CN106190980A - A kind of special culture media and cultural method being used for In vitro culture esophageal carcinoma tumor organoid based on Human Esophageal Carcinoma - Google Patents

A kind of special culture media and cultural method being used for In vitro culture esophageal carcinoma tumor organoid based on Human Esophageal Carcinoma Download PDF

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CN106190980A
CN106190980A CN201610548084.1A CN201610548084A CN106190980A CN 106190980 A CN106190980 A CN 106190980A CN 201610548084 A CN201610548084 A CN 201610548084A CN 106190980 A CN106190980 A CN 106190980A
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张云霞
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Abstract

The present invention relates to the cultivation field of tumor organoid, be specifically related to a kind of based on Human Esophageal Carcinoma for the special culture media of In vitro culture esophageal carcinoma tumor organoid and cultural method.Described special culture media includes the agonist of the path of FZ inhibitors of kinases, Metabolism, Vitamins and Hormones, antioxidant and the differentiation of suppression cell that receptor tyrosine kinase part, Rho are relevant.The substep culture technique that the present invention is dedifferented by condition, it is achieved in the tumor cell short time, rapid amplifying becomes organoid.Technology that this substep is cultivated makes primary cell can quickly form organoid, and obtains abundant cell within the time effectively and carry out subsequent experimental operation, improves cultivation speed and the success rate of organoid.

Description

A kind of based on Human Esophageal Carcinoma special for In vitro culture esophageal carcinoma tumor organoid With culture medium and cultural method
Technical field
The present invention relates to the cultivation field of tumor organoid, be specifically related to one based on Human Esophageal Carcinoma for external training Support special culture media and the cultural method of esophageal carcinoma tumor organoid.
Background technology
The esophageal carcinoma is common malignant tumor of digestive tract, accounts for the 2% of all malignant tumor.The whole world there are about 20～30 every year Ten thousand people die from the esophageal carcinoma.The esophageal carcinoma should ranked first position in China's digestive system tumor, position in whole tumor sorts Secondary the highest, about 45 levels.Compared with other countries, the Incidence of esophageal cancer of China is that comparison is high, the whole world nearly one The esophageal carcinoma of half occurs in China.China is Incidence of esophageal cancer and the highest country of mortality rate, annual Incidence of Esophageal Cancer and dead Number of dying all account for the whole world number more than half.Visible, the esophageal carcinoma can be considered one of malignant tumor of most distinct Chinese characteristics.Esophagus The morbidity of cancer is relevant with E&H factor, but exact cause and pathogenesis are not clear.With gastric cancer, hepatocarcinoma is similar to, by Relatively low in Hesperian sickness rate, cause the tumor occurred frequently compared with other wests of studying of the esophageal carcinoma in world wide to fall After.And the highest as China's incidence rate, one of several tumors that fatality rate is the highest, strengthen study of pathogenesis and the medicine of the esophageal carcinoma Thing research and development have very major and immediate significance.
Existing esophageal carcinoma cell line is mainly by long-term cultivation, and spontaneous immortalization or transfection promote normal cell immortality The oncogene changed obtains.Such as Het-1A is that a strain is resisted by people's normal esophageal epithelial cell, the big T viral by transfection Sv40 Former, make cell obtain immortalization and obtain.These methods change the genetic background of cell, the cell line of long-term cultivation, also hold Easily cause genomic instability, may cause the phenotype of tumor cell line and interior tumor cell that artificial change, impact occur Esophageal carcinoma scientific research and the accuracy of medicine method research and development.
Esophageal carcinoma original cuiture pattern before, after tumor cell and other cell separation, plants in Tissue Culture Dish In, cell loses original microenvironment.Therefore cell is difficult to steady in a long-term cultivation.Therefore, tumor growth is simulated in vitro Microenvironment, it is provided that the three-dimensional that tumor is depended on for existence supports and multiple existence path, it is possible to realize training the most steady in a long-term Support tumor tissues.
The cultivation of vitro in primary tumor cell organoid, it is desirable to provide simulation tumor microenvironment in vivo.These micro-loop Border includes maintaining the extracellular matrix of 3 D stereo cultivation conditions, the somatomedin of important path of surviving, suppression cell to divide The factor changed, various vitamin, trace element, hormone, energy metabolism, Antioxidative Factors, fine chemical synchronizing signal and anti- Tune is died material.Each tumor type, even not same tumor cell hypotype, these microenvironment factors all may differ widely, Therefore, the cell primary organoid of different tumor types is cultivated, and is required for the type of this tumor, optimizes one by one.
Original organoid culture technique is applied to the cultivation of human esophagus cancer organoid following problem: first generation incubation time is relatively Long, need just to complete the first generation 2 to 6 weeks and cultivate；The vigor passing on rear organoid declines rapidly, and the speed of growth substantially slows down； Frozen rear recovery efficiency is low, and easily dead, vigor reduces, and the speed of growth slows down；The success rate cultivated is the highest, and it is new that operation obtains Fresh insufficient sample 60%；For biopsy specimen, original organoid culture technique almost cannot effectively be cultivated.
The tumor organoid of esophageal cancer cell is cultivated, and makes on the conditioned basic that mice and human stem cell organoid are cultivated, In conjunction with the feature of esophageal cancer cell, further optimal conditions, to utilize less tissue, within the shorter time, it is thus achieved that more preferably Cell viability and higher success rate.
Summary of the invention
Present invention aim to address above-mentioned the deficiencies in the prior art, it is provided that one is used for external based on Human Esophageal Carcinoma Cultivate special culture media and the cultural method of esophageal carcinoma tumor organoid.
The present invention is achieved by the following technical solutions:
A kind of special culture media being used for In vitro culture esophageal carcinoma tumor organoid based on Human Esophageal Carcinoma, described special Culture medium includes following components:
(1) receptor tyrosine kinase part,
(2) FZ kinases (ROCK1, ROCK2) inhibitor relevant for Rho,
(3) Metabolism, Vitamins and Hormones,
(4) antioxidant,
(5) agonist of the path of suppression cell differentiation.
Preferably, described receptor tyrosine kinase part include the epithelical cell growth factor (EGF) of human or animal, people or One or more in animal liver cell somatomedin (HGF) and human or animal's basic fibroblast growth factor (bFGF).
Preferably, described
The concentration range of the epithelical cell growth factor of human or animal is 2ng/mL～100ng/mL；
The concentration range of human or animal's hepatocyte growth factor is 10ng/mL～200ng/mL；
The concentration range of human or animal's basic fibroblast growth factor is 5ng/mL～100ng/mL.
Preferably, relevant for described Rho FZ inhibitors of kinases include Fasudil, Y-27632, RKI-1447 and One or more in GSK429286A.
Preferably, during described Metabolism, Vitamins and Hormones includes putrescine, Adrenalone, nicotiamide and vitamin A acetate Plant or multiple.
Preferably, described
The concentration range of putrescine is 0.1 μ g/mL～100 μ g/mL；
The concentration range of Adrenalone is 10ng/mL～10 μ g/mL；
The concentration range of nicotiamide is 1 μ g/mL～2mg/mL；
The concentration range of vitamin A acetate is at 10ng/mL～500ng/mL.
Preferably, described antioxidant includes reduced glutathion (GSH), vitamin C, vitamin E and β-sulfydryl second One or more in alcohol.
Preferably, the concentration range of described beta-mercaptoethanol is 0.01mM～1.5mM.
Preferably, the agonist of path of described suppression cell differentiation include people's activin A albumen, Wnt pathway agonist, One or more in Hedgehog pathway agonist.
Preferably, described
The concentration range of people's activin A (activin A) albumen is 5ng/mL～200ng/mL；
Wnt pathway agonist includes recombinate the Wnt3a albumen of human or animal, restructuring human or animal's RSPO-1 albumen, GSK-3 One or more in beta inhibitor；
Hedgehog pathway agonist includes human or animal's SHH albumen of recombinating, the one in the agonist SAG of SMO albumen Or it is multiple.
Preferably, described special culture media includes following components:
ECRM-I:DMEM/F12 culture medium, adds 2.5% hyclone (FBS), 1x B27 additive, ascorbic acid, dimension Raw element A acetate, Adrenalone, putrescine, nicotiamide, insulin, hydrocortisone, people's activin A, FGF2, Wnt3a, EGF, Tretinoin and Y27632；Or
ECRM-II:DMEM/F12 culture medium, adds 2.5% hyclone, 1x B27 additive, nicotiamide, adrenal gland Ketone, BMP4, EGF, Wnt3a, IGF, sodium butyrate, putrescine, vitamin A acetate, insulin, dexamethasone, beta-mercaptoethanol, FGF2, people's activin A, tretinoin and Y27632；Or
ECRM-III:DMEM/F12 culture medium, adds 1% bovine serum albumin (BSA), 1x B27 additive, peroxidating Hydrogen is mould, vitamin A acetate, nicotiamide, Adrenalone, sodium butyrate, insulin, dexamethasone, putrescine, reduced form gluathione Peptide, people's activin A, EGF, BMP4, FGF1, NOG, SAG, RSPO1, Wnt3a and Y27632；Or
EDM-1:DMEM/F12 culture medium, add 2.5% hyclone, 1x B27 additive, HGF, IGF, Wnt3a, EGF, people's activin A, Adrenalone, vitamin A acetate, nicotiamide, insulin, reduced glutathion.
A kind of training utilizing the special culture media being used for In vitro culture esophageal carcinoma tumor organoid based on Human Esophageal Carcinoma Breeding method, comprises the following steps:
(1) by the esophageal carcinoma specimen tissue of fresh acquisition, clean with tissue-wash solution；
(2) once purged esophageal carcinoma specimen tissue is transferred in tissue transfer liquid, is placed in 0 to 4 degrees Celsius of transfers or short Phase preserves；
(3) from tissue transfer liquid, take out piece of tissue, in tissue-wash solution after rinsing, transfer to steril cell culture dish In, sop up surplus liquid；
(4) according to the size of piece of tissue, add tissue digestion liquid, cut with sterilizing and repeatedly shred tissue；
(5) tissue shredded is transferred in sterile centrifugation tube, continuously add 2～10mL tissue digestion liquid, clean transfer Tumor tissues in culture dish, is incorporated in sterile centrifugation tube；
(6) sterile centrifugation tube being placed in 37 degrees Celsius of shaking tables digestion, frequently take out observation, how digestion is to few cell mass Or till unicellular；
(7) by the suspension in sterile centrifugation tube through 70 μm cell screen clothes, filtered solution is transferred in 50mL centrifuge tube；
(8) adding 10～30mL PBS in 50mL centrifuge tube, 100～300g are centrifuged 2～5 minutes, abandon supernatant, with 10～ 30mL PBS is resuspended, again 100～300g is centrifuged 2～5 minutes；
(9) suck the supernatant in centrifuge tube carefully, add the special culture media of 3～8mL esophageal carcinoma tumor organoids, Re-suspended cell；
(10) take 50～200 μ L cell suspension and add trypan blue, calculate the quantity of living cells by platelet counter；
(11) it is close that the special culture media of the cell suspension esophageal carcinoma tumor organoid after counting carries out resuspended adjustment cell Degree, adds the Matrigel of 4 DEG C of defrostings of cell suspension 0.1 to 1 times；
(12), after cell suspension is resuspended with Matrigel, it is inoculated in 24 orifice plates, is placed in 37 DEG C of cell culture incubators, 15～30 After minute, treat that Matrigel solidifies, add the special culture media of esophageal carcinoma tumor organoid, change liquid every other day；
After (13) 4～10 days, esophageal carcinoma organoid grows up to many spheroid forms, has epithelioid cell layer.
Preferably, the compound method of step (1) and (3) described tissue-wash solution is: in normal saline or phosphate-buffered Liquid adds the penicillin of 500U/mL, the amphotericin B of 2.5 μ g/mL and the streptomycin of 500 μ g/mL, filtration sterilization.
Preferably, the compound method of step (2) described tissue transfer liquid is: add 5% tire Sanguis Bovis seu Bubali in DMEM culture medium Clearly, the penicillin of 500U/mL, the amphotericin B of 2.5 μ g/mL and the streptomycin of 500 μ g/mL are added.
Preferably, the compound method of step (4) and (5) described tissue digestion liquid is:
EM-BS-1: add 2.5% hyclone, 1 μ g/mL～the IX Collagen Type VI of 10 μ g/mL, 50 μ in DMEM culture medium The neutral proteolytic enzyme of g/mL-500 μ g/mL；Or
EM-BS-2: add 2.5% hyclone, 1x penicillin and streptomycin, 0.01mg/mL-in DMEM culture medium The IV Collagenase Type of 1mg/mL, the hyaluronidase of 0.001mg/mL～0.2mg/mL and 0.001mg/mL's～0.2mg/mL DNA enzymatic I；Or
EM-3: add 2.5% hyclone, 1x penicillin and streptomycin, 20U/mL～200U/mL in DMEM culture medium The DNA of Bacillus polymyxa Neutral proteinase neutral proteolytic enzyme I and 0.01mg/mL～1mg/mL of XI Collagenase Type, 1 μ g/mL～100 μ g/mL Enzyme I.
The beneficial effects of the present invention is:
The substep culture technique that the present invention is dedifferented by condition, it is achieved in the tumor cell short time, rapid amplifying becomes class device Official.The technology that this substep is cultivated makes primary cell can quickly form organoid, and obtains enough within the time effectively Many cells carry out subsequent experimental operation.Esophageal carcinoma primary cell is realized ECRM-I, ECRM-II, ECRM-of rapid amplifying III culture medium and the EDM-1 culture medium used when amplification culture and drug susceptibility-types detect are used in combination.
In tumor organoid incubation, tumor metabolic is vigorous, can produce oxygen-derived free radicals and to cell damage, fall Low cell viability, causes differentiation, apoptosis.The esophageal carcinoma of the present invention primary organoid culture fluid with the addition of antioxidant, gluathione Peptide or vitamin C or vitamin E are all the natural metabolism products of the most retrievable material of cell or cell, Ke Yiwei Hold environment and intracellular oxidative pressure balance, scavenging activated oxygen, there is the effect of protection cell membrane integrity.β-sulfydryl second Alcohol is also additive common in cell cultivation, particularly stem cell incubation.Research shows, the shortage of antioxidant will make The cell reactive enhancing to oxidative pressure, makes organoid vigor decline, and the most aging, functional rehabilitation postpones.Long-term cultivation The problems such as esophageal carcinoma organoid has the more fragment of a certain proportion of appearance, occurs vesicle in cell, the speed of growth substantially reduction. Antioxidant can improve the vigor of cell, reduces and produces aging ratio.
The present invention can improve the speed of growth of organoid, by shortening to for 1 to 2 week 2 to 6 original weeks.
The present invention improves the success rate that organoid is cultivated, and the tumor specimen of excision improves to more than 80%.
The present invention passes through formula and the using method of optimizing tissue Digestive system, obtains enough cultivations from small specimen Tumor cell alive so that the biopsy specimen originally cannot cultivated is possibly realized.
The present invention is passed on and frozen formula and technology by optimization so that organoid keeps good after passing on and recovering Vigor.
In the present invention, optimize the concentration of glucose under some specified conditions, match, by adding antioxidation freely Base reagent, reduces the cell ageing caused in long-term cultivation.
Accompanying drawing explanation
Fig. 1 is the statistical result that several additives formula In vitro culture forms organoid rate number.
Wherein, culture medium culturing based on B, BW be on the basis of basal medium add Wnt3a cultivate, B27 be Adding B27 additive on the basis of basal medium to cultivate, BM is to add BMP4 on the basis of basal medium to cultivate, and BE is Adding EGF on the basis of basal medium to cultivate, BF is to add FGF2 on the basis of basal medium to cultivate, and BH is at base Adding HGF on the basis of basal culture medium to cultivate, BEW is to add Wnt3a and EGF on the basis of basal medium to cultivate, EDM- I, ECRM-I, ECRM-II, ECRM-III are respectively corresponding culture medium culturing.
Detailed description of the invention
For being best understood from the present invention, below in conjunction with embodiment and accompanying drawing, the invention will be further described, following example It is only that the present invention will be described rather than is limited it.
The preparation of embodiment one special culture media
Culture medium of the present invention includes
One or more receptor tyrosine kinase parts: include the epithelical cell growth factor of human or animal, concentration range At 2ng/mL to 100ng/mL；Human or animal's hepatocyte growth factor, concentration range is at 10ng/mL to 200ng/mL；People or dynamic Thing basic fibroblast growth factor, concentration range is 5ng/mL to 100ng/mL；
The FZ inhibitors of kinases that Rho is relevant: selected from Fasudil, Y-27632, RKI-1447 and GSK429286A；
Multivitamin and hormone: putrescine, concentration range is at 0.1 μ g/mL to 100 μ g/mL；Adrenalone, concentration range At 10ng/mL to 10 μ g/mL；Nicotiamide, concentration range is at 1 μ g/mL to 2mg/mL；Vitamin A acetate, concentration range exists 10ng/mL to 500ng/mL；
Antioxidant: selected from reduced glutathion；Vitamin C；Vitamin E；(0.01mM is extremely for beta-mercaptoethanol 1.5mM)；
The agonist of the path of suppression cell differentiation: people's activin A albumen, concentration range is 5n/mL to 200ng/mL； Wnt pathway agonist, including the Wnt3a albumen of restructuring human or animal, human or animal's RSPO-1 albumen of recombinating；GSK-3 β suppresses Agent, selected from CHIR-99021 (No. CAS: 252917-06-9), SB216763 (No. CAS: 280744-09-4) etc.；Hedgehog leads to Road agonist, including restructuring human or animal SHH (Sonic hedgehog) albumen, the agonist SAG of SMO albumen (No. CAS: 912545-86-9)。
Embodiment two esophageal carcinoma primary cell realize rapid amplifying ECRM-I, ECRM-II, ECRM-III culture medium and The preparation of the EDM-1 culture medium used when amplification culture and drug susceptibility-types detect
The formula of ECRM-I includes DMEM/F12 culture medium, adds 2.5%FBS, 1x B27 additive, ascorbic acid, dimension Raw element A acetate, Adrenalone, putrescine, nicotiamide, insulin, hydrocortisone, people's activin A, FGF2, Wnt3a, EGF, Tretinoin and Y27632.
The formula of ECRM-II culture medium includes DMEM/F12 culture medium, adds 2.5%FBS, 1x B27 additive, nicotinoyl Amine, Adrenalone, BMP4, EGF, Wnt3a, IGF, sodium butyrate, putrescine, vitamin A acetate, insulin, dexamethasone, β-mercapto Base ethanol, FGF2, people's activin A, tretinoin and Y27632.
The formula of ECRM-III culture medium includes DMEM/F12 culture medium, adds 1%BSA, 1x B27 additive, peroxidating Hydrogen is mould, vitamin A acetate, nicotiamide, Adrenalone, sodium butyrate, insulin, dexamethasone, putrescine, reduced form gluathione Peptide, people's activin A, EGF, BMP4, FGF1, NOG, SAG, RSPO1, Wnt3a and Y27632.
EDM-I culture medium prescription include in DMEM/F12 culture medium add 2.5%FBS, B27 additive, HGF, IGF, Wnt3a, EGF, people's activin A, Adrenalone, vitamin A acetate, nicotiamide, insulin, reduced glutathion.
The preparation of embodiment three tissue digestion liquid
Optimize operation and biopsy Digestive system, the maximum acquisition rate in the case of holding tumor vigor can be realized.
EM-BS-1 organizes the formula of enzymolysis solution as follows: add 2.5%FBS, IX Collagen Type VI (1 μ g/ in DMEM culture medium ML-10 μ g/mL), neutral proteolytic enzyme (50 μ g/mL-500 μ g/mL).
EM-BS-2 organizes the formula of enzymolysis solution as follows: add 2.5%FBS, 1x penicillin and strepto-in DMEM culture medium Element, IV Collagenase Type (0.01mg/mL-1mg/mL), hyaluronidase (0.001mg/mL-0.2mg/mL) and DNA enzymatic I (0.001mg/mL-0.2mg/mL)。
The formula of EM-3 enzymolysis solution is as follows: add 2.5%FBS, 1x penicillin and streptomycin, XI type glue in DMEM culture medium Protoenzyme (20U/mL-200U/mL), Bacillus polymyxa Neutral proteinase neutral proteolytic enzyme I (1 μ g/mL-100 μ g/mL) and DNA enzymatic I (0.01mg/ mL-1mg/mL)。
The cultural method of embodiment four esophageal carcinoma tumor organoid
The present invention obtains specifically comprising the following steps that of process human esophageal carcinoma and (is wrapped by the esophageal carcinoma specimen tissue of fresh acquisition Include the human esophageal carcinoma obtained in excision tissue, biopsy or PDX model), clean with tissue-wash solution, clean three times After be transferred to tissue transfer liquid in, be placed in 0 to 4 degrees Celsius transfer or short-term preservation.It is organized in the cell of air cleaner In culturing room, operation in more than two grades Biohazard Safety Equipments.To bigger surgical tissue, the doctor containing 70%～75% ethanol Soak tissue 10 seconds with in disinfectant solution, take out and be placed in PBS, clean 3 times, remove residual ethanol, transfer to steril cell training Support in ware, sop up surplus liquid, with sterilization eye scissors place to go connective tissue, select knub position harder, that color is white, take 10mm and see Side's left and right tumor tissues.Add 0.5mL tissue digestion liquid, repeatedly shred tissue more than 50 times with sterilizing eye scissors, cut into 1mm Square fritter, can suck smoothly to 1mL pipettor gun head.The tissue shredded is transferred to, in sterile centrifugation tube, continuously add 2 ～10mL enzymolysis solution, clean the tumor tissues in transfer culture dish, be incorporated in centrifuge tube.It is placed in 37 degrees Celsius of shaking tables digestion, Frequently take out observation, digestion to how with widow's cell mass or unicellular.Through 70 μm cell screen clothes, filtered solution transfers to 50mL In centrifuge tube, adding 20mL PBS, 200g and be centrifuged 3 minutes, abandon supernatant, resuspended with 20mL PBS, 200g is centrifuged 3 minutes again. Suck supernatant carefully, add the 5mL esophageal carcinoma of the present invention organoid and cultivate special culture solution, re-suspended cell.Take 100 μ L thin Born of the same parents' suspension adds trypan blue, calculates the quantity of living cells by platelet counter.
The cultivation stage concrete operation step of esophageal cancer cell organoid of the present invention: human esophageal carcinoma is through mechanical shear Cut with enzymic digestion after, it is thus achieved that unicellular and few cell mass suspension, with culture medium resuspended adjustment cell density after counting, the body such as addition Long-pending 4 DEG C of Matrigel mixing thawed, in certain embodiments, Marigel adds volume and is adjusted to 0.1 times of cell suspension To 1 times.After cell suspension is resuspended with Matrigel, it is inoculated in 24 orifice plates, is placed in 37 DEG C of cell culture incubators, after 20 minutes, treats Matrigel solidifies, and adds culture medium, changes liquid every other day.After 4-10 days, esophageal carcinoma organoid grows up to many spheroid forms, has Epithelial Cellular layer.
Esophageal carcinoma primary cell collection of specimens is the successful committed step of primitive cell culture, and collection of specimens and transport are not When, it will directly affecting the success or not of tumor organoid, basic demand and the points for attention of tumor specimen collection are described below: (1) prevention and control antibacterial and fungal contamination.Operation technique environment not sterile working, environment and internal have a lot of microorganism, correctly Collection and processing mode can reduce pollution to greatest extent.Preparation tissue-wash solution: normal saline (0.9% sodium chloride) or Person's phosphate buffer (PBS) adds penicillin (500U/mL), amphotericin B (2.5 μ g/mL) and streptomycin (500 μ g/ ML), filtration sterilization.Preparation tissue transfer liquid: add 5% hyclone in DMEM culture medium, add penicillin (500U/mL), Amphotericin B (2.5 μ g/mL) and streptomycin (500 μ g/mL).After the apparatus autoclavings such as the shears drawn materials, tweezers, blade Dry.(2) collection of specimens to choose peplos completely as far as possible, eugonic region, it is to avoid rot tissue, slough, fat And connective tissue.Structural blood, fat, slough and connective tissue is carefully removed during pretreatment.(3) operation transport Process will as quickly as possible, and the time after organizing in vitro is the most long, and the success rate of external structure tumor organoid is the lowest, typically after in vitro Complete in 4 hours to cultivate operation.If desired, specimen can store by 4 C overnight in specimen transfer liquid, but can be substantially reduced Success rate, increases and pollutes probability.
The substep culture technique dedifferented by condition, it is achieved in the tumor cell short time, rapid amplifying becomes organoid.This The technology that one substep is cultivated makes primary cell can quickly form organoid, and obtains abundant thin within the time effectively Born of the same parents carry out subsequent experimental operation.In some embodiments, the unicellular or few cell prepared from acquisition operation or biopsy specimen Use ECRM-I, ECRM-II, ECRM-III culture medium culturing cell, it is achieved the part of tumor cell is dedifferented and rapid amplifying, This process was at 3-10 days.Such as 0-7 day, 0-6 day, 0-5 day, 0-4 day, 0-3 day, 0-2 day, 0- 1,0th be wherein that day that cell separates from its derived tissues, and the 1st is that day subsequently, or uses EDM-I Culture medium culturing cell 1 week or more long, such as 2,3,4 weeks or more long, or 1,2,3,4,5,6,7,8,9,10,11,12 months Or it is more long.In some embodiments, EDM-I culture medium can use cultivate on the 1st, is using EDM-I culture medium Under the conditions of, carry out tumor organoid biology or the experiment of drug effect.
Embodiment five tests the impact on organoid In vitro culture formation rate of the several additives formula
2.5%FBS, Adrenalone, nicotiamide, ascorbic acid and antibiotic is added as base in DMEM/F12 culture medium Basal culture medium (B), adds Wnt3a (BW) the most respectively, adds B27 additive (B27), interpolation BMP4 (BM), interpolation EGF (BE), interpolation FGF2 (BF), interpolation HGF (BH), interpolation Wnt3a and EGF (BEW).In addition with as described above ECRM-I, ECRM-II, ECRM-III and EDM-I condition of culture.Under these culture conditions, test P1 is for the formation of organoid Rate, the unicellular 5x10 of P0 band organoid4It is layered on after mixing with matrigel in 24 moistening in advance orifice plates, static at 37 degrees Celsius After 10 minutes, after matrigel solidifies, in different holes, add the above-mentioned culture medium of 1mL respectively.Respectively cultivate the 5th day and The organoid number of record amplification in the 7th day.
As it is shown in figure 1, under ECRM-I, ECRM-II, ECRM-III and EDM-I condition of culture, hence it is evident that be better than B, BW, B27, BM, BF, BH, BEW condition of culture, wherein the condition of culture best results of ECRM-II.
The above embodiment is only to be described the preferred embodiment of the present invention, the not model to the present invention Enclose and be defined, on the premise of designing spirit without departing from the present invention, the those of ordinary skill in the art technical side to the present invention Various deformation that case is made and improvement, all should fall in the protection domain that claims of the present invention determines.

Claims (10)

1. be used for a special culture media for In vitro culture esophageal carcinoma tumor organoid based on Human Esophageal Carcinoma, its feature exists Following components is included in, described special culture media:
(1) receptor tyrosine kinase part；
(2) FZ inhibitors of kinases relevant for Rho；
(3) Metabolism, Vitamins and Hormones；
(4) antioxidant；
(5) agonist of the path of suppression cell differentiation；
Described receptor tyrosine kinase part includes the epithelical cell growth factor of human or animal, human or animal's hepatocyte growth factor One or more in son and human or animal's basic fibroblast growth factor；
The concentration range of the epithelical cell growth factor of human or animal is 2ng/mL～100ng/mL；
The concentration range of human or animal's hepatocyte growth factor is 10ng/mL～200ng/mL；
The concentration range of human or animal's basic fibroblast growth factor is 5ng/mL～100ng/mL；
FZ inhibitors of kinases relevant for described Rho includes in Fasudil, Y-27632, RKI-1447 and GSK429286A One or more.
The most according to claim 1 a kind of based on Human Esophageal Carcinoma special for In vitro culture esophageal carcinoma tumor organoid By culture medium, it is characterised in that: described Metabolism, Vitamins and Hormones includes in putrescine, Adrenalone, nicotiamide and vitamin A acetate One or more.
The most according to claim 2 a kind of based on Human Esophageal Carcinoma special for In vitro culture esophageal carcinoma tumor organoid By culture medium, it is characterised in that: described
The concentration range of putrescine is 0.1 μ g/mL～100 μ g/mL；
The concentration range of Adrenalone is 10ng/mL～10 μ g/mL；
The concentration range of nicotiamide is 1 μ g/mL～2mg/mL；
The concentration range of vitamin A acetate is at 10ng/mL～500ng/mL.
The most according to claim 1 a kind of based on Human Esophageal Carcinoma special for In vitro culture esophageal carcinoma tumor organoid By culture medium, it is characterised in that: described antioxidant includes reduced glutathion, vitamin C, vitamin E and β-sulfydryl second One or more in alcohol.
The most according to claim 4 a kind of based on Human Esophageal Carcinoma special for In vitro culture esophageal carcinoma tumor organoid By culture medium, it is characterised in that: the concentration range of described beta-mercaptoethanol is 0.01mM～1.5mM.
The most according to claim 1 a kind of based on Human Esophageal Carcinoma special for In vitro culture esophageal carcinoma tumor organoid By culture medium, it is characterised in that: the agonist of the path of described suppression cell differentiation includes that people's activin A albumen, Wnt path swash One or more in dynamic agent, Hedgehog pathway agonist.
The most according to claim 6 a kind of based on Human Esophageal Carcinoma special for In vitro culture esophageal carcinoma tumor organoid By culture medium, it is characterised in that: described
The concentration range of people's activin A albumen is 5ng/mL～200ng/mL；
Wnt pathway agonist includes that the Wnt3a albumen of restructuring human or animal, restructuring human or animal's RSPO-1 albumen, GSK-3 β press down One or more in preparation；
Hedgehog pathway agonist includes human or animal's SHH albumen of recombinating, the one or many in the agonist SAG of SMO albumen Kind.
The most according to claim 1 a kind of based on Human Esophageal Carcinoma special for In vitro culture esophageal carcinoma tumor organoid By culture medium, it is characterised in that described special culture media includes following components:
DMEM/F12 culture medium, adds on 2.5% hyclone, 1xB27 additive, ascorbic acid, vitamin A acetate, kidney Gland ketone, putrescine, nicotiamide, insulin, hydrocortisone, people's activin A, FGF2, Wnt3a, EGF, tretinoin and Y27632；Or Person
DMEM/F12 culture medium, add 2.5% hyclone, 1xB27 additive, nicotiamide, Adrenalone, BMP4, EGF, Wnt3a, IGF, sodium butyrate, putrescine, vitamin A acetate, insulin, dexamethasone, beta-mercaptoethanol, FGF2, people's activin A, tretinoin and Y27632；Or
DMEM/F12 culture medium, add that 1% bovine serum albumin, 1xB27 additive, hydrogen peroxide be mould, vitamin A acetate, Nicotiamide, Adrenalone, sodium butyrate, insulin, dexamethasone, putrescine, reduced glutathion, people's activin A, EGF, BMP4, FGF1, NOG, SAG, RSPO1, Wnt3a and Y27632；Or
DMEM/F12 culture medium, add 2.5% hyclone, 1xB27 additive, HGF, IGF, Wnt3a, EGF, people's activin A, Adrenalone, vitamin A acetate, nicotiamide, insulin, reduced glutathion.
9. one kind utilize claim 1～8 arbitrary described based on Human Esophageal Carcinoma for In vitro culture esophageal carcinoma tumor class The cultural method of the special culture media of organ, it is characterised in that comprise the following steps:
(1) by the esophageal carcinoma specimen tissue of fresh acquisition, clean with tissue-wash solution；
(2) once purged esophageal carcinoma specimen tissue is transferred in tissue transfer liquid, is placed in 0 to 4 degrees Celsius of transfers or short-term is protected Deposit；
(3) from tissue transfer liquid, take out piece of tissue, in tissue-wash solution after rinsing, transfer in steril cell culture dish, Sop up surplus liquid；
(4) according to the size of piece of tissue, add tissue digestion liquid, cut with sterilizing and repeatedly shred tissue；
(5) tissue shredded is transferred in sterile centrifugation tube, continuously add 2～10mL tissue digestion liquid, clean transfer and cultivate Tumor tissues in ware, is incorporated in sterile centrifugation tube；
(6) sterile centrifugation tube being placed in 37 degrees Celsius of shaking tables digestion, frequently take out observation, how digestion is to few cell mass or list Till cell；
(7) by the suspension in sterile centrifugation tube through 70 μm cell screen clothes, filtered solution is transferred in 50mL centrifuge tube；
(8) adding 10～30mL PBS in 50mL centrifuge tube, 100～300g are centrifuged 2～5 minutes, abandon supernatant, with 10～30mL PBS is resuspended, again 100～300g is centrifuged 2～5 minutes；
(9) suck the supernatant in centrifuge tube carefully, add the special culture media of 3～8mL esophageal carcinoma tumor organoids, resuspended Cell；
(10) take 50～200 μ L cell suspension and add trypan blue, calculate the quantity of living cells by platelet counter；
(11) special culture media of the cell suspension esophageal carcinoma tumor organoid after counting carries out resuspended adjustment cell density, adds Enter the Matrigel of 4 DEG C of defrostings of cell suspension 0.1 to 1 times；
(12), after cell suspension is resuspended with Matrigel, it is inoculated in 24 orifice plates, is placed in 37 DEG C of cell culture incubators, 15～30 minutes After, treat that Matrigel solidifies, add the special culture media of esophageal carcinoma tumor organoid, change liquid every other day；
After (13) 4～10 days, esophageal carcinoma organoid grows up to many spheroid forms, has epithelioid cell layer.
The most according to claim 9 a kind of based on Human Esophageal Carcinoma for In vitro culture esophageal carcinoma tumor organoid The cultural method of special culture media, it is characterised in that the compound method of step (1) and (3) described tissue-wash solution is: at physiology Saline or phosphate buffer add the penicillin of 500U/mL, the amphotericin B of 2.5 μ g/mL and the strepto-of 500 μ g/mL Element, filtration sterilization；
The compound method of step (2) described tissue transfer liquid is: adds 5% hyclone in DMEM culture medium, adds The penicillin of 500U/mL, the amphotericin B of 2.5 μ g/mL and the streptomycin of 500 μ g/mL；
The compound method of step (4) and (5) described tissue digestion liquid is:
2.5% hyclone, 1 μ g/mL～the IX Collagen Type VI of 10 μ g/mL, 50 μ g/mL-500 μ g/ are added in DMEM culture medium The neutral proteolytic enzyme of mL；Or
DMEM culture medium adds the IV Collagen Type VI of 2.5% hyclone, 1x penicillin and streptomycin, 0.01mg/mL-1mg/mL Enzyme, the hyaluronidase of 0.001mg/mL～0.2mg/mL and DNA enzymatic I of 0.001mg/mL～0.2mg/mL；Or
DMEM culture medium adds the XI Collagen Type VI of 2.5% hyclone, 1x penicillin and streptomycin, 20U/mL～200U/mL DNA enzymatic I of Bacillus polymyxa Neutral proteinase neutral proteolytic enzyme I and 0.01mg/mL～1mg/mL of enzyme, 1 μ g/mL～100 μ g/mL.
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CN114231490B (en) * 2021-12-23 2024-06-04 北京基石生命科技有限公司 Method for culturing tumor organoids by permeable hydrogel scaffold
CN114736865B (en) * 2022-03-09 2024-05-17 上海交通大学医学院附属第九人民医院 Salivary gland malignant polymorphic adenoma 3D organoid and culture method and application thereof
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CN114807039A (en) * 2022-06-22 2022-07-29 杭州艾名医学科技有限公司 Culture medium and culture method for esophageal cancer tumor organoid culture
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CN116064395A (en) * 2023-01-14 2023-05-05 郑乐民 Method for constructing brain glioma organoids
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