Method for culturing lung fibroblasts

Abstract

The invention relates to a lung fibroblast culture method, and belongs to the technical field of cell culture. Comprising the following steps: S1, organization preparation; s2, tissue treatment; s3, primary culture: the treated tissue blocks are attached to a culture dish, a culture solution is added, the culture solution is a primary culture system, and culture is performed in an incubator for several days until the culture dish is full of fibroblasts; s4, subculture: digesting the cells subjected to primary culture by adopting conventional 0.25% trypsin, then adding a culture solution which is a subculture system for subculture, and carrying out subculture to a fourth generation; and S5, cell identification. According to the invention, primary and subculture are respectively purified and amplified by virtue of a double culture system of fibroblasts, so that the purification of target cells is facilitated, the cell quality is good, a low-serum growth additive is used as a nutrient substance, the low-serum growth additive is more easily utilized by the cells, the proliferation of the cells is facilitated, and good proliferation activity and phenotypic stability are still kept when the cells are subcultured to the fourth generation; no obvious differentiation phenomenon occurs.

Description

Technical Field

[0001] This invention relates to a method for culturing lung fibroblasts, belonging to the field of cell culture technology. Background Technology

[0002] Pulmonary fibrosis is the end-stage manifestation of interstitial lung disease, characterized by fibroblast proliferation and massive extracellular matrix deposition, leading to the destruction of lung tissue structure. Idiopathic pulmonary fibrosis (IPF) is a common clinical type, mainly affecting middle-aged and elderly individuals, and its etiology remains unclear. Occupational or home environmental pollution, medications, radiation therapy, high-concentration oxygen therapy, smoking, underlying diseases, and genetic factors can all induce pulmonary fibrosis. Patients often present with dry cough, progressive dyspnea, which worsens with activity, and some may also exhibit clubbing of the fingers / toes. The disease is insidious and progresses slowly, and is prone to complications such as pulmonary hypertension, emphysema, lung cancer, and gastroesophageal reflux, seriously threatening patients' lives and health.

[0003] Excessive proliferation of fibroblasts and abnormal expression of extracellular matrix proteins are key steps in the development and progression of pulmonary fibrosis. Therefore, in vitro culture of pulmonary fibroblasts (PFs) has become an important tool for studying the pathogenesis of lung diseases and developing treatment options. Although commercially available fibroblast cell lines are available, primary and passaged fibroblasts within five generations remain the preferred research materials for studying cell growth, differentiation, and physiological and pathological regulatory mechanisms.

[0004] The main method for isolating lung fibroblasts is the tissue block adhesion method. This method yields a diverse cell population containing various cell types, including vascular endothelial cells, mesenchymal stem cells, epithelial cells, smooth muscle cells, and fibroblasts. However, this method results in low cell purity, hindering subsequent purification and leading to poor cell quality. Furthermore, the use of conventional culture systems results in slow proliferation and prolonged culture times that cause cell differentiation. Therefore, this invention provides a method for culturing lung fibroblasts. Summary of the Invention

[0005] In view of this, the present invention provides a method for culturing lung fibroblasts, which relies on a dual culture system for fibroblasts to purify and expand primary and passaged cells separately, which is conducive to the purification of target cells and results in good cell quality. At the same time, the use of low serum growth additives as nutrients makes them easier for cells to utilize, which is conducive to cell proliferation. Even after passage to the fourth generation, the cells still maintain good proliferative activity and phenotypic stability, and no obvious differentiation phenomenon is observed.

[0006] This invention provides a method for culturing lung fibroblasts, the proposed technical solution comprising the following steps: S1: Tissue preparation: Select normal tissue adjacent to the cancerous tissue after surgical removal from lung cancer patients; S2: Tissue processing: After washing away bloodstains from the prepared adjacent normal tissue, cut it into tissue blocks and rinse repeatedly with PBS solution; S3: Primary culture: Place the processed tissue blocks into a culture dish, add culture medium for primary culture, and culture in an incubator for several days until the fibroblasts cover the culture dish. S4: Subculture: Primary cultured cells were digested with 0.25% trypsin and then subcultured in culture medium. The culture medium was used as the subculture system and the cells were passaged to the fourth generation. S5: Cell identification: After passage to the third generation, the cells are identified as fibroblasts.

[0007] Furthermore, in step S3, the primary culture system is a mixture of fibroblast AOF culture medium, AOF additives, and 100X antibiotic-antifungal agent liquid.

[0008] Furthermore, the fibroblast AOF culture medium is FABM culture medium; the AOF additive is dFAS.

[0009] Furthermore, in step S2, the PBS solution includes 100 U / mL penicillin and 100 mg / L streptomycin.

[0010] Furthermore, in step S2, the size of the tissue block is 1mm × 1mm × 1mm.

[0011] Furthermore, in step S3, the diameter of the petri dish is 6 cm.

[0012] Furthermore, in step S3, the culture is carried out in an incubator under 5% CO2, 37°C and saturated humidity conditions for 10 days.

[0013] Furthermore, in step S4, the passage culture system is a mixture of human fibroblast expansion culture medium, low serum growth additive, and penicillin-streptomycin 1X.

[0014] Furthermore, the human fibroblast expansion culture medium is formerly Medium 106; the low serum growth additive is LSGS.

[0015] Furthermore, the fibroblasts were identified using vimentin and keratin immunohistochemistry.

[0016] The beneficial effects of this invention are as follows: This invention uses a dual culture system for fibroblasts, which purifies and expands primary and passaged cells separately. This further restricts the adhesion of vascular endothelial cells and epithelial cells, which is conducive to the purification of target cells and results in good cell quality. At the same time, the use of low serum growth additives as nutrients makes them easier for cells to utilize, avoiding proliferative senescence caused by a nutrient-rich environment. This promotes cell proliferation, and even after passage to the fourth generation, the cells still maintain good proliferative activity and phenotypic stability, without showing obvious differentiation. Attached Figure Description

[0017] Figure 1 This is a schematic flowchart of a method for culturing lung fibroblasts according to the present invention.

[0018] Figure 2 This is a 4× identification diagram of lung fibroblasts-P1- of the present invention.

[0019] Figure 3 This is a diagram illustrating the identification of lung fibroblasts-P3-4× in this invention.

[0020] Figure 4 This is an identification diagram of vimentin-P1-4× in lung fibroblasts according to the present invention.

[0021] Figure 5 This is an identification diagram of vimentin-P3-4× in lung fibroblasts according to the present invention.

[0022] Figure 6 This is a comparative table of lung fibroblast growth data at different generations, as presented in this invention. Detailed Implementation

[0023] The preferred embodiments of the present invention will now be described in detail. Example

[0024] According to the appendix Figure 1-5 As shown, the present invention provides a method for culturing lung fibroblasts, which includes the following steps: S1: Tissue preparation: Select normal tissue adjacent to the cancerous tissue after surgical removal from lung cancer patients; S2: Tissue processing: After washing away bloodstains from the prepared adjacent normal tissue, cut it into 1mm×1mm×1mm tissue blocks and rinse repeatedly with PBS solution containing 100U / mL penicillin and 100mg / L streptomycin. S3: Primary Culture: The treated tissue blocks were placed in 6cm diameter culture dishes, and culture medium was added for primary culture. The culture medium was the primary culture system. Culture was carried out in an incubator at 5% CO2, 37℃, and saturated humidity for 10 days, until the fibroblasts completely covered the culture dish. The primary culture system consisted of: fibroblast AOF medium + AOF additive + 100X antibiotic-antifungal agent liquid (AA) - 5 mL; the AOF medium was FABM medium, catalog number M-206-500, brand: Thermo Fisher Scientific; the AOF additive was dFAS, catalog number S-019-5, brand: Thermo Fisher Scientific. S4: Passaging: Primary cultured cells were digested with 0.25% trypsin as usual, followed by the addition of culture medium for passage culture. The culture medium was used as the passage culture system, and the cells were passaged to the fourth generation. The passage culture system consisted of Human Fibroblast Expansion Basal Medium (formerly Medium 106) + Low Serum Growth Supplement (LSGS) + Penicillin-Streptomycin 1X. The Human Fibroblast Expansion Basal Medium was formerly Medium 106, catalog number M106500, brand: Thermo Fisher Scientific. The Low Serum Growth Supplement was LSGS, catalog number S00310, brand: Thermo Fisher Scientific.

[0025] S5: Cell Identification: After passage to the third generation, fibroblast identification was performed using vimentin and keratin immunohistochemistry, as per the instruction manual. Figure 2-5 As shown. Example

[0026] Cell proliferation capacity detection Resuspend P0 to P3 generation fibroblasts and seed them into 6-well plates at a density of 2 × 10⁴ cells / mL. For 24-well plates, seed 1 mL per well. Every 2 days, remove culture medium from 3 wells, count the cells in each well, calculate the average value, and plot a growth curve. Results are shown in the attached instruction manual. Figure 6 As shown, P < 0.05 indicates a significant difference compared to the reference; P < 0.001 indicates a highly significant difference compared to the reference. The P0 cell proliferation curve of this invention is flat, and P1-P3 show a significant increase in number throughout the entire culture period. In contrast, the P0 cells in the reference have a longer latency period, and the overall proliferation of P1-P3 is slower than that of this invention, without significant logarithmic growth, and the cell number remains relatively low. Furthermore, both are significantly different from this invention (P < 0.05). This indicates that the culture system of this invention is superior to existing culture systems, more effectively supporting the expansion of lung fibroblasts and facilitating the construction of clinical-grade cell models for disease research using fibroblasts.

[0027] In summary, compared with existing technologies, this invention uses a dual culture system for fibroblasts, purifying and expanding primary and passaged cells separately. This further restricts the adhesion of vascular endothelial cells and epithelial cells, which is beneficial for the purification of target cells and results in good cell quality. At the same time, the use of low-serum growth additives as nutrients makes them easier for cells to utilize, avoiding proliferative senescence caused by a nutrient-rich environment, and promoting cell proliferation. Even after passage to the fourth generation, cells maintain good proliferative activity and phenotypic stability, without showing obvious differentiation.

[0028] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention; the actual structure is not limited thereto. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the invention, such designs should fall within the protection scope of the present invention.

Claims

1. A method for culturing lung fibroblasts, characterized in that: Includes the following steps: S1: Tissue preparation: Select normal tissue adjacent to the cancerous tissue after surgical removal from lung cancer patients; S2: Tissue processing: After washing away bloodstains from the prepared adjacent normal tissue, cut it into tissue blocks and rinse repeatedly with PBS solution; S3: Primary culture: Place the processed tissue blocks into a culture dish, add culture medium for primary culture, and culture in an incubator for several days until the fibroblasts cover the culture dish. S4: Subculture: Primary cultured cells were digested with 0.25% trypsin and then subcultured in culture medium. The culture medium was used as the subculture system and the cells were passaged to the fourth generation. S5: Cell identification: After passage to the third generation, the cells are identified as fibroblasts.

2. The method for culturing lung fibroblasts according to claim 1, characterized in that: In step S3, the primary culture system is a mixture of fibroblast AOF culture medium, AOF additives, and 100X antibiotic-antifungal agent liquid.

3. The method for culturing lung fibroblasts according to claim 2, characterized in that: The fibroblast AOF culture medium is FABM medium; the AOF additive is dFAS.

4. The method for culturing lung fibroblasts according to claim 1, characterized in that: In step S2, the PBS solution includes 100 U / mL penicillin and 100 mg / L streptomycin.

5. The method for culturing lung fibroblasts according to claim 1, characterized in that: In step S2, the size of the tissue block is 1mm × 1mm × 1mm.

6. The method for culturing lung fibroblasts according to claim 1, characterized in that: In step S3, the diameter of the petri dish is 6 cm.

7. The method for culturing lung fibroblasts according to claim 1, characterized in that: In step S3, the cells are cultured in an incubator with 5% CO2, 37°C and saturated humidity for 10 days.

8. The method for culturing lung fibroblasts according to claim 1, characterized in that: In step S4, the passage culture system is a mixture of human fibroblast expansion culture medium, low serum growth additive, and penicillin-streptomycin 1X.

9. A method for culturing lung fibroblasts according to claim 8, characterized in that: The human fibroblast expansion medium is formerly Medium 106; the low serum growth additive is LSGS.

10. A method for culturing lung fibroblasts according to claim 1, characterized in that: The fibroblasts were identified using vimentin and keratin immunohistochemistry.

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