon washing lotion;
3) add 0.05% pancreatin/EDTA predigestion cell 30-40 and discard after second, add 0.05% pancreatin/EDTA once more and digested 30 seconds, pat petridish cell is disperseed, add perfect medium (DMEM that contains 10% foetal calf serum) neutralization reaction then;
4) low-speed centrifugal (1000rpm) removes supernatant, obtains cell precipitation;
5) cell that precipitates can directly be used as feeder cell, perhaps stores subsequent use;
A) directly as the situation of feeder cell, operation steps is following:
-add the warm 1xPBS that bathes to wash cell precipitation 1 time, recentrifuge
-cell is suspended from the HL substratum
-in the proper ratio (as 1: 3) epithelial cell of feeder cell and some amount is suspended in the HL substratum, the righttest inoculum density of epithelial cell is 2.5x10 5The petridish of individual/100mm;
B) feeder cell are second day subsequent use situation, and operation steps is following:
-add the warm 1xPBS that bathes to wash cell precipitation 1 time, recentrifuge
-cell is suspended from complete DMEM substratum (DMEM that contains 10% foetal calf serum)
-be inoculated in the petridish overnight cultures with 1: 3 ratio
The 1xPBS that bathed with temperature in-the second day washes cell 2 times, changes the HL substratum into
-again epithelial cell being inoculated in the petridish that contains feeder cell, the righttest inoculum density of epithelial cell is 2.5x10 5The petridish of individual/100mm;
C) feeder cell are to store subsequent use situation, and operation steps is following:
-add the warm 1xPBS that bathes 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 frozen pipes
-frozen under liquid nitrogen or-150 ℃ of conditions
-face the time spent to get 1 recovery, add complete DMEM substratum (DMEM that contains 10% foetal calf serum) and be inoculated in the 100mm petridish
Remarks: the 3T3 cell organizine that is used as feeder cell splits the plain C of enzyme handle after, need in 3-5 days, to use or low temperature frozen subsequent use.
The preparation of embodiment 4. feeder cell (losing the Swiss 3T3 cell of splitting ability) (radioactive rays are handled swiss 3T3 cell)
As the swiss 3T3 cell of feeder cell, should adopt goes down to posterity cultivates early stage cell, and these cell cultures grow to the nearly abundance that expires, with not containing Ca in the DMEM substratum 2+/ Mg 2+ 1xPBS wash cell 1 time; Add 0.05% trypsin digestion cell 20-40 second of 1ml; Add in the complete DMEM substratum of 9ml (DMEM that contains 10% foetal calf serum) then and digestion reaction,, add 10ml DMEM re-suspended cell in 4 ℃ of low-speed centrifugal 1000rmp collecting cell depositions; The radiation exposure cell suspension, promptly Cesium-137 alpha cellulose a gage (JL Shepherd Mark) is handled 3000Rad (30Grey).These are inoculated in the HL substratum directly as feeder cell through the swiss 3T3 cell that radioactive rays are handled; Perhaps be incubated at complete DMEM substratum or place 4 ℃ to store and within 1-7 days, use; Perhaps long-term frozen subsequent use in-80 ℃ or liquid nitrogen.
Separation and Culture epithelial cell of former generation in embodiment 5. flesh tissues
Under the situation of patient or patient care people informed consent, collector's normal or neoplasmic tissue sample.The concrete operations step is following:
1. the preparation of Digestive system: the HL substratum that contains collagenase/Unidasa (final concentration is the mixed solution of 1x) (is DMEM: 3: 1 mixed culture medium of F12 volume ratio), add 5%FBS.
According to the consumption of the size of flesh tissue decision Digestive system, need 10 times usually to the consumption of the Digestive system of sample volume, for example 2-3 mouse prostate gland need 2-5ml Digestive system;
2. wash isolating flesh tissue 1 time with the ethanol of 95-100%, wash 2 times with PBS again, then tissue is put into the sterile petri dish that contains precooling PBS, under dissecting microscope, remove residual fat in the tissue with dissecting tweezers and scissors.
3. tissue sample is put into 14ml or the 50ml centrifuge tube that contains above-mentioned Digestive system, 37 ℃ digested 1-3 hour.
4. organize low-speed centrifugal (1000rpm) 5 minutes with postdigestive, remove supernatant.
5. cell precipitation is resuspended among 0.25% pancreatin/EDTA of 2-5mL, placed on ice 1 hour or room temperature 10 minutes.
6. add 10ml then and contain the DMEM substratum of 10%FBS, centrifugal 5 minutes of low speed 1000rmp.
7. supernatant is removed clean as far as possible.
8. add the 5mg/mL Dispase II of 2ml temperature bath and the 1mg/mL DNase I of 200 μ L, blew and beat sample repeatedly 1 minute with aseptic P1000 disposable plastic rifle head.
9. add the DMEM that 10ml contains 10%FBS, with the strainer filtration cell suspension in 40-70 μ m aperture, collect the cell suspension after filtering, centrifugal 5 minutes of low speed 1000rmp removes supernatant.
10. re-suspended cell is deposited in the HL substratum, is inoculated in the culturing bottle of T25 or T75 to cultivate by arbitrary scheme of the present invention (seeing Fig. 1-4).
Embodiment 6. feeder cell/epithelial directly cultivate altogether (seeing Fig. 4 and Fig. 6)
The feeder cell (losing the Swiss3T3 cell of splitting ability) that above-mentioned silk splits plain C processing of enzyme or radiation exposure are undertaken by following three kinds of modes as feeder layer and epithelial the cultivation altogether:
1. directly feeder cell/epithelial cell is cultivated altogether.Add the 1xPBS that temperature bathes and wash the feeder cell deposition 1 time, recentrifuge is suspended from cell in the HL substratum, is suspended in the HL substratum with 1: 3 ratio epithelial cell with feeder cell and some amount, and the righttest inoculum density of epithelial cell is 2.5x10 5The petridish of individual/100mm places 37 ℃ with culturing bottle, 5%CO 2Cultivate under the condition.
2. feeder cell are adherent in advance, add epithelial cell again and cultivate altogether.The feeder cell that above-mentioned silk split plain C processing of enzyme or radiation exposure are suspended from complete DMEM substratum (DMEM that contains 10% foetal calf serum); Be inoculated in the petridish with 1: 3 ratio; Cultivated 2-3 hour or spent the night for 37 ℃, the 1xPBS that bathes with temperature washes cell 2 times, changes the HL substratum into; Epithelial cell is inoculated in the petridish that contains feeder cell, the righttest inoculum density of epithelial cell is 2.5x10 again 5The petridish of individual/100mm, adherent feeder cell can use in 3-5 days any times.Culturing bottle is placed 37 ℃, 5%CO 2Cultivate under the condition.
3. feeder cell are subsequent use at 4 ℃ or liquid nitrogen storage.Above-mentioned silk is split that the plain C of enzyme handles or the feeder cell of radiation exposure are suspended from complete DMEM substratum (DMEM that contains 10% foetal calf serum) and are stored in 4 ℃ (1-7 days) or standing storage in liquid nitrogen, 2 carry out and epithelial cultivate altogether (in the HL substratum) as stated above after the recovery.The righttest inoculum density of epithelial cell is 2.5x10 5The petridish of individual/100mm.Culturing bottle is placed 37 ℃, cultivate under the 5%CO2 condition.
No matter with the common cultivation of above-mentioned which kind of mode, should change fresh HL substratum in second day.When coming off more than 50% feeder cell, should replenish fresh above-mentioned silk and split that the plain C of enzyme handles or the feeder cell of radiation exposure, up to epithelial cell growth when the 70-90% abundance need go down to posterity.
Embodiment 7. feeder cell/epithelial separation is gone down to posterity
1. epithelial cell growth is to the 70-90% abundance, and the sucking-off substratum is washed 1 time with the warm PBS that bathes,
2. sucking-off PBS adds the PBS washed cell that contains 0.02%EDTA (0.68mM) that the 10-12mL temperature is bathed, to remove feeder cell Swiss 3T3; Pay special attention to, washed cell need be tried one's best fast, earlier EDTA solution is added to the cell of petridish periphery, rotation wave and culture ware, and then make the circle round cell in to the petridish week of EDTA solution, washed cell 10 times repeat to circle round.
3. sucking-off EDTA washing lotion adds 0.02%EDTA once more to the inboard cell of petridish, and rotation wave and culture ware makes EDTA solution circle round to the cell in the petridish outside then, and washed cell 30 times repeat to circle round.Usually people's Eponychium cell needs 40 EDTA washings, and other cell is different according to adherent tightness degree, can suitably adjust washing times.
4. sucking-off EDTA washing lotion adds the warm PBS that bathes and washes 3 times, remains petridish.Before the last washings sucking-off, examine under a microscope, confirm that all feeder cell Swiss 3T3 are washed off fully, otherwise, the EDTA washing times need be increased once more.
5. adopt conventional pancreatin method digestive epithelium cell, for the Eponychium cell, pancreatin normal temperature digestion 2 minutes, the sucking-off Digestive system once more in 37 ℃ of trysinizations 5 minutes, is patted petridish cell is disperseed, and re-suspended cell is in the HL substratum.Inoculate epithelial cell then on the feeder cell that ametycin or radioactive rays were handled, the optimal cell inoculum density is 2.5x10 5The petridish of individual/100mm;
7. in case of necessity can be with 1x10 6Epithelial cell is resuspended in the cells frozen storing liquid (90% foetal calf serum and 10%DMSO) of 1-2ml, is stored in the liquid nitrogen subsequent use.
The preparation and the epithelial cultivation of embodiment 8. feeder cell conditioned mediums go down to posterity (seeing Fig. 2 and Fig. 6)
According to Fig. 2, the feeder cell that above-mentioned silk split plain C processing of enzyme or radiation exposure are incubated at (T75 culturing bottle: 2x10 in the HL substratum 6Cell, 15ml HL substratum; T175: 5x10 6Cell, the 35mlHL substratum) at 37 ℃, 5%CO 2Incubator in cultivated 48 hours.Collect substratum, centrifugal 15 minutes of 2000rpm, through 0.22 μ membrane filtration, (can be by 1: 99-99: 1 optimization) mix with freshly prepared HL substratum and promptly obtain the HL conditioned medium by 1: 5 ratio.The primary cell suspension or the passage cell that separate preparation are directly cultivated in the HL conditioned medium, and culture condition is 37 ℃, 5%CO 2When cell proliferation to 70-90% abundance, 1xPBS washed cell twice, 0.05% pancreatin/EDTA digested monolayer 2-5 minute; Among the DMEM and digestion reaction, centrifugal 5 minutes of 1000rmp removes supernatant to 10ml completely; With certain proportion (1: 2,1: 3,1: 4; 1: 5 all can, as long as can keep cell attachment and normal growth) re-suspended cell is deposited in inoculation culture in the 10ml HL conditioned medium.In case of necessity can be with 1x10 6Epithelial cell is resuspended in the cells frozen storing liquid (90% foetal calf serum and 10%DMSO) of 1-2ml, is stored in the liquid nitrogen subsequent use.
Embodiment 9. epithelial cells/feeder cell are cultivated (Transwell culture systems) and go down to posterity (seeing Fig. 3 and Fig. 6) indirectly altogether
According to Fig. 3; The feeder cell that above-mentioned silk split plain C processing of enzyme or radiation exposure place Transwell basket (Transwell insert; Corning company) in, separate the primary cell suspension or the passage cell of preparation and can directly in the HL substratum, cultivate, culture condition is 37 ℃, 5%CO 2When cell proliferation to 70-90% abundance, remove the Transwell basket earlier, twice of 1xPBS washing epithelial cell; 0.05% pancreatin/EDTA digestion monolayer 2-5 minute, 10ml be completely among the DMEM and digestion reaction, centrifugal 5 minutes of 1000rpm; Remove supernatant, with certain proportion (1: 2,1: 3; 1: 4,1: 5 all can, as long as can keep cell attachment and normal growth) re-suspended cell is deposited in 10ml HL inoculation of medium and cultivates.In case of necessity can be with 1x10 6Epithelial cell is resuspended in the cells frozen storing liquid (90% foetal calf serum and 10%DMSO) of 1-2ml, is stored in the liquid nitrogen subsequent use.
Embodiment 10. contains blood serum medium and serum free medium is used for epithelial cultivation (see figure 5)
DMEM substratum: DMEM completely, 10% foetal calf serum (FBS), 100U/ml penicillium mould (penicillin) and 100 μ g/ml Streptomycin sulphates (streptomycin)
Serum free medium: the epithelial cell substratum SFM (GIBCO#10744-019) of serum-free, qingfengmeisu qiong (10 μ g/m1)
Trysinization liquid: 0.05% pancreatin/EDTA
No calcium magnesium phosphoric acid buffer: Ca 2+, Mg 2+Free PBS
Separate the primary cell suspension or the passage cell of preparation and directly in above-mentioned complete DMEM substratum or serum free medium, cultivate, culture condition is 37 ℃, 5%CO 2When cell proliferation to 70-90% abundance; Twice of 1xPBS washed cell; 0.05% pancreatin/EDTA digestion monolayer 2-5 minute, 10ml be completely in the DMEM substratum and digestion reaction, centrifugal 5 minutes of 1000rmp; Remove supernatant, be deposited in complete DMEM of 10ml or the serum free medium and cultivate with certain proportion (normally 1: 2 or 1: 3) re-suspended cell.In case of necessity can be with 1x10 6Cell is resuspended in the cells frozen storing liquid (90% foetal calf serum and 10%DMSO) of 1-2ml, is stored in the liquid nitrogen subsequent use.
Also need to prove; Can implement and the not obvious prerequisite of running counter to purport of the present invention under, the combination as described arbitrary technical characterictic of the component part of a certain technical scheme or technical characterictic equally also goes for other technical scheme in this manual; And, can implement and the not obvious prerequisite of running counter to purport of the present invention under, as also making up between the described technical characterictic of the component part of different technologies scheme, constitute other technical scheme with any-mode.The present invention is also contained in the technical scheme that obtains through combination under the above-mentioned situation, and these technical schemes are equivalent to record in this manual.
More than describe the present invention through embodiment and embodiment, but it will be understood by those skilled in the art that these are not that intention limits scope of the present invention, scope of the present invention should be confirmed by claims.
Industrial applicibility
Substratum of the present invention, cell cultures can be widely used in every field such as basic RESEARCH ON CELL-BIOLOGY, tumor research, aging research, new drug development, gene therapy, external transgenic and gene knockout research, tissue regeneration, clinical regenerative medicine, clinical personalized treatment, molecule and genetic epidemiology research with test kit, epithelial cell cultural method and the epithelial cell that obtains through this method.

Claims (8)

1. substratum, it comprises
Serum,
Calcium component and
The ROCK suppressor factor.
2. substratum according to claim 1, wherein, this substratum also comprises feeder cell.
3. substratum according to claim 1 wherein, comprises conditioned medium in this substratum.
4. test kit, it comprises substratum, serum and ROCK suppressor factor.
5. test kit according to claim 4, it also comprises feeder cell.
6. test kit, it comprises each described substratum and feeder cell in the claim 1～3.
7. cultivate epithelial method for one kind, this method comprises
Steps A: each described substratum is cultivated epithelial cell and feeder cell altogether in the use claim 1～3.
8. cultivate epithelial method for one kind, this method comprises
Step B: use claim 2 or 3 described culture medium culturing epithelial cells.
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