Primary cell culture method for constructing lung cancer cell line by using micro-tumor PTC (Positive Temperature Coefficient) in-vitro model

Abstract

The invention provides a primary cell culture method for constructing a lung cancer cell line by using a micro-tumor PTC (Positive Temperature Coefficient) in-vitro model. PTC is a 3D tumor microsphere which is formed by performing digestion pretreatment on a tumor tissue from a patient and performing self-assembly again in an in-vitro liquid culture medium. The PTC micro-tumor comprises a plurality of cell types such as tumor cells, fibroblasts and immune cells derived from epithelium in original tissues, and has the advantages of short self-assembly period, high culture success rate and the like. The human-derived lung cancer cell line is constructed by utilizing PTC, so that the time cost can be shortened while bacterial pollution is reduced, the culture success rate is improved, and the characteristics and multiplication capacity of a primary lung cancer cell line are reserved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to primary cell culture of a lung cancer cell line. Background Technology

[0002] Lung cancer has the highest mortality rate among malignant tumors worldwide and is also the leading cause of death from malignant tumors in my country. Tumor cell lines are fundamental in vitro tumor models, playing a crucial role in research on tumorigenesis and development mechanisms, drug resistance mechanisms, and the screening of novel biomarkers. Traditional cell lines, after multiple in vitro passages, have lost many personalized molecular characteristics and cannot meet the ever-evolving research needs. Therefore, establishing patient-derived primary tumor cell lines is essential. Commonly used primary cell culture methods include tissue block culture and digestion culture. Tissue block culture is relatively simple to operate, and pretreatment does not damage cells, but culture is relatively time-consuming, and the different growth capacities of different cells may result in the final cultured cells not being the target cells. Digestion culture uses digestive enzymes to pretreat tissue blocks into single-cell suspensions. Its advantage is that monolayer cells can more easily absorb nutrients and expel metabolic waste, resulting in faster growth. However, the operation is more cumbersome, and the digestion process may damage cells and cannot avoid interference from other cells. Therefore, developing new technologies for the construction and culture of primary cells, especially primary tumor cell lines, is of great significance.

[0003] PTC (Proton Tumor Cells) is a novel technique for constructing in vitro tumor models. PTCs are 3D tumor microspheres formed by the self-assembly of patient-derived tumor tissue through digestion pretreatment and in vitro liquid culture medium. PTC microtumors contain various cell types from the original tissue, including epithelial-derived tumor cells, fibroblasts, and immune cells, offering advantages such as short self-assembly cycles and high culture success rates. This invention, based on PTC technology, utilizes PTCs to construct human lung cancer cell lines, reducing bacterial contamination, shortening time and costs, improving culture success rates, and preserving the characteristics and proliferative capacity of primary lung cancer cell lines. Summary of the Invention

[0004] This invention provides a primary cell culture method for constructing lung cancer cell lines using the PTC microtumor model. This method can shorten the cell line culture time and improve the cell line culture success rate.

[0005] The technical solution of the present invention:

[0006] Step 1: Construct a PTC model derived from lung cancer patients;

[0007] Step 2: Constructing primary lung cancer cell lines based on the PTC microtumor model

[0008] Step 3: Identify the cultured cell line as an epithelial-derived lung cancer cell line.

[0009] Furthermore, the specific method for constructing a lung cancer PTC in step one includes the following steps:

[0010] 1.1 Fresh lung cancer patient-derived tissue was minced into a paste using sterilized medical scissors, mixed thoroughly with digestive solution, and digested at 37°C. The mixture was agitated every 15-20 minutes for a total digestion time of 30-90 minutes. The digestive solution was prepared by mixing collagenase I and collagenase IV at final concentrations of 100-200 U / mL, 200-300 U / mL, and 100-200 U / mL, respectively, and diluted with PBS.

[0012] 1.2 After digestion is terminated, the suspension is filtered through a 40 μm filter screen, the filtrate is collected, and primary lung cancer cells are obtained; or fresh pleural and peritoneal fluid specimens are centrifuged to settle tumor cells, resuspended, and primary lung cancer cells are obtained.

[0013] 1.3 Centrifuge the filtrate from step 1.2, discard the supernatant, resuspend the precipitate in lung cancer PTC-specific medium, and inoculate into low-adsorption plates at 37°C.

[0014] The lung cancer PTC model was obtained by culturing under 5% CO2 conditions and changing the culture medium every 2-4 days.

[0015] Furthermore, the specific method for constructing lung cancer cell lines based on the PTC model in step two includes the following steps:

[0016] 1.1 Take cultured PTC microtumor cells, centrifuge to remove supernatant, resuspend in adherent culture medium, mix thoroughly, and place in a culture dish. Incubate at 37℃ and 5% CO2, changing the culture medium every 2-4 days. The adherent culture medium is prepared by adding 5%-20% fetal bovine serum to PTC culture medium.

[0017] 1.2 Once the adherent proliferating cells have reached a confluence of 80-90%, they are digested with 0.25% trypsin and passaged 1:3. Stably passaged cells are then identified and cryopreserved.

[0018] Furthermore, the specific method for identifying the cultured cell line as an epithelial-derived lung cancer cell line in step three includes the following steps:

[0019] 1.1 After sterilizing the cell slides, place them into 24-well plates. Digest the adherent cells with 0.25% trypsin, resuspend them in culture medium, and add them to the wells, with 10,000-50,000 cells per well. After culturing for 24-48 hours, remove the cell slides and fix them with 4% paraformaldehyde for 1 hour.

[0020] 1.2 Remove the slides and wash them three times with PBS, soaking for 2 minutes each time. Add 1% Triton 100 and treat at room temperature for 20 minutes. After washing with PBS, incubate the slides at room temperature in 3% H2O2 for 10 minutes.

[0021] 1.3 Wash 3 times with PBS, soaking for 2 min each time, then block with 10% normal goat serum at room temperature for 30 min-60 min, add primary antibody (CK8 / 18), and incubate overnight at 4 degrees Celsius;

[0022] 1.4 Incubate with secondary antibody (goat anti-rabbit / mouse HRP-labeled polymer) for 30 min, wash 3 times with PBS, add DAB for 3-10 min for color development, counterstain with hematoxylin for 1-2 min, and stop when the cell nucleus turns blue.

[0023] 1.5 The cell smears were soaked and dehydrated sequentially with 50% alcohol for 5 min, 75% alcohol for 5 min, 85% alcohol for 5 min, 95% alcohol for 5 min, anhydrous ethanol for 5 min, xylene I for 10 min, and xylene II for 10 min.

[0024] 1.6 Add a drop of neutral resin, cover with a coverslip, and examine under a microscope after the slide is prepared. Attached Figure Description

[0025] Figure 1 Patient-derived lung cancer PTC model

[0026] Figure 2 Lung cancer cell lines constructed based on the PTC model Detailed Implementation

[0027] The technical solution of the present invention will be further described below, but the present invention is not limited thereto.

[0028] Example 1

[0029] Fresh lung cancer patient-derived tissue, approximately the size of a soybean and weighing 0.1g, was collected and minced into a paste using sterilized medical scissors. Digestive fluid was added and thoroughly mixed. Digestion was carried out at 37°C, with the mixture being agitated every 15 minutes for a total digestion time of 60 minutes, dispersing the tissue mass into single cells. After digestion was complete, the suspension was filtered through a 40μm filter, the filtrate was collected, centrifuged, and the supernatant was discarded to obtain primary lung cancer cells. These cells were resuspended in a lung cancer-specific culture medium and cultured at 10... 5 / cm 2 Cells were seeded at a low adsorption surface in culture dishes and cultured at 37°C under 5% CO2 conditions. The culture medium was changed every 2-3 days, and cell morphology was observed and recorded under a microscope until a stable cell microsphere structure was formed.

[0030] Example 2

[0031] After centrifuging to remove the supernatant, the cultured PTC microspheres were resuspended in adherent medium, thoroughly mixed, and placed in culture dishes. Incubation was carried out at 37°C and 5% CO2, with the medium changed every 2-4 days. Once the adherent cells reached 80-90% confluence, they were digested with 0.25% trypsin and passaged 1:3. Stably passaged cells were identified as epithelial-derived lung cancer cell lines using standard cell slide and fixation staining methods.

[0032] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A method for primary cell culture to construct lung cancer cell lines using a microtumor PTC in vitro model, wherein, The lung cancer cell line culture method only involves construction using PTC, and is characterized by including the following steps: Step 1: Collect fresh samples to construct a PTC model derived from lung cancer patients; Step 2: Constructing primary lung cancer cell lines based on the PTC microtumor model Step 3: Identify the cultured cell line as an epithelial-derived lung cancer cell line.

2. The method according to claim 1, characterized in that, The available experimental specimens in step one include, but are not limited to, surgical tissue, puncture tissue, or pleural and peritoneal fluid.

3. The PTC construction method according to claim 1, characterized in that, Includes the following steps: Step 1: Using physical shearing or enzymatic digestion, the experimental specimens derived from lung cancer patients are dispersed into single cells; Step 2: In a lung cancer-specific culture medium, epithelial tumor cells, fibroblasts, and immune cells self-assemble to form microspheres, thus obtaining the PTC model.

4. The lung cancer cell line for identification and culture according to claim 1, characterized in that, Includes the following steps: Step 1: Stable passaged lung cancer cells are prepared into identification samples for subsequent experiments by cell smearing. Step 2: After washing with PBS, add 1% Triton 100, treat at room temperature for 20 min, and incubate with 3% H2O2 at room temperature for 10 min; Step 3: Non-specific blocking is performed using 10% normal goat serum at room temperature, followed by the addition of primary and secondary antibodies for an immune response; Step 4: Add DAB to stain the cells, counterstain with hematoxylin for 1-2 minutes until the cell nuclei turn blue, then stop. Afterwards, soak the slides in ethanol (50%-75%-85%-95%-100%) and xylene in sequence to dehydrate them, and then add neutral resin to mount the slides. Step 5: Microscopic examination.

5. The lung cancer-specific culture medium according to claim 3, characterized in that, Using Advanced DMEM (dulbecco's modified eagle medium) as the substrate, additional components included 1 mM hydrogen ion buffer (HEPES), 1×GlutaMAX, 100 U / mL penicillin-streptomycin, 1×B27, 1×insulin-transferrin-selenoethanolamine (ITS-X), 1×non-essential amino acids, 40 ng / mL epidermal growth factor (EGF), 20 ng / mL fibroblast growth factor (FGF-basic), 10 μM ROCK inhibitor (Y-27632-07), 30 ng / mL hepatocyte growth factor (HGF), and 20 ng / mL human recombinant protein (MSP).

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