CN103352027A - Tumor stem cell suspension culture method - Google Patents
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Abstract
The invention belongs to the technical field of modern biotechnology cell culture. A purpose of the invention is to provide a tumor stem cell suspension culture method, which can be used for culture of tumor cells with different tissue sources, and tumor stem cells obtained through the culture method have characteristics of self-renewal capacity and enrichment. The tumor stem cell suspension culture method comprises: carrying out digestion centrifugation collection on tumor cells requiring culture, adopting PBS to carry out resuspending centrifugation, adding to a prepared suspension culture medium of the invention, carrying out beating-up into a single cell suspension, inoculating into a paved ultra-low absorption culture dish, and carrying out cell culture. The tumor stem cell suspension culture method has the following characteristics that: ball forming rates of various in vitro cultured tumor cells are high, multi-function factors are expressed, the OCT4 proportion and the NANOG proportion are increased, rich and comprehensive experimental materials are provided for tumor stem cell researches, and broad application prospects are provided in basic researches and clinical applications.
Description
Technical field
The invention belongs to the cell culture technology field of modern biotechnology, be specifically related to a kind of suspension culture method of tumor stem cell.
Background technology
Tumour is comprised of the heterogeneous cell with different propagation and invasive ability.There are some researches show, tumour is also followed the system structure of normal tissue cell, and the minimum cell of a group ratio is wherein arranged, and possesses long-term self and differentiation capability, causes tumour constantly to occur and progress.This subgroup because with the functional similarity of healthy tissues stem cell, correspondingly be called tumor stem cell (cancer stem cell), or tumour initiator cell (tumor initiating cell).Participating in growth and the transfer of primary tumo(u)r, have the potential of similar stem cell height self and multi-direction differentiation, is the source of different tumour cell Differentiation and proliferations, so the research of its functional mechanism has far reaching significance to the clinical prevention of tumor disease.
Understand mechanics, function and the control methods of tumor stem cell from the cell aspect, must have reliable method that they are separated from daughter cell.But because the tumor stem cell number is rare, and the tumor stem cell mark not yet obtains the clear and definite of unification, can only it be separated from other tumour cells by the difference on the function, namely produce artificially the environment that is conducive to tumor stem cell self and differentiation, make its daughter cell with differentiation show obvious difference.
Suspension culture is tumor stem cell study in vitro commonly used at present.The method be with tumour cell to be inoculated in than low density in liquid or the semisolid serum-free medium, the culture vessel that adopts ultralow absorption during cultivation with reduce cell attachment (
ELVIRA G, ZAPATA AG, et al.Mesenchymal stem cells:biological properties and clinical applications[J] .Expert Opin Biol Ther, 2010,10 (10): 1453-1468).Because the adherent of composition and cell can promote cytodifferentiation in the serum, and serum-free medium and ultralow absorption culture vessel can be kept cell and be in undifferentiated state, the cell that only possesses stem cell potential could be bred by long-term survival under the environment of serum-free, the cells that differentiation degree is higher is dead gradually, therefore suspension culture can be used for purifying tumor stem cell subgroup, but not tumor stem cell can not long-term surviving under such environment.But because the low support that lacks peripheral cell paracrine and signal transduction of the cell density of inoculation, apoptosis very easily, thus in nutrient solution, add the somatomedin of heavy dose, to keep cell proliferation.If the tumour cell of inoculation sum is certain, suspension culture is through after repeatedly going down to posterity, the cell that only possesses self-renewal capacity can survive, can be according to the number of the tumour ball that forms and ratio and the functional status of size reflection tumor stem cell, estimate intuitively the ability of cell self-renewal and propagation from the level of individual cells, can be widely used in the correlative study of tumor stem cell.
Singh in 2003 is with children's medulloblastoma, the star spongiocyte knurl becomes single cell suspension with tissue digestions such as ependymomas, adopt special serum free medium to add various growth factors and promote its increment, and keep its undifferentiated state, find that the part cell has formed the spheroid sample, this spheroid is rich in tumor stem cell, the substitute that can be used as tumor stem cell is used for experiment in vitro research (Singh, S.K., I.D.Clarke, et al. (2003) .Identification of a cancer stem cell in human brain tumors.Cancer Res63 (18): 5821-5828.).The tumour cell spheroid has propagation and self-renewal capacity, and also the isolation identification for tumor stem cell provides a feasible method.Because cultivating, the suspension cloning can show fairly simple, intuitively self and the differentiation capability of individual cells from cell aspect upper body, thus be widely used in the research of other types tissue stem cell, and the preliminary evaluation of Multidrug-resistant.Suetsugua etc. are isolated CD133 from hepatoma cell strain Huh-7
+Cell is than CD133
-Cell tumorigenicity is strong, has proved that the CD133+ liver cancer cell is in relatively naivety and similar etap of ancestral cells (Suetsugu, A., M.Nagaki, etal. (2006) .Characterization of CD133
+Hepatocellular carcinoma cells as cancer stem/progenitor cells.Biochem Biophys Res Commun351 (4): 820-824.).Except CD133, also have CD44, CD30, ABCB5 also to can be used as the surface marker (Fang of tumor stem cell, D., T.K.Nguyen, et al. (2005) .A tumorigenic subpopulation with stem cell properties in melanomas.Cancer Res65 (20): 9328-9337; Herszfeld, D., E.Wolvetang, et al. (2006) .CD30is a survival factor and a biomarker for transformed human pluripotent stem cells.Nat Biotechnol24 (3): 351-357; Pries, R., N.Witrkopf, et al. (2008) .Potential stem cell marker CD44is constitutively expressed in permanent cell lines of head and neck cancer.In Vivo22 (1): 89-92; ).The core transcription factor, OCT4, NANOG, SOX2, TCF3 and to keep the versatility of embryonic stem cell closely related.Recently, a large amount of evidences are closely related with them and malignant tumour, and its relationship between expression of crossing the ability that recurs behind tumour generation, transfer even the chemicotherapy at a distance.These transcription factors play the crucial effect of regulating and determine that tumor stem cell is in the destiny of tumor development the self-renewal capacity of tumor stem cell.Research is found, the increasing expression of the upper timing tumor stem cell of NANOG surface markers CD133/ABCG2/ALDH1A1/CD44 in prostate cancer.Illustrated that these factors are indispensable to the performance of keeping tumor stem cell, and can select tumor stem cell based on the expression of these transcription factors.
After the cell inoculation nude mice of suspension culture forms transplanted tumor, the draw materials tissue of tumour of can reducing better than the tumour cell of cellar culture is arranged and cellular form, gene expression profile is also more approaching with former tumour, and the transplanted tumor that tumor cell line inoculation forms often differentiation state is poor, the morphological structure indistinguishable.Reason may be, stem cell possesses hereditary fidelity, and therefore the heritable variation that the fast breeding of having avoided having the serum condition to cultivate lower tumour cell causes can be used for setting up more desirable animal model.
But, the reagent that is used at present the tumor stem cell suspension culture, do not have concrete company to produce, the cultural method that proposes in the document is disunity also, also exists different for the used basic medium of the tumour cell of different tissue sources and the kind of somatomedin with dosage.
Summary of the invention
The suspension culture method that the purpose of this invention is to provide a kind of tumor stem cell can be common to the cultivation of the tumour cell of different tissue sources, and the tumor stem cell that this cultural method obtains has self-renewal capacity and enrichment.
The technical solution used in the present invention is as follows:
The present invention adopts the serum-free suspension culture, can inoculate different types of tumour cell, at first in ultralow absorption culture vessel, cell mass digestion is become unicellular after, again be inoculated in the special-purpose culture environment of the present invention's research and development.
The invention provides a kind of suspension culture method of tumor stem cell, may further comprise the steps:
A, ultralow absorption culture dish paved
(a) preparation of ultralow absorption glue: polymethyl acrylic acid 2-hydroxyl second fat (Poly (2-hydroxyethyl methacrylate), PHEMA), 95% alcohol mixing, final concentration are 12mg/ml;
(b) with pipettor ultralow absorption glue is covered in the culture dish bottom, blots residual gum, whole density is 0.8mg/cm
2, to dry to complete drying (generally wanting more than 10 hours), uv irradiating 1 hour is for subsequent use;
(c) the dry ventilation of lucifuge preserved 1-2 month;
The preparation of B, suspension medium
(a) in Bechtop, in basic medium DMEM/F12, add rhIGF-1 (IGF), Prostatropin (bFGF), Urogastron (EGF), piping and druming mixing; Described IGF concentration is 10-30ng/ml, and bFGF concentration is 1.0-3ng/ml, and EGF concentration is 20-30ng/ml, and concentration is all for basic medium DMEM/F12;
(b) 4 ℃ kept in Dark Place 1-2 month;
C, cell cultures
After the tumour cell of required cultivation digested centrifugal collection, with PBS resuspended centrifugal 5-10 minute 2-3 time, adding step B prepared suspension medium, blows and beats into single cell suspension, is inoculated in the paved good ultralow absorption culture dish of steps A; Cultivate and collected whole substratum in per three days from the outset, centrifugally change once fresh all kinds of substratum, incubation time is 7 days, uses the accutase digestive ferment after 7 days, and piping and druming is digested to the unicellular 1:2 of carrying out and goes down to posterity gently, repeats the aforementioned operation culturing process that goes down to posterity;
Culture environment: temperature is 35-38 ℃, CO
2Concentration is the 5-8%(volumn concentration), humidity is saturated humidity.
The basic medium DMEM/F12 that the present invention is used is the mixed culture medium of DMEM and F12, combines the advantage of these two kinds of substratum, can be fit to how tissue-derived tumor stem cell.
The present invention adds the cytokine of high density--rhIGF-1 (IGF), and Prostatropin (bFGF), Urogastron (EGF) can be kept a small set of cell and be in undifferentiated state.
In the steps A, preferably, weighing PHEMA, 95% alcohol mixing, 65 ℃ of shaking tables shake low speed slowly, dissolve 8 hours; The selection of described culture dish size is decided according to final required cell concentration, such as 2.5 * 10
5Individual cell selects hole of 6 orifice plates to get final product, and it is 1ml that 6 holes of 6 orifice plates spread the glue amount.
Among the step B, preferably, the preferred 20ng/ml of described IGF concentration; The preferred 1.0ng/ml of bFGF concentration; The preferred 20ng/ml of EGF concentration.
Among the step C, be inoculated in the paved good ultralow absorption culture dish of steps A the cell concentration with respect to suspension medium: 10
4Individual/ml/cm
2
Preferred 37 ℃ of described culture temperature, CO
2Concentration is preferably 5%.
Described basic medium DMEM/F12, polymethyl acrylic acid 2-hydroxyl second fat, rhIGF-1, Prostatropin and Urogastron are the commercially available prod, all can buy from company to obtain.
Positively effect of the present invention:
1, the present invention is simple to operate, the convenient use, and the cytokine of utilization is few, and expense is low, the characteristic of the good and maximum analogue body inner cell of cell that increases of security.
2, the amplification in vitro cultural method of tumor stem cell suspension culture need not specific installation, operation is simple and feasible, prepare in advance the basic medium of large volume, make the volume of the few cells factor of adding more accurate, the perfect medium for preparing can save backup 4 degrees centigrade of long periods, the required cytokine of disposable adding also greatly reduces repeatedly the contamination probability that liquid feeding causes, has shortened the time, has improved the cell cultures success ratio.
3, in the fundamental research of tumor stem cell suspension culture and clinical application, will have widely prospect.
4, the balling ratio of all kinds of tumour cells of perfect medium vitro culture of the present invention is high, expresses the versatility factor, and OCT4, NANOG ratio all increase to some extent, for the research of tumor stem cell provides abundant comprehensively experiment material.
Description of drawings
Fig. 1 is different tumor cell line suspension culture growthhabit figure (200 times in microscope);
After Fig. 2 is the former culture of liver cancer tissue, adherent growth aspect graph and suspension culture aspect graph (200 times in microscope);
Wherein A is the adherent growth aspect graph, and B is the suspension culture aspect graph;
Fig. 3 is the PT-PCR result of hepatoma cell line CSQT-2, Hep3B, Huh7, liver cancer tissue adherent culture of former generation and suspension culture multipotency factor OCT4, NANOG;
Wherein A is the PT-PCR result of NANOG, and B is the PT-PCR result of OCT4;
Fig. 4 is the PT-PCR result of U251, Hela, A549, U87, Hep2, MCF-7 adherent culture and suspension culture multipotency factor OCT4, NANOG.
Embodiment
Now in conjunction with the embodiments and accompanying drawing, the invention will be further described, but enforcement of the present invention is not limited in this.
Following embodiment if no special instructions method therefor is ordinary method, and institute responds and all at room temperature carries out, and centrifugal speed is 500g, 5 minutes.
The reagent of using in this experiment is as follows: DMEM/F12 is Gibco company, DMEM is Gibco company, FBS is Gibco company, IGF is PeproTech company, bFGF is Gibco company, EGF is AbD Serotec Kidlington company, polymethyl acrylic acid 2-hydroxyl second fat (Poly (2-hydroxyethyl methacrylate), PHEMA) for Sigma buys, culture dish Corning company, Accutase is Gibco company, 0.25% pancreatin is Sigma company, CD133-PE is Miltenyi Biotec company, and it is Invitrogen company that total RNA extracts TRIZOL, and PT-PCR is Takara company.PBS(8.0g NaCl, 0.2g KCl, 0.2g KH
2PO
4, 1.44g Na
2HPO
412H
2O is dissolved in the 1000ml deionized water, adjusts pH value to 7.35.Non-special pointing out, PBS used herein is all this prescription)
Used β-actin, Nanog, Oct4 are synthetic by following primer PCR among the embodiment, and primer is that Invitrogen company is synthetic, and concrete sequence is as follows:
Embodiment 1: hepatoma cell line Huh7 stem cell is cultivated
Hepatoma cell line Huh7 is purchased from the Shanghai Chinese Academy of Sciences.
1. the alcohol final concentration that the PHEMA that joins glue: 0.12g is dissolved in 20ml95% is 12mg/ml, and the low speed jog is 8 hours in 65 ℃ of shaking tables.
2. bed board: in super clean bench, get 1ml Jiao Pu to the 6 orifice plate bottom for preparing with pipettor, cover and want evenly avoid repeatable operation, once finish.Exhaust unnecessary glue, blowing is 10 hours in super clean bench, uv irradiating 1 hour, and dry ventilation of lucifuge preserved 1-2 month.
3. substratum preparation: add IGF1ug among the DMEM/F12 of 49.9ml, bFGF50ng, EGF1ug blows and beats mixing, preserves 1-2 month for 4 ℃.
4.Huh7 go down to posterity with the DMEM culture medium culturing that contains 10%FBS is stable, adherently go down to posterity when growing to 90%.Blot the interpolation substratum, PBS washes 2 times, 37 ℃ of digestion of pancreatin 3 minutes, and 10% DMEM substratum stops digestion, and is centrifugal, abandons supernatant, resuspended rear centrifugal 2 times of PBS.
5.Huh7 the DMEM/F12 substratum that adds the factor is resuspended with containing, counting.
6.6 2.5*10 is added in the every hole of orifice plate
5Individual cell, substratum are supplied the 2.5ml/ hole.37 ℃, 5%CO
2Cultivate in the incubator.
7. half amount is changed liquid every other day, and full dose was changed liquid in 3 days, collected whole substratum and cell centrifugation, and abandoned supernatant in the 7th day, PBS is resuspended rear centrifugal, adds accutase37 ℃ of digestion, blows and beats into unicellular, stop digestion, centrifugal, interpolation newly contains the DMEM/F12 substratum 1:2 that adds the factor and goes down to posterity.Repeat appeal and operate the culturing process that goes down to posterity.
8. morphologic observation: Fig. 1 is the cellular form photo when suspension culture method is cultivated 7 days, and visible cell is the bright spheroid of round and smooth consolidation, and after the pipettor piping and druming, spheroid is not loose, keeps form constant.
9.PT-PCR detect multipotency factor expression situation: total RNA adopts TRIZOL reagent to extract, and requires the synthetic cDNA of reverse transcription take it as template in strict accordance with reverse transcription test kit specification sheets, utilizes primer to carry out PCR according to following condition, 95 ℃ 10 seconds, 60 ℃ 15 seconds, 72 ℃ 15 seconds, totally 40 circulations.Take β-actin as confidential reference items, adopt 2
-Δ Δ CtMethod is calculated the relative expression quantity of gene Nanog, Oct4, the difference of expression amount after comparison adherent culture and the suspension culture, Fig. 3 has shown that the Nanog after the suspension culture, Oct4 value are higher than adherent culture as a result, proved absolutely suspension culture after versatility factor expression amount obviously raise.
Embodiment 2: glioma cell line U251 stem cell is cultivated
Glioma cell line U251 is purchased from the Shanghai Chinese Academy of Sciences.
1. the alcohol final concentration that the PHEMA that joins glue: 0.12g is dissolved in 20ml95% is 12mg/ml, and the low speed jog is 8 hours in 65 ℃ of shaking tables.
2. bed board: in super clean bench, get 1ml Jiao Pu to the 6 orifice plate bottom for preparing with pipettor, cover and want evenly avoid repeatable operation, once finish.Exhaust unnecessary glue, blowing is 10 hours in super clean bench, uv irradiating 1 hour, and dry ventilation of lucifuge preserved 1-2 month.
3. substratum preparation: add IGF1.25ug among the DMEM/F12 of 49.9ml, bFGF75ng, EGF1ug blows and beats mixing, preserves 1-2 month for 4 ℃.
4.U251 go down to posterity with the DMEM culture medium culturing that contains 10%FBS is stable, adherently go down to posterity when growing to 90%.Blot the interpolation substratum, PBS washes 2 times, 37 ℃ of digestion of pancreatin 3 minutes, and 10% DMEM substratum stops digestion, and is centrifugal, abandons supernatant, resuspended rear centrifugal 2 times of PBS.
5.U251 the DMEM/F12 substratum that adds the factor is resuspended with containing, counting.
6.6 2.5*10 is added in the every hole of orifice plate
5Individual cell, substratum are supplied the 2.5ml/ hole.37 ℃, 5%CO
2Cultivate in the incubator.
7. half amount is changed liquid every other day, and full dose was changed liquid in 3 days, collected whole substratum and cell centrifugation, and abandoned supernatant in the 7th day, PBS is resuspended rear centrifugal, adds accutase37 ℃ of digestion, blows and beats into unicellular, stop digestion, centrifugal, interpolation newly contains the DMEM/F12 substratum 1:2 that adds the factor and goes down to posterity.Repeat appeal and operate the culturing process that goes down to posterity.
8. morphologic observation: Fig. 1 is the cellular form photo when suspension culture method is cultivated 7 days, and visible cell is the bright spheroid of round and smooth consolidation, and after the pipettor piping and druming, spheroid is not loose, keeps form constant.
9.PT-PCR detect multipotency factor expression situation: total RNA adopts TRIZOL reagent to extract, and requires the synthetic cDNA of reverse transcription take it as template in strict accordance with reverse transcription test kit specification sheets, utilizes primer to carry out PCR according to following condition, 95 ℃ 10 seconds, 60 ℃ 15 seconds, 72 ℃ 15 seconds, totally 40 circulations.Take β-actin as confidential reference items, adopt 2
-Δ Δ CtMethod is calculated the relative expression quantity of gene Nanog, the difference of expression amount after adherent culture and the suspension culture relatively, Fig. 4 has shown that the Nanog value after the suspension culture is higher than adherent culture as a result, proved absolutely suspension culture after versatility factor expression amount obviously raise.
Embodiment 3: the stem cell behind the former culture of liver cancer tissue is cultivated
Liver cancer tissue derives from clinical liver resection.
1. the alcohol final concentration that the PHEMA that joins glue: 0.12g is dissolved in 20ml95% is 12mg/ml, and the low speed jog is 8 hours in 65 ℃ of shaking tables.
2. bed board: in super clean bench, get 1ml Jiao Pu to the 6 orifice plate bottom for preparing with pipettor, cover and want evenly avoid repeatable operation, once finish.Exhaust unnecessary glue, blowing is 10 hours in super clean bench, uv irradiating 1 hour, and dry ventilation of lucifuge preserved 1-2 month.
3. substratum preparation: add IGF1.5ug among the DMEM/F12 of 49.9ml, bFGF75ng, EGF1.5ug blows and beats mixing, preserves 1-2 month for 4 ℃.
4. the liver cancer tissue primary cultured cell goes down to posterity with the DMEM culture medium culturing that contains 10%FBS is stable, adherently goes down to posterity when growing to 90%.Blot the interpolation substratum, PBS washes 2 times, 37 ℃ of digestion of pancreatin 3 minutes, and 10% DMEM substratum stops digestion, and is centrifugal, abandons supernatant, resuspended rear centrifugal 2 times of PBS.
5. the liver cancer tissue primary cultured cell is resuspended with containing the DMEM/F12 substratum that adds the factor, counting.
6.6 2.5*10 is added in the every hole of orifice plate
5Individual cell, substratum are supplied the 2.5ml/ hole.37 ℃, 5%CO
2Cultivate in the incubator.
7. half amount is changed liquid every other day, and full dose was changed liquid in 3 days, collected whole substratum and cell centrifugation, and abandoned supernatant in the 7th day, PBS is resuspended rear centrifugal, adds accutase37 ℃ of digestion, blows and beats into unicellular, stop digestion, centrifugal, interpolation newly contains the DMEM/F12 substratum 1:2 that adds the factor and goes down to posterity.Repeat appeal and operate the culturing process that goes down to posterity.
8. morphologic observation: Fig. 2 is the cellular form photo when suspension culture method is cultivated 7 days, and visible cell is the bright spheroid of round and smooth consolidation, and after the pipettor piping and druming, spheroid is not loose, keeps form constant.
9.PT-PCR detect multipotency factor expression situation: total RNA adopts TRIZOL reagent to extract, and requires the synthetic cDNA of reverse transcription take it as template in strict accordance with reverse transcription test kit specification sheets, utilizes primer to carry out PCR according to following condition, 95 ℃ 10 seconds, 60 ℃ 15 seconds, 72 ℃ 15 seconds, totally 40 circulations.Take β-actin as confidential reference items, adopt 2
-Δ Δ CtMethod is calculated the relative expression quantity of gene Nanog, Oct4, the difference of expression amount after comparison adherent culture and the suspension culture, Fig. 3 has shown that the Nanog after the suspension culture, Oct4 value are higher than adherent culture as a result, proved absolutely suspension culture after versatility factor expression amount obviously raise.
Above demonstration and described ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; that describes in above-described embodiment and the specification sheets just illustrates principle of the present invention; the present invention also has various changes and modifications without departing from the spirit and scope of the present invention, and these changes and improvements all fall in the claimed scope of the invention.The claimed scope of the present invention is defined by appending claims and equivalent thereof.
Claims (6)
1. the suspension culture method of a tumor stem cell is characterized in that, the method may further comprise the steps:
A, ultralow absorption culture dish paved
(a) preparation of ultralow absorption glue: polymethyl acrylic acid 2-hydroxyl second fat, the alcohol mixing with 95%, final concentration are 12mg/ml;
(b) with pipettor ultralow absorption glue is covered in the culture dish bottom, blots residual gum, whole density is 0.8mg/cm
2, to dry to complete drying, uv irradiating 1 hour is for subsequent use;
(c) the dry ventilation of lucifuge preserved 1-2 month;
The preparation of B, suspension medium
(a) in Bechtop, in basic medium DMEM/F12, add insulin-like growth factor I GF, Prostatropin bFGF, Urogastron EGF, the piping and druming mixing; Described IGF concentration is 10-30ng/ml, and bFGF concentration is 1.0-3ng/ml, and EGF concentration is 20-30ng/ml, and concentration is all for basic medium DMEM/F12;
(b) 4 ℃ kept in Dark Place 1-2 month;
C, cell cultures
After the tumour cell of required cultivation digested centrifugal collection, with PBS resuspended centrifugal 5-10 minute, 2-3 time, add the suspension medium that step B prepares, blow and beat into single cell suspension, be inoculated in the paved good ultralow absorption culture dish of steps A; Cultivate and collected whole substratum in per three days from the outset, centrifugally change once fresh all kinds of substratum, incubation time is 7 days, uses the accutase digestive ferment after 7 days, and piping and druming is digested to the unicellular 1:2 of carrying out and goes down to posterity gently, repeats the aforementioned operation culturing process that goes down to posterity;
Culture environment: temperature is 35-38 ℃, CO
2Concentration is that 5-8%, humidity are saturated humidity.
2. the suspension culture method of a kind of tumor stem cell according to claim 1 is characterized in that,
In the steps A wherein, weighing polymethyl acrylic acid 2-hydroxyl second fat, the alcohol mixing with 95%, 65 ℃ of shaking tables shake low speed slowly, dissolve 8 hours.
3. the suspension culture method of a kind of tumor stem cell according to claim 1 and 2 is characterized in that, in the steps A wherein, final required cell concentration is 2.5 * 10
5During individual cell, culture dish is selected hole of 6 orifice plates, and spreading the glue amount is 1ml.
4. the suspension culture method of a kind of tumor stem cell according to claim 1 and 2 is characterized in that, among the step B wherein, described IGF concentration is 20ng/ml; BFGF concentration is 1.0ng/ml; EGF concentration is 20ng/ml.
5. the suspension culture method of a kind of tumor stem cell according to claim 1 and 2 is characterized in that, among the step C wherein, is inoculated in that the cell concentration with respect to suspension medium is 10 in the paved good ultralow absorption culture dish of steps A
4Individual/ml/cm
2
6. the suspension culture method of a kind of tumor stem cell according to claim 1 and 2 is characterized in that, among the step C wherein, described culture environment: temperature is 37 ℃, CO
2Concentration is 5%.
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| CN111321119A (en) * | 2020-03-18 | 2020-06-23 | 四川大学 | Liver cancer cell line suitable for large-scale serum-free adherent culture and establishment method and application thereof |
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| WO2022134159A1 (en) * | 2020-12-25 | 2022-06-30 | 中国科学院广州生物医药与健康研究院 | Chip for integrated tumor cell behavior experiments |
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