CN110447633A

CN110447633A - A kind of lung cancer solid tumor mass Sample preservation liquid

Info

Publication number: CN110447633A
Application number: CN201810427138.8A
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 lung cancer solid tumor mass Sample preservation liquid.Sample preservation liquid of the invention is made of fetal calf serum, dual anti-P/S, HEPES and HBSS；Wherein, the final concentration of 1-5% (volumn concentration) of the fetal calf serum；The final concentration of 100-200U/mL of penicillin in the dual anti-P/S；The final concentration of 100-200 μ g/mL of streptomysin in the dual anti-P/S；The final concentration of 8-12mM of the HEPES；Surplus is HBSS.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 and Sample preservation liquid provided by the present invention are with a wide range of applications in the research of lung cancer and clinical conditions field.

Description

A kind of lung cancer solid tumor mass Sample preservation liquid

Technical field

The present invention relates to field of biotechnology, and in particular to a kind of lung cancer solid tumor mass Sample preservation liquid.

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.

Separation of the fresh tumor tissue sample taken in art since primary cell cannot be carried out at once, good sample Liquid is saved for there is sample to maintain in the short time activity of cell in sample most important after in vitro, it may be said that this be influence it is primary Tumour cell separation and an important link in culture effect.

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 Art and matched reagent, present invention employs a kind of new Sample preservation liquid, can maintain sample in sample in vitro rear short time The activity of middle cell.

In a first aspect, a kind of claimed lung cancer solid tumor mass Sample preservation liquid.

Lung cancer solid tumor mass Sample preservation liquid provided by the present invention is by fetal calf serum, dual anti-P/S, HEPES and HBSS (Hank's balanced salt solution) composition.

Wherein, (such as 2%, % indicates volume hundred to final concentration of 1-5% of the fetal calf serum in the Sample preservation liquid Divide content)；Final concentration of 100-200U/mL (such as 100U/ of the penicillin in the Sample preservation liquid in the dual anti-P/S mL)；Final concentration of 100-200 μ g/mL (such as 100 μ g/s of the streptomysin in the Sample preservation liquid in the dual anti-P/S mL)；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.

The Sample preservation liquid existence form can be two kinds:

First, the Sample preservation liquid is by the fetal calf serum, the dual anti-P/S, the HEPES and the HBSS The solution that (Hank's balanced salt solution) mixes.

It can be reserved for 1 month for 4 DEG C after the Sample preservation liquid prepares.

Second, each component individualism in the Sample preservation liquid, when use, is prepared according to formula.

Second aspect, the claimed Sample preservation liquid are saving the application in lung cancer solid tumor mass.

The third aspect, a kind of claimed method for saving lung cancer solid tumor mass.

The method of the preservation lung cancer solid tumor mass of institute's body of the present invention, specifically may include following steps: will be just in vitro Lung cancer solid tumor mass (such as the fresh lung cancer solid tumor mass just taken out in surgical procedure from patient's body) be placed in institute above It is saved in the Sample preservation liquid stated, the holding time is within 2 hours.

Fourth aspect, claimed one kind dissociate lung cancer solid tumor primary cell from lung cancer solid tumor mass Method.

The method provided by the present invention that lung cancer solid tumor primary cell is dissociateed from lung cancer solid tumor mass, specifically may be used Include the following steps:

(1) just in vitro lung cancer solid tumor mass is placed in previously described Sample preservation liquid and is saved, within 2 hours Dissociation pre-treatment is carried out to the lung cancer solid tumor mass in accordance with the following steps: with volumn concentration be 70-75% (such as 75%) ethyl alcohol cleans lung cancer solid tumor mass sample surface 10 to 30 seconds；The lung cancer solid tumor is cleaned with sample cleaning solution Tissue samples 5-10 times (such as 5 times), the lung cancer solid tumor mass sample is cleaned 5-10 times (such as 5 times) with sterile PBS solution； Then it is primary thin to remove the influence such as impurity, connective tissue, adipose tissue, necrotic tissue in the lung cancer solid tumor mass sample The ingredient of born of the same parents' culture.

(2) lung is obtained to step (1) treated the lung cancer solid tumor mass carries out dissociation processing with sample dissociation solution Cancer solid tumor primary cell.

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

Clostridiopetidase A I and clostridiopetidase A IV therein can -20 DEG C of long-term preservations, concretely 10 times of storages in the form of liquid storage (mother liquor) Liquid (mother liquor).10 × clostridiopetidase A I liquid storage is made of the clostridiopetidase A I and PBS；Wherein the clostridiopetidase A I's is final concentration of 2000U/mL；Surplus is PBS.10 × clostridiopetidase A IV liquid storage is made of the clostridiopetidase A IV and PBS；The wherein clostridiopetidase A IV Final concentration of 2000U/mL；Surplus is PBS.The definition of the enzyme activity of the clostridiopetidase A I and the clostridiopetidase A IV sees above.

Further, the sample cleaning solution is made of dual anti-P/S (Pen .- Strep) and PBS；Wherein, described double Final concentration of 100-200U/mL (such as 100U/mL) of the penicillin in the sample cleaning solution in anti-P/S；The dual anti-P/S In 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.

Wherein, the step of carrying out dissociation pre-treatment to the lung cancer solid tumor mass needs to operate on ice, whole operation Step needs are completed in 10 minutes.

Further, being dissociated using the sample dissociation solution to the lung cancer solid tumor mass can be according to including as follows The method of step carries out: by the dosage of the every mg tissue of 0.1-0.3mL (such as 0.1mL) described sample dissociation solution, the institute after shredding It states lung cancer solid tumor mass and (is such as cut into 0.8-1.2mm³Fritter) with prior 37 DEG C preheat the sample dissociation solution at Reason, progress sample dissociation under the conditions of 37 DEG C, Dissociation time 15 minutes to 3 hours.Sample is observed under the microscope within every 15 minutes Dissociation situation, until observing a large amount of individual cells.

Further, may also include after carrying out dissociation processing to the lung cancer solid tumor mass with the sample dissociation solution Following steps: dissociation reaction is terminated with the digestion terminate liquid of 8-15 times of (such as 10 times) volume, collects cell suspension；With 100 μm or Cell suspension described in 40 μm of steril cell strainer filterings removes tissue relic and adhesion cells；800-1000g (such as 800g) room temperature 10-15 minutes (such as 10 minutes) are centrifuged, are discarded supernatant；Cell is resuspended with 3-5mL (such as 5mL) sterile PBS afterwards；800-1000g again (such as 800g) room temperature is centrifuged 10-15 minutes (such as 10 minutes), discards supernatant.

Wherein, the digestion terminate liquid (can be saved one month at 4 DEG C after preparing) by fetal calf serum, dual anti-P/S and DMEM culture medium composition；Wherein, final concentration of 8-12% (such as 10%, % of the fetal calf serum in the digestion terminate liquid Indicate volumn concentration)；Final concentration of 100-200U/ of the penicillin in the digestion terminate liquid in the dual anti-P/S ML (such as 100U/mL)；Final concentration of 100-200 μ g/mL of the streptomysin in the digestion terminate liquid in the dual anti-P/S (such as 100 μ g/mL)；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.

5th aspect, it is claimed a kind of primary for dissociateing lung cancer solid tumor from lung cancer solid tumor mass The reagent set of cell.

The complete examination provided by the present invention for being used to dissociate lung cancer solid tumor primary cell from lung cancer solid tumor mass Agent contains at least one of previously described Sample preservation liquid, the sample dissociation solution and following reagent: previously described sample This cleaning solution and the digestion terminate liquid.

6th aspect, claimed previously described reagent set are dissociateing lung from lung cancer solid tumor mass Application in cancer solid tumor primary cell.

In first aspect into the 6th aspect, concretely primary lung cancer, pathological staging are II phase or III to the lung cancer 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.

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 present invention provides a kind of to extract the side for cultivating lung cancer solid tumor primary cell from fresh lung cancer solid tumor mass Method and matched reagent, present invention employs a kind of new Sample preservation liquid, can maintain sample in sample in vitro rear short time The activity of middle cell, using Sample preservation liquid provided by the present invention and combine the method for the present invention, can reach it is following the utility model has the advantages 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 this cultural method and sample provided by the present invention This preservation liquid is with a wide range of applications in the research of lung cancer and clinical conditions field.

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 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 18.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 18

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 19:

The different Sample preservation liquid culture situations of table 19

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.

Claims

1. a kind of lung cancer solid tumor mass Sample preservation liquid, it is characterised in that: the Sample preservation liquid is by fetal calf serum, dual anti-P/ S, HEPES and HBSS composition；Wherein, final concentration of 1-5% (volume basis of the fetal calf serum in the Sample preservation liquid Content)；Final concentration of 100-200U/mL of the penicillin in the Sample preservation liquid in the dual anti-P/S；The dual anti-P/ Final concentration of 100-200 μ g/mL of the streptomysin in the Sample preservation liquid in S；The HEPES is in the Sample preservation liquid In final concentration of 8-12mM；Surplus is HBSS.

2. Sample preservation liquid described in claim 1 is saving the application in lung cancer solid tumor mass.

3. a kind of method for saving lung cancer solid tumor mass, includes the following steps: to set just in vitro lung cancer solid tumor mass It is saved in Sample preservation liquid described in claim 1, the holding time is within 2 hours.

4. a kind of method for dissociateing lung cancer solid tumor primary cell from lung cancer solid tumor mass, includes the following steps:

(1) just in vitro lung cancer solid tumor mass is placed in Sample preservation liquid described in claim 1 and is saved, 2 hours with Dissociation pre-treatment inside is carried out to the lung cancer solid tumor mass in accordance with the following steps: the second for being 70-75% with volumn concentration Alcohol cleans lung cancer solid tumor mass surface；The lung cancer solid tumor group is successively cleaned with sample cleaning solution and sterile PBS solution It knits；

(2) it is real that lung cancer is obtained to step (1) treated the lung cancer solid tumor mass carries out dissociation processing with sample dissociation solution Body tumor primary cell；

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.

5. according to the method described in claim 4, it is characterized by: the sample cleaning solution is made of dual anti-P/S and PBS；Its In, final concentration of 100-200U/mL of the penicillin in the sample cleaning solution in the dual anti-P/S；In the dual anti-P/S Final concentration of 100-200 μ g/mL of the streptomysin in the sample cleaning solution；Surplus is PBS.

6. method according to claim 4 or 5, it is characterised in that: using the sample dissociation solution to the lung cancer entity Tumor tissue, which carries out dissociation, to be carried out according to the method included the following steps: 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.

7. according to the method any in claim 4-6, it is characterised in that: with the sample dissociation solution to the lung cancer Solid tumor mass further includes following steps after carrying out dissociation processing: terminating dissociation reaction with digestion terminate liquid, collects cell suspension； The cell suspension is filtered, tissue relic and adhesion cells are removed；

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.

8. it is a kind of for dissociateing the reagent set of lung cancer solid tumor primary cell from lung cancer solid tumor mass, it is wanted containing having the right Ask Sample preservation liquid described in 1, at least one of sample dissociation solution and following reagent described in claim 4: right It is required that digestion terminate liquid described in sample cleaning solution described in 5 and claim 7.

9. reagent set according to any one of claims 8 in lung cancer solid tumor mass from dissociateing in lung cancer solid tumor primary cell Using.

10. any the Sample preservation liquid or method or reagent set or application, feature exist in -9 according to claim 1 In: the lung cancer is primary lung cancer, and pathological staging is II phase or III phase, and pathological is non-small cell lung cancer or cellule Lung cancer.