CN104531620A - Method for culturing lung cancer stem cells under 3D culture conditions - Google Patents
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Abstract
The invention discloses a method for culturing lung cancer stem cells under 3D culture conditions and belongs to the field of cell culture methods. The method comprises the following steps of placing human lung adenocarcinoma cell line A549 in an incubator for culturing with a RPMI-1640 complete culture medium containing 10% fetal calf serum, taking a cell suspension in a logarithm growth period, adjusting the concentration of the cells to 5*10<4>cells/ml and adding 200 mu L/well into a 96-well plate in which 50 mu L/well BME is spread; on the third day, changing the culture medium for the A549 cells with a RPMI-1640 complete culture medium containing IGF-1 and FGF and further culturing; digesting BME through a proteolytic enzyme, recovering the A549 cells from a recovery liquid and finally identifying the cells. By such design, the A549 cells form cloned mass similar to in-vivo tumor mass in vitro, which is closer to the three-dimensional growth state in the human body and thus the stable separation and effective amplification of stem cells are achieved. The cancer stem cells capable of resisting anticancer drugs are screened.
Description
Technical field
The present invention relates to a kind of condition 3D cell culture processes, be a kind of can the method for high efficiency separation and amplification lung cancer stem cell, can be used for the in-vitro screening system setting up tumour original new drug.
Technical background
Lung cancer is the malignant tumour that sickness rate and case fatality rate are all occupied the forefront in worldwide at present, and serious threat human health, wherein nonsmall-cell lung cancer (NSCLC) accounts for the 80%-85% of lung cancer.In recent years along with the popularization of the progress of surgical technic and the chemicotherapy scheme of specification, the clinical levels of NSCLC has had large increase, but 5 of NSCLC years survival rates are not effectively improved.Trace it to its cause, except NSCLC high aggressive and the easy malignant characteristics recurred, the limitation of clinical drug therapy is also the important factor of restriction NSCLC result for the treatment of.The mankind occur NSCLC and the deep understanding of developer molecule mechanism is that original new drug exploitation filters out numerous valuable molecular target, but most of alternative original new drug cannot repeat out the satisfactory effect that clinical precursor is tested outward in clinical trial, therefore greatly limit research and development speed and the clinical therapeutic efficacy of NSCLC new medicine.
Cause the major cause of above-mentioned phenomenon to be growth pattern and the behavioral characteristic that tumor cell in vitro model conventional in existing medicament sifting motion system is difficult to tumor tissues in analogue body, therefore cause clinical test results and preliminary in vitro experiments result far from each other.The outer tumor models of clinical precursor is based upon on traditional 2D cell cultures basis usually, this cultural method makes cell monolayer adherent growth, contact with each other, mutual suppression, be difficult to simulation in-vivo tumour three-dimensional growth pattern, not easily form the local microenvironment of short tumor growth, therefore also cannot reflect the biological behaviour of in-vivo tumour and the reactivity to medicine strictly according to the facts.In addition, traditional 2D cell culture processes is not suitable for growth and the amplification of the tumor stem cell in tumor tissues with extremely strong tumorigenicity.Tumor stem cell is that in tumour, a very little part has the cell promoting tumour generation, maintain tumor growth and maintenance Tumor Heterogeneity, has self, Multidirectional Differentiation and infinite multiplication potential, carries unique stem-cell marker.More and more research at present confirms that tumor stem cell is the key reason causing tumor drug resistance and relapse and metastasis, and has the natural tolerance to chemotherapeutics due to it, is also the one of the main reasons of obtained resistance.Therefore, set up the tumor cell in vitro model being rich in tumor stem cell and contribute to filtering out there is more powerful antitumor activity, lower drug resistance, effectively control the tumour original new drug of progression of disease and tumour metastasis and recurrence.But because tumor stem cell quantity in tumour cell is few, and many places are stationary phase, utilize traditional 2D cell cultures to be difficult to effectively be separated in vitro and increase, be therefore also difficult to the external pharmacology model of tumor stem cell setting up the screening of applicable tumour original new drug.
Document confirms, in cancer research 3D cell culture processes can make up 2D cell cultures cannot the defect of living environment in analog cell body, and in-vitro separation and the amplification of tumor stem cell can be realized.3D cell culture processes be by stereoscopic culture pattern for tumour cell provides the microenvironment of life condition in close proximity to body more: first, tumour cell in the mutually isolated growth of cloning cluster, can not come in contact suppression each other in 3D cell culture system; Secondly, the inner intercommunication of matrigel of 3D cell culture system, in growth of tumour cell process, various cytokine can freely exchange with soluble chemical material, forms the local living environment of tumor microenvironment in analog, promote tumour cell, the especially propagation of tumor stem cell; Finally, in 3D cell culture system, tumour cell spatially but not horizontal distribution, closer to the form of in-vivo tumour agglomerate, independent studies of being therefore more convenient for original new drug, on the impact of signal path, biological function in tumour cell, makes the situation of drug reaction practical application in close proximity to body more.At present, 3D cell culture processes be not only applied to cell high-flux medicaments sifting, poisonous substance screening and other screening, multiple internal milieu simulated experiment can also be applied to.
Summary of the invention
The object of the invention is to solve tumor stem cell quantity in tumour cell few, and many places are stationary phase, traditional 2D cell cultures is utilized to be difficult to effectively be separated in vitro and increase, therefore be also difficult to the problem of the external pharmacology model of tumor stem cell setting up the screening of applicable tumour original new drug, and the condition 3D cultural method of a kind of high efficiency separation and amplification lung cancer stem cell is provided.
The present invention realizes according to following technology.
A kind of lung cancer stem cell condition 3D cultural method, its method is:
By human lung adenocarcinoma cell line A549 with the RPMI-1640 perfect medium containing 10% foetal calf serum, be placed in 37 DEG C, 5%CO
2, relative humidity 90% incubator in cultivate, cell suspension in vegetative period of taking the logarithm, adjustment cell concn be 5 × 10
4individual cell/ml, gets 200 μ L(1 × 10
4individual cell)/hole be added to complete 50 μ L/ holes basal medium of Eagle (BME) 96 orifice plates in; In the 3rd day, change liquid to A549 cell, 37 DEG C, 5%CO with the RPMI-1640 perfect medium containing 25 ~ 75 ng/ml insulin-like growth factor-is and 10 ~ 30ng/ml fibroblast growth factor
2cultivation is continued 4 days under condition; Through neutral metalloprotease lytic enzyme (Dispase, BD
tM, 354235) and digest BME, Ethylene recov (Cell Recovery Solution, BD
tM, 354253) and reclaim A549 cell, finally carry out a series of stem cell identification experiments such as cell proliferation, self, stem cell markers qualification, Invasion and Metastasis activity and Resistance detection.
Described lung cancer stem cell condition 3D cultural method, the RPMI-1640 perfect medium changing liquid to described in it A549 cell is mainly containing 10% foetal calf serum and the insulin-like growth factor-i of 50ng/ml and the fibroblast growth factor of 20ng/ml.
The present invention of such design, the cloning cluster of tumor mass in analog is formed in vitro through the A549 cell of condition 3D cell cultures, three-dimensional growth state in close proximity to body more, BME is utilized to make between A549 cell without any contact, and add the factor of short stem cell growth, make the separation of stem cell more stable, and realize effectively amplification.Lung cancer stem cell through the present invention's cultivation is 2.82 times of conventional 2D culturing cell, similar with experiment in vivo result, implied condition 3D cell culture processes can filter out the tumor stem cell to cancer therapy drug opposing, provides experiment basis for setting up more effective original new drug screening system.
Accompanying drawing explanation
Fig. 1 is IGF-1 and FGF concentration comparison diagram in 3D cultivation group and 2D group cultivation group;
Fig. 2 is the lung carcinoma cell surface dry cell marker CD326 that condition 3D cultivates
+cD44
+cD24
-ratio display figure;
Fig. 3 is the balling ratio comparison diagram of 3D-3L group and other A549 cells organized;
Fig. 4 be 3D-3L group and other organize the invasive ability comparison diagram of cell;
Fig. 5 is 3D-3L group and other resistance comparison diagrams organized.
Embodiment
One, material
Human lung adenocarcinoma cell line A549: purchased from Shanghai Inst. of Life Science, CAS cell centre, is preserved by this laboratory;
10% foetal calf serum: purchased from American Hyclone company;
RPMI-1640 perfect medium: purchased from American Hyclone company;
Ethylene recov: Cell Recovery Solution, 354253, purchased from American BD companies;
BME(basal medium of Eagle): purchased from American BD company;
PBS: phosphate buffered saline buffer (1 ×), 0.0067M(PO
4);
Neutral metalloprotease lytic enzyme: Dispase, 354235, purchased from American BD companies;
Insulin-like growth factor-i (IGF-1), purchased from Proteck company;
Fibroblast growth factor (FGF) is purchased from Proteck company.
Two, method
(1) cell cultures
Arranging 6 groups of cells, is the 0th day high density cytokine group (3D-H), the 0th day lower concentration cytokine group (3D-L), the 3rd day high density cytokine group (3D-3H), the 3rd day lower concentration cytokine group (3D-3L), original 3D cultivation group (3D) and 2D control group (2D) respectively.Be 3D cultivation group.
By RPMI 1640 perfect medium of A549 cell containing 10% foetal calf serum, be placed in 37 DEG C, 5%CO
2, relative humidity 90% incubator in cultivate.To take the logarithm cell suspension in vegetative period, with 1 × 10
4the concentration 200 μ L of individual cells/well is inoculated in common 96 orifice plates, abandon old substratum subsequently, PBS washs 2 times, add 0.25% trysinization, basis of microscopic observation cellular form change after room temperature 1 ~ 2min, when observing that intercellular substance becomes large, attached cell protrusion collapses time, discard trypsinase, RPMI 1640 perfect medium added containing 10% foetal calf serum stops digestion, and piping and druming re-suspended cell goes down to posterity, reclaims cell, is 2D control group.
Get A549 logarithmic phase cell suspension, adjustment cell concn is 5 × 10
4individual cell/ml, gets 200 μ L/ holes and is added to and completes in 96 orifice plates of BME, in the 7th day, with Dispase digest BME, Ethylene recov reclaims cell, is 3D cultivation group.
The 0th day and the 3rd day is cultivated at 3D, respectively with containing the insulin-like growth factor-i (IGF-1) of 100ng/ml and the FGF(high density group of 20ng/ml), the FGF(low concentration group of IGF-1 and 20ng/ml of 50ng/ml) RPMI-1640 complete culture solution, to cell change liquid continue culturing cell to the 7th day.
Wherein in the 3rd day, change liquid with the RPMI-1640 perfect medium of the FGF of IGF-1 and 20ng/ml containing 50ng/ml to A549 cell, continue cultivation 4 days; Reclaim A549 cell be condition 3D group through neutral metalloprotease lytic enzyme digestion BME, Ethylene recov.
(2) PCR screens cytokine
1. Trizol method extracts total serum IgE:
1. collect cultured cells, add appropriate Trizol (1ml Trizol/1 × 10
6individual cell), move into without in enzyme Eppendorf pipe after piping and druming.Guarantee the complete cracking of cell, liquid is clarified substantially.
2. above-mentioned Eppendorf pipe room temperature is left standstill 5min, add chloroform (200 μ l/1ml Trizol), mixing of turning upside down, room temperature leaves standstill 10min, 4 DEG C, 12000g, centrifugal 15min.
3. carefully draw upper strata aqueous phase and be placed in that another is new for enzyme Eppendorf pipe, add equal-volume Virahol, mixing of turning upside down, room temperature leaves standstill 10min, 4 DEG C, 12000g, centrifugal 10min.Abandon supernatant.
4. use 75% ethanol (1ml/1mlTrizol) to wash precipitation, 4 DEG C, 7500g, centrifugal 5min, abandons supernatant, and room temperature leaves standstill several minutes, makes precipitation seasoning.The DDW adding appropriate DEPC process makes it dissolve, and-80 DEG C save backup.
5. the concentration of determined by ultraviolet spectrophotometry RNA and purity; 1% agarose gel electrophoresis detects the integrity of RNA.
2. synthesize cDNA, carry out real-time quantitative PCR:
3. application of sample
A. the film on PCR Array is carefully opened.
B. 20 μ L mixed solutions are added in each hole corresponding to PCR Array.
C. careful cover lid sealing PCR Array.
D. before PCR program is set, ready PCR Array is placed on ice.
E. real-time quantitative PCR programming complete after, PCR Array is placed in real-time PCR and carries out PCR reaction.Set program is as follows:
(3) flow cytomery stem cell specific antibody
The mark of 1 streaming antibody
Collect the cell that 6 groups are cultivated 3,7,11,14,20 days, make cell suspension respectively, adjustment cell concn is 1 × 10
5individual cell/100 μ L, respectively with antibody labeling thing CD24, CD326, CD44 mark, in contrast, 4 DEG C of lucifuges hatch 30 minutes for flag F ITC-IgG2b, PE-IgG1, APC-IgG1 simultaneously.After PBS washed cell twice, add and be fixed with 1% paraformaldehyde 100 μ L, flow cytometer can be used to detect.
The detection of 2 flow cytometers
1. cover before opening, selects suitable nozzle, implants the nozzle with 0 circle in flow detection pond.
2. voltage stabilized source, instrument primary source and laser apparatus are opened successively, run software interface BD FACSDiva software in workstation.
3. find software interface menu " Instrument ", select " Fluidics Start up " option.Then perform " Turn on stream " order, open liquid stream.
4. after waiting liquid stream to stablize gradually, close the front cover of flow cytometer, start Quality Control afterwards and detect, if Quality Control detects normal, upper machine can be started and carry out sample tests.
5. use 488nm wavelength to detect laser, be set up sample pipe on Stage microscope, select Load to start loading, carry out flow stream velocity adjustment according to the standard of particulate 100-300 p.s. by detection cell.Regulate the parameter value of FSC and SSC to allow most particulate fall in scatter diagram centre rectangular door as far as possible, the threshold voltage of different fluorescence channel is regulated again according to Isotype control, and pass through to control the compensation between different fluorescence channel thus the interference as much as possible reduced each other, Record is selected to record the detected result of 10000-20000 sample cell, carry out immunophenotype detection, qualification stem cell.
(4) external balling-up experiment
6 groups of culturing cells are contained the DMEM/F12 cell culture medium of bFGF, EGF, BSA and Insulin with 5000/3ml MFM() density be inoculated in 6 orifice plates that non-processor crosses, the balling ratio of observation of cell after 7 days.
(5) vitro invasion experiment
Utilize Matrigel with Transwell to build invasion and attack cell and carry out being separated (poly-carbon cruel film diameter 6.smm, micropore size 8.0 μm) of high aggressive and low aggressive cell mass.The Matrigel being 1.17mg/ml by 20 μ L/ hole concentration is laid on the cruel film of poly-carbon of room on Transwell culture plate, 37 DEG C, 30min, allows Matrigel be polymerized plastic.Unicellular outstanding 1.0 × 10 are prepared in conventional manner with serum free medium
5individual/ml, every hole adds 200 μ L cell suspensions.Under Transwell culture plate, room adds 400 μ L foetal calf serums, 37 DEG C, 5% CO
2, 95% humidity cultivates 48h.Take out room methyl alcohol on Transwell culture plate after 48h and fix 30min, 1% violet staining 30min, just put counted under microscope, imaging for 200 times.
(6) drug responsiveness experiment
The A549 cell that condition 3D, 3D and 2D cultivate is made single cell suspension respectively, and adjustment cell density is 1 × 10
5/ ml, is inoculated in 96 orifice plates, and every hole 100 μ L, blank control wells only adds nutrient solution.Be placed in 37 DEG C, 5% CO
2in saturated humidity incubator, treat that cell is completely adherent, add the substratum of 100 μ L containing cis-platinum next day respectively, concentration gradient is 16 μ g/ml, 8 μ g/ml, 4 μ g/ml, 2 μ g/ml, 1 μ g/ml, and every hole cumulative volume is 200 μ L.Often group does 3 parallel holes, and after 48h, every hole adds the MTT 20 μ L of 5mg/ml, continues to cultivate 4h, and centrifugal culture plate, abandons supernatant, and every hole adds DMSO 150 μ L, and micro-oscillator slight oscillatory 10min is dissolving crystallized.Each hole absorbancy (OD value) is detected under microplate reader 490nm wavelength.By following formulae discovery cell survival rate, cell survival rate=experimental group OD value/blank group OD value × 100%, MTT experiment is not repeating 3 times on the same day.Cell inhibitory rate=1-cell survival rate, does linear regression by the logarithmic value of drug level and inhibitory rate of cell growth, obtains the half-inhibition concentration IC50 of cis-platinum to cell.
Three, result
1. in 3D cultured cells the concentration of IGF-1 and FGF two short stem cell factors apparently higher than 2D group
Utilize pcr chip to detect, in the A549 cell of 3D cell cultures, find that the Cytokine Expression Level of multiple promotion stem cell growth raises, especially IGF-1 (20.12) and FGF (10.24) changes particularly evident, see Fig. 1.
2. the lung carcinoma cell surface dry cell marker positive rate that condition 3D cultivates obviously raises
Detect 6 groups of A549 cell surfaces CD44, CD24 and CD326 respectively with flow cytometer, result shows, the A549 cell surface CD44 of 3D-3L group
+, CD326
+, CD44
+cD24
-and CD326
+cD44
+cD24
-ratio be significantly higher than 3D cultivation group (53.97 ± 4.49% vs 23.37 ± 2.41%, 16.10 ± 1.21% vs 10.73 ± 2.32%, 58.90 ± 6.08% vs 34.03 ± 10.46%, 4.17 ± 1.01% vs 2.73 ± 0.71%,
p<0.01).CD326
+cD44
+cD24
-ratio is see Fig. 2, CD24
-ratio and 3D no significant difference (93.13 ± 3.62% vs 88.20 ± 7.21%).
3. the balling ratio of the A549 cell of 3D-3L group is higher than other groups
Stem cell can form mono-clonal microsphere, and diameter thinks a mono-clonal microsphere more than 70 μm.A549 cell cultures is after 7 days, the balling ratio after the cytokine promoting stem cell growth is added all higher than the 3D cultivation group of routine in 3D cultivates, wherein the balling ratio of the A549 cell of 3D-3L group is higher than other groups, the balling ratio that the conventional 3D of obvious ratio cultivates is high see Fig. 3, (30.58 ± 1.44% vs 19.20 ± 0.76%
p<0.01), and go down to posterity through twice, balling ratio is still higher than other groups, and it is as shown in the table, illustrates that improved condition 3D-3L group has turned out more a high proportion of stem cells.
4. the invasive ability of 3D-3L group cell is higher than other groups
The A549 cell extracting 6 groups is respectively inoculated in room on 24 hole transwell flat boards, serum deprivation cultivates 48 hours, the cell count contrast of 6 groups of transwell cell counterdies is counted under microscope, result shows that 3D is all strong than the invasive ability of conventional 3D cell cultures after cultivating the cytokine adding promotion stem cell growth, wherein the invasive ability of 3D-3L group cell is higher than other groups, stronger (363.33 ± 56.37 vs 167.33 ± 29.69 of more conventional 3D cultured cells invasiveness, P<0.05, see Fig. 4, for 2.17 times of conventional 3D culturing cell invasive ability, for 5.17 times of conventional 2D cultivation group.
5. the resistance of 3D-3L group will apparently higher than other groups
Extract 3 groups of A549 cells cultivated respectively and be inoculated in 96 orifice plates, add 8 μ g/ml cis-platinums, mtt assay detects two kinds of cultured cells survival states simultaneously.Result is that 3 groups of cells add at 48h after cis-platinum, condition 3D group cell survival rate all higher than other groups (71.37 ± 8.65% vs 55.90 ± 5.12%, 71.37 ± 8.65% vs 33.63 ± 1.39%,
p< 0.05) see Fig. 5, show that condition 3D cultured cells has stronger resistance.At 48h, the half-inhibition concentration IC50 of A549 to DDP cultivated through condition 3D and 3D, 2D is respectively (15.58 ± 1.25 μ g/ml vs 8.36 ± 0.96 μ g/ml, 15.58 ± 1.25 μ g/ml vs5.52 ± 0.74 μ g/ml,
p< 0.05).
In sum, the cloning cluster of tumor mass in analog is formed in vitro through the A549 cell of condition 3D cell cultures, three-dimensional growth state in close proximity to body more, BME is utilized to make between A549 cell without any contact, and add the factor of short stem cell growth, make the separation of stem cell more stable, and realize effectively amplification.Detect for cell phenotype, find that in the A549 cell in the cell culture processes source of 3D-3L group, stem cell markers C326 and CD44 expression level raise, the CD24 as maturation differentiation mark then significantly declines, and wherein carries stem-cell marker CD326
+cD44
+cD24
-cell proportion obviously raise, implied condition 3D cell culture processes has turned out more a high proportion of lung cancer stem cell.Stem cell functional experiment shows that the A549 cells in vitro balling ratio of condition 3D cell cultures significantly raises, invasive ability strengthens, and the expression level of the stem cell genes involved of the A549 cell of condition 3D cell cultures significantly raises, declaration condition 3D cell culture processes can be separated in vitro and increase has the NSCLC cell of cells and characteristic of stem and functionally active.Further pharmacodynamic experiment confirms, A549 cell through condition 3D cell cultures increases the resistivity of cancer therapy drug, the half-inhibition concentration of cis-platinum is increased to 1.86 times of conventional 3D culturing cell, it is 2.82 times of conventional 2D culturing cell, similar with experiment in vivo result, implied condition 3D cell culture processes can filter out the tumor stem cell to cancer therapy drug opposing, provides experiment basis for setting up more effective original new drug screening system.
Claims (3)
1. a lung cancer stem cell condition 3D cultural method, is characterized in that:
By human lung adenocarcinoma cell line A549 with the RPMI-1640 perfect medium containing 10% foetal calf serum, be placed in incubator and cultivate, cell suspension in vegetative period of taking the logarithm, adjustment cell concn is 5 × 10
4individual cell/ml, get 200 μ L/ holes be added to complete 50 μ L/ hole BME 96 orifice plates in; In the 3rd day, change liquid with the RPMI-1640 perfect medium containing 25 ~ 75 ng/ml insulin-like growth factor-is and 10 ~ 30ng/ml fibroblast growth factor to A549 cell, continue cultivation 4 days; Reclaim A549 cell through neutral metalloprotease lytic enzyme digestion BME, Ethylene recov, finally carry out cellular identification.
2. lung cancer stem cell condition 3D cultural method according to claim 1, is characterized in that: the described RPMI-1640 perfect medium changing liquid to A549 cell is mainly containing 10% foetal calf serum and the insulin-like growth factor-i of 50ng/ml and the fibroblast growth factor of 20ng/ml.
3. lung cancer stem cell condition 3D cultural method according to claim 1, is characterized in that: described perfect medium is cultivated in incubator, and the perfect medium after changing liquid to A549 cell continues to cultivate, and is be placed in 37 DEG C, 5%CO
2, relative humidity 90% condition under to carry out.
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| CN106967672A (en) * | 2017-03-24 | 2017-07-21 | 四川大学华西医院 | Lung and lung cancer tissue culture method and method for constructing lung cancer mouse animal model by using same |
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