CN104531620A - Method for culturing lung cancer stem cells under 3D culture conditions - Google Patents

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

Conditioned 3D culture method of lung cancer stem cells

technical field

The invention relates to a conditional 3D cell culture method, which is a method capable of efficiently separating and expanding lung cancer stem cells, and can be used to establish an in vitro screening system for innovative tumor drugs.

technical background

Lung cancer is a malignant tumor with the highest morbidity and mortality rate in the world, which seriously threatens human health. Among them, non-small cell lung cancer (NSCLC) accounts for about 80%-85% of lung cancer. In recent years, with the advancement of surgical techniques and the promotion of standardized radiotherapy and chemotherapy regimens, the clinical treatment level of NSCLC has been greatly improved, but the 5-year survival rate of NSCLC has not been effectively improved. The reason is that in addition to the malignant characteristics of NSCLC, which are highly invasive and prone to recurrence, the limitation of clinical drug treatment is also an important factor restricting the therapeutic effect of NSCLC. Human's in-depth understanding of the molecular mechanism of NSCLC occurrence and development has screened many valuable molecular targets for the development of innovative drugs, but most of the candidate innovative drugs cannot repeat the satisfactory curative effect of preclinical in vitro experiments in clinical trials, which greatly limits The research and development speed and clinical treatment effect of NSCLC innovative drugs have been greatly improved.

The main reason for the above phenomenon is that the in vitro tumor cell model commonly used in the existing drug screening system is difficult to simulate the growth mode and behavior characteristics of tumor tissue in vivo, which leads to a large discrepancy between the results of clinical trials and the results of previous in vitro experiments. Preclinical in vitro tumor cell models are usually established on the basis of traditional 2D cell culture. This culture method allows cells to grow in a single layer, contact with each other, and inhibit each other. environment, and therefore cannot faithfully reflect the biological behavior and responsiveness of tumors in vivo. In addition, traditional 2D cell culture methods are not suitable for the growth and expansion of tumor stem cells with strong tumorigenicity in tumor tissues. Cancer stem cells are a small part of tumor cells that can promote tumorigenesis, maintain tumor growth and maintain tumor heterogeneity, have self-renewal, multidirectional differentiation and unlimited proliferation potential, and carry unique stem cell markers. At present, more and more studies have confirmed that cancer stem cells are the key cause of tumor drug resistance and recurrence and metastasis, and because of their natural resistance to chemotherapy drugs, they are also one of the main reasons for tumor acquired drug resistance. Therefore, the establishment of an in vitro tumor cell model enriched in tumor stem cells is helpful for screening innovative tumor drugs with stronger anti-tumor activity, lower drug resistance, and effective control of disease progression and tumor metastasis and recurrence. However, due to the very small number of tumor stem cells in tumor cells and most of them are in the quiescent phase, it is difficult to effectively separate and expand in vitro using traditional 2D cell culture, so it is also difficult to establish in vitro pharmacology of tumor stem cells suitable for innovative tumor drug screening. Model.

The literature has confirmed that in cancer research, the 3D cell culture method can make up for the defect that 2D cell culture cannot simulate the living environment of cells in vivo, and can realize the isolation and expansion of tumor stem cells in vitro. The 3D cell culture method is to provide tumor cells with a microenvironment that is closer to the living conditions in vivo through the three-dimensional culture mode: first, tumor cells grow in 3D cell culture system as clonal clusters isolated from each other, and there is no contact inhibition between each other; secondly, , The matrigel of the 3D cell culture system communicates with each other, various cytokines and soluble chemical substances can freely exchange during the growth of tumor cells, forming a local living environment similar to the tumor microenvironment in vivo, and promoting the proliferation of tumor cells, especially tumor stem cells; Finally, the tumor cells in the 3D cell culture system are three-dimensional rather than planar, which is closer to the shape of the tumor mass in the body, so it is easier to independently study the influence of innovative drugs on the signaling pathways and biological functions of tumor cells, making the drug response closer The case of practical application in vivo. At present, the 3D cell culture method is not only applied to high-throughput drug screening, toxicant screening and other screening of cells, but also can be applied to various in vivo environment simulation experiments.

Contents of the invention

The purpose of the present invention is to solve the problem that the number of tumor stem cells in tumor cells is extremely small, and most of them are in the quiescent phase. It is difficult to effectively separate and amplify in vitro by using traditional 2D cell culture, so it is also difficult to establish a tumor cell that is suitable for innovative drug screening. To address the problem of in vitro pharmacological models of cancer stem cells, and provide a conditional 3D culture method for efficiently isolating and expanding lung cancer stem cells.

The present invention is achieved according to the following techniques.

A conditional 3D culture method for lung cancer stem cells, the method comprising:

The human lung adenocarcinoma cell line A549 was cultured in RPMI-1640 complete medium containing 10% fetal bovine serum in an incubator at 37°C, 5% CO ₂ , and 90% relative humidity, and the cells in the logarithmic growth phase were suspended. solution, adjust the cell concentration to 5×10 ⁴ cells/ml, take 200 μL (1×10 ⁴ cells)/well and add to a 96-well plate on which 50 μL/well of Eagle’s basal medium (BME) has been laid; On the third day, replace the medium of A549 cells with RPMI-1640 complete medium containing 25-75 ng/ml insulin-like growth factor-1 and 10-30 ng/ml fibroblast growth factor, at 37°C, 5% CO ₂ Continue to culture for 4 days under the same conditions; digest BME with neutral metalloproteinase (Dispase, BD ^TM , 354235), recover A549 cells with recovery solution (Cell Recovery Solution, BD ^TM , 354253), and finally carry out cell proliferation, self-renewal, stem cell A series of stem cell identification experiments such as marker identification, invasion and metastasis activity and drug resistance detection.

In the conditioned 3D culture method for lung cancer stem cells, the RPMI-1640 complete medium for A549 cell replacement mainly contains 10% fetal bovine serum, 50ng/ml insulin-like growth factor-1 and 20ng/ml adult Fibroblast growth factor.

In the present invention designed in this way, the A549 cells cultured in conditioned 3D form a clonal group similar to the tumor mass in the body in vitro, which is closer to the three-dimensional growth state in the body, and the BME is used to prevent any contact between the A549 cells, and the growth-promoting stem cells are added Factors that make the isolation of stem cells more stable and achieve efficient expansion. The lung cancer stem cells cultured by the present invention are 2.82 times that of conventional 2D cultured cells, which is similar to the results of in vivo experiments, suggesting that the conditional 3D cell culture method can screen out tumor stem cells resistant to anticancer drugs, providing a basis for establishing a more effective innovative drug screening system the basis of the experiment.

Description of drawings

Fig. 1 is a comparison diagram of IGF-1 and FGF concentrations in the 3D culture group and the 2D culture group;

Figure 2 is a graph showing the proportion of stem cell marker CD326 ⁺ CD44 ⁺ CD24 ^- on the surface of lung cancer cells cultured in conditional 3D;

Figure 3 is a comparison chart of the sphere formation rate of A549 cells between the 3D-3L group and other groups;

Figure 4 is a comparison of the invasion ability of cells in the 3D-3L group and other groups;

Figure 5 is a comparison chart of drug resistance between the 3D-3L group and other groups.

Detailed ways

1. Materials

Human lung adenocarcinoma cell line A549: purchased from the Cell Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and preserved by our laboratory;

10% fetal bovine serum: purchased from Hyclone Company of the United States;

RPMI-1640 complete medium: purchased from Hyclone Company of the United States;

Recovery solution: Cell Recovery Solution, 354253, purchased from BD Company in the United States;

BME (Eagle basal medium): purchased from BD Company in the United States;

PBS: Phosphate buffered saline (1×), 0.0067M (PO ₄ );

Neutral metalloproteolytic enzyme: Dispase, 354235, purchased from BD Company of the United States;

Insulin-like growth factor-1 ( IGF-1), purchased from Proteck;

Fibroblast growth factor (FGF) was purchased from Proteck.

2. Method

(1) Cell culture

Six cell groups were set up, namely high-concentration cytokine group on day 0 (3D-H), low-concentration cytokine group on day 0 (3D-L), high-concentration cytokine group on day 3 (3D-3H), Low concentration cytokine group (3D-3L), original 3D culture group (3D) and 2D control group (2D) on day 3. This is the 3D culture group.

A549 cells were cultured in RPMI 1640 complete medium containing 10% fetal bovine serum in an incubator at 37°C, 5% CO ₂ , and 90% relative humidity. Take the cell suspension in the logarithmic growth phase, inoculate 200 μL of 1× ¹⁰⁴ cells/well in an ordinary 96-well plate, then discard the old medium, wash twice with PBS, add 0.25% trypsin to digest, room temperature for 1~ After 2 minutes, observe the changes in cell morphology under the microscope. When the cell gap becomes larger and the protrusions of adherent cells shrink, discard the trypsin, add RPMI 1640 complete medium containing 10% fetal bovine serum to terminate the digestion, and resuspend the cells by pipetting. Subcultured and recovered cells were used as the 2D control group.

Take the A549 logarithmic growth phase cell suspension, adjust the cell concentration to 5× ¹⁰⁴ cells/ml, take 200 μL/well and add it to a 96-well plate that has been covered with BME. On the 7th day, digest the BME with Dispase and recover The cells are recovered from the liquid, which is the 3D culture group.

On day 0 and day 3 of 3D culture, insulin-like growth factor-1 (IGF-1) containing 100ng/ml and 20ng/ml FGF (high concentration group), 50ng/ml IGF-1 and 20ng /ml of FGF (low concentration group) in RPMI-1640 complete culture medium, change the medium for the cells and continue to culture the cells until the 7th day.

Wherein on the 3rd day, with the RPMI-1640 complete culture medium that contains the IGF-1 of 50ng/ml and the FGF of 20ng/ml to give A549 cell medium change medium, continue to cultivate 4 days; Digest BME through neutral metalloproteinase, recover The A549 cells recovered from the liquid were the conditional 3D group.

(2) PCR screening of cytokines

1. Extraction of total RNA by Trizol method:

① Collect the cultured cells, add an appropriate amount of Trizol (1ml Trizol/1×10 ⁶ cells), pipette and transfer into an enzyme-free Eppendorf tube. Make sure the cells are completely lysed and the fluid is mostly clear.

② Put the above-mentioned Eppendorf tube at room temperature for 5 minutes, add chloroform (200 μl/1ml Trizol), mix it upside down, let it stand at room temperature for 10 minutes, centrifuge at 12000g for 15 minutes at 4°C.

③ Carefully pipette the upper aqueous phase into another new enzyme-free Eppendorf tube, add an equal volume of isopropanol, mix up and down, let stand at room temperature for 10 minutes, 4°C, 12000g, centrifuge for 10 minutes. Discard the supernatant.

④ Wash the precipitate with 75% ethanol (1ml/1ml Trizol), centrifuge at 7500g at 4°C for 5min, discard the supernatant, and let stand at room temperature for several minutes to allow the precipitate to dry naturally. Add an appropriate amount of DEPC-treated DDW to dissolve it, and store it at -80°C for later use.

⑤ UV spectrophotometry was used to measure the concentration and purity of RNA; 1% agarose gel electrophoresis was used to detect the integrity of RNA.

2. Synthesize cDNA for real-time quantitative PCR:

3. Add sample

a. Carefully open the membrane on the PCR Array.

b. Add 20μL of the mixture to each well corresponding to the PCR Array.

c. Carefully close the lid to seal the PCR Array.

d. Put the prepared PCR Array on ice before setting the PCR program.

e. After the real-time quantitative PCR program is set, put the PCR Array in the real-time quantitative PCR instrument for PCR reaction. The set procedure is as follows:

(3) Detection of stem cell-specific antibodies by flow cytometry

1 Labeling of Flow Cytometry Antibodies

Collect 6 groups of cells cultured for 3, 7, 11, 14, and 20 days, make cell suspensions respectively, adjust the cell concentration to 1×10 ⁵ cells/100 μL, and label with antibody markers CD24, CD326, and CD44 respectively. Label FITC-IgG2b, PE-IgG1, and APC-IgG1 as controls, and incubate at 4°C in the dark for 30 minutes. After the cells were washed twice with PBS, 100 μL of 1% paraformaldehyde was added for fixation, and then the cells could be detected by flow cytometry.

2 Detection by flow cytometry

① Open the front cover, select the appropriate nozzle, and implant the nozzle with 0 rings in the flow detection cell.

② Turn on the stabilized power supply, the main power supply of the instrument and the laser in turn, and run the software interface BD FACSDiva software in the workstation.

③ Find the "Instrument" item in the software interface menu and select "Fluidics Start up" option. Then execute the "Turn on stream" command to turn on the liquid flow.

④ After the liquid flow gradually stabilizes, close the front cover of the flow cytometer, and then start the quality control test. If the quality control test is normal, you can start the machine for sample measurement.

⑤ Use a wavelength of 488nm to detect the laser, place the sample loading tube on the stage, select Load to start loading the sample, and adjust the liquid flow rate according to the standard of 100-300 particles per second passing through the detection cell. Adjust the parameter values of FSC and SSC so that most of the particles fall into the central rectangular gate of the scattergram as much as possible, and then adjust the threshold voltages of different fluorescent channels according to the same type control, and control the compensation between different fluorescent channels to make the best possible To reduce mutual interference, select Record to record the detection results of 10,000-20,000 sample cells, perform immunophenotyping detection, and identify stem cells.

(4) In vitro sphere formation experiment

Six groups of cultured cells were inoculated into untreated 6-well plates at a density of 5000/3ml MFM (DMEM/F12 cell culture medium containing bFGF, EGF, BSA and Insulin), and the sphere formation rate of the cells was observed after 7 days.

(5) In vitro invasion test

Matrigel and Transwell were used to construct invasion chambers for the separation of highly invasive and low invasive cell populations (polycarbonate membrane diameter 6.smm, pore size 8.0 μm). Spread 20 μL/well of Matrigel with a concentration of 1.17 mg/ml on the polycarbonate membrane in the chamber of the Transwell culture plate, and let Matrigel polymerize into a gel at 37°C for 30 minutes. A single cell suspension of 1.0×10 ⁵ cells/ml was prepared in a serum-free medium by a conventional method, and 200 μL of the cell suspension was added to each well. Add 400 μL fetal bovine serum to the lower chamber of the Transwell culture plate, and incubate for 48 hours at 37°C, 5% CO ² , and 95% humidity. After 48 hours, take out the Transwell plate and fix it with methanol for 30 minutes, stain with 1% crystal violet for 30 minutes, count and image under a 200-fold upright microscope.

(6) Drug Reactivity Experiment

A549 cells cultured in 3D, 3D and 2D conditions were made into single-cell suspensions, and the cell density was adjusted to 1×10 ⁵ /ml, and seeded in 96-well plates, 100 μL per well, and only culture solution was added to blank control wells. Place in a 37°C, 5% _CO2 saturated humidity incubator. After the cells are completely attached to the wall, add 100 μL of cisplatin-containing medium the next day, with a concentration gradient of 16 μg/ml, 8 μg/ml, 4 μg/ml, and 2 μg/ml. ml, 1 μg/ml, the total volume of each well is 200 μL. Make 3 parallel wells in each group, add 20 μL of 5 mg/ml MTT to each well after 48 hours, continue to incubate for 4 hours, centrifuge the culture plate, discard the supernatant, add 150 μL of DMSO to each well, shake gently with a micro shaker for 10 minutes, and dissolve the crystals. The absorbance (OD value) of each well was detected at a wavelength of 490 nm on a microplate reader. The cell survival rate was calculated according to the following formula, cell survival rate = OD value of the experimental group/OD value of the blank group × 100%, and the MTT experiment was repeated 3 times on different days. Cell inhibition rate=1-cell survival rate, and linear regression was made between the logarithmic value of the drug concentration and the cell growth inhibition rate to obtain the half inhibitory concentration IC50 of cisplatin on cells.

3. Results

1. The concentration of IGF-1 and FGF in the cells cultured in 3D was significantly higher than that in the 2D group

Using PCR chip detection, in A549 cells cultured in 3D cells, it was found that the expression levels of various cytokines that promote stem cell growth were increased, especially the changes of IGF-1 (20.12) and FGF (10.24), see Figure 1.

2. The positive rate of stem cell markers on the surface of lung cancer cells cultured in conditional 3D was significantly increased

The CD44, CD24 and CD326 on the surface of A549 cells in the 6 groups were detected by flow cytometry, and the results showed that the ratios of CD44 ⁺ , CD326 ⁺ , CD44 ⁺ CD24 ^- and CD326 ⁺ CD44 ⁺ CD24 ^- on the surface of A549 cells in the 3D-3L group were significantly higher than 3D culture 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 see Figure 2, CD24 ^- ratio was not significantly different from 3D (93.13±3.62% vs 88.20±7.21%).

3. The sphere formation rate of A549 cells in 3D-3L group was higher than that in other groups

Stem cells can form monoclonal microspheres, and those with a diameter of more than 70 μm are considered a monoclonal microsphere. After A549 cells were cultured for 7 days, the sphere formation rate of A549 cells in the 3D culture group was higher than that of the conventional 3D culture group after adding cytokines that promote stem cell growth in the 3D culture, and the sphere formation rate of A549 cells in the 3D-3L group was higher than that of other groups, significantly Compared with conventional 3D culture, the sphere formation rate is higher (30.58±1.44% vs 19.20±0.76%, P <0.01), and after two passages, the sphere formation rate is still higher than other groups, as shown in the table, indicating The improved condition 3D-3L group cultured a higher proportion of stem cells.

4. The invasive ability of 3D-3L group cells was higher than that of other groups

The A549 cells of 6 groups were extracted and inoculated in the upper chamber of 24-well transwell plate, and cultured for 48 hours without serum. The invasive ability of conventional 3D cell culture is strong, and the invasive ability of cells in 3D-3L group is higher than that of other groups, which is stronger than that of conventional 3D cultured cells (363.33±56.37 vs 167.33±29.69, P<0.05, see Figure 4 , which was 2.17 times that of conventional 3D cultured cells and 5.17 times that of conventional 2D cultured cells.

5. The drug resistance of the 3D-3L group was significantly higher than that of other groups

Three groups of cultured A549 cells were extracted and inoculated in 96-well plates, and 8 μg/ml cisplatin was added at the same time, and the survival status of the two cultured cells was detected by MTT method. The results showed that at 48 hours after cisplatin was added to the cells in the three groups, the cell survival rate in the conditional 3D group was higher than that in the 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, showing that cells cultured in conditional 3D have stronger drug resistance. At 48h, the IC50 of A549 cultured in conditioned 3D, 3D and 2D on DDP were (15.58±1.25μg/ml vs 8.36±0.96μg/ml, 15.58±1.25μg/ml vs 5.52±0.74μg/ml ml, P <0.05).

In summary, A549 cells cultured in conditioned 3D form clonal clusters in vitro similar to tumor masses in vivo, which is closer to the three-dimensional growth state in vivo. BME is used to prevent A549 cells from contacting each other, and factors that promote stem cell growth are added. , to make the isolation of stem cells more stable and to achieve efficient expansion. For the detection of cell phenotype, it was found that the expression levels of stem cell markers C326 and CD44 in the A549 cells derived from the cell culture method of the 3D-3L group increased, while CD24, which is a marker of mature differentiation, decreased significantly, and the expression levels of stem cell markers CD326 ⁺ CD44 were significantly decreased. The proportion of ⁺ CD24 ^- cells was significantly increased, suggesting that the conditional 3D cell culture method cultivated a higher proportion of lung cancer stem cells. Stem cell function experiments showed that the in vitro sphere formation rate of A549 cells cultured in conditional 3D cells was significantly increased, and the invasion ability was enhanced, and the expression level of stem cell-related genes in A549 cells cultured in conditional 3D cells was significantly increased, indicating that the conditional 3D cell culture method can be used in In vitro isolation and expansion of NSCLC cells with stem cell characteristics and functional activity. Further pharmacodynamic experiments confirmed that the resistance of A549 cells cultured in conditioned 3D cells to anticancer drugs increased, and the half inhibitory concentration of cisplatin increased to 1.86 times that of conventional 3D cultured cells and 2.82 times that of conventional 2D cultured cells. The results of in vivo experiments are similar, suggesting that the conditional 3D cell culture method can screen out tumor stem cells resistant to anticancer drugs, which provides an experimental basis for establishing a more effective innovative drug screening system.

Claims (3)

1. A lung cancer stem cell conditional 3D culture method, characterized in that: Human lung adenocarcinoma cell line A549 was cultured in RPMI-1640 complete medium containing 10% fetal bovine serum in an incubator, and the logarithmic growth phase cell suspension was taken, and the cell concentration was adjusted to 5× ¹⁰⁴ cells/ ml, add 200 μL/well to a 96-well plate that has been covered with 50 μL/well BME; on the third day, grow with fibroblasts containing 25-75 ng/ml insulin-like growth factor-1 and 10-30 ng/ml The RPMI-1640 complete medium of the factor was used to change the medium of A549 cells, and the culture was continued for 4 days; the BME was digested by neutral metalloproteinase, the recovered solution was used to recover the A549 cells, and finally the cell identification was carried out. 2. The conditional 3D culture method for lung cancer stem cells according to claim 1, characterized in that: the RPMI-1640 complete medium for A549 cell replacement mainly contains 10% fetal bovine serum and 50ng/ml insulin-like growth factor -1 and 20ng/ml of fibroblast growth factor. 3. The lung cancer stem cell conditional 3D culture method according to claim 1, characterized in that: the complete medium is cultivated in an incubator, and the complete medium after the A549 cell is changed to continue to cultivate is placed at 37 °C, 5% CO ₂ , and 90% relative humidity.