CN110452877A

CN110452877A - A kind of cultural method of lung cancer solid tumor primary cell

Info

Publication number: CN110452877A
Application number: CN201810426591.7A
Authority: CN
Inventors: 席建忠; 尹申意; 李娟�
Original assignee: Beijing Gishanley De Biological Science And Technology Co Ltd
Current assignee: BEIJING GENEX HEALTH TECHNOLOGY Co.,Ltd.
Priority date: 2018-05-07
Filing date: 2018-05-07
Publication date: 2019-11-15

Abstract

The invention discloses a kind of cultural methods of lung cancer cell.The present invention provides a kind of lung cancer solid tumor primitive cell culture method and matched reagents, and the core of the technology is: (1) handling lung cancer solid tumor mass with bland cell dissociation reagent, ensure that the vigor of cancer cell in tissue to the greatest extent；(2) special serum free medium is prepared, in vitro culture is carried out using tumour cell of the suspension culture system to lung cancer solid tumor source, guarantees the interference for excluding normal cell while cancer cell normally expands to greatest extent.The lung cancer cell culture obtained using the method for the present invention can be used for the experiment in vitro of various kinds of cell level, the sequencing of two generations, building animal model, building cell line etc..It is contemplated that this cultural method is with a wide range of applications in the research of lung cancer and clinical conditions field.

Description

A kind of cultural method of lung cancer solid tumor primary cell

Technical field

The present invention relates to field of biotechnology, and in particular to a kind of cultural method of lung cancer solid tumor primary cell.

Background technique

Lung cancer is the highest cancer of morbidity and mortality in the world, and wherein male lung cancer morbidity and mortality are all Highest in malignant tumour, and female lung cancer morbidity and mortality are second.Meanwhile lung cancer morbidity growth rate and death increase Long rate highest in all malignant tumours.It may be said that lung cancer is to one of human health and the maximum malignant tumour of life threat.

Although there are very the research of the cause of disease and occurrence and development process of lung cancer in the scientific research of countries in the world and medical institutions The investment of great dynamics, but the mankind still know little about it to this disease.Lung cancer is a kind of complex disease, is occurred, development is One dynamic process is related to many signaling molecule interactions, forms a complicated molecular regulation network, simultaneously also It is influenced by outside environmental elements.The cause of disease and occurrence and development process of lung cancer have very strong individual difference, cannot without exception and By.Therefore using lung cancer solid tumor primary cell culture as model progress individuation precisely study be lung cancer research field or even The trend of pulmonary cancer diagnosis therapy field.

Existing primary tumor cell culture technique mainly has 2D culture, 3D culture, several classes such as reprogramming culture, these sides Cultivation cycle is extremely long, and culture success ratio is low, and heteroproteose cell is difficult to the problems such as removing for method all different degrees of facing.

Summary of the invention

In order to effectively solve above-mentioned technical problem, the present invention provides a kind of new lung cancer solid tumor primitive cell culture skills The core of art and matched reagent, the technology is: (1) handling lung cancer solid tumor mass with bland cell dissociation reagent, utmostly The vigor that ensure that cancer cell in tissue；(2) special serum free medium is prepared, using suspension culture system to lung cancer reality The tumour cell in body tumor source carries out in vitro culture, guarantees to exclude normal cell to greatest extent while cancer cell normally expands Interference.

In a first aspect, a kind of claimed method for cultivating lung cancer solid tumor primary cell.

The method of culture lung cancer solid tumor primary cell provided by the present invention, specifically may include following steps:

(1) dissociation processing is carried out to lung cancer solid tumor mass with sample dissociation solution, obtains lung cancer solid tumor primary cell；

The sample dissociation solution is made of clostridiopetidase A I, clostridiopetidase A IV and PBS；Wherein, the clostridiopetidase A I is in the sample Final concentration of 150-250U/mL (such as 200U/mL) in dissociation solution；End of the clostridiopetidase A IV in the sample dissociation solution is dense Degree is 150-250U/mL (such as 200U/mL)；Surplus is PBS.

Wherein, the unit U:In of clostridiopetidase A (the clostridiopetidase A I or the clostridiopetidase A IV) is defined with the enzyme activity of protease It 37 DEG C,, can be with 1U Protease Treatment clostridiopetidase A (the clostridiopetidase A I or the clostridiopetidase A IV) 5 hours under conditions of pH 7.5 Discharge 1 μm of ol of L-Leu.

In a specific embodiment of the present invention, the brand article No. of the clostridiopetidase A I is Gibco#17100-017；The glue The brand article No. of protoenzyme IV is Gibco#17104-019；The brand article No. of the PBS is Gibco#21-040-CVR.

(2) using lung cancer solid tumor primitive cell culture base suspension incubation step (1) dissociate come lung cancer solid tumor it is former For cell；

The lung cancer solid tumor primitive cell culture base is by dual anti-P/S (Pen .- Strep), HEPES, non-essential amino Acid solution, GlutaMax, human recombination protein EGF, human recombination protein bFGF, human recombination protein MSP, cortisol, B27, ITS-X (Insulin, Transferrin, Selenium, Ethanolamine Solution), Y-27632 and Advanced DMEM/ F12 culture medium composition；Wherein, end of the penicillin in the dual anti-P/S in the lung cancer solid tumor primitive cell culture base Concentration is 100-200U/mL (such as 100U/mL)；Streptomysin in the dual anti-P/S is trained in the lung cancer solid tumor primary cell Support the final concentration of 100-200 μ g/mL (such as 100 μ g/mL) in base；The HEPES is trained in the lung cancer solid tumor primary cell Support the final concentration of 8-12mM (such as 10mM) in base；The nonessential amino acid solution is trained in the lung cancer solid tumor primary cell Support the final concentration of 0.8-1.2% in base (such as 1%, % indicates volumn concentration)；The GlutaMax is real in the lung cancer Final concentration of 0.8-1.2% in body tumor primitive cell culture base (such as 1%, % indicates volumn concentration)；People's recombination Final concentration of 10-100ng/mL of the albumen EGF in the lung cancer solid tumor primitive cell culture base；The human recombination protein Final concentration of 10-50ng/mL of the bFGF in the lung cancer solid tumor primitive cell culture base；The human recombination protein MSP exists Final concentration of 5-25ng/mL in the lung cancer solid tumor primitive cell culture base；The cortisol is in the lung cancer solid tumor Final concentration of 20-50ng/mL in primitive cell culture base；The B27 is in the lung cancer solid tumor primitive cell culture base Final concentration of 1.5-2.5% (such as 2%, % indicate volumn concentration)；The ITS-X is primary thin in the lung cancer solid tumor Final concentration of 0.8-1.2% in born of the same parents' culture medium (such as 1%, % indicates volumn concentration)；The Y-27632 is in the lung cancer Final concentration of 5-20 μM in solid tumor primitive cell culture base；Surplus is Advanced DMEM/F12 culture medium.

Further, the solvent of the nonessential amino acid solution is water, and solute and concentration are as follows: glycine 10mM；L- Alanine 10mM；Altheine 10mM；L-Aspartic acid 10mM；Pidolidone 10mM；L-PROLINE 10mM；Serine 10mM.The B27 is " B-27^TMSupplement (50X), minus vitamin A " (such as Gibco#12587010, or and its Form other identical products)." the B-27^TMContain biotin in Supplement (50X), minus vitamin A " (Biotin), DL- alpha-tocopherol acetate (DL Alpha Tocopherol Acetate), DL- alpha-tocopherol (DL Alpha- Tocopherol), BSA (fatty acid free Fraction V), catalase (Catalase), biosynthetic human insulin (Human Recombinant Insulin), human transferrin (Human Transferrin), superoxide dismutase (Superoxide Dismutase), cortisone (Corticosterone), D- galactolipin (D-Galactose), ethanolamine salt Acid (Ethanolamine HCl), reduced glutathione (Glutathione (reduced)), L-carnitine hydrochloric acid (L- Carnitine HCl), linoleic acid (Linoleic Acid), linolenic acid (Linolenic Acid), progesterone (Progesterone), putrescine (Putrescine 2HCl), sodium selenite (Sodium Selenite), triiodo thyroid gland original ammonia Sour (T3 (triodo-I-thyronine)).The solvent of the ITS-X is EBSS solution (Earle's balanced salt solution), solute And concentration is as follows: insulin 1g/L；Transferrins 0.55g/L；Sodium selenite 0.00067g/L；Ethanol amine 0.2g/L.It is described GlutaMAX is a kind of advanced cell culture additive, can directly substitute the L-Glutamine in cell culture medium.It is described GlutaMAX is " GlutaMAX^TMSupplement " (such as Gibco#35050061, or form other identical products with it).Institute State " GlutaMAX^TMThe ingredient of Supplement " is L-alanyl-L-glutamine, is the substitute of L-glutamine, dense Degree is 200nM, and solvent is 0.85%NaCl solution.The Y-27632 is a kind of " Y-27632 dihydrochloride (ATP Emulative ROCK-I and ROCK-II inhibitor, Ki are respectively 220nM and 300nM) " (such as MCE#129830-38-2, or and its Form other identical products).

In a specific embodiment of the present invention, the brand article No. of the dual anti-P/S (Pen .- Strep) is Gibco# 15140122；The brand article No. of the HEPES is Gibco#15630080；The brand article No. of the nonessential amino acid solution is Gibco#11140-050；The brand article No. of the GlutaMAX is Gibco#35050061；The product of the human recombination protein EGF Board article No. is Peprotech AF-100-15-100；The brand article No. of the human recombination protein bFGF is Peprotech AF- 100-18B-50；The brand article No. of the human recombination protein MSP is R&D#352-MS-050；The brand article No. of the cortisol is Sigma#H0888；The brand article No. of the B27 is Gibco#12587010；The brand article No. of the ITS is Gibco# 51500056；The brand article No. of the Y-27632 is MCE#129830-38-2；The Advanced DMEM/F12 culture medium Brand article No. is Gibco#12634010.

Further, in step (1), can according to the method included the following steps with the sample dissociation solution to the lung cancer Solid tumor mass is dissociated: by the dosage of the every mg tissue of 0.1-0.3mL (such as 0.1mL) the sample dissociation solution, after shredding The lung cancer solid tumor mass (be such as cut into 0.8-1.2mm³Fritter) with prior 37 DEG C preheat the sample dissociation solution into Row processing, progress sample dissociation under the conditions of 37 DEG C, Dissociation time 15 minutes to 3 hours.It observes under the microscope within every 15 minutes The dissociation situation of sample, until observing a large amount of individual cells.

Further, it in step (2), can be trained according to the method included the following steps the lung cancer solid tumor primary cell It supports base suspension and cultivates the lung cancer solid tumor primary cell: using with low adsorption surface (low-attachment-surface) Culture vessel, suspended using the lung cancer solid tumor primitive cell culture base and cultivate the lung cancer solid tumor primary cell, 37 DEG C, 5%CO₂Under the conditions of cultivated, 2-4 days every (such as 3 days) replace a subculture, until cell formed 80-120 μm of diameter The agglomerate of (such as 100 μm).

Wherein, Initial seeding density can be 10⁵A/cm²Container bottom area, by taking six orifice plates as an example, by every hole 10⁶A cell Density bed board.

Further, before step (1), it may also include and dissociation pre-treatment is carried out to the lung cancer solid tumor mass as follows The step of: lung cancer solid tumor mass sample surface 10 to 30 is cleaned with the ethyl alcohol that volumn concentration is 70-75% (such as 75%) Second；The lung cancer solid tumor mass sample is cleaned 5-10 times (such as 5 times) with sample cleaning solution, cleans institute with sterile PBS solution State lung cancer solid tumor mass sample 5-10 times (such as 5 times)；Then impurity in the lung cancer solid tumor mass sample, connective are removed The ingredient of the influence primitive cell culture such as tissue, adipose tissue, necrotic tissue.

Wherein, the sample cleaning solution is made of dual anti-P/S (Pen .- Strep) and PBS；Wherein, the dual anti-P/S In final concentration of 100-200U/mL (such as 100U/mL) of the penicillin in the sample cleaning solution；In the dual anti-P/S Final concentration of 100-200 μ g/mL (such as 100 μ g/mLs) of the streptomysin in the sample cleaning solution；Surplus is PBS.

In a specific embodiment of the present invention, the brand article No. of the dual anti-P/S is Gibco#15140122；The PBS Brand article No. be Gibco#21-040-CVR.

The step of carrying out dissociation pre-treatment to the lung cancer solid tumor mass needs to operate on ice, and whole operation step needs It to be completed in 10 minutes.

Further, the isolated time for carrying out the lung cancer solid tumor mass sample of the dissociation pre-treatment need to be small for 2 When within, and be stored in always in Sample preservation liquid before being handled before carrying out the dissociation.

Wherein, the Sample preservation liquid is by fetal calf serum, dual anti-P/S, HEPES and HBSS (Hank's balanced salt solution) group At；Wherein, (such as 2%, % indicates that volume basis contains to final concentration of 1-5% of the fetal calf serum in the Sample preservation liquid Amount)；Final concentration of 100-200U/mL (such as 100U/mL) of the penicillin in the Sample preservation liquid in the dual anti-P/S； Final concentration of 100-200 μ g/mL (such as 100 μ g/mLs) of the streptomysin in the Sample preservation liquid in the dual anti-P/S；Institute State final concentration of 8-12mM (such as 10mM) of the HEPES in the Sample preservation liquid；Surplus is HBSS.

In a specific embodiment of the present invention, the brand article No. of the fetal calf serum is Gibco#16000-044；It is described double The brand article No. of anti-P/S is Gibco#15140122；The brand article No. of the HEPES is Gibco#15630080；The HBSS Brand article No. be Gibco#14170161.

Further, in step (1), dissociation processing is carried out to the lung cancer solid tumor mass with the sample dissociation solution It may also include the steps of: afterwards and terminate dissociation reaction with the digestion terminate liquid of 8-15 times of (such as 10 times) volume, collect cell suspension； The cell suspension described in 100 μm or 40 μm of steril cell strainer filterings removes tissue relic and adhesion cells；800-1000g is (such as 800g) room temperature is centrifuged 10-15 minutes (such as 10 minutes), discards supernatant；Cell is resuspended with 3-5mL (such as 5mL) sterile PBS afterwards；Again 800-1000g (such as 800g) room temperature is centrifuged 10-15 minutes (such as 10 minutes), discards supernatant；Then former with the lung cancer solid tumor Cell precipitation is resuspended for cell culture medium, observes cell state under the microscope, carries out cell count.

Wherein, the digestion terminate liquid is made of fetal calf serum, dual anti-P/S and DMEM culture medium；Wherein, the tire ox blood Final concentration of 8-12% clearly in the digestion terminate liquid (such as 10%, % indicates volumn concentration)；In the dual anti-P/S Penicillin it is described digestion terminate liquid in final concentration of 100-200U/mL (such as 100U/mL)；Chain in the dual anti-P/S Final concentration of 100-200 μ g/mL (such as 100 μ g/mLs) of the mycin in the digestion terminate liquid；Surplus is DMEM culture medium.

In a specific embodiment of the present invention, the brand article No. of the fetal calf serum is Gibco#16000-044；It is described double The brand article No. of anti-P/S is Gibco#15140122；The brand article No. of the DMEM culture medium is Gibco#11965-092.

Further, in step (2), it may also include the steps of: and formed directly to the lung cancer solid tumor primary cell When the agglomerate of 80-120 μm of diameter (such as 100 μm), the lung cancer solid tumor primary cell is passed on.

Wherein, the cell dissociation buffer composition used when carrying out the passage is as follows: containing in cell dissociation buffer described in every 10mL There are the EDTA (i.e. 10 μ L 0.5M EDTA) of 4-6mL (such as 5mL) Accutase, final concentration of 5mM, 1.5-2.5mL (such as 2mL) TrypLE Express, surplus PBS.

Further, the Accutase is " StemPro^TMAccutase^TMCell Dissociation Reagent” (such as Gibco#A11105-01, or form other identical products with it).The Accutase is a kind of enzyme of single component, It is dissolved in D-PBS, 0.5mM EDTA solution.The TrypLE Express is " TrypLE^TMExpress Enzyme(1X), No phenol red " (such as Gibco#12604013, or form other identical products with it)." the TrypLE^TMExpress The KH of KCl, 200mg/L containing 200mg/L in Enzyme (1X), no phenol red "₂PO₄, 8000mg/L NaCl, The Na of 2160mg/L₂HPO₄·7H₂O, the EDTA of 457.6mg/L；Also contain recombinant protease.

In a specific embodiment of the present invention, the brand article No. of the Accutase is Gibco#A11105-01；It is described The brand article No. of 0.5M EDTA is Invitrogen#AM9261；The brand article No. of the TrypLE Express is Gibco# 12604013；The brand article No. of the PBS is Gibco#21-040-CVR.

Further, the digestion temperature used when carrying out the passage is 37 DEG C.

Further, the digestion terminate liquid used when carrying out the passage is previously described digestion terminate liquid.

More specifically, it carries out the step of passage: collecting cell mass to be passed on, with sterile PBS after centrifugation Solution cleans cell mass, then is centrifuged, and cell mass then is resuspended with the cell dissociation buffer, disappears under the conditions of 37 DEG C Change, until cell mass is all digested as individual cells, with the digestion terminate liquid, (its dosage can be 5-10 times, such as 10 times of bodies Product) digestion reaction is terminated, collect cell suspension；Cell is hanged with the lung cancer solid tumor primitive cell culture base weight after centrifugation to sink It forms sediment, counts, then (Initial seeding density can be 10 using the culture vessel suspended culture cell with low adsorption surface⁵A/cm² Container bottom area, by taking six orifice plates as an example, by every hole 10⁶The density bed board of a cell), condition of culture is 37 DEG C, 5%CO₂.It is above-mentioned All centrifugations concretely 800-1000g (such as 800g) room temperature passed in step is centrifuged 10-20 minutes (such as 10 minutes).

Further, the method may also include primary to the lung cancer solid tumor after 2-3 passage amplification thin Born of the same parents freeze and/or the step of recoveries.

Wherein, the cells frozen storing liquid used when being frozen described in progress by Advanced DMEM/F12 culture medium, DMSO and 1% methocel solution composition；Wherein, the Advanced DMEM/F12 culture medium, the DMSO and 1% methyl The volume proportion of cellulose solution is 20:2:(0.8-1.2), such as 20:2:1；1% methocel solution is that concentration is The methylated cellulose aqueous solution of 1g/100ml.

In a specific embodiment of the present invention, the brand article No. of the Advanced DMEM/F12 culture medium is Gibco# 12634010；The brand article No. of the DMSO is Sigma#D2438；The brand article No. of the methylcellulose is Sigma# M7027。

Further, the specific steps frozen described in progress: collecting cell mass to be frozen, with sterile after centrifugation PBS solution cleans cell mass, then is centrifuged, and cell mass then is resuspended with the cell dissociation buffer, carries out under the conditions of 37 DEG C Digestion, until cell mass is all digested as individual cells, with the digestion terminate liquid, (its dosage can be 5-10 times, such as 10 times Volume) digestion reaction is terminated, collect cell suspension；With the cells frozen storing liquid after centrifugation, by 0.5-2 × 10⁶/ mL (such as 10⁶/ ML cell precipitation is resuspended in density), and gradient cooling box is transferred in liquid nitrogen after being frozen overnight and saves for a long time.In above-mentioned cryopreservation step All centrifugations concretely 800-1000g (such as 800g) room temperature be centrifuged 10-20 minutes (such as 10 minutes).

Further, the specific steps of the recovery are carried out: will be taken from liquid nitrogen equipped with the cryopreservation tube to recovery cell Out, melt cell rapidly in 37-39 DEG C of (such as 37 DEG C) sterile water；Centrifugation (such as 800-1000g, as 800g room temperature is centrifuged 5-10 Minute, such as 10 minutes) cell precipitation is hanged with the lung cancer solid tumor primitive cell culture base weight afterwards, then using with low adsorption (Initial seeding density can be 10 to the culture vessel suspended culture cell on surface⁵A/cm²Container bottom area), every solencyte (10⁶ It is a) recover to 3.5cm culture dish), condition of culture is 37 DEG C, 5%CO₂。

Second aspect, it is claimed a kind of for cultivating the reagent set of lung cancer solid tumor primary cell.

The reagent set provided by the present invention for being used to cultivate lung cancer solid tumor primary cell, concretely following any:

(A) it is made of sample dissociation solution described previously and the lung cancer solid tumor primitive cell culture base；

(B) by sample dissociation solution described previously, the lung cancer solid tumor primitive cell culture base and following reagent at least A kind of composition: Sample preservation liquid, the cell dissociation buffer, the sample cleaning solution, the digestion terminate liquid and institute described previously State cells frozen storing liquid.

Further, described (B) specifically can be trained such as by sample dissociation solution described previously, the lung cancer solid tumor primary cell Support base, the Sample preservation liquid and cell dissociation buffer composition.

The Sample preservation liquid can be used for sample it is in vitro after temporary preservation, can have sample it is in vitro after, in the short time Maintain the activity of cell in sample.It can be reserved for 1 month for 4 DEG C after the Sample preservation liquid prepares.

The sample cleaning solution can be used for the cleaning and disinfection of sample.The sample cleaning solution needs ready-to-use.

The sample dissociation solution can be used for the dissociation of sample, can be by the lung cancer solid tumor primary cell in sample from tissue In dissociate come.The sample dissociation solution need to be ready-to-use, and clostridiopetidase A I and clostridiopetidase A IV therein can be with liquid storage (mother liquor) shapes - 20 DEG C of long-term preservations of formula, concretely 10 times of liquid storages (mother liquor).10 × clostridiopetidase A I liquid storage is made of the clostridiopetidase A I and PBS； The wherein final concentration of 2000U/mL of the clostridiopetidase A I；Surplus is PBS.10 × clostridiopetidase A IV liquid storage is by the clostridiopetidase A IV It is formed with PBS；The wherein final concentration of 2000U/mL of the clostridiopetidase A IV；Surplus is PBS.The clostridiopetidase A I and the glue The enzyme activity definition of protoenzyme IV sees above.

The cell dissociation buffer can be used for the digestion and passage of cell mass, lung cancer tumor agglomerate can be digested to individually Cell.The cell dissociation buffer needs ready-to-use.

The digestion terminate liquid can be used for terminating sample dissociation or cell dissociation process.After the digestion terminate liquid prepares It can be saved one month at 4 DEG C.

The lung cancer solid tumor primitive cell culture base can be used for the culture of lung cancer solid tumor primary cell.The lung cancer is real Body tumor primitive cell culture basigamy is needed after making with 0.22 μM of syringe filter (Millipore SLGP033RS) filtration sterilization, It can be saved at 4 DEG C two weeks.Human recombination protein EGF, human recombination protein bFGF, human recombination protein MSP, cortisol and Y- therein 27632 can in the form of liquid storage (mother liquor) -80 DEG C of long-term preservations, concretely 1000 times of liquid storages (mother liquor).1000 × people recombinates egg White EGF liquid storage is made of human recombination protein EGF, BSA and PBS, wherein the final concentration of 20 μ g/mL of the human recombination protein EGF, The final concentration of 0.01g/mL of the BSA, surplus are PBS.1000 × human recombination protein bFGF liquid storage is by human recombination protein BFGF, BSA and PBS composition, wherein the final concentration of 20 μ g/mL of the human recombination protein bFGF, the BSA's is final concentration of 0.01g/mL, surplus are PBS.1000 × human recombination protein MSP liquid storage is made of human recombination protein MSP, BSA and PBS, wherein The final concentration of 20 μ g/mL of the human recombination protein MSP, the final concentration of 0.01g/mL of the BSA, surplus is PBS.It is above-mentioned In three kinds of 1000 times of liquid storages, the BSA is that can have (ready-to-use) in the form of 100 times of liquid storages (mother liquor), specifically by BSA and PBS composition, wherein the final concentration of 0.1g/mL of BSA (Sigma#A1933), surplus are PBS.In addition, 1000 × cortisol stores up Liquid is made of cortisol, dehydrated alcohol and ultrapure water, wherein the final concentration of 25 μ g/mL of the cortisol, the dehydrated alcohol Final concentration of 5% (volumn concentration), surplus is ultrapure water.1000 × Y-27632 is by Y-27632 and ultrapure water group At wherein the final concentration of 10mM of Y-27632, surplus are ultrapure water.

The cells frozen storing liquid needs ready-to-use.Wherein, 1% methocel solution can be in 4 DEG C of long-term preservations.

The third aspect, claimed previously described reagent set is in culture lung cancer solid tumor primary cell Using.

Further, concretely primary lung cancer, pathological staging are II phase or III phase, pathological to the lung cancer For non-small cell lung cancer or Small Cell Lung Cancer, lung cancer specimen weight is more than the sample of 20mg.

In the present invention, the above all of PBS can be 1 × PBS, pH7.3-7.5.Its concrete composition is as follows: molten Agent is water, solute and concentration are as follows: KH₂PO₄144mg/L, NaCl 9000mg/L, Na₂HPO₄·7H₂O 795mg/L。

The method that the present invention provides a kind of to extract culture lung cancer primary tumor cell from fresh lung cancer solid tumor mass And matched reagent, this method have the advantage that

1, tissue samples dosage is few, it is only necessary to the operation of lung cancer sample of 20mg or so；

2, cultivation cycle is short, it is only necessary to can be obtained 10 within 3-10 days⁷The lung cancer primary tumor cell of the order of magnitude；

3, culture stability is high, is up to the success rate that this method carries out in vitro culture to qualified operation of lung cancer sample 70%；

4, cell purity is high, and in the lung cancer cell culture obtained using this method, the ratio of cancer cell can reach To 70%-95%, heteroproteose cell interference is less.

The lung cancer cell culture obtained using the method for the present invention can be used for various kinds of cell level experiment in vitro, The sequencing of two generations, building animal model, building cell line etc..It is contemplated that research and clinic of this cultural method in lung cancer Diagnoses and treatment field is with a wide range of applications.

Detailed description of the invention

Fig. 1 obtains unicellular after treatment for cancerous lung tissue.Scale is 200 μm, 10 times of amplifications.

Fig. 2 is obtained cell mass after cancerous lung tissue originally culture.Scale is 100 μm, 10 times of amplifications.

Fig. 3 is obtained lung carcinoma cell HE colored graph after cancerous lung tissue originally culture.Scale is 50 μm, 40 times of amplifications.

Fig. 4 is obtained cancer cell agglomerate immunofluorescence dyeing figure after cancerous lung tissue originally culture.

Fig. 5 is to carry out copy number analysis of variance (CNV) according to sequencing result to show each generation lung cancer cell culture (P1, P2, P3, P4) is consistent with the copy number variation situation height of primary lung cancer tumor tissue (Tumor).

Specific embodiment

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

Embodiment 1, the reagent for being formulated for culture lung cancer cell

1, Sample preservation liquid (100mL)

The specific formula of Sample preservation liquid (100mL) is as shown in table 1.

1 Sample preservation liquid (100mL) of table

After the completion of Sample preservation liquid is prepared, dispensed with 15mL centrifuge tube, every pipe 5mL.After packing 1 can be saved in 4 DEG C A month.

2, sample cleaning solution (100mL)

The specific formula of sample cleaning solution (100mL) is as shown in table 2.

2 sample cleaning solution (100mL) of table

Sample cleaning solution needs ready-to-use.

3, sample dissociation solution (10mL)

The specific formula of sample dissociation solution (10mL) is as shown in table 3.

3 sample dissociation solution (10mL) of table

Note: sample dissociation solution is ready-to-use.

In table 3, the preparation of clostridiopetidase A liquid storage is as shown in table 4 and table 5.

Table 4 10 × clostridiopetidase A I liquid storage (100mL)

After 10 × clostridiopetidase A I liquid storage is prepared, dispensed with 1.5mL sterile centrifugation tube, every pipe 1mL.The liquid storage can be long at -20 DEG C Phase saves.

Table 5 10 × clostridiopetidase A IV liquid storage (100mL)

After 10 × clostridiopetidase A IV liquid storage is prepared, dispensed with 1.5mL sterile centrifugation tube, every pipe 1mL.The liquid storage can be at -20 DEG C Long-term preservation.

In table 4 and table 5, the list of clostridiopetidase A (the clostridiopetidase A I or the clostridiopetidase A IV) is defined with the enzyme activity of protease Position U: small with 1U Protease Treatment clostridiopetidase A (the clostridiopetidase A I or the clostridiopetidase A IV) 5 under conditions of 37 DEG C, pH 7.5 When, 1 μm of ol of L-Leu can be discharged.

4, cell dissociation buffer (10mL)

The specific formula of cell dissociation buffer (10mL) is as shown in table 6.

6 cell dissociation buffer of table (10mL)

Cell dissociation buffer is ready-to-use.

5, terminate liquid (100mL) is digested

The specific formula for digesting terminate liquid (100mL) is as shown in table 7.

Table 7 digests terminate liquid (100mL)

After digesting terminate liquid preparation, it can be saved one month at 4 DEG C.

6, lung cancer solid tumor primitive cell culture base (100mL)

The specific formula of lung cancer solid tumor primitive cell culture base (100mL) is as shown in table 8.

8 lung cancer solid tumor primitive cell culture base (100mL) of table

After the completion of lung cancer solid tumor primitive cell culture basigamy system, with 0.22 μM of syringe filter (Millipore SLGP033RS) filtration sterilization can save two weeks at 4 DEG C.

In table 8, the preparation of human recombination protein liquid storage is as shown in table 9- table 12, the preparation of cortisol liquid storage liquid storage such as 13 institute of table Show, the preparation of Y-27632 liquid storage is as shown in table 14.

9 100 × BSA of table solution (1mL)

100 × BSA solution is ready-to-use.

Table 10 1000 × human recombination protein EGF liquid storage (5mL)

After 1000 × human recombination protein EGF liquid storage is prepared, dispensed with 1.5mL sterile centrifugation tube, which can be long at -80 DEG C Phase saves.

Table 11 1000 × human recombination protein bFGF liquid storage (2.5mL)

After 1000 × human recombination protein bEGF liquid storage is prepared, dispensed with 1.5mL sterile centrifugation tube, which can be at -80 DEG C Long-term preservation.

Table 12 1000 × human recombination protein MSP liquid storage (2.5mL)

After 1000 × human recombination protein MSP liquid storage is prepared, dispensed with 1.5mL sterile centrifugation tube, which can be long at -80 DEG C Phase saves.

13 1000 × cortisol of table liquid storage (100mL)

After 1000 × cortisol liquid storage is prepared, dispensed with 1.5mL sterile centrifugation tube, which can be in -80 DEG C of long-term preservations.

14 1000 × Y-27632 of table liquid storage (3.125mL)

It after 1000 × Y-27632 liquid storage is prepared, is dispensed with 0.5mL sterile centrifugation tube, which can protect -80 DEG C long-term It deposits.

7, cells frozen storing liquid

The specific formula of cells frozen storing liquid is as shown in Table 15.

15 cells frozen storing liquid of table

Cells frozen storing liquid is ready-to-use.

In table 15, the preparation of 1% methocel solution is as shown in table 16.

16 1% methocel solution (10mL) of table

1% methocel solution can be in 4 DEG C of long-term preservations after preparing.

The acquisition of embodiment 2, lung cancer Postoperative Specimen

1, regular Medical Ethics has been passed through with Grade A hospital cooperation, the development of cooperation and has examined.

2, attending physician doctor selects patient in group according to clinical indication as defined in Medical guidelines, and is referred to according to clinic in art Sign selects suitable sample to be used in vitro culture, the selection standard of sample are as follows: primary lung cancer, pathological staging are II phase or III Phase, pathological are non-small cell lung cancer or Small Cell Lung Cancer, and lung cancer specimen weight is more than the sample of 20mg.

3, gender, age, medical history, family history, smoking history, pathological staging parting, the clinic that attending physician provides patient are examined The basic clinical information such as disconnected.The information relevant to patient privacy such as name, identification card number of patient is concealed, is compiled with unified experiment Number replace, the nomenclature principles of experiment numbers be collecting sample eight-digit number word date+patient's admission number after four.Such as 2018 The sample that January 1 provided, patient's admission number are T001512765, then sample experiment numbers are 201801012765.

4, fresh specimens are acquired in operating room gnotobasis by surgeon in art, is placed in preprepared sample It saves in liquid (see embodiment 1).It is kept on ice after sample is in vitro, is transported in two hours to laboratory and carry out next step operation.

Embodiment 3, cancerous lung tissue sample dissociate pre-treatment

Operations described below needs to operate on ice, and whole operation step needs are completed in 10 minutes.

The operating equipment used in operations described below is both needed to prior autoclave sterilization, could use after drying.

1, sample is weighed.

2, sample surface is cleaned 10 to 30 seconds with 75% (volumn concentration) ethyl alcohol.

3, it is cleaned sample five times with sample cleaning solution, is cleaned sample 5 times with sterile PBS solution.

4, with equipment such as eye scissors, ophthalmic tweezers, scalpels, carefully by the adipose tissue in sample, connective tissue, downright bad group Knit removing.

Embodiment 4, the dissociation of cancerous lung tissue sample

The operating equipment used in following embodiments is both needed to prior autoclave sterilization, could use after drying.

1, tissue shear is broken into 1mm with eye scissors³The fritter of left and right.

2, by the dosage of the every mg tissue of 0.1mL sample dissociation solution (see embodiment 1), the sample preheated with prior 37 DEG C is dissociated The tissue samples that liquid processing shreds, progress sample dissociation under the conditions of 37 DEG C, Dissociation time 15 minutes to 3 hours.Every 15 minutes The dissociation situation for observing sample under the microscope, until observing a large amount of individual cells.

3, dissociation reaction is terminated with the digestion terminate liquid (see embodiment 1) of 10 times of volumes, collects cell suspension.

4, with 100 μm of steril cell strainer filtering cell suspensions, tissue relic and adhesion cells are removed.

5,800g room temperature is centrifuged 10 minutes, is discarded supernatant.

6, cell is resuspended with the sterile PBS of 5mL, 800g room temperature is centrifuged 10 minutes, discards supernatant.

7, cell precipitation is resuspended with lung cancer solid tumor primitive cell culture base (see embodiment 1), observation is thin under the microscope Born of the same parents' state carries out cell count.

As shown in Figure 1, also mixing a large amount of various types other than tumour cell in the single cell suspension that dissociation obtains Other cells, such as red blood cell, lymphocyte, fibrocyte etc..One of advantage of this method is exactly to cultivate subsequent Cheng Zhong, only cancer cell can carry out massive amplification, and the ratio of other cells is gradually decreased and even disappeared, and finally obtains purity Higher lung cancer primary tumor cell.

Embodiment 5, lung cancer cell culture

1, lung cancer cell suspension culture is carried out using low adsorption surface (low-attachment-surface), it is used Culture medium is the lung cancer solid tumor primitive cell culture base in embodiment 1, by taking six orifice plates as an example, by every hole 10⁶A cell Density bed board, 37 DEG C, 5%CO₂Under the conditions of cultivated in cell incubator.

2, cell state, every 3 days one subcultures of replacement are observed daily, until cell forms 100 μm of diameter or so of group Block.

As shown in Fig. 2, cancer cell massive amplification formed the cell mass of 100 μm of sizes of diameter by culture in 3-10 days, Tumour cell total quantity can be more than 10⁷, other kinds of cell quantity, which significantly reduces, even to disappear.This method passes through a large amount of samples This test, lung cancer primary tumor cell in vitro culture success rate can achieve 70%.

Embodiment 6, lung cancer cell passage

1, the cell mass in culture dish is collected, 800g room temperature is centrifuged 10 minutes, discards supernatant.

2, cell mass is cleaned with sterile PBS solution, 800g room temperature is centrifuged 10 minutes, discards supernatant.

3, cell mass is resuspended with cell dissociation buffer (see embodiment 1), is digested under the conditions of 37 DEG C.Every 5 minutes The case where microscopically observation cell mass digests, until cell mass is all digested as individual cells.

4, dissociation reaction is terminated with the digestion terminate liquid (see embodiment 1) of 10 times of volumes, collects cell suspension.

5,800g room temperature is centrifuged 10 minutes, is discarded supernatant.

6, cell precipitation, cell count are hanged with lung cancer solid tumor primitive cell culture base weight.

7, lung cancer cell culture, culture used are carried out using low adsorption surface (low-attachment-surface) Base is the lung cancer solid tumor primitive cell culture base in embodiment 1, by taking six orifice plates as an example, by every hole 10⁶The density of a cell Bed board, 37 DEG C, 5%CO₂Under the conditions of cultivated in cell incubator.

Embodiment 7, lung cancer cell freeze

The lung cancer cell cultivated suspend after 2-3 passage amplification, can be frozen:

3, cell mass is resuspended with cell dissociation buffer (see embodiment 1), is digested under the conditions of 37 DEG C.Every 15 minutes The case where microscopically observation cell mass digests, until cell mass is all digested as individual cells.

4, dissociation reaction is terminated with the digestion terminate liquid (see embodiment 1) of 10 times of volumes, collects cell suspension, cytometer Number.

5,800g room temperature is centrifuged 10 minutes, is discarded supernatant.

6, with cells frozen storing liquid (see embodiment 1), by 10⁶Cell precipitation, the every pipe 1mL of 2mL cryopreservation tube is resuspended in the density of/mL Cell suspension, gradient cooling box are transferred in liquid nitrogen after being frozen overnight and save for a long time.

The recovery of embodiment 8, lung cancer cell

The lung cancer cell saved in liquid nitrogen can recover:

1, shift to an earlier date the 37 DEG C of sterile waters of preparation in five minutes.

2, cryopreservation tube is removed from liquid nitrogen, melts cell rapidly in 37 DEG C of sterile waters.

3,800g room temperature is centrifuged 10 minutes, is discarded supernatant.

4, with lung cancer solid tumor primitive cell culture base (see embodiment 1) be resuspended cell precipitation, using low adsorption surface into Row lung cancer cell culture, every solencyte are recovered into 3.5cm culture dish, and 37 DEG C, 5%CO₂Under the conditions of in cell incubator In cultivated.

The HE dyeing identification of embodiment 9, lung cancer cell

The reagent consumptive material explanation used in following embodiments:

HE staining kit (Beijing Suo Laibao Biotechnology Co., Ltd, #G1120)；

Cationic anticreep slide (Beijing Biotechnology Co., Ltd, Zhong Shan Golden Bridge)；

Dimethylbenzene, methanol, acetone (Beijing chemical reagents corporation analyzes pure)；

Resinene glue (Beijing Yili Fine Chemicals Co., Ltd.).

1, concentration is made in suspension cell is 10⁴The cell suspension of/mL, is added dropwise 10 μ L on cationic anticreep slide, from So dry.

2,50 μ L are carefully added dropwise on air-dried cell through 4 DEG C of precooled methanol/acetone mixed liquors (volume ratio 1:1), Then slide is put into the fixed 10mins of 4 DEG C of refrigerators.

3, the slide of fixed cell, room temperature naturally dry are taken out.

4, slide is cleaned twice with 200 μ L PBS.

5,100 μ L hematoxylin dye liquors dyeing 1mins is added when moisture on slide is micro- dry.

6, hematoxylin dye liquor is sucked, is cleaned slide 3 times with 200 μ L tap water.

7,100 μ L differentiation liquid is added dropwise and breaks up 1mins.

8, differentiation liquid is sucked, is successively cleaned slide 2 times with tap water, distilled water cleans slide 1 time.

9, surface of glass slide moisture is sucked, 200 μ L eosin stains are added dropwise and dye 40s.

10, suck eosin stain, successively with 75%, 80%, 90%, 100% ethyl alcohol rinse dehydration 20s, 20s, 40s, 40s。

Etc. 11, after ethyl alcohol dry, 50 μ L dimethylbenzene is added dropwise and carry out cell-permeant.

Etc. 12, after dimethylbenzene dry completely, a drop resinene glue is added dropwise and is observed under the microscope with coverslip mounting And it takes pictures.

Fig. 3 illustrates the lung cancer primary tumor cell HE dyeing effect figure that in vitro culture obtains, it can be seen that these cells Generally there is nucleocytoplasmic ratio height, nuclear hyperchromatism, the cancer cells feature such as chromatic agglutination, multicore, cell size be inhomogenous in core.

The immunofluorescence dyeing identification of embodiment 10, lung cancer cell

The reagent explanation used in following embodiments:

Paraformaldehyde (Beijing chemical reagents corporation analyzes pure), dissolves paraformaldehyde powder with ultrapure water, is made 4% (4g/100mL) paraformaldehyde solution；

Methanol, dimethyl sulfoxide (Beijing chemical reagents corporation analyzes pure)；

Hydrogen peroxide (Beijing chemical reagents corporation, 35%)；

Methanol, dimethyl sulfoxide, 35% hydrogen peroxide are mixed and made into Dan Shi rinsing liquid according to the ratio of 4:4:1 (volume ratio)；

Bovine serum albumin(BSA) (Sigma, #A1933) dissolves bovine serum albumin(BSA) with PBS solution, 3% (3g/ is made BSA solution 100mL)；

One antiantibody of immunofluorescence (Abcam, #ab17139)；

Two antiantibody of immunofluorescence (CST, #4408)；

Hoechst dye liquor (Beijing Suo Laibao Biotechnology Co., Ltd, #C0021)；

Immunofluorescence dyeing is carried out to lung carcinoma cell agglomerate according to the following steps, primary antibody CK8+CK18 characterizes epithelial origin Cell.

1, collect culture dish in cell mass, after clean one time with PBS, with 4% paraformaldehyde resuspension cell precipitation, 4 It is DEG C fixed overnight.

2,800g centrifugation discards supernatant, and cell precipitation is resuspended with the methanol solution of pre-cooling, places 1 hour on ice.

3,800g centrifugation discards supernatant, and cell precipitation is resuspended in Dan Shi rinsing liquid, is placed at room temperature for 2 hours.

4,800g centrifugation discards supernatant, successively with 75%, 50%, 25% (volumn concentration) the diluted methanol of PBS Solution cleaning cell, 10 minutes every time.

5,800g centrifugation discards supernatant, and with 3%BSA solution suspension cell precipitation, room temperature is closed 2 hours.

6, in the ratio of 1:500, primary antibody is diluted with 3%BSA solution, and be resuspended carefully with antibody diluent (3%BSA solution) Born of the same parents' precipitating, 4 DEG C of primary antibodies are stayed overnight.

7,800g centrifugation discards supernatant, and is cleaned cell precipitation 5 times, every time 20 minutes with PBS solution.

8, in the ratio of 1:2000, secondary antibody is diluted with 3%BSA solution, and be resuspended with antibody diluent (3%BSA solution) Cell precipitation, room temperature secondary antibody 2 hours.

9,800g centrifugation discards supernatant, and is cleaned cell precipitation 5 times, every time 20 minutes with PBS solution.

10,100 × Hoechst dye liquor is added by 1/100 volume ratio, room temperature dyes 20 minutes.

11, it is cleaned cell precipitation 2 times, every time 10 minutes with PBS solution.Use confocal laser scanning microscope cell mass The staining conditions of block.

Fig. 4 illustrates the effect picture of the lung cancer primary tumor cell agglomerate immunofluorescence dyeing of in vitro culture, it can be seen that The cell of composition cell mass is all the CK8/CK18 positive, is epithelial origin, it was confirmed that this method culture obtained is purity Higher tumour cell.Immunofluorescence dyeing identification is carried out to 20 lung cancer sample primary cultures, statistical result showed is through this In the lung cancer cell that method obtains, the ratio of tumour cell reaches 75%-95% (table 17).

The identification of 17 lung cancer sample primary culture immunofluorescence dyeing of table

Embodiment 11, lung cancer cell culture and primary tumor tissue

The DNA referred in following embodiments extracts process using Tiangeng blood/tissue/cellular genome extracts kit (DP304) it carries out.

The Library development flow referred in following embodiments builds library kit (E7645) progress using NEB DNA sequencing.

The high-flux sequence referred in following embodiments refers to Illumina HiSeq X-ten microarray dataset.

1, lung cancer solid tumor sample is obtained, before carrying out in vitro culture operation, lung cancer solid tumor sample 10mg is first taken to carry out DNA is extracted, and builds library and full-length genome high-flux sequence (WGS), and sequencing depth 300 ×, remaining solid tumor sample is former for lung cancer For cell injuring model.

2, it forms 100 μm of diameter or more of cell mass through culture after a period of time after cancerous lung tissue processing and is denoted as P0 For cell, P1, P2 ..., Pn are successively denoted as by the number of passage later.From the lung cancer primary tumor cell in P1, P2, P3, P4 generation 10 are respectively taken in culture⁶A cell carries out DNA extraction, builds library and full-length genome high-flux sequence (WGS), and depth 300 is sequenced ×。

3, each group sequencing result carries out copy number analysis of variance (CNV) respectively, more primary lung cancer tumor tissue and each generation Copy number variation between lung cancer cell culture, as shown in figure 5, respectively for lung cancer cell culture (P1, P2, P3, P4) consistent with the copy number variation situation height of primary lung cancer tumor tissue (Tumor), therefore the lung cancer obtained through this method is former The truth of patient's primary tumo(u)r can be represented for cell.

Embodiment 12, different primitive cell culture base culture success ratios compare

The operating method process of all sample originally cultures is completely the same (with reference to the foregoing) in the present embodiment, only trains Support based formulas different from.The various primitive cell culture bases tested are shown in Table 18.Wherein used in the scheme D present invention Formula, is specifically shown in Table 8.

Primitive cell culture based formulas (100 mL) is used in the test of table 18

After the completion of primitive cell culture basigamy system, filtered with 0.22 μM of syringe filter (Millipore SLGP033RS) Degerming can save two weeks at 4 DEG C.

Four kinds of primitive cell culture base schemes respectively handle 20 samples, carry out sample by method described in embodiment 3,4,5 Processing and culture operation, statistics lung cancer solid tumor primitive cell culture success rate is as shown in table 19 after culture 10 days:

19 different culture medium culture situation of table

It can be seen that primitive cell culture base influences greatly the culture success ratio of lung cancer cell, the present invention is used Lung cancer solid tumor primitive cell culture base (table 8) cancer cell can be stimulated to increase to the greatest extent in lung cancer solid tumor mass sample It grows, improves the success rate of lung cancer solid tumor primitive cell culture.

Embodiment 13, different Sample preservation liquid culture success ratios compare

The operating method process of all sample originally cultures is completely the same (with reference to the foregoing) in the present embodiment, only sample This preservation formula of liquid different from.The various Sample preservation liquid tested are shown in Table 20.Wherein used in the scheme E present invention Formula, is specifically shown in Table 1.

Sample preservation formula of liquid (100mL) is used in the test of table 20

After the completion of various Sample preservation liquid are prepared in upper table, dispensed with 15mL centrifuge tube, every pipe 5mL.It can after packing It is saved 1 month in 4 DEG C.

Five kinds of Sample preservation liquid schemes respectively handle 20 samples, after sample is in vitro in Sample preservation liquid 4 DEG C it is temporary, in vitro After 2 hours, sample process is carried out by method described in embodiment 3,4,5 and culture operates, count lung cancer entity after culture 10 days Tumor primitive cell culture success rate such as table 21:

The different Sample preservation liquid culture situations of table 21

It can be seen that Sample preservation formula of liquid has large effect to the success rate of lung cancer solid tumor primitive cell culture, The Sample preservation liquid (table 1) that the present invention uses can protect the work of cancer cell in lung cancer solid tumor mass sample to the greatest extent Property, improve the success rate of culture.

Embodiment 14, different sample dissociation solution culture success ratios compare

The operating method process of all sample originally cultures is completely the same (with reference to the foregoing) in the present embodiment, only sample This dissociation formula of liquid different from.The various sample dissociation solutions tested are shown in Table 22.Wherein used in the scheme D present invention Formula, is specifically shown in Table 3.

22 test of table dissociates formula of liquid (10mL) with sample

Sample dissociation solution is ready-to-use.

The sample that 20 lung cancer solid tumor mass block weight are more than 100mg is chosen, four parts are divided into, respectively with above-mentioned four Kind sample dissociation solution carries out sample process by method described in embodiment 3,4,5 and culture operates.Culture counted lung cancer after 10 days Solid tumor primitive cell culture success rate such as the following table 23:

The different sample dissociation solution culture situations of table 23

It can be seen that sample dissociation formula of liquid has a great impact to the success rate of lung cancer solid tumor primitive cell culture, The sample dissociation solution (table 3) that the present invention uses can utmostly separate the cancer cell in lung cancer solid tumor mass, improve lung cancer The success rate of solid tumor primitive cell culture.

Embodiment 15, different cell dissociation buffer passage success rates compare

All sample primary cell passage operating method processes are completely the same (with reference to the foregoing) in the present embodiment, only Cell dissociation formula of liquid different from.The various sample dissociation solutions tested are shown in Table 24.Wherein scheme D is to use in the present invention Formula, be specifically shown in Table 6.

Cell dissociation formula of liquid (10mL) is used in the test of table 24

Cell dissociation buffer is ready-to-use.

Choose 20 successful lung cancer samples of culture, the lung cancer solid tumor primary cell that culture is obtained, respectively with above-mentioned Four kinds of cell dissociation buffers carry out continuous passage operation by method described in embodiment 6.100 μ of diameter is formed whenever cancer cell expands It is just passed on and (is no more than 10 times) when the cell mass of m size, record maximum passage number.Statistical result such as table 25:

The different cell dissociation buffer culture situations of table 25

It can be seen that the success rate that cell dissociation formula of liquid passes on lung cancer solid tumor primary cell has a great impact, The cell dissociation buffer (table 6) that the present invention uses can cancer cell in mild dissociated cell agglomerate, carry out sample continuously It passes on and keeps lung cancer solid tumor primary cell active.

Claims

1. a kind of method for cultivating lung cancer solid tumor primary cell, includes the following steps:

The sample dissociation solution is made of clostridiopetidase A I, clostridiopetidase A IV and PBS；Wherein, the clostridiopetidase A I is dissociated in the sample Final concentration of 150-250U/mL in liquid；Final concentration of 150-250U/ of the clostridiopetidase A IV in the sample dissociation solution mL；Surplus is PBS；

(2) using lung cancer solid tumor primitive cell culture base suspension incubation step (1) dissociate come lung cancer solid tumor it is primary thin Born of the same parents；

The lung cancer solid tumor primitive cell culture base is by dual anti-P/S, HEPES, nonessential amino acid solution, GlutaMax, people Recombinant protein EGF, human recombination protein bFGF, human recombination protein MSP, cortisol, B27, ITS-X, Y-27632 and Advanced DMEM/F12 culture medium composition；Wherein, the penicillin in the dual anti-P/S is in the lung cancer solid tumor primitive cell culture base Final concentration of 100-200U/mL；Streptomysin in the dual anti-P/S is in the lung cancer solid tumor primitive cell culture base Final concentration of 100-200 μ g/mL；Final concentration of 8- of the HEPES in the lung cancer solid tumor primitive cell culture base 12mM；Final concentration of 0.8-1.2% of the nonessential amino acid solution in the lung cancer solid tumor primitive cell culture base (volumn concentration)；Final concentration of 0.8- of the GlutaMax in the lung cancer solid tumor primitive cell culture base 1.2% (volumn concentration)；Final concentration of the human recombination protein EGF in the lung cancer solid tumor primitive cell culture base For 10-100ng/mL；Final concentration of 10- of the human recombination protein bFGF in the lung cancer solid tumor primitive cell culture base 50ng/mL；Final concentration of 5-25ng/mL of the human recombination protein MSP in the lung cancer solid tumor primitive cell culture base； Final concentration of 20-50ng/mL of the cortisol in the lung cancer solid tumor primitive cell culture base；The B27 is described Final concentration of 1.5-2.5% (volumn concentration) in lung cancer solid tumor primitive cell culture base；The ITS-X is in the lung Final concentration of 0.8-1.2% (volumn concentration) in cancer solid tumor primitive cell culture base；The Y-27632 is in the lung Final concentration of 5-20 μM in cancer solid tumor primitive cell culture base；Surplus is Advanced DMEM/F12 culture medium.

2. according to the method described in claim 1, it is characterized by: in step (1) being used according to the method included the following steps The sample dissociation solution dissociates the lung cancer solid tumor mass: by the every mg group of sample dissociation solution described in 0.1-0.3mL The dosage knitted, the sample dissociation solution that the lung cancer solid tumor mass after shredding is preheated with prior 37 DEG C are handled, Progress sample dissociation under the conditions of 37 DEG C, Dissociation time 15 minutes to 3 hours.

3. method according to claim 1 or 2, it is characterised in that: be according to the side included the following steps in step (2) Method is suspended with the lung cancer solid tumor primitive cell culture base and cultivates the lung cancer solid tumor primary cell: using with low suction The culture vessel in subordinate list face, being suspended using the lung cancer solid tumor primitive cell culture base, it is primary thin to cultivate the lung cancer solid tumor Born of the same parents, 37 DEG C, 5%CO₂Under the conditions of cultivated, every 2-4 days one subcultures of replacement.

4. method according to claim 1 to 3, it is characterised in that: further include as follows to institute before step (1) It states the step of lung cancer solid tumor mass carries out dissociation pre-treatment: cleaning lung cancer reality with the ethyl alcohol that volumn concentration is 70-75% Body tumor tissue sample surface；The lung cancer solid tumor mass sample is successively cleaned with sample cleaning solution and sterile PBS solution；

Specifically, the sample cleaning solution is made of dual anti-P/S and PBS；Wherein, the penicillin in the dual anti-P/S is described Final concentration of 100-200U/mL in sample cleaning solution；End of the streptomysin in the sample cleaning solution in the dual anti-P/S Concentration is 100-200 μ g/mL；Surplus is PBS.

5. according to the method described in claim 4, it is characterized by: carrying out the lung cancer solid tumor group of the dissociation pre-treatment The isolated time for knitting sample is within 2 hours, and is stored in always in Sample preservation liquid before handling before carrying out the dissociation；

Specifically, the Sample preservation liquid is made of fetal calf serum, dual anti-P/S, HEPES and HBSS；Wherein, the fetal calf serum Final concentration of 1-5% (volumn concentration) in the Sample preservation liquid；Penicillin in the dual anti-P/S is in the sample Final concentration of 100-200U/mL in this preservation liquid；End of the streptomysin in the Sample preservation liquid in the dual anti-P/S is dense Degree is 100-200 μ g/mL；Final concentration of 8-12mM of the HEPES in the Sample preservation liquid；Surplus is HBSS.

6. any method in -5 according to claim 1, it is characterised in that: in step (1), with the sample dissociation solution Further include following steps after carrying out dissociation processing to the lung cancer solid tumor mass: terminating dissociation reaction with digestion terminate liquid, receive Collect cell suspension；The cell suspension is filtered, tissue relic and adhesion cells are removed；Cell is resuspended with sterile PBS after centrifugation；Again Then centrifugation hangs cell precipitation with the lung cancer solid tumor primitive cell culture base weight；

Specifically, the digestion terminate liquid is made of fetal calf serum, dual anti-P/S and DMEM culture medium；Wherein, the fetal calf serum Final concentration of 8-12% (volumn concentration) in the digestion terminate liquid；Penicillin in the dual anti-P/S is described Digest the final concentration of 100-200U/mL in terminate liquid；End of the streptomysin in the digestion terminate liquid in the dual anti-P/S Concentration is 100-200 μ g/mL；Surplus is DMEM culture medium.

7. any method in -6 according to claim 1, it is characterised in that: further include following steps in step (2): When the lung cancer solid tumor primary cell forms 80-120 μm of diameter of agglomerate, the lung cancer solid tumor primary cell is carried out Passage；

Specifically, it is as follows to carry out the cell dissociation buffer used when the passage composition: containing in cell dissociation buffer described in every 10mL 4-6mL Accutase, the EDTA of final concentration of 5mM, 1.5-2.5mL TrypLE Express, surplus PBS；And/or

The digestion terminate liquid used when the passage is carried out as the digestion terminate liquid described in claim 6；

And/or

The method also includes to by 2-3 times passage amplification after the lung cancer solid tumor primary cell frozen and/or The step of recovery；

The cells frozen storing liquid used when specifically, freezing described in carrying out is by Advanced DMEM/F12 culture medium, DMSO and 1% Methocel solution composition；Wherein, the Advanced DMEM/F12 culture medium, the DMSO and 1% Methyl cellulose The volume proportion of plain solution is 20:2:(0.8-1.2)；1% methocel solution is the methyl that concentration is 1g/100ml Cellulose aqueous solution.

8. it is a kind of for cultivating the reagent set of lung cancer solid tumor primary cell, be following any:

(A) by claim 1-7 it is any described in sample dissociation solution and the lung cancer solid tumor primitive cell culture base form；

(B) as claim 1-7 it is any described in sample dissociation solution, the lung cancer solid tumor primitive cell culture base and following examination At least one of agent composition: Sample preservation liquid described in claim 1-7 is any, the cell dissociation buffer, sample cleaning Liquid, the digestion terminate liquid and the cells frozen storing liquid.

9. application of the reagent set according to any one of claims 8 in culture lung cancer solid tumor primary cell.

10. any method or reagent set or application in -9 according to claim 1, it is characterised in that: the lung cancer is Primary lung cancer, pathological staging are II phase or III phase, and pathological is non-small cell lung cancer or Small Cell Lung Cancer.