CN102559579A - Novel multi-cell three-dimensional co-culture system for in-vitro detection of newly born blood vessel and kit thereof - Google Patents
Novel multi-cell three-dimensional co-culture system for in-vitro detection of newly born blood vessel and kit thereof Download PDFInfo
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Abstract
The invention discloses a novel multi-cell three-dimensional co-culture system for in-vitro detection of a newly born blood vessel and a kit thereof. The co-culture system disclosed by the invention mainly comprises two layers, wherein the first layer comprises substrate gel cells or a substrate gel cell tissue mixture, or substrate gel tissue mixture, the first layer is tightly adhered to the bottom of a culture ware, blood vessel endothelial cells are uniformly distributed in a gel layer, and tumor cell aggregates or a biopsy sample of a tumor tissue or a biopsy sample of a pancreas islet tissue, a biopsy sample of an endometrium tissue or biopsy samples of other tissues suspend in the gel layer; and the second layer comprises a cell culture liquid which is arranged on the first layer. The kit disclosed by the invention is a high-flux medicine-selecting kit or a standardized individual kit. After the co-culture system and the kit are applied, a new medicine and a special medicine for inhibiting regeneration of a blood vessel and promoting the regeneration of the blood vessel are screened in high flux through observing the blood vessel regeneration level of a blood vessel endothelial cell, and the co-culture system and the kit can be applied to the monitoring of individual treatment and drug resistance of a cancer patient.
Description
Technical field
The present invention relates to a kind of various kinds of cell 3 D stereo co-culture system and test kit thereof of novel vitro detection new vessel.
Background technology
Mutual coordinative role between living organism dependence its intravital various cells, various growth factor and the body fluid is kept the normal function of body.Revascularization is exactly wherein a kind of synergistic bioprocess of various kinds of cell that relates to, and its characteristic shows as the blood vessel that existing revascularization makes new advances in certain organ or tissue.Through further investigation for many years, we find that new vessel has very important effect to the incidence and development of tissue, vascular disease and cancer.Under the normal circumstances, the human and animal only just has revascularization to take place in certain specified phase.For example, in fetation and after the Mammals birth, revascularization plays crucial effects in growth course, corpus luteum forming process and the wound healing stage of ovary, uterine endometrium, placenta.
Revascularization is a process of being regulated by many revascularization regulatory factors (Folkman, J.Nature Med., 1:27-31,1995).These factors comprise the factor that promotes revascularization and inhibition revascularization, and whether the balance decision revascularization between these two kinds of factors takes place.Abnormal revascularization usually is accompanied by the generation of some diseases.These diseases comprise the incidence and development of cancer, mellitus, sacroiliitis, rheumatoid arthritis, vascularization of cornea, atheroma formation, psoriasis and diabetic retinopathy.
Osteo-arthritis belongs to non-bacteria inflammation, and the pathological characteristic of main morbidity is because the disturbance of blood circulation of joint tissue causes histiocytic nutrition supply bad, causes histocyte generation pathology.If can improve blood circulation, promote the immunization of white corpuscle, scavenger cell etc., realize anti-inflammatory and repercussive effect.
The pancreas islet endotheliocyte is the important component part of islet cell populations; Have heterogeneous strong, permeability is high, be characteristic such as blood sugar concentration dependency ground secretion vaso-active substance. pancreas islet endotheliocyte and islet function are in close relations; Except to pancreatic islet endocrine supply oxygen and nutritive substance, also interact with endocrine cell, participate in pathophysiological processes such as the generation of pancreas islet blood vessel, pars endocrina pancreatis growth, insulin gene induced expression, pancreas islet Inflammatory response damage and islet transplantation revascularization; Be the important target spot (Li Xin of research islet function; Yuan Li, 200929 (1), international endocrine metabolism magazine).The diabetic vascular endotheliocyte has to be regulated vascular tone, vascular permeability and promotes the effect that blood vessel takes place.Therefore; The blood vessel inner skin cell function obstacle is considered to the key factor of the caused chronic vascular complication morbidity of mellitus; But the blood vessel inner skin cell function obstacle is influenced by the various performance of insulin resistant also can; As: (getting in touch of insulin resistant and endotheliocyte pathology, Chinese mellitus magazine, 2008 (3)) such as hypertension, blood fat disorder and hyperglycemias.
Tumour is through the various growth factor of secretion; For example, VEGF (VEGF) and Basic Fibroblast Growth Factor (bFGF) thus the induced tumor tissue blood vessel regeneration provides the tumour more nutrition and removes the quick growth that metabolic waste in the tumour promotes tumour.Research shows that revascularization plays crucial effects to metastases simultaneously, and statistic data shows that metastases is the high key factor of cancer patient mortality ratio.Evidence suggests that the regenerated blood vessel is rambling in the tumour, and vessel wall not only comprises vascular endothelial cell and pericyte as long blood vessel, some blood vessel also possibly be integrated with tumour cell, therefore is the mosaic structure.This mosaic structure makes tumour cell invade blood at an easy rate, thereby metastases takes place.1971, Folkman proposed the theory of famous anti-angiogenic regenerative therapy cancer.From that time, scientific research circle has just started the upsurge of studying and seek anti-angiogenic regeneration mechanism and medicine.Recently, U.S. food and drugs administration approved the monoclonal antibody drug Avastin of anti-vascular endothelial growth factor of the treatment people rectum cancer be exactly an instance.
Summary of the invention
Technical problem to be solved by this invention provides a kind of various kinds of cell 3 D stereo co-culture system and test kit thereof of novel vitro detection new vessel.Through using the revascularization level that the present invention observes the extracorporeal blood vessel endotheliocyte; Can high-throughput ground screening suppress the new drug of revascularization, special medicine and promote the new drug and the special medicine of revascularization; Can be applied to cancer patients's personalized treatment and chemical sproof monitoring thereof; Can be for patient make the most suitable this patient's efficient medicine to measure, thus improve therapeutic efficiency and shorten the treatment phase.
Technical scheme provided by the invention is: a kind of various kinds of cell 3 D stereo co-culture system of novel vitro detection new vessel; It comprises two-layer; Wherein, the first layer is the matrix gel cell, or matrix gel cell tissue mixture; Or matrix gel mixed tissue thing; Be close to the bottom of used cultivation vessel, mammiferous vascular endothelial cell is dispersed in the gel coat, and mammiferous or the tumour cell class cell mass or the biopsy samples of tumor tissues or the biopsy samples of the biopsy samples of islet tissue, endometrial tissue biopsy samples or other histoorgans is suspended in the gel coat; The second layer is a cell culture fluid, on the first layer.
The three-dimensional co-culture system of above-mentioned external various kinds of cell is cultivated observation in cell culture incubator; Observe following at least a kind of index: in the first layer glue-line, the generating state of the upgrowth situation of vascular endothelial cell, vascular endothelial cell tubular structure or the level of pancreatic islet endocrine excreting insulin or the situation of its hetero-organization function corresponding.
Described vascular endothelial cell is the vascular endothelial cell of a kind of GFP of permanent transfection expression; For example; Vascular endothelial cell by transfection red fluorescent protein expression plasmid pDsRed, filter out the vascular endothelial cell of stably express red fluorescent protein through using G418.Then, use selected by flow cytometry apoptosis to go out the vascular endothelial cell of high expression level red fluorescent protein.
Said tumour cell or tumor tissues biopsy samples are the tumour cells of a kind of GFP of permanent transfection expression; And the emission spectrum of the GFP that tumour cell is expressed is different with the GFP emission spectrum of the permanent transfection expression of vascular endothelial cell.For example, tumour cell by transfection blue fluorescent protein expression plasmid pCFP, filter out the tumour cell of stably express blue fluorescent protein through using G418.Then, use selected by flow cytometry apoptosis to go out the tumour cell of high expression level blue fluorescent protein.
Also comprise other at least a kind of mammal cell line in the said second layer.
Described co-culture system also comprises other a kind of mammal cell line at least, and this clone can a kind of GFP of permanent transfection expression; The emmission spectrum of the GFP that the emmission spectrum of this GFP and vascular endothelial cell and tumour cell are expressed is all different.For example, select for use pericyte to be added on this co-culture system as other a kind of cell.Pericyte by transfection yellow fluorescence protein expression plasmid pYFP, filter out the pericyte of stably express yellow fluorescence protein through using G418.Then, use selected by flow cytometry apoptosis to go out the pericyte of high expression level yellow fluorescence protein.
Above-mentioned other a kind of mammal cell line is a kind of in the following cell: immunocyte is scavenger cell and mastocyte for example; Mesenchymal cell is inoblast and adipocyte for example; Or required pericyte takes place in blood vessel.
The biopsy samples of said islet tissue biopsy samples or other histoorgans is a kind of GFPs of permanent transfection expression; The emission spectrum of the GFP that the emission spectrum of this kind GFP and vascular endothelial cell and other a kind of mammiferous clone that possibly include are expressed is different.
Described co-culture system in the first layer or the second layer, has at least one deck to contain reagent to be tested.
Described co-culture system, wherein said reagent to be tested are known or unknown but have the reagent of anti-angiogenic regeneration or the reagent of promotion revascularization.
Said mammiferous tumour cell derives from some main bodys, simultaneously, first or the second layer have at least one deck to contain a kind of reagent to be tested, this reagent once was used for treating its tumour by this main body.
Said mammiferous biopsy samples derives from some main bodys, and this main body is people or animal.
Said animal is wild-type animal or transgenic animal.
Said transgenic animal are the transgenic animal of expressing a kind of GFP; The emission spectrum of the GFP that the emission spectrum of expressed GFP and vascular endothelial cell and other a kind of mammal cell line that possibly comprise are expressed is different.For example, use the gfp transgene big white mouse.After isolating islet tissue, islet tissue is cut into the fritter about 1 cubic millimeter, add in the matrix gel solution cell mixture that contains the endotheliocyte of expressing red fluorescent protein.
The present invention also provides a kind of stdn test kit, comprises described co-culture system.
Described stdn test kit is the personalized diagnoses and treatment test kit of stdn high-throughput sieve medicine test kit or stdn.
Wherein, stdn high-throughput sieve medicine test kit is that screening has the test kit that promotes revascularization and the medicine that suppresses revascularization; The personalized diagnoses and treatment test kit of stdn is for the selected cancer therapy drug with efficient therapeutic action of cancer patients, exempts the test kit of the blindness of chemotherapy.
The said stdn test kit of a kind of application carries out high-throughput antineoplastic vascular regenerating medicine method for screening, and its process is following:
A kind of monoclonal cell spheroid of tumor cell line or certain cancer patients's cancerous tissue biopsy samples are applied in the described stdn high-flux medicaments sifting test kit; Cultivate altogether with vascular endothelial cell; Candidate agent to be tested is added in each hole of cell cultures dish; Set up the negative control group of adding 2% foetal calf serum simultaneously and add 3% methyl-sulphoxide as positive controls; Observation test group reagent vascular endothelial cell forms capillary structure; Compare with the result of negative control group and positive controls,, show that the anti-angiogenic regenerated effectiveness of this candidate agent is very weak or almost do not have anti-angiogenic regenerated effectiveness if the result of revascularization is similar with negative control group or close; If the result of revascularization and positive controls result are similar, show that the anti-angiogenic regenerated effectiveness of this candidate agent is more intense or have anti-angiogenic regenerated effectiveness.
A kind of preparation method of described co-culture system, it comprises the steps:
(1) preparation of matrix gel solution cell mixture
A. use cell cultures flask culture vascular endothelial cell and pericyte; When vascular endothelial cell and pericyte are that stand density is when reaching 70% left and right sides; Use the proteopepsis enzyme that the cell of adherent growth is separated from wall of container, be suspended in neutral phosphonic acid buffer low-speed centrifugal washing several times, at last cell is suspended in the EBM2 grown cultures liquid again; And use the density of blood-counter system pair cell to count, preserve for use then;
B. matrix gel solution of preserving after will thawing and vascular endothelial cell or vascular endothelial cell are blended in the matrix gel solution together with the cell of pericyte or other kinds equably;
(2) preparation of the processing of cell cultures dish and co-culture system the first layer
A. the processing of cell cultures dish: the cell cultures dish is a porous, and the NaOH solution soaking of process 4N 1 hour is cleaned several times with sterile distilled water then, then after the HCL solution-treated with 4N; Clean several times with sterile distilled water again, last, use aseptic neutral phosphonic acid buffer to clean several times, with the culture plate after handling air-dry after; Put into the cell culture incubator inner equilibrium, preferred temperature is 37 ℃ in the cell culture incubator, then culture plate is sealed; Preserve, subsequent use, preferred 4 ℃ of storage temperature;
B. the preparation of co-culture system the first layer: in each hole of above-mentioned ready culture plate, add the first layer of the matrix gel solution cell mixture of 30-50 μ l, after interpolation finishes, preserve for use as co-culture system;
(3) preparation of tumour cell monoclonal cell spheroid or biopsy sample
A. the preparation of tumour cell monoclonal cell spheroid: use the Tissue Culture Flask culture of tumor cell; When growth of tumour cell density reaches the 60-80% left and right sides; Use the proteopepsis enzyme that the cell of adherent growth is separated from wall of container, be suspended in neutral phosphonic acid buffer low-speed centrifugal washing several times, at last cell is suspended in the grown cultures liquid again; And the density of pair cell counts, and preserves for use then; In the nutrient solution of tumour cell, add foetal calf serum, microbiotic, 1.5% agar-agar soln and above-mentioned tumour cell; This cell mixture is added on the agar gel layer that solidifies refrigerative 1.5%; Then, it is transferred to cultivate 14 days～21 days in the cell culture incubator of 100% humidity to obtain tumour cell mono-clonal spheroid; With long be tumour monoclonal cell spheroid centrifugation from low-density agar gel of 0.5 millimeter～1 millimeter to diameter, through the washing of several times neutral phosphonic acid buffer; The antibiotic neutral phosphonic acid buffer of use interpolation suspends sedimentary tumour monoclonal cell spheroid again and transfers in the Micro-Organism Culture Dish then; The picking diameter is that 0.5～0.7 millimeter tumour spheroid is put into the hole central authorities that added cold matrix gel solution cell mixture in advance; This alveolar disk transferred to make cold matrix gel solution solidifies in the cell culture incubator that contains 100% humidity, afterwards, take out this alveolar disk; Nutrient solution is added in each hole; At last, culture plate is placed on cultivates in the incubator that contains 100% humidity and observations, preferred temperature is 37 ℃ in the cell culture incubator;
B. the preparation of biopsy sample
The biopsy sample retention of gathering is subsequent use in preserving liquid; After from preserve liquid, taking out biopsy samples; Spirituous solution through 70% embathes several times, after the neutral phosphonic acid buffer embathes several times, transfers in the Tissue Culture Dish; And be divided into fritter, place the hole central authorities of the alveolar disk that has added cold matrix gel solution cell mixture in advance; Then, this alveolar disk is transferred to made cold matrix gel solution solidifies in the cell culture incubator that contains 100% humidity; Take out this alveolar disk, nutrient solution is added in each hole; At last, culture plate is placed in the incubator that contains 100% humidity cultivates, observe, preferred temperature is 37 ℃ in the cell culture incubator.
Described preparation method is added with reagent to be tested in said matrix gel solution cell mixture or the nutrient solution.
Described preparation method, the use density of vascular endothelial cell is 140,000-1,000,000 cell/ml, pericyte or other kind cells are according to adding in the matrix gel solution with 1: 50～1: 5 ratio of vascular endothelial cell; Said matrix gel solution is the class matrix gel product of the matrix gel that extracts in the animal body that is fit to cell cultures or synthetic or both mixtures according to 1: 3～1: 1 ratio.
Cold matrix gel solution of the present invention is 4 ℃ matrix gel solution.
The clone that the present invention uses, matrix gel solution, porous cell culture plate, cell culture fluid are bought-in article.People's vascular endothelial cell of former generation purchase (HMVEC) from Shanghai the general Bioisystech Co., Ltd that flies, human brain perivascular cell (HBVP) is available from the rich Science and Technology Ltd. of the abundant perseverance in Beijing.Through cultivating and the transfection human telomerase gene, through the human vascular endothelial system and the pericyte system of being immortalized property of Xin Meisu G418 screening.Vascular endothelial cell system and pericyte system use EBM2 basic culture solution and growth factor thereof to add test kit, available from the good scientific instrument ltd that fits in Shanghai.Various tumor cell lines use ATCC clone to cultivate the corresponding nutrient solution of being advised available from U.S. histocyte engineering corporation (ATCC).These nutrient solutions and foetal calf serum are available from general Bioisystech Co., Ltd and the Biolab China Branch of flying in Shanghai.Red (pDsRed), yellow (pYFP), blue (pCFP) and green (pIRES-EGFP) fluorescence protein expression carrier/plasmid are available from Jin Weike (China) biotechnology center.The various clones of the different GFPs of permanent expression be through traditional electrotransfection method transfection corresponding fluorescent protein expression plasmid, obtain the clone of the corresponding GFP of permanent expression through the further screening of Xin Meisu G418 screening and flow cytometer.
The present invention has following beneficial effect:
The novel external various kinds of cell 3 D stereo culture system of the present invention mainly comprises matrix gel cell mixture and cell culture fluid.Matrix gel can be a collagen protein, Matrigel, and Hydrogel, the class matrix gel product of matrix gel that extracts in the animal body of suitable cell cultures such as Geltrex or synthetic or both are according to the mixture of 1: 3～1: 1 ratio.Cell is mammalian blood endothelial cell line or wild-type mammalian blood endothelial cell line and tumour cell, tumor biopsy sample, pancreatic tissue biopsy samples of permanent transfection expression GFP etc.This novel external various kinds of cell 3 D stereo culture system is applied in the various porous cell culture plates as an applying unit, can be prepared into the corresponding standard test kit.Test kit is stdn high-throughput sieve medicine test kit or the personalized test kit of stdn.Use the present invention, through observing the revascularization level of vascular endothelial cell, can the screening of high-throughput ground suppress the new drug and the special medicine of the new drug of revascularization, special medicine and promotion revascularization.And, adding the biopsy samples of the cancerous tissue of using the cancer patients, the present invention can be applied to cancer patients's personalized treatment and chemical sproof monitoring thereof.If in this stdn test kit, use different patients' biopsy sample, can also make the most suitable this patient's efficient medicine to measure for patient, thereby improve therapeutic efficiency and shorten the treatment phase; Meanwhile, the partner treatment process, this stdn test kit can predict in advance that whether patient has produced resistance to the existing medicine that uses, and provides scientific basis for the medical worker adjusts regimen timely and reasonably.
Embodiment
Come further to illustrate the present invention through the detailed description of embodiment below, but be not limitation of the present invention, only make example description.
Embodiment 1: the preparation of various kinds of cell 3 D stereo culture system
1, the preparation of matrix gel solution cell mixture
Use 150 centimetres
2Cell cultures flask culture vascular endothelial cell; 75 centimetres
2Cell cultures flask culture pericyte.When vascular endothelial cell and pericyte are that stand density is when reaching 70% left and right sides; Use the proteopepsis enzyme that the cell of adherent growth is separated from wall of container; Be suspended in neutral phosphonic acid buffer low-speed centrifugal washing 2-3 time; At last cell is suspended in the EBM2 grown cultures liquid of 500 μ l again, and uses the density of blood-counter system pair cell to count, being placed on then wet preserve on ice for use.
The matrix gel solution that will be kept at 4 ℃ after will thawing (comprises collagen protein; Matrigel; Hydrogel, the class matrix gel product of matrix gel that extracts in the animal body of suitable cell cultures such as Geltrex or synthetic or both are according to the mixture of 1: 2 ratio) be blended in equably in the matrix gel solution together with the cell that the plan of pericyte or other kinds adds in this three-dimensional co-culture system with vascular endothelial cell or vascular endothelial cell.The use density of vascular endothelial cell is 140,000-1, and 000,000 cell/ml is (such as 140; 000 cell/ml, 1000,000 cells/ml, 600,000 cells/ml etc., in the present embodiment; The density of using of vascular endothelial cell is g00,000 cell/ml), pericyte or other kind cells according to the application target of test kit according to 1: 50～1: 5 ratio of vascular endothelial cell (such as 1: 50; 1: 40,1: 30,1: 10; 1: 5 etc. in the present embodiment, is 1: 30 ratio) add in the matrix gel solution.At this moment, reagent to be tested can add in this matrix gel solution cell mixture.With this matrix gel solution cell mixture be placed on wet preserve on ice for use.
2, the preparation of the processing of cell cultures dish and novel external many cells 3 D stereo culture system the first layer thereof
Porous cell culture plate as carrying platform can be the vessel that 384-hole, 96-hole, 48-hole, 24 holes, 12-hole, 4-hole etc. are similar, be suitable for cell cultures.Is example at this porous cell culture plate with the 96-porose disc.The NaOH solution soaking of 96-porocyte culture plate process 4N one hour is cleaned three times with sterile distilled water then.Then with the HCL solution-treated of 4N after one hour, again with sterile distilled water cleaning three times.At last, use aseptic neutral phosphonic acid buffer to clean three times.With the culture plate after handling be placed in the aseptic technique platform air-dry after, put into the cell culture incubator inner equilibrium 12 hours, 37 ℃ of cell cultures the temperature inside the box.Then culture plate is sealed with Parafilm, be placed under the room temperature or under 4 ℃ and preserve.During use, Parafilm is removed, be placed on culture plate and wet on ice, in each hole of culture plate, add the first layer of the matrix gel solution cell mixture of 30-50 μ l as this novel external many cells 3 D stereo culture system.Add finish after, 96-porocyte culture plate still be placed on wet preserve on ice for use.
3, the preparation of tumour cell monoclonal cell spheroid or biopsy sample
(1) preparation of tumour cell monoclonal cell spheroid
Use 75 centimetres
2The Tissue Culture Flask culture of tumor cell.When growth of tumour cell density reaches 70% left and right sides; Use the proteopepsis enzyme that the cell of adherent growth is separated from wall of container; Be suspended in neutral phosphonic acid buffer low-speed centrifugal washing 2-3 time; At last cell is suspended in 1 milliliter the corresponding grown cultures liquid again, and uses the density of blood-counter system pair cell to count, being placed on then wet preserve on ice for use.The lower melting point agar-agar soln of preparation 1.5% used the high-pressure sterilizing pot sterilization 30 minutes.Can adopt any suitable cell cultures vessel to prepare tumour cell monoclonal cell spheroid, for example adopt the 6-porose disc.When agar-agar soln is cooled to 45 ℃ of left and right sides, in every hole of aseptic 6-porose disc, add the 1.5ml agar-agar soln.Waiting for 20 minutes solidifies agar.Meanwhile, add foetal calf serum, microbiotic (penicillium mould and Streptomycin sulphate), 1.5% agar-agar soln and tumour cell in the employed nutrient solution of tumour cell.The concentration of foetal calf serum is 6%, and microbiotic is 6 times and cultivates employed concentration to normal cell, and agar concentration is 0.22-0.3%, and the concentration of tumour cell is that ten thousand cell/3ml of 1-5 (are 30,000 cells/3ml) in the present embodiment.This cell mixture is added on the agar gel layer that solidifies refrigerative 1.5%, and every hole addition is 3ml.Then, the 6-porose disc is carefully transferred to cultivated 14 days～21 days in the cell culture incubator of 100% humidity to obtain tumour cell mono-clonal spheroid, 37 ℃ of cell cultures the temperature inside the box.
With long be tumour monoclonal cell spheroid centrifugation from low-density agar gel of 0.5 millimeter～1 millimeter to diameter, through three neutral phosphonic acid buffers washings.Use adding then that antibiotic neutral phosphonic acid buffer suspends sedimentary tumour monoclonal cell spheroid again and transfer to diameter is in 100 centimetres the Micro-Organism Culture Dish.The Micro-Organism Culture Dish that fills tumour monoclonal cell spheroid is transferred under the anatomical lens; Use Drummond microscale sampler picking diameter under anatomical lens is that 0.5～0.7 millimeter tumour spheroid is put into the hole central authorities that added cold matrix gel solution cell mixture in advance, the spheroid in every hole.Behind the end of operation, this alveolar disk transferred to make cold matrix gel solution solidifies in the cell culture incubator that contains 100% humidity.After 45 minutes, take out this alveolar disk, carefully 50-100 μ l (being 80 μ l in the present embodiment) nutrient solution is added in each hole.If reagent to be tested does not add in the matrix gel solution cell mixture of the first layer in advance, at this moment can it be added in the nutrient solution (EBM2) of the second layer.At last, culture plate is placed in the incubator that contains 100% humidity cultivates.In general, 7-9 days (being 8 days in the present embodiment) can observations and taken pictures and compare analysis then.Time length is different according to the difference of cell category.
(2) preparation of biopsy sample
But in the various kinds of cell 3 D stereo culture system of the present invention also the using-system biopsy samples substitute tumour monoclonal cell spheroid, the preparation of biopsy sample is following:
With the biopsy sample retention that collects cold, contain 2-3 doubly in (in the present embodiment being 2 times) microbiotic, the high sugared RPMI1640 nutrient solution, being placed on that wet to be transported to preparation room on ice for use.After from preserve liquid, taking out biopsy samples, the spirituous solution through 70% embathes twice, and after the neutral phosphonic acid solution embathed three times, it was in 100 centimetres the Tissue Culture Dish that biopsy samples is transferred to a diameter.Use disposable tweezers and No. 10 scalpels that biopsy samples is divided into the fritter about 1 cubic millimeter.Biopsy samples after the disposable aspiration needle of use 25G will cut is placed on the hole central authorities of the alveolar disk that has added cold matrix gel solution cell mixture in advance.Every Kong Jiayi biopsy samples.Then, this alveolar disk is transferred to made cold matrix gel solution solidifies in the cell culture incubator that contains 100% humidity.After 45 minutes, take out this alveolar disk, carefully 50-100 μ l (80 μ l) nutrient solution is added in each hole.If reagent to be tested does not add in the matrix gel solution cell mixture of the first layer in advance, at this moment can it be added in the cultivation of the second layer.At last, culture plate is placed in the incubator that contains 100% humidity cultivates 37 ℃ of cell cultures the temperature inside the box.In general, 3-9 days (being 7 days in the present embodiment) can observations and taken pictures and compare analysis then.
Embodiment 2: the preparation of stdn test kit
This novel external various kinds of cell 3 D stereo culture system of the present invention as a unit application in 384-hole that cell cultures is used, 96-porose disc, 48-porose disc, 24-porose disc, 12-porose disc, 6-porose disc, 4-porose disc and similarly, be suitable in the vessel that cell cultures uses, thereby can be prepared into a kind of stdn high-flux medicaments sifting test kit or the personalized diagnoses and treatment test kit of a kind of stdn.A unit is a 3 D stereo (3D) co-culture system.The first layer in unit is the matrix gel that one deck solidifies; Be close to the bottom of used cultivation vessel; Wherein, Mammiferous vascular endothelial cell is dispersed in the gel coat, and mammiferous or tumour cell class cell mass or the biopsy samples of the biopsy samples of the biopsy samples of tumor tissues or islet tissue, endometrial tissue biopsy samples or other histoorgans is suspended in the glue-line.
This stdn test kit not only can be used for carrying out the anti-angiogenic regeneration of screening of high-throughput ground and promote the new drug and the special medicine of revascularization; What is more important; If in this stdn test kit, use different patients' biopsy sample; Can also be for patient make the most suitable this patient's efficient medicine to measure, thus improve therapeutic efficiency and shorten the treatment phase; Meanwhile, the partner treatment process, this stdn test kit can predict in advance that whether patient has produced resistance to the existing medicine that uses, and provides scientific basis for the medical worker adjusts regimen timely and reasonably.
Embodiment 3: the standard reagent box carries out the screening of high-throughput antineoplastic vascular regenerating medicine
A kind of monoclonal cell spheroid of tumor cell line is applied in the stdn high-flux medicaments sifting test kit of novel external various kinds of cell 3 D stereo culture system of this invention, cultivates altogether with vascular endothelial cell.Candidate agent to be tested is added in each hole, set up simultaneously the negative control group of adding 2% foetal calf serum with add 3% methyl-sulphoxide (DMSO) as positive controls (Koizumi, et.al.).Observation test group reagent vascular endothelial cell forms capillary structure, comes comparison with the result of negative and positive controls.If the result of revascularization is similar with negative control group or close, just shows that the anti-angiogenic regenerated effect of this reagent is very weak or almost do not have anti-angiogenic regenerated effectiveness; If the result of revascularization and positive controls result similarly talk about, just show that the anti-angiogenic regenerated effectiveness of this reagent is more intense or have anti-angiogenic regenerated effectiveness.Therefore, just can further study and test, perhaps can find the special medicine of antineoplastic vascular regenerated new drug having anti-angiogenic regenerated reagent.At first, because the various kinds of cell 3 D stereo co-culture system described in this invention is very near the environment of in-vivo tumour revascularization, therefore, it is just very high to use the medicine of this culture system screening finally to have a probability that obtains the medicine lot number.Secondly, be example to use the 96-porose disc as the sieve medicine test kit of carrying platform preparation, as negative control group, rightmost row are as positive controls with 8 holes of Far Left one row, other 80 different reagent to be tested of holes interpolation.So, a 96-porose disc test kit just can once screen and observe 80 kinds of reagent.Therefore, this test kit can high-throughput ground screening of medicaments.The 3rd, the most important thing is that also this test kit is a kind of standardized product, therefore have high duplication and safety.The 4th because external various kinds of cell 3 D stereo co-culture system that this test kit adopted, can parody in the microenvironment of tumor tissues revascularization, so the medicine of this test kit screening has very high confidence level.
Embodiment 4: the stdn test kit carries out the tumour patient personalized treatment
Certain cancer patients's cancerous tissue biopsy samples is applied in the personalized diagnoses and treatment test kit of stdn of external various kinds of cell 3 D stereo culture system of this invention, cultivates altogether with vascular endothelial cell.Existing drug candidate is added in each hole, set up simultaneously the negative control group of adding 2% foetal calf serum with add 3% methyl-sulphoxide (DMSO) as positive controls (Koizumi, et.al.).Observe drug test group vascular endothelial cell and form capillary structure, come comparison with the result of negative and positive controls.If the result of revascularization is similar with negative control group or close, just shows that the anti-angiogenic regenerated effect of this medicine is very weak or almost do not have anti-angiogenic regenerated effectiveness; If the result of revascularization and positive controls result similarly talk about, just show that the anti-angiogenic regenerated effectiveness of this medicine is more intense or have anti-angiogenic regenerated effectiveness.At first; Because the various kinds of cell 3 D stereo co-culture system described in this invention is very near the environment of in-vivo tumour revascularization; Add and added the cancerous tissue biopsy samples that carries cancer patient cancer characteristic " signature ", therefore, using this stdn individual character diagnoses and treatment test kit to detect the medicine with high efficiency anti-tumor revascularization will have very original efficient therapeutic action to this cancer patients; And concerning the patient, removed the blindness of chemotherapy.Secondly, be example as the personalized diagnoses and treatment test kit of carrying platform preparation, have 3 kinds, each medicine of patient's cancerous tissue biopsy samples is carried out two repeated tests if having drug candidate now to use the 96-porose disc.So, a 96-porose disc test kit once just can carry out three kinds of drug tests to five cancer patientss at least.Therefore, this test kit is suitable for diagnostic detection in batches.The 3rd, the most important thing is that also this test kit is a kind of standardized product, therefore have high duplication and safety.The 4th; For combined with chemotherapy process better; Use this test kit regularly cancer patients's cancer biopsy samples to be carried out; This stdn test kit can also predict in advance that whether patient has produced resistance to the existing chemotherapeutics that uses, and provides scientific basis for the medical worker adjusts regimen timely and reasonably.
Claims (19)
1. the various kinds of cell 3 D stereo co-culture system of a novel vitro detection new vessel; It is characterized in that: it comprises two-layer; Wherein, the first layer is the matrix gel cell, or matrix gel cell tissue mixture; Or matrix gel mixed tissue thing; Be close to the bottom of used cultivation vessel, mammiferous vascular endothelial cell is dispersed in the gel coat, and mammiferous or the tumour cell class cell mass or the biopsy samples of tumor tissues or the biopsy samples of the biopsy samples of islet tissue, endometrial tissue biopsy samples or other histoorgans is suspended in the gel coat; The second layer is a cell culture fluid, on the first layer.
2. according to the described co-culture system of claim 1, it is characterized in that: described vascular endothelial cell is the vascular endothelial cell of a kind of GFP of permanent transfection expression; Said tumour cell or tumor tissues biopsy samples are the tumour cells of a kind of GFP of permanent transfection expression; And the emission spectrum of the GFP that tumour cell is expressed is different with the GFP emission spectrum of the permanent transfection expression of vascular endothelial cell.
3. according to the described co-culture system of claim 1, it is characterized in that: also comprise other at least a kind of mammal cell line in the said second layer.
4. according to the described co-culture system of claim 1, it is characterized in that: also comprise other a kind of mammal cell line at least, a kind of GFP of the permanent transfection expression of this clone; The emmission spectrum of the GFP that the emmission spectrum of this GFP and vascular endothelial cell and tumour cell are expressed is all different.
5. according to the described confession culture system of claim 4, it is characterized in that: said other a kind of mammal cell line is a kind of in the following cell: immunocyte is scavenger cell and mastocyte for example; Mesenchymal cell is inoblast and adipocyte for example; Or required pericyte takes place in blood vessel.
6. according to the described co-culture system of claim 1, it is characterized in that: the biopsy samples of said islet tissue biopsy samples or other histoorgans is a kind of GFPs of permanent transfection expression; The emission spectrum of the GFP that the emission spectrum of this kind GFP and vascular endothelial cell and other a kind of mammiferous clone that possibly include are expressed is different.
7. according to the described co-culture system of claim 1, it is characterized in that: in the first layer or the second layer, have at least one deck to contain reagent to be tested.
8. according to the described co-culture system of claim 7, it is characterized in that: said reagent to be tested is known or unknown but has the reagent of anti-angiogenic regeneration or the reagent of promotion revascularization.
9. according to the described co-culture system of claim 1; It is characterized in that: said mammiferous tumour cell derives from some main bodys; Simultaneously, first or the second layer have at least one deck to contain a kind of reagent to be tested, this reagent once was used for treating its tumour by this main body.
10. according to the described co-culture system of claim 1, it is characterized in that: said mammiferous biopsy samples derives from some main bodys, and this main body is people or animal.
11. according to the described co-culture system of claim 10, it is characterized in that: said animal is wild-type animal or transgenic animal.
12. according to the described co-culture system of claim 11, it is characterized in that: said transgenic animal are the transgenic animal of expressing a kind of GFP; The emission spectrum of the GFP that the emission spectrum of expressed GFP and vascular endothelial cell and other a kind of mammal cell line that possibly comprise are expressed is different.
13. a stdn test kit is characterized in that: comprise each described co-culture system of claim 1 to 12.
14., be the personalized diagnoses and treatment test kit of stdn high-throughput sieve medicine test kit or stdn according to the described stdn test kit of claim 13.
15. according to the described stdn test kit of claim 14, wherein, stdn high-throughput sieve medicine test kit is that screening has the test kit that promotes revascularization and the medicine that suppresses revascularization; The personalized diagnoses and treatment test kit of stdn is for the selected cancer therapy drug with efficient therapeutic action of cancer patients, exempts the test kit of the blindness of chemotherapy.
16. an application rights requires 15 said stdn test kits to carry out high-throughput antineoplastic vascular regenerating medicine method for screening, it is characterized in that:
A kind of monoclonal cell spheroid of tumor cell line or certain cancer patients's cancerous tissue biopsy samples are applied in the described stdn high-flux medicaments sifting test kit; Cultivate altogether with vascular endothelial cell; Candidate agent to be tested is added in each hole of cell cultures dish; Set up the negative control group of adding 2% foetal calf serum simultaneously and add 3% methyl-sulphoxide as positive controls; Observation test group reagent vascular endothelial cell forms capillary structure; Compare with the result of negative control group and positive controls,, show that the anti-angiogenic regenerated effectiveness of this candidate agent is very weak or almost do not have anti-angiogenic regenerated effectiveness if the result of revascularization is similar with negative control group or close; If the result of revascularization and positive controls result are similar, show that the anti-angiogenic regenerated effectiveness of this candidate agent is more intense or have anti-angiogenic regenerated effectiveness.
17. the preparation method of the described co-culture system of claim 1 is characterized in that comprising the steps:
(1) preparation of matrix gel solution cell mixture
A. use cell cultures flask culture vascular endothelial cell and pericyte; When vascular endothelial cell and pericyte are that stand density is when reaching 70% left and right sides; Use the proteopepsis enzyme that the cell of adherent growth is separated from wall of container, be suspended in neutral phosphonic acid buffer low-speed centrifugal washing several times, at last cell is suspended in the EBM2 grown cultures liquid again; And use the density of blood-counter system pair cell to count, preserve for use then;
B. matrix gel solution of preserving after will thawing and vascular endothelial cell or vascular endothelial cell are blended in the matrix gel solution together with the cell of pericyte or other kinds equably;
(2) preparation of the processing of cell cultures dish and co-culture system the first layer
A. the processing of cell cultures dish: the cell cultures dish is a porous, and the NaOH solution soaking of process 4N 1 hour is cleaned several times with sterile distilled water then; Then after the HCL solution-treated with 4N, clean several times with sterile distilled water again, last; Use aseptic neutral phosphonic acid buffer to clean several times, with the culture plate after handling air-dry after, put into the cell culture incubator inner equilibrium; Then culture plate is sealed, preserve subsequent use;
B. the preparation of co-culture system the first layer: in each hole of above-mentioned ready culture plate, add the first layer of the matrix gel solution cell mixture of 30-50 μ l, after interpolation finishes, preserve for use as co-culture system;
(3) preparation of tumour cell monoclonal cell spheroid or biopsy sample
A. the preparation of tumour cell monoclonal cell spheroid: use the Tissue Culture Flask culture of tumor cell; When growth of tumour cell density reaches the 60-80% left and right sides; Use the proteopepsis enzyme that the cell of adherent growth is separated from wall of container, be suspended in neutral phosphonic acid buffer low-speed centrifugal washing several times, at last cell is suspended in the grown cultures liquid again; And the density of pair cell counts, and preserves for use then; In the nutrient solution of tumour cell, add foetal calf serum, microbiotic, 1.5% agar-agar soln and above-mentioned tumour cell; This cell mixture is added on the agar gel layer that solidifies refrigerative 1.5%; Then, it is transferred to cultivate 14 days～21 days in the cell culture incubator of 100% humidity to obtain tumour cell mono-clonal spheroid; With long be tumour monoclonal cell spheroid centrifugation from low-density agar gel of 0.5 millimeter～1 millimeter to diameter, through the washing of several times neutral phosphonic acid buffer; The antibiotic neutral phosphonic acid buffer of use interpolation suspends sedimentary tumour monoclonal cell spheroid again and transfers in the Micro-Organism Culture Dish then; The picking diameter is that 0.5～0.7 millimeter tumour spheroid is put into the hole central authorities that added cold matrix gel solution cell mixture in advance; This alveolar disk transferred to make cold matrix gel solution solidifies in the cell culture incubator that contains 100% humidity; Afterwards, take out this alveolar disk, nutrient solution is added in each hole; At last, culture plate is placed on cultivates in the incubator that contains 100% humidity and observations;
B. the preparation of biopsy sample
The biopsy sample retention of gathering is subsequent use in preserving liquid; After from preserve liquid, taking out biopsy samples; Spirituous solution through 70% embathes several times, after the neutral phosphonic acid buffer embathes several times, transfers in the Tissue Culture Dish; And be divided into fritter, place the hole central authorities of the alveolar disk that has added cold matrix gel solution cell mixture in advance; Then, this alveolar disk is transferred to made cold matrix gel solution solidifies in the cell culture incubator that contains 100% humidity; Take out this alveolar disk, nutrient solution is added in each hole; At last, culture plate is placed in the incubator that contains 100% humidity cultivates, observe.
18. preparation method according to claim 17 is characterized in that: be added with reagent to be tested in said matrix gel solution cell mixture or the nutrient solution.
19. preparation method according to claim 17; It is characterized in that: the use density of vascular endothelial cell is 140,000-1,000; 000 cell/ml, pericyte or other kind cells are according to adding in the matrix gel solution with 1: 50～1: 5 ratio of vascular endothelial cell; Said matrix gel solution is the class matrix gel product of the matrix gel that extracts in the animal body that is fit to cell cultures or synthetic or both mixtures according to 1: 3～1: 1 ratio.
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