CN108913662B - A kind of method of the functional high-throughput medication screening of lung cancer - Google Patents
A kind of method of the functional high-throughput medication screening of lung cancer Download PDFInfo
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Abstract
It is different from previous methods the present invention provides a kind of method of the functional high-throughput medication screening of lung cancer, accurate individualized treatment can be provided for cancer patient and select medicine.The survival rate of tumour cell can be improved by the concentration of gentamicin and cholera mycin in control tumor cell culture liquid in the present invention, and cellular morphology is effectively maintained to promote absorption of the tumour cell to nutriment.Pass through the concentration of insulin and the EGF in control tumor cell culture liquid, the growth rate of tumour cell can be improved, and improve cell activity, available a large amount of high activity tumour cell, the usage amount of fetal calf serum can be reduced using the insulin of the ratio and growth factor simultaneously, since fetal calf serum price is more expensive, usage amount reduction is conducive to economize on resources, and half-inhibitory concentration (IC50) error of the cell cultivated using the present invention for the medication of drug screening measurement is small, it is possible to reduce detection number.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of method of the functional high-throughput medication screening of lung cancer.
Background technique
Lung cancer is the highest tumour of Chinese's death rate, seriously threatens the health of compatriots.Lung cancer patients in China is in diagnosis
Most of (about 75%) belongs to advanced stage, cannot perform the operation.Primary treatment includes radiotherapy, chemotherapy, targeted drug treatment and
The comprehensive therapies such as immunization therapy.
The accurate individualized treatment of tumour proposed that, that is, according to tumour individual biological data, gene was prominent in recent years
The case where change, Tumor cell select optimal the integrated informations such as the function and effect of various tumour medicines for tumor patient
Therapeutic agent scheme is different from traditional empirical property therapeutic scheme.Because there are many traditional empirical treatment schemes, due to
Tumour is a kind of different substantiality disease, and the different patients of same tumour are very big to the difference on effect of same therapeutic scheme,
Each Conventional wisdom cancer immunotherapies effective percentage is widely different, efficient 20% to 40%, invalid therapeutic agent
Scheme cannot kill tumour cell, and have many side effects, especially inhibit the immunity of body, promote tumour cell
Diffusion.Genetic test has been achieved with good result in terms of lung cancer therapy selects targeted drug, but only 40% or so lung
Cancer patient can find targeted drug, and effective percentage can all generate drug resistance in 45%-75% or so, and at 1 year or so.ROHS
(Reprogramed organoid Based High-throughput screening reprograms organoid high-throughput drug
Screening, is to be separated the tumour cell of patient oneself from tumor tissues with specific process, is expanded in laboratory fidelity, raw
A kind of high-throughput Composition analyzed technology that drug screening is carried out at a large amount of minimal neoplastics, realizes that the accurate individuation of tumor patient is controlled
It treats, this technology is different from the technology of genetic test, and application range is more extensive.Pole is brought to improve the therapeutic effect of tumor patient
Hope.
It is maximum to population health and life threat as the deterioration lung cancer of environment is most fast as morbidity and mortality growth
One of malignant tumour, many countries all report that the morbidity and mortality of lung cancer obviously increase in recent decades, male's lung
Cancer morbidity and the death rate account for first of all malignant tumours, and women disease incidence accounts for second, and the death rate accounts for second.Lung
The cause of disease of cancer is not completely clear so far, and great mass of data shows that long-term a large amount of smokings have close relationship with lung cancer.?
Research have shown that: the probability that long-term a large amount of smokers suffer from lung cancer is 10-20 times of non-smoker, and the age for starting to smoke gets over
Small, the probability for suffering from lung cancer is higher.In addition, smoking not only directly affects my health, the also health to the crowd of surrounding
It has a adverse impact, involuntary smoker's lung cancer illness rate is caused to obviously increase.The disease incidence of city dweller's lung cancer is higher than rural area,
This may be related with carcinogenic substance is contained in urban atmospheric pollution and flue dust.Due to combinatorial chemistry appearance and and it is fast-developing, make
Must synthesize largely has the compound speed of potential clinical treatment function also to greatly improve, however assesses these medical compounds
Stability, and to various drugs to the treatment condition of cancer cell, is often used some intracorporal evaluation methods at present, but due to
Operating difficulty is big, somewhat expensive, and the application in terms of drug screening is restricted very much.
The screening of anti-tumor drug is very important link in anti-tumor drug research work, establishes reasonable screen body
System, finds new anti-tumor drug, is an important topic of tumour medicine research, and the screening technique of anti-tumor drug is various,
Generally it is divided into internal and external two class of method, every kind of method has its advantage and limitation, still lack effective technology at present
Means are for screening and identifying anti-tumor drug.Therefore, establish it is new it is quick, conveniently, accurately, high-throughput and efficient inspection
Survey method has great importance.
With the further investigation to apoptosis of tumor cells mechanism, drug-induced apoptosis of tumor cells has become current tumour and controls
One of important channel for the treatment of.High throughput screening drug is the new technology body for organically combining multiple technologies method and being formed
System, it is based on the test method of molecular level or cellular level, using microplate format as experimental tool carrier, with automation
Operating system executes experimentation, acquires experimental data with sensitive, quick detecting instrument, the number obtained with computer to experiment
According to being analyzed and processed, sample of the logarithm in terms of thousand, ten thousand is detected within the same time, and whole with the support of corresponding database
The normal operation of a technical system.
According to relevant report, phase early 1990s, a laboratory use conventional methods, and make by more than 20 drugs
With target position, 7.5 ten thousand samples of screening are only capable of in 1 year.And at the initial stage of the development of high flux screening in 1997, using more than 100 targets
Position, can screen 1,000,000 samples every year.Further perfect due to high flux screening by 1999, daily screening amount is just high
Up to 100,000 kinds of compounds.
High-flux medicaments sifting is segmented into four parts: medicaments sifting model, the sample library of high capacity, Automation workstation
With efficient data processing system, wherein medicaments sifting model is crucial, and the sample library of high capacity is the prerequisite of drug screening
Condition.
With the development of high-flux medicaments sifting, when drug screening can be accelerated by being applied to the screening of cancer drug
Between, manpower and material resources are reduced, and then mitigate the slight illness of patient, finds the drug that can effectively treat, such as China in a short time
Patent application 201510829192.1 discloses a kind of high-throughput screening method of anti-hydatid drugs, specifically discloses a kind of benefit
The high-throughput screening method of anti-hydatid drugs is carried out with the secondary Germinal Cells From Hidatid Cyst of Echinococcus Granulosus of mouse.The scheme of use is:
With Echinococcus Granulosus Cysts protoscolex infection experiment mouse, after establishing echinococcosis secondary infection model 8-10 months, dissect disease mouse is taken out
Its internal hydatidocystis separates germinal layer cell and establishes cell line.It adjusts the cell concentration and is carried out in 96 well culture plates
Bed board evaluates drug effectiveness using cell activity variation and cellular endogenous substance release situation after drug effect,
This invention greatly reduces the drug screening periods, and not only the dosage of drug to be measured is few, but also improve screening accuracy, reduce
Screening cost.
For another example, the foundation of lung-cancer medicament screening cell strain, the Shen are disclosed in Chinese patent application 201110245360.4
A kind of human lung carcinoma cell line please be disclose, the construction method and purposes of the cell strain, the human lung carcinoma cell line are also disclosed
Have the characteristics that high chromosomal integration degree, high fluorescent, stabilization characteristics of genetics and sensitive to lung-cancer medicament, is a kind of ideal
The model of lung-cancer medicament screening is studied, but this application does not specifically describe the selection of drug and therapeutic effect.
To sum up, due to today's society various chemical substances using and environmental pollution cause the disease incidence of cancer more next
Higher, the drug for how fast and effectively selecting treatment lung cancer, which improves therapeutic effect, becomes an important class of tumour medicine research
Topic.
Summary of the invention
In order to solve the above-mentioned technical problem, the present invention is intended to provide a kind of side of the functional high-throughput medication screening of lung cancer
Method includes the following steps:
(1) acquisition of lung carcinoma cell: from fresh surgical or sample acquisition lung cancer tumor tissue is punctured, digests tumor group
It knits, the lung carcinoma cell of isolated dispersion.
(2) fidelity amplification training the amplification cultivation of lung carcinoma cell: is carried out in laboratory to lung carcinoma cell obtained in step (1)
It supports, generates minimal neoplastic to get to lung cancer and reprogram organoid；
(3) by the reprogramming organoid dispersion of lung cancer obtained in step (2), 384 holes the adhere-wall culture of lung carcinoma cell: are inoculated with
Plate, adhere-wall culture obtain attached cell；
(4) drug screening: the attached cell prepared in step (3) is subjected to drug candidate processing, and detects cells survival
Rate carries out drug screening.
Digestion described in above-mentioned steps (1) is: with digestion enzymic digestion.The digestive ferment is by 225 units/mL clostridiopetidase A
Or hyaluronidase and 13 units/mL dispase form, then using is 4 times of volumes of lung cancer tumor tissue containing 10% small ox blood
Clear DMEM cell culture fluid stops digestion, collects digestive juice.
Culture described in above-mentioned steps (2) is: debulk tumor cell obtained in step (1) is placed in tumour cell training
In 37 DEG C of CO in nutrient solution2It is cultivated in incubator, culture obtains tumor cell line in 2-3 weeks；
The tumor cell culture liquid is that every 500mL contains following component:
Complete DMEM (fetal calf serum containing 6-10%) indicates in mentioned component: the volume hundred of fetal calf serum and complete DMEM
Divide than being 6-10%.
In a preferred embodiment, the concentration of the amphotericin B and hydrocortisone ratio is 9-12: 1；It is preferred that
For the concentration ratio of the amphotericin B and hydrocortisone is 9-11: 1；It is further preferably the amphotericin B and hydrogenation
The concentration ratio of cortisone is 9-10: 1.
Culture described in above-mentioned steps (3) is: by lung cancer obtained in step (2) reprogram organoid digestive ferment into
Row digestion, is then diluted with tumor-selective culture solution, adherent to be incubated overnight；The digestive ferment is 0.05% pancreas enzyme -EDTA.
The tumor-selective culture solution is that every 500mL contains following component:
In a further preferred embodiment, insulin described in the selective tumor cell culture liquid and turn iron
The concentration ratio of albumen is 1: 10-30.
1640 culture medium of RPMI (fetal calf serum containing 2-5%) indicates in mentioned component: fetal calf serum and RPMI 1640 are trained
The percent by volume for supporting base is 2-5%.
Specifically, the technical scheme is that a kind of method of the functional high-throughput medication screening of lung cancer, specific to grasp
Make step are as follows:
(1) acquisition of lung carcinoma cell: the lung carcinoma cell of acquisition is aseptically removed into its adipose tissue, uses digestive ferment
Digestion, the digestive ferment are made of 225 units/mL clostridiopetidase A or hyaluronidase and 13 units/mL dispase, then use
Stop digestion for the DMEM cell culture fluid containing 10% (percent by volume) calf serum of 4 times of volumes of lung cancer tumor tissue, receives
Collect digestive juice, precipitating is collected after centrifugation, the lung carcinoma cell dispersed；
(2) amplification cultivation of lung carcinoma cell: use tumor cell culture liquid by the lung carcinoma cell of the dispersion obtained in step (1)
Resuspension is deposited in laboratory and carries out fidelity amplification cultivation, is placed in culture bottle and is placed in 37 DEG C of CO2It is cultivated in incubator,
Culture obtains minimal neoplastic for 2-3 weeks and reprograms organoid to get to lung cancer；(3) adhere-wall culture of lung carcinoma cell: by step (2)
Middle obtained lung cancer reprogramming organoid of cultivating is digested with 0.05% pancreas enzyme -EDTA, and is carried out with automated cell calculating instrument
It counts, is inoculated with 384 orifice plates, then with adhere-wall culture after the dilution of tumor-selective culture solution overnight to get adherent lung carcinoma cell.
(4) drug screening: by the various classic chemotherapy drugs of lung carcinoma cell adherent obtained in step (3), medicine is targeted
The candidate drug-treated such as object or several drugs combination detected cells survival with ATP chemoluminescence method (Promeaga) after 72 hours
Rate carries out drug screening.
The tumor cell culture liquid is that every 500mL contains following component two
Complete DMEM (fetal calf serum containing 6-10%) indicates in mentioned component: the volume hundred of fetal calf serum and complete DMEM
Divide than being 6-10%.
In a preferred embodiment, the concentration of the amphotericin B and hydrocortisone ratio is 9-12: 1；It is preferred that
For the concentration ratio of the amphotericin B and hydrocortisone is 9-11: 1；It is further preferably the amphotericin B and hydrogenation
The concentration ratio of cortisone is 9-10: 1.
Tumor-selective culture solution described in above-mentioned steps (3) is that every 500mL contains following component:
In a further preferred embodiment, insulin described in the selective tumor cell culture liquid and turn iron
The concentration ratio of albumen is 1: 10-30.
1640 culture medium of RPMI (fetal calf serum containing 2-5%) indicates in mentioned component: fetal calf serum and RPMI 1640 are trained
The percent by volume for supporting base is 2-5%.
The beneficial effects of the present invention are:
1, the present invention, which is disclosed, cultivates tumour cell using tumor cell culture liquid provided by the invention, passes through control
The survival rate of tumour cell can be improved in the concentration of gentamicin and cholera mycin, effectively cellular morphology is maintained to promote tumour thin
Absorption of the born of the same parents to nutriment.
2, the present invention, which is disclosed, cultivates tumour cell using tumor cell culture liquid provided by the invention, passes through control
The concentration of insulin and the EGF, can be improved the growth rate of tumour cell, and improve cell activity, available big
The high activity tumour cell of amount, while the usage amount of fetal calf serum can be reduced using the insulin of the ratio and growth factor,
Since fetal calf serum price is more expensive, usage amount reduction is conducive to economize on resources.
3, the present invention is diluted processing to tumor cell line using tumor-selective culture solution in implementation process, and controls
The concentration ratio of insulin described in tumor-selective culture solution processed and transferrins is 1: 10-30, is incubated overnight to obtain
The good attached cell of survival state, so as to further increase the accuracy of drug screening.
4, with drug screening method provided by the invention, can be used for screening, the tradition of Small side effects maximally efficient to patient
Chemotherapeutics and targeted drug.
Detailed description of the invention
Fig. 1 is targeting pharmaceutical quantities response curve
Fig. 1: --- 4 Ponatinib (wave pa replaces Buddhist nun)；--- 5 Vandetanib (Fan Detani)；--- 8 Ah Fas replace
You；--- 9 Gefitinibs；--- 10 grams of azoles replace Buddhist nun；--- 11 is difficult to understand uncommon for Buddhist nun；--- 12 Tarcevas；--- 21 angstroms grams are replaced
Buddhist nun；--- 37 everolimus；--- 39 Bortezomib (bortezomib)；--- 40 Bosutinib (Bosutinib)；——
41 Cabozantinib (kappa, which is praised, to be replaced)；--- 42 Ceritinib (Ceritinib)；--- 43 Cobimetinib (Ke's ratio
Mention Buddhist nun)；--- 44 Dasatinib (Dasatinib)；--- 45 Ibrutinib (she replaces Buddhist nun by cloth)；——46 Idelalisib
(Ai Dailalisi)；--- 47 Rapamycin (rapamycin)；--- 48 Vismodegib (Wei Simodaiji).
Fig. 2 is non-targeted pharmaceutical quantities response curve
Fig. 2: --- 6 gemcitabines；--- 7 Etoposides；--- 13 docetaxels；--- 14 taxols；--- 15 tables
Adriamycin；--- 17 pemetrexeds；--- 38 vinorelbines.
Fig. 3 is drug combination pharmaceutical quantities response curve
Fig. 3: --- 1 carboplatin+taxol；--- 2 carboplatins+gemcitabine；--- 3 carboplatins+pemetrexed；--- 16 table Ah
Mycin+taxol；--- 18 Epi-ADMs+cyclophosphamide；--- 19 cis-platinums+Etoposide；--- 20 carboplatins+support pool
Glycosides；--- 22 cis-platinums+vinorelbine；--- 23 cis-platinums+taxol；--- 24 cis-platinums+gemcitabine；--- 25 cis-platinum+Yi Li
For health；--- 26 cis-platinums+Afatinib；--- 27 cis-platinums+Gefitinib；--- 28 cis-platinums+Lapatinib；--- 29 cis-platinums+
Ao Xi replaces Buddhist nun；--- 30 cis-platinums+pemetrexed；--- 31 cis-platinums+Etoposide；--- 32 cis-platinums+Etoposide+ring phosphinylidyne
Amine；--- 33 carboplatins+Irinotecan；--- 34 carboplatins+Etoposide；--- 35 carboplatins+docetaxel；--- 36 adriamycins+
Vincristine+cyclophosphamide.
Fig. 4 is drug relative potencies dose-effect curve
Fig. 4: --- 4 Ponatinib (wave pa replaces Buddhist nun)；--- 5 Vandetanib (Fan Detani)；--- 8 Ah methods replace
Buddhist nun；--- 9 Gefitinibs；--- 10 grams of azoles replace Buddhist nun；--- 11 is difficult to understand uncommon for Buddhist nun；--- 15 Epi-ADMs；--- 16 Epi-ADMs+
Taxol；--- 18 Epi-ADMs+cyclophosphamide；--- 39 Bortezomib (bortezomib).
Fig. 5 is drug (number 1-24) dose-effect curve
Fig. 5: --- 1 carboplatin+taxol；--- 2 carboplatins+gemcitabine；--- 3 carboplatins+pemetrexed；——4
Ponatinib (wave pa replaces Buddhist nun)；--- 5 Vandetanib (Fan Detani)；--- 6 gemcitabines；--- 7 rely on pool
Glycosides；--- 8 Afatinibs；--- 9 Gefitinibs；--- 10 grams of azoles replace Buddhist nun；--- 11 is difficult to understand uncommon for Buddhist nun；--- it replaces 12 Lip rivers in distress
Buddhist nun；--- 13 docetaxels；--- 14 taxols；--- 15 Epi-ADMs；--- 16 Epi-ADMs+taxol；--- 17 trainings
Beautiful Qu Sai；--- 18 Epi-ADMs+cyclophosphamide；--- 19 cis-platinums+Etoposide；--- 20 carboplatins+Etoposide；——21
Conmana；--- 22 cis-platinums+vinorelbine；--- 23 cis-platinums+taxol；--- 24 cis-platinums+gemcitabine.
Fig. 6 is drug (number 25-48) dose-effect curve
Fig. 6: --- 25 cis-platinums+Irinotecan；--- 26 cis-platinums+Afatinib；--- 27 cis-platinums+Gefitinib；——
28 cis-platinums+Lapatinib；--- 29 cis-platinums+Austria is uncommon to replace Buddhist nun；--- 30 cis-platinums+pemetrexed；--- 31 cis-platinums+support pool
Glycosides；--- 32 cis-platinums+Etoposide+cyclophosphamide；--- 33 carboplatins+Irinotecan；--- 34 carboplatins+Etoposide；——
35 carboplatins+docetaxel；--- 36 adriamycins+vincristine+cyclophosphamide；--- 37 everolimus；--- 38 Changchun are auspicious
Shore；--- 39 Bortezomib (bortezomib)；--- 40 Bosutinib (Bosutinib)；——41 Cabozantinib
(kappa, which is praised, to be replaced)；--- 42 Ceritinib (Ceritinib)；--- 43 Cobimetinib (Ke Bitini)；——44
Dasatinib (Dasatinib)；--- 45 Ibrutinib (she replaces Buddhist nun by cloth)；--- 46 IdeIaIisib (Ai Dailali
Department)；--- 47 Rapamycin (rapamycin)；--- 48 Vismodegib (Wei Simodaiji).
Specific technical solution
The lung carcinoma cell mentioned in following embodiment is derived from the lung on the ground such as Yinchuan of Ningxia Province, Shanghai, Zhangjiakou, Beijing, Shanxi
Cancer patient's body, frozen section report turns out to be lung cancer in art.
First group of embodiment
A kind of method of the functional high-throughput medication screening of the lung cancer of embodiment 1
Concrete operation step are as follows:
(1) acquisition of lung carcinoma cell: the lung carcinoma cell of acquisition is aseptically removed into its adipose tissue, is cut into 1mm
Then size is used digestion enzymic digestion 2 hours of 4mL, the digestive ferment by 225 units/mL clostridiopetidase A or hyaluronidase and
13 units/mL dispase composition, then using is 4 times of volumes of lung cancer tumor tissue containing 10% (percent by volume) calf serum
DMEM cell culture fluid stop digestion, collect digestive juice, precipitating is collected after centrifugation, the lung carcinoma cell dispersed；
(2) amplification cultivation of lung carcinoma cell: use tumor cell culture liquid by the lung carcinoma cell of the dispersion obtained in step (1)
Resuspension is deposited in laboratory and carries out fidelity amplification cultivation, is placed in culture bottle and is placed in 37 DEG C of CO2It is cultivated in incubator,
It cultivates and obtains within 3 weeks minimal neoplastic to get lung cancer reprogramming organoid is arrived；
(3) lung cancer that culture obtains in step (2) adhere-wall culture of lung carcinoma cell: is reprogrammed into 0.05% pancreas of organoid
Enzyme-EDTA is digested, and is counted with automated cell calculating instrument, and 384 orifice plates are inoculated with, and then uses tumor-selective culture solution
Dilution, adhere-wall culture is overnight to get adherent lung carcinoma cell.
(4) drug screening: by the various classic chemotherapy drugs of lung carcinoma cell adherent obtained in step (3), medicine is targeted
The candidate drug-treated such as object or several drugs combination detected cells survival with ATP chemoluminescence method (Promeaga) after 72 hours
Rate carries out drug screening.
The tumor cell culture liquid is that every 500mL contains following component:
Tumor-selective culture solution described in above-mentioned steps (3) is that every 500mL contains following component:
Tumour culture solution provided by the invention and selective tumour culture are used in the present embodiment 1 in implementation process
Liquid, in the present embodiment in tumor cell culture liquid the concentration range of amphotericin B and hydrocortisone not it is of the invention preferably
In range, so needing to be added 10% fetal calf serum in culture solution, the culture for having carried out 3 weeks to tumour cell is available
The good adherent lung carcinoma cell of growth performance, can satisfy the requirement of drug screening.
A kind of method of the functional high-throughput medication screening of the lung cancer of embodiment 2
Concrete operation step are as follows:
(1) acquisition of lung carcinoma cell: the lung carcinoma cell of acquisition is aseptically removed into its adipose tissue, is cut into 3mm
Then size is used digestion enzymic digestion 3 hours of 5mL, the digestive ferment by 225 units/mL clostridiopetidase A or hyaluronidase and
13 units/mL dispase composition, then with the DMEM cell culture containing 10% (percent by volume) calf serum of 4 times of volumes
Liquid stops digestion, collects digestive juice, precipitating is collected after centrifugation, the lung carcinoma cell dispersed；
(2) amplification cultivation of lung carcinoma cell: use tumor cell culture liquid by the lung carcinoma cell of the dispersion obtained in step (1)
Resuspension is deposited in laboratory and carries out fidelity amplification cultivation, is placed in culture bottle and is placed in 37 DEG C of CO2It is cultivated in incubator,
It cultivates and obtains within 2 weeks minimal neoplastic to get lung cancer reprogramming organoid is arrived；
(3) lung cancer that culture obtains in step (2) adhere-wall culture of lung carcinoma cell: is reprogrammed into 0.05% pancreas of organoid
Enzyme-EDTA is digested, and is counted with automated cell calculating instrument, is then diluted with tumor-selective culture solution, adherent training
It supports overnight to get adherent lung carcinoma cell.
(4) drug screening: by the various classic chemotherapy drugs of lung carcinoma cell adherent obtained in step (3), medicine is targeted
The candidate drug-treated such as object or several drugs combination detected cells survival with ATP chemoluminescence method (Promeaga) after 72 hours
Rate carries out drug screening.
The tumor cell culture liquid is that every 500mL contains following component:
Tumor-selective culture solution described in above-mentioned steps (3) is that every 500mL contains following component:
In embodiment 2 use the preferred embodiment of the invention, control tumour culture solution in amphotericin B and hydrogenation can
The concentration ratio of pine is 10: 1, is disclosed in concentration range in the present invention；And it controls insulin in tumor-selective culture solution and turns iron
The concentration ratio of albumen is 1: 20 in the open scope of the present invention, and being found surprisingly that can be in reducing culture solution using the culture solution
Fetal calf serum content (in tumour culture solution plus containing 6% fetal calf serum, in tumor-selective culture solution plus containing 2% tire
Cow's serum) in the case where can still turn out satisfactory tumour cell in a relatively short period of time, and tumour cell
Various performances are all relatively good, can be adapted for drug screening.
A kind of method of the functional high-throughput medication screening of the lung cancer of embodiment 3
Concrete operation step are as follows:
(1) acquisition of lung carcinoma cell: the lung carcinoma cell of acquisition is aseptically removed into its adipose tissue, is cut into 2mm
Size, digestion enzymic digestion 2.5 hours for then using 5mL, the digestive ferment is by 225 units/mL clostridiopetidase A or hyaluronidase
It is formed with 13 units/mL dispase, then using is 4 times of volumes of lung cancer tumor tissue containing 10% (percent by volume) small ox blood
Clear DMEM cell culture fluid stops digestion, collects digestive juice, precipitating is collected after centrifugation, the lung carcinoma cell dispersed；
(2) amplification cultivation of lung carcinoma cell: use tumor cell culture liquid by the lung carcinoma cell of the dispersion obtained in step (1)
Resuspension is deposited in laboratory and carries out fidelity amplification cultivation, is placed in culture bottle and is placed in 37 DEG C of CO2It is cultivated in incubator,
It cultivates and obtains within 2.5 weeks minimal neoplastic to get lung cancer reprogramming organoid is arrived；(3) adhere-wall culture of lung carcinoma cell: by step (2)
Middle obtained lung cancer reprogramming organoid of cultivating is digested with 0.05% pancreas enzyme -EDTA, and is carried out with automated cell calculating instrument
It counts, is inoculated with 384 orifice plates, then diluted with tumor-selective culture solution, rear adhere-wall culture is thin to get adherent lung cancer overnight
Born of the same parents.
(4) drug screening: by the various classic chemotherapy drugs of lung carcinoma cell adherent obtained in step (3), medicine is targeted
The candidate drug-treated such as object or several drugs combination detected cells survival with ATP chemoluminescence method (Promeaga) after 72 hours
Rate carries out drug screening.
The tumor cell culture liquid is that every 500mL contains following component:
Tumor-selective culture solution described in above-mentioned steps (3) is that every 500mL contains following component:
The preferred embodiment of the invention has equally been used in embodiment 3, controls amphotericin B and hydrogen in tumour culture solution
The concentration ratio for changing cortisone is 10: 1, is disclosed in concentration range in the present invention；And control insulin in tumor-selective culture solution
Concentration ratio with transferrins is 1: 15 in the open scope of the present invention, but since the increase of component content each in culture medium is anticipated
Outer discovery can be in the content for reducing the fetal calf serum in culture solution (in tumour culture solution plus containing 8% using the culture solution
Fetal calf serum, in tumor-selective culture solution plus containing 3% fetal calf serum) in the case where can still train in a relatively short period of time
Satisfactory tumour cell is raised, and the various performances of tumour cell are all relatively good, can be adapted for drug screening.
In order to prove that culture effect that tumor cell culture liquid provided by the present application has had, each embodiment take 100 parts
Lung cancer tumor cell carries out tumor cell culture using the cultural method of above-described embodiment 1-3 respectively, monitors cell culture feelings
Condition.
Second group of embodiment
A kind of method of the functional high-throughput medication screening of the lung cancer of embodiment 4
The difference from embodiment 1 is that: it swells described in tumor cell culture liquid described in step (2) and step (3)
The content of fetal calf serum is different in tumor selectivity culture solution.
The content of fetal calf serum is 8% in tumor cell culture liquid described in step (2)；It swells described in step (3)
The content 4% of fetal calf serum in tumor selectivity culture solution.
A kind of method of the functional high-throughput medication screening of the lung cancer of embodiment 5
The difference from embodiment 1 is that: it swells described in tumor cell culture liquid described in step (2) and step (3)
The content of fetal calf serum is different in tumor selectivity culture solution.
The content of fetal calf serum is 6% in tumor cell culture liquid described in step (2)；It swells described in step (3)
The content 3% of fetal calf serum in tumor selectivity culture solution.
A kind of method of the functional high-throughput medication screening of the lung cancer of embodiment 6
The difference from embodiment 1 is that: it swells described in tumor cell culture liquid described in step (2) and step (3)
The content of fetal calf serum is different in tumor selectivity culture solution.
The content of fetal calf serum is 4% in tumor cell culture liquid described in step (2)；It swells described in step (3)
The content 2% of fetal calf serum in tumor selectivity culture solution.
A kind of method of the functional high-throughput medication screening of the lung cancer of embodiment 7
The difference from example 2 is that: it swells described in tumor cell culture liquid described in step (2) and step (3)
The content of fetal calf serum is different in tumor selectivity culture solution.
The content of fetal calf serum is 7% in tumor cell culture liquid described in step (2)；It swells described in step (3)
The content 3% of fetal calf serum in tumor selectivity culture solution.
A kind of method of the functional high-throughput medication screening of the lung cancer of embodiment 8
The difference from example 2 is that: it swells described in tumor cell culture liquid described in step (2) and step (3)
The content of fetal calf serum is different in tumor selectivity culture solution.
The content of fetal calf serum is 8% in tumor cell culture liquid described in step (2)；It swells described in step (3)
The content 2% of fetal calf serum in tumor selectivity culture solution.
A kind of method of the functional high-throughput medication screening of the lung cancer of embodiment 9
Difference with embodiment 3 is: swelling described in tumor cell culture liquid described in step (2) and step (3)
The content of fetal calf serum is different in tumor selectivity culture solution.
The content of fetal calf serum is 6% in tumor cell culture liquid described in step (2)；It swells described in step (3)
The content 3% of fetal calf serum in tumor selectivity culture solution.
A kind of method of the functional high-throughput medication screening of the lung cancer of embodiment 10
Difference with embodiment 3 is: swelling described in tumor cell culture liquid described in step (2) and step (3)
The content of fetal calf serum is different in tumor selectivity culture solution.
The content of fetal calf serum is 7% in tumor cell culture liquid described in step (2)；It swells described in step (3)
The content 2% of fetal calf serum in tumor selectivity culture solution.
In order to further prove that culture effect that tumor cell culture liquid provided by the present application has had, each embodiment take
100 parts of lung cancer tumor cells carry out tumor cell culture, monitoring cell training using the cultural method of above-described embodiment 4-10 respectively
Support situation.
According to above-mentioned test data can be seen that when in cell culture fluid nutritional ingredient i.e. amphotericin B and hydrogenation can
When the concentration of pine and insulin and transferrins is compared in the preferred range, the appropriate content culture for reducing fetal calf serum
The success rate of cell is not in be substantially reduced, and cell can still keep its original form, but work as the dense of nutritional ingredient
When degree is compared not in the preferred range, the success rate of cell culture can be reduced, and the cell cultivated cannot keep original
Form, economically culture solution provided by the invention is economic and environment-friendly, economizes on resources.
Test example drug screening test
Breast cancer tumour drug screening is carried out with the tumour cell cultivated in above-described embodiment 1-3
1, types of medicines is screened
2, the half-inhibitory concentration (IC50) of drug is screened
Half-inhibitory concentration refers to drug concentration corresponding when inhibitory effect is 50%, and half inhibition is for measuring medicine
The value of the index of object sensitivity, half-inhibitory concentration is lower, illustrates that the action concentration of drug is lower, to the lethality of tumour cell
It is stronger.
The present invention is by a series of screening experiment by the detection curve of Fig. 1: No. 39 in targeted drug
Bortezomib (bortezomib), No. 4 Ponatinib (wave pa replaces Buddhist nun), No. 5 Vandetanib (Fan Detani), No. 10 grams frustrate
It wishes for Buddhist nun, No. 11 drug Austria for Buddhist nun successively to the tumour cell of the sample with good inhibiting effect, wherein No. 39 drugs
Ortezomib (bortezomib) is especially pronounced to the inhibiting effect of cancer cell.
By the detection curve of Fig. 2: No. 15 Epi-ADMs of drug, No. 38 vinorelbines are successively in non-targeted drug
There is good inhibiting effect to the tumour cell of the sample, wherein No. 15 drug Epi-ADMs are outstanding to the inhibiting effect of cancer cell
It is significant.
By the detection curve of Fig. 3: in drug combination No. 18 Epi-ADM+cyclophosphamide, No. 16 Epi-ADMs+
Taxol, No. 36 adriamycins+vincristine+C successively have good inhibiting effect to the tumour cell of the sample, wherein No. 18
Pharmaceutical composition Epi-ADM+cyclophosphamide is the most effective.
By Fig. 5,6 detection curve by: in all drugs No. 39 Bortezomib (bortezomib), No. 18 table Ah
Mycin+cyclophosphamide, No. 15 Epi-ADMs successively have stronger lethal effect to the sample tumour cell, wherein No. 39 drugs
Bortezomib (bortezomib) is the most significant to the inhibiting effect of the sample cancer cell.
The above description is only an embodiment of the present invention, is not intended to limit the scope of the invention, all to utilize this hair
Equivalent structure or equivalent flow shift made by bright specification and accompanying drawing content is applied directly or indirectly in other relevant skills
Art field, is included within the scope of the present invention.
Claims (6)
1. a kind of method of the functional high-throughput medication screening of lung cancer, characterized by the following steps:
(1) acquisition of lung carcinoma cell: from fresh surgical or puncturing sample acquisition lung cancer tumor tissue, digest tumor tissues, point
From the lung carcinoma cell dispersed；
(2) amplification cultivation of lung carcinoma cell: carrying out fidelity amplification cultivation in laboratory to lung carcinoma cell obtained in step (1),
It generates minimal neoplastic and reprograms organoid to get to lung cancer；
(3) adhere-wall culture of lung carcinoma cell: by lung cancer obtained in step (2) reprogramming organoid dispersion, adhere-wall culture is obtained
Attached cell；
(4) drug screening: carrying out drug candidate processing for the attached cell prepared in step (3), and detect cells survival rate, into
Row drug screening；
Amplification cultivation described in step (2) is: debulk tumor cell obtained in step (1) is placed in tumor cell culture liquid
In in 37 DEG C of CO2It is cultivated in incubator, culture obtains tumor cell line in 2-3 weeks；
The tumor cell culture liquid is that every 500mL contains following component:
Fetal calf serum 373mL of the complete DMEM containing 6-10%；
F12 culture medium 125mL；
Insulin 5-10 μ g/mL；
Amphotericin B 250-300ng/mL；
Gentamicin 10-15 μ g/mL；
Cholera toxin 0.1-0.5nM；
EGF 0.125-0.25ng/mL；
Hydrocortisone 25-30ng/mL；
ROCK inhibitor Y-27632 10-15 μm ol/L；
Adhere-wall culture described in step (3) is: lung cancer obtained in step (2) reprogramming organoid is disappeared with digestive ferment
Change, is incubated overnight after then being diluted with tumor-selective culture solution；The digestive ferment is 0.05% pancreas enzyme -EDTA；
The tumor-selective culture solution is that every 500mL contains following component:
1640 culture mediums of RPMI fetal calf serum containing 2-5% 485mL；
Gentamicin 10-15 μ g/mL；
Hydrocortisone 10-15nM；
Insulin 5-10 μ g/mL；
Transferrins 100-150 μ g/mL；
17 beta- estradiol 10-15nM；
Sodium selenite 30-40nM.
2. the method for the functional high-throughput medication screening of lung cancer according to claim 1, it is characterised in that: in step (1)
The digestion is: use digestion enzymic digestion, then use is that the DMEM containing 10% calf serum of 4 times of volumes of lung cancer tumor tissue is thin
Born of the same parents' culture solution stops digestion, collects digestive juice.
3. the method for the functional high-throughput medication screening of lung cancer according to claim 2, it is characterised in that: the digestive ferment
It is made of 225 units/mL clostridiopetidase A or hyaluronidase and 13 units/mL dispase.
4. the method for the functional high-throughput medication screening of lung cancer according to claim 1, it is characterised in that: the both sexes
The concentration of mycin B and hydrocortisone ratio is 9-12:1.
5. the method for the functional high-throughput medication screening of lung cancer according to claim 1, it is characterised in that: the selection
Property tumor cell culture liquid described in the concentration ratio of insulin and transferrins be 1:10-30.
6. the method for the functional high-throughput medication screening of lung cancer according to claim 1-5, it is characterised in that: tool
Body operating procedure are as follows:
(1) acquisition of lung carcinoma cell: the lung carcinoma cell of acquisition is aseptically removed into its adipose tissue, is disappeared with digestive ferment
Change, the digestive ferment is made of 225 units/mL clostridiopetidase A or hyaluronidase and 13 units/mL dispase, then with for
The DMEM cell culture fluid containing 10% calf serum of 4 times of volumes of lung cancer tumor tissue stops digestion, digestive juice is collected, after centrifugation
Collect precipitating, the lung carcinoma cell dispersed；
(2) amplification cultivation of lung carcinoma cell: the lung carcinoma cell of the dispersion obtained in step (1) is resuspended with tumor cell culture liquid
It is deposited in laboratory and carries out fidelity amplification cultivation, be placed in culture bottle and be placed in 37 DEG C of CO2It is cultivated, is cultivated in incubator
It obtains within 2-3 weeks minimal neoplastic and reprograms organoid to get to lung cancer；
(3) lung cancer that culture obtains in step (2) adhere-wall culture of lung carcinoma cell: is reprogrammed into organoid 0.05% pancreatin-
EDTA is digested, and is counted with automated cell calculating instrument, and 384 orifice plates are inoculated with, then dilute with tumor-selective culture solution
It releases, adhere-wall culture is overnight to get adherent lung carcinoma cell；
(4) drug screening: by the various classic chemotherapy drugs of lung carcinoma cell adherent obtained in step (3), targeted drug or
The candidate drug-treated such as several drugs combination detects cells survival rate with ATP chemoluminescence method, carries out drug sieve after 72 hours
Choosing.
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| JP7510998B2 (en) * | 2019-08-05 | 2024-07-04 | 北京基石生命科技有限公司 | Method and kit for culturing primary cells from solid lung cancer tumors and primary tumor cells from lung cancer pleural effusion |
| CN113481162B (en) * | 2021-07-01 | 2023-02-24 | 丹望医疗科技(上海)有限公司 | Culture medium, method and kit for rapidly culturing tumor organoid |
| CN115960815A (en) * | 2021-10-13 | 2023-04-14 | 合肥中科普瑞昇生物医药科技有限公司 | Culture medium, culture method and application of lung cancer pleural effusion source organoid |
| WO2023060684A1 (en) * | 2021-10-13 | 2023-04-20 | 合肥中科普瑞昇生物医药科技有限公司 | Culture medium for lung cancer organoids, culture method and application thereof |
| CN118272312B (en) * | 2024-05-30 | 2024-09-17 | 山东大学 | Culture medium and related reagent for culturing cancer cells in microgravity environment and application of culture medium and related reagent |
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| CN102321587A (en) * | 2011-08-25 | 2012-01-18 | 上海吉凯基因化学技术有限公司 | Construction of lung cancer drug screening cell line |
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