Method for constructing immortalized thymoma primary epithelial cells

Abstract

The invention relates to the technical field of biology, and discloses a method for constructing immortalized thymoma primary epithelial cells. The method comprises the following steps: step 1, tissue dissociation and mixed cell population acquisition; step 2, performing differential liquid change to remove immune cells; step 3, immortalization treatment; step 4, carrying out monoclonal screening; step 5, amplification and verification; and step 6, passage and storage. The method is mainly used for establishing a stable immortalized cell line in a laboratory for subsequent scientific research. The specific application comprises tumor biology research; anti-tumor drug screening; cancer-related gene function research; and cell signal transduction mechanism research and the like. For tumors which do not have mature cell lines or are difficult to establish primary cell lines, such as thymoma, the invention is beneficial to improving the success rate of establishing immortalized tumor primary cell lines and promoting the development of subsequent basic scientific research.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and more specifically, to a method for constructing immortalized thymoma primary epithelial cells. Background Art

[0002] At present, the methods for constructing primary tumor cell lines mainly include isolating and purifying primary epithelial cells from tumor tissues, and then immortalizing these cells (such as transfecting SV40 large T antigen or hTERT, etc.). However, the separation and purification process of primary cells is complicated and time-consuming. In tumors, fibroblast proliferation is often dominant, and tumor epithelial cells are difficult to separate. Since tumor tissue contains many different types of cells, including immune cells, fibroblasts, stromal cells, etc., how to efficiently and accurately separate tumor epithelial cells is one of the primary challenges in constructing immortalized tumor primary epithelial cells. The purification process of primary tumor epithelial cells usually has problems such as many impurity cells, poor activity of primary epithelial cells, and difficulty in proliferation and cloning. This makes it very difficult to passage and amplify primary epithelial cells, and it is difficult to obtain sufficient numbers of high-purity cells for immortalization or for subsequent research. These challenges are more obvious in thymomas with a higher degree of inertness. Therefore, in view of the current situation that it is difficult to purify and expand primary tumor epithelial cells, especially in relatively indolent tumors such as thymoma, there is an urgent need to develop an immortalization construction method with strong operability and high success rate. Summary of the invention

[0003] The purpose of the present invention is to provide a method for purifying and immortalizing primary tumor epithelial cells with a higher success rate and stronger operability, and to introduce thymoma as an example. By proposing a new process and strategy, the bottlenecks of difficult purification, poor activity and difficulty in cloning and amplification of primary tumor cells are solved.

[0004] In order to achieve the above invention objectives, this application adopts the following technical solutions:

[0005] In a first aspect, the present application provides a method for constructing immortalized thymoma primary epithelial cells, comprising:

[0006] Step 1: Tissue dissociation and mixed cell population acquisition: obtain fresh samples from thymoma tissue, use digestive enzymes to dissociate the tissue, obtain a mixed cell population suspension and plate it for culture;

[0007] Step 2: Use differential fluid replacement to remove cells that are difficult to survive in the short term in the mixed cell population and remove immune cells;

[0008] Step 3: Immortalization treatment: continue to culture the remaining mixed cell population for 72 hours, and after the cell state is stable, perform immortalization treatment at the same time;

[0009] Step 4: Monoclonal screening: After the immortalized cell population has gradually and stably proliferated, the screened cells are diluted and cloned, and then passaged and amplified to gradually purify and screen out high-purity thymoma primary epithelial cell clones;

[0010] Step 5: Amplification and verification: functional verification of the amplified epithelial cells, detection of epithelial cell markers and immortalization markers;

[0011] Step 6: Passaging and storage.

[0012] Furthermore, in step 1, a mixed digestion solution of 0.1% collagenase IV and trypsin (volume ratio of 1:1) is used to dissociate the tissue. After digestion, the mixed solution is filtered and the cells are washed 2-3 times with pre-cooled PBS containing 10% double antibody.

[0013] Furthermore, in step 2, the cells are incubated for 4-6 hours for the first fluid change, and the second fluid change is performed after 36-48 hours.

[0014] Furthermore, in step 3, lentivirus-mediated hTERT gene transfection is used to achieve the unlimited proliferation ability of cells.

[0015] Furthermore, in step 5, epithelial cell-specific markers are detected using immunofluorescence staining or immunohistochemistry.

[0016] Furthermore, in step 5, total cell RNA is extracted, RT-PCR is performed to detect the expression level of the hTERT gene, and the cDNA of hTERT is amplified using specific primers to analyze the success rate of immortalization. If the RT-PCR results show that hTERT expression is high and stable, immortalization can be considered successful.

[0017] Furthermore, in step 6, the epithelial cell clones that have passed functional verification are continued to be subcultured, the medium is changed every 3-4 days, and subculture is performed when the cell confluence reaches 70-80%.

[0018] Furthermore, in step 6, the expanded cells are collected in the logarithmic growth phase, and cryopreserved using a cryopreservation solution (10% DMSO + 90% FBS), with 1×10 6 cells per tube, pre-frozen at -80°C for 2 hours in a gradient cooling box, and then transferred to liquid nitrogen for long-term storage.

[0019] In a second aspect, the present application provides applications of the method for constructing immortalized thymoma primary epithelial cells described in the first aspect, including applications in tumor biology research, anti-tumor drug screening, cancer-related gene function research, and cell signal transduction mechanism research.

[0020] Existing methods for purifying and immortalizing primary tumor cells have significant limitations, especially in relatively inert tumor tissues such as thymoma, where the number of primary epithelial cells is small and the proliferation ability is poor, making it difficult to purify or the number after purification is small and the activity is low, making it difficult to amplify and obtain cells of sufficient quantity and purity for subsequent experiments. The present invention is based on the following two principles: avoiding complex purification processes: by simultaneously immortalizing mixed cell populations, the cell purification steps can be simplified and the loss of cells in the initial processing can be reduced; using clone screening to improve purity: primary tumor cells are easier to be screened for monoclonal clones after immortalization, and a sufficient number of high-purity tumor epithelial cells can be finally obtained, overcoming the difficulty of initial separation and purification.

[0021] In summary, this application has the following beneficial effects:

[0022] (1) Simplify the cell purification process: no need for complex primary cell purification, reduce cell loss, and improve the success rate of immortalization.

[0023] (2) Enhanced expansion and passage capabilities: Immortalized epithelial cells proliferate stably and can achieve long-term passage and expansion, facilitating subsequent research.

[0024] (3) Strong applicability: It is applicable to various tumor types and other normal tissues, especially in other tumor tissues that are difficult to purify, such as thymomas with strong inertness, and has broad application prospects.

[0025] (4) Cost and time savings: It reduces the multiple purification and screening processes, shortens the time for cell line construction, and reduces experimental costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : Primary adherent cell culture after thymoma tissue dissociation;

[0027] Figure 2 : The proliferation status of immortalized primary tumor cells after culture for different time periods. DETAILED DESCRIPTION

[0028] The technical scheme and effects of the present application are further described in detail below in conjunction with the embodiments. It is to be understood that the specific embodiments described herein are only used to explain the invention, rather than to limit the invention. It should also be noted that the materials used in the following embodiments are commercially available unless otherwise specified.

[0029] Example 1: Method for constructing immortalized thymoma primary epithelial cells

[0030] Step 1: Tissue dissociation and mixed cell population acquisition

[0031] (1) Tissue collection and preliminary processing:

[0032] Obtain fresh samples from surgically removed tumor tissue from patients with thymoma. Cut the tissue into approximately 1 cm 3 The small pieces were removed and placed in pre-cooled PBS solution containing 10% double antibody.

[0033] Prepare a mixed digestion solution of 0.1% collagenase IV and trypsin (volume ratio of 1:1) and add it to the tissue block to fully immerse it.

[0034] Shake at 120 rpm for 30 min on a 37°C constant temperature shaker to ensure uniform enzymatic hydrolysis.

[0035] Observe the tissue digestion. If the lumpy tissue has not been completely dissociated, the digestion time can be extended to 1 hour.

[0036] (2) Filtration and single cell suspension preparation:

[0037] After digestion is complete, filter the mixture using a 70 μm cell strainer to remove undigested tissue fragments and leave a single cell suspension.

[0038] The cells were gently washed 2-3 times with pre-cooled PBS containing 10% double antibody (centrifugation at 1000 rpm for 5 minutes each time), and the supernatant was removed.

[0039] Finally, the cells were resuspended in a complete culture medium for epithelial cells, evenly divided into several portions according to the cell amount, plated in culture flasks and incubated in a cell culture incubator at 37°C for subsequent experiments.

[0040] Step 2: Differential fluid replacement to remove impurity cells such as immune cells ( Figure 1 )

[0041] After incubation for about 4 hours, change the medium for the first time and gently wash the cells 2-3 times with pre-cooled PBS containing 10% double antibody. This step can remove most of the immune cells and a small number of interstitial cells.

[0042] Add an appropriate amount of complete culture medium to continue incubating the remaining adherent cells. After 48 hours, change the medium for the second time and wash thoroughly with PBS. This step can remove cells that die due to poor condition, as well as immune cells that were not washed thoroughly in the first step.

[0043] Step 3: Immortalization: Preparation of lentivirus and cell transfection

[0044] HEK293T cells were used to produce hTERT lentivirus (pLV-hTERT-pVSV-G-pGag-Pol-puromycin). The lentiviral system consists of the pLV-hTERT plasmid carrying the hTERT gene, the auxiliary plasmids pVSV-G and pGag-Pol, and includes the puromycin resistance gene for screening. These plasmids were co-transfected into HEK293T cells, and the viral supernatant was collected 48 hours later.

[0045] Infect the mixed cell population: add hTERT lentiviral supernatant to the mixed cell culture medium, set the multiplicity of infection (MOI) to 50, that is, every 50 virus particles infect 1 cell. Add Polybrene (5 μg / mL) to treat the cells to enhance the efficiency of virus infection.

[0046] The infection was carried out in a 37°C incubator for 24 hours without changing the culture medium.

[0047] After 24 hours, the medium was replaced with fresh medium to remove uninfected viral particles.

[0048] After continuing to culture for 48 hours, puromycin was added for antibiotic selection, first with a large dose shock, and then with a small dose concentration for continuous screening for 3-7 days until the uninfected control group cells died completely and the infected group cells survived and began to proliferate.

[0049] Step 4: Monoclonal screening: dilution cloning single cell isolation and expansion

[0050] The screened cells are diluted and cloned, and diluted to the clone formation level in a culture flask or diluted to 1-2 cells per well in a 96-well plate (in this case, the clone formation is performed by dilution in a culture flask). The growth of monoclonal cells is regularly observed.

[0051] Culture in appropriate culture medium, changing the medium every 2-3 days until cell clones are formed ( Figure 2 ).

[0052] Observe the morphology of cell clones in each culture flask or well, select single clones with epithelial cell morphology, and transfer them to a 6-well plate or a new culture flask for expansion.

[0053] Step 5: Testing and Verification

[0054] Detection of epithelial cell markers:

[0055] Use immunofluorescence staining or immunohistochemistry to detect epithelial cell-specific markers, such as E-cadherin, EPCAM, CK19, etc.

[0056] Observe the staining results under a fluorescence microscope to confirm whether the cells show typical markers of epithelial cells.

[0057] Verification of hTERT gene expression:

[0058] Total RNA was extracted from cells and RT-PCR was performed to detect the expression level of hTERT gene. Specific primers were used to amplify the cDNA of hTERT and analyze the success rate of immortalization.

[0059] If the RT-PCR results show that hTERT expression is high and stable, immortalization can be considered successful.

[0060] Step 6: Passaging and storage

[0061] The epithelial cell clones that have passed functional verification are subcultured, the medium is changed every 3-4 days, and the cells are subcultured when the confluence reaches 70-80%.

[0062] Observe the cell growth curve and record the number of cell passages to ensure that the immortalized cells have the ability to be passaged for a long time.

[0063] The expanded cells were collected in the logarithmic growth phase and cryopreserved using cryopreservation solution (10% DMSO + 90% FBS). Each tube was cryopreserved with 1×10 6 The cells were pre-frozen at -80°C for 2 hours in a gradient cooling box and then transferred to liquid nitrogen for long-term storage.

[0064] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A method for constructing immortalized thymoma primary epithelial cells, characterized in that: include: Step 1: Tissue dissociation and mixed cell population acquisition: obtain fresh samples from thymoma tissue, use digestive enzymes to dissociate the tissue, obtain a mixed cell population suspension and plate it for culture; Step 2: Use differential fluid replacement to remove cells that are difficult to survive in the short term in the mixed cell population and remove immune cells; Step 3: Immortalization treatment: continue to culture the remaining mixed cell population for 72 hours, and after the cell state is stable, perform immortalization treatment at the same time; Step 4: Monoclonal screening: After the immortalized cell population has gradually and stably proliferated, the screened cells are diluted and cloned, and then passaged and amplified to gradually purify and screen out high-purity thymoma primary epithelial cell clones; Step 5: Amplification and verification: functional verification of the amplified epithelial cells, detection of epithelial cell markers and immortalization markers; Step 6: Passaging and storage.

2. The method for constructing immortalized thymoma primary epithelial cells according to claim 1, characterized in that: In step 1, a mixed digestion solution of 0.1% collagenase IV and trypsin (volume ratio of 1:1) is used to dissociate the tissue. After digestion, the mixed solution is filtered and the cells are washed 2-3 times with pre-cooled PBS containing 10% double antibody.

3. The method for constructing immortalized thymoma primary epithelial cells according to claim 2, characterized in that: In step 2, the cells are incubated for 4-6 hours for the first fluid change, and the second fluid change is performed after 36-48 hours.

4. The method for constructing immortalized thymoma primary epithelial cells according to claim 3, characterized in that: In step 3, lentivirus-mediated hTERT gene transfection is used to achieve the unlimited proliferation ability of cells.

5. The method for constructing immortalized thymoma primary epithelial cells according to claim 4, characterized in that: In step 5, epithelial cell-specific markers are detected using immunofluorescence staining or immunohistochemistry.

6. The method for constructing immortalized thymoma primary epithelial cells according to claim 5, characterized in that: In step 5, total cell RNA is extracted, RT-PCR is performed to detect the expression level of the hTERT gene, specific primers are used to amplify the cDNA of hTERT, and the success rate of immortalization is analyzed. If the RT-PCR results show that hTERT expression is high and stable, it can be considered that immortalization is successful.

7. The method for constructing immortalized thymoma primary epithelial cells according to claim 6, characterized in that: In step 6, the epithelial cell clones that have passed functional verification are continued to be subcultured, the medium is changed every 3-4 days, and subculture is performed when the cell confluence reaches 70-80%.

8. The method for constructing immortalized thymoma primary epithelial cells according to claim 6, characterized in that: In step 6, the amplified cells are collected in the logarithmic growth phase and cryopreserved using a cryopreservation solution (10% DMSO + 90% FBS), with 1×106 cells cryopreserved in each tube. The cells are pre-frozen at -80°C in a gradient cooling box for 2 hours and then transferred to liquid nitrogen for long-term storage.

9. Use of the method for constructing immortalized thymoma primary epithelial cells according to any one of claims 1 to 8, characterized in that: Including applications in tumor biology research, anti-tumor drug screening, cancer-related gene function research, and cell signal transduction mechanism research.

Images