A porcine fibroblast cell line for long-term subculture of cultured meat and its application

Abstract

The present invention discloses a porcine fibroblast cell line capable of long-term passage and its application. The present invention uses porcine fibroblast tissue as a source, obtains porcine fibroblasts from different parts through tissue block separation, performs long-term in vitro passage, and induces the cells to break through the Hayflick limit. Further, monoclonal screening is performed through limiting dilution to obtain a single-source porcine fibroblast cell line. After screening, porcine fibroblasts from two parts, ear margin fibroblasts and hind leg fibroblasts, can be long-term passaged in vitro and have the ability to secrete collagen. These cells have been deposited with the China Center for Type Culture Collection under the accession numbers CCTCC NO: C202511 and CCTCC NO: C202512. This cell line has broken through the Hayflick limit and has been passaged to 42 generations. The cells exhibit a healthy, plump, long spindle shape. Long-term passage does not affect proliferation levels, and the cell line can be used in the preparation of cell-cultured meat.

Description

Technical Field

[0001] The present invention belongs to the technical field of stem cells and biological cell lines, and in particular relates to a porcine fibroblast cell line capable of long-term passage for cell culture of meat and its application. Background Art

[0002] Fibroblasts are the primary cell type responsible for extracellular matrix (ECM) synthesis and play a crucial role in tissue repair and regeneration. During the production of cultured meat, fibroblasts provide structural support for the meat by synthesizing ECM components such as collagen and elastin. The production of cultured meat requires the formation of tissues through the proliferation of dividing and differentiated cells. However, most cells have a finite number of divisions before natural death, known as the Hayflick limit. This limits the large-scale cultivation of cell tissues in the laboratory. Therefore, obtaining adult cells capable of unlimited proliferation is crucial for the industrialization of cultured meat.

[0003] In the current research on cell-cultured meat, seed cells are mainly obtained by extracting and isolating high-purity muscle stem cells from animals such as cattle and pigs. However, after activation, these cells differentiate into myoblasts, which means they lose their stemness and cannot continue to grow permanently. The cell propagation capacity still faces many challenges. In addition, continuously obtaining cells from animal bodies is also contrary to animal welfare. The rapid development of cell-cultured meat technology has promoted the industrialization process of cell-cultured meat, and is also facing the demand for a large number of seed cell preparations. Therefore, it is urgent to construct an efficient and sustainable seed cell preparation method. Cell immortalization technology can enable cells to acquire the ability to proliferate continuously, be propagated for a long time, and have the growth characteristics of unlimited proliferation, overcoming the situation that general primary cells age and die after a limited number of propagations. It has been reported that a chicken fibroblast cell line that can be propagated for a long time has been screened out. This cell line can grow in the form of a single cell suspension, and its cell density reaches 10 using serum-free culture medium. 8 ×10 6 Cells are used to produce cell-cultured meat. Long-term cell culture is essentially a process of chromosome breakage and rearrangement, a very rare occurrence. Studies have shown that the heavier the species, the lower the probability of this phenomenon. Therefore, the probability of this phenomenon in large mammals such as pigs and cattle is much lower than in small animals such as fish and poultry. Therefore, there are currently no long-term cell cultures from pigs or cattle, further limiting the production of cultured meat.

[0004] At the genetic level, current methods for enabling long-term cell culture include the SV40 large T antigen method, the telomerase reverse transcriptase method, the HPV16 E6 method, and methods involving proto-oncogenes and tumor suppressor genes. Each of these methods has its pros and cons. For food science research, seed cells used in cell-based meat production must first be free of human factors while ensuring cell viability and functionality. Crucially, the induction of long-term cell culture must not interfere with the normal use of the seed cells, i.e., introduce no undesirable factors. Reports have demonstrated that inducing long-term cell culture by inducing lentivirus carrying hTERT via viral infection can increase exogenous hTERT expression in primary cells, promote telomerase activity, maintain telomere length, and extend the growth cycle of primary cells that normally die rapidly, even allowing them to be cultured for extended periods. Reports have also demonstrated that overexpressing TERT and using CRISPR to knock out p15 and p16 expression in chicken skeletal muscle cells have enabled long-term cell culture. Long-term cell culture has been shown to have no adverse effects on other cell types and to prevent the primary cells from rapidly senescent. However, currently, these methods are limited to basic research, and existing long-term cell lines are mostly mouse or human-derived, making them incapable of direct application in cell-based meat systems, let alone food science research. Furthermore, the aforementioned construction methods all involve the introduction of exogenous genes, which are not spontaneous phenomena. Therefore, it is necessary to provide more porcine fibroblast cell lines that can be spontaneously immortalized and applied to cultured meat systems to fill the technological gap in this field. Summary of the Invention

[0005] The present invention addresses the problem that there is a gap in the research of porcine fibroblast cell lines that can be passaged for a long time in the prior art, and provides a porcine fibroblast cell line that can be passaged for a long time for cell-cultured meat. The porcine fibroblast cell line is isolated and screened from porcine subcutaneous tissue, and no exogenous genes are introduced. It can break the Hayflick limit and can be passaged for a long time without special in vitro culture conditions. It has been passaged to 42, and the cells are still healthy, plump, and spindle-shaped. The proliferation level is not affected after long-term passage, and it can be used in the preparation of cell-cultured meat.

[0006] In order to solve the above problems, the technical solutions adopted by the present invention are as follows:

[0007] The first object of the present invention is to provide a long-term passaged porcine fibroblast cell line, which can be passaged for a long time in vitro and maintain a proliferative state. The porcine fibroblast cell line is derived from the pig ear edge or the pig hind leg. The long-term passaged porcine fibroblast cell line derived from the pig ear edge has a preservation number of CCTCC NO: C202511, and the long-term passaged porcine fibroblast cell line derived from the pig hind leg has a preservation number of CCTCC NO: C202512. The preservation date is February 26, 2025.

[0008] Furthermore, the long-term passage is no less than 42 generations.

[0009] In certain specific embodiments, the long-term passaged porcine fibroblast cell line is passaged in vitro and maintains a proliferation state for a number of passages of not less than 2, 18, 20, 32, or 42.

[0010] A second object of the present invention is to provide a method for constructing the aforementioned porcine fibroblast cell line capable of long-term passage, the method comprising the following steps:

[0011] S1: Cell separation and sorting: Isolate fibroblast tissue from the pig ear margin and / or pig hind leg to obtain pig ear margin fibroblasts and / or pig hind leg fibroblasts;

[0012] S2: Cell culture and passaging: The porcine fibroblasts obtained in S1 were cultured at a rate of 1.5 ± 0.5 × 10 cells per 10 cm dish. 5 The number of cells was inoculated into fibroblast growth medium and subcultured for a long time with medium replacement;

[0013] S3: After continuous culture for no less than 24 generations, select fibroblasts with rapid proliferation and good vitality, dilute the cell concentration to 100 ± 10 cells / ml, culture, select densely growing single cell clones, and digest with 0.25% trypsin to obtain the aforementioned porcine fibroblast cell line that can be passaged long-term.

[0014] In a specific embodiment, in S1, pig ear margin fibroblasts and pig hind leg fibroblasts are obtained by tissue block separation method.

[0015] In a specific embodiment, the cells are diluted to a concentration of 100 ± 10 cells / ml using limiting dilution in S3.

[0016] Furthermore, the long-term passage porcine fibroblast cell line derived from the pig ear margin is deposited with CCTCC NO: C202511, and the long-term passage porcine fibroblast cell line derived from the pig hind leg is deposited with CCTCC NO: C202512.

[0017] Furthermore, the fibroblast growth medium includes 10 vol% fetal bovine serum, 89 vol% DMEM / F12 culture medium, and 1 vol% penicillin-streptomycin double antibody solution.

[0018] Furthermore, in the penicillin-streptomycin dual antibiotic solution, the content of penicillin is 10,000 U / ml, and the content of streptomycin is 10 mg / ml.

[0019] The third object of the present invention is to provide the use of the aforementioned long-term passaged porcine fibroblast cell line in the preparation of cell-cultured meat.

[0020] In a specific embodiment, the above application is to use a long-term permuted porcine fibroblast cell line as a raw material for cultured meat, or based on the ability of the long-term permuted porcine fibroblast cell line to secrete extracellular matrix (collagen I, elastin and fibronectin, especially collagen), the extracellular matrix secreted by the long-term permuted porcine fibroblast cell line is used to increase the tenderness of cell-cultured meat, or the above application is to determine the ability of the above-mentioned long-term permuted porcine fibroblast cell line to secrete extracellular matrix, so as to use it in the production of cell-cultured meat to increase the tenderness of the meat.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention provides a porcine fibroblast cell line capable of long-term passage and its application. The porcine fibroblast cell line is isolated and screened from porcine fibroblast tissues of different locations without the introduction of exogenous genes. After 15 consecutive generations of culture in conventional porcine fibroblast growth medium, the fibroblast growth rate significantly slows. However, the fibroblasts isolated from the ear margin and hind leg of the present invention are able to overcome aging and continue to proliferate, exceeding the Hayflick limit. After 42 generations, the cells still exhibit a healthy, plump, long spindle shape. Long-term passage does not affect the proliferation level, and the cells can be used in the preparation of cell-cultured meat. Furthermore, there are no special restrictions on culture conditions during in vitro culture; the long-term passage state can be maintained using conventional proliferation medium, making it suitable for industrial production applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The figures are the growth curves of primary culture of porcine fibroblasts from different parts and batches obtained in the present invention.

[0024] Figure 2 These are pictures of different generations of long-term passaged porcine fibroblast cell lines obtained from two parts of the present invention.

[0025] Figure 3 The present invention provides the results of immunoblotting experiments on the identification genes vimentin (Vim) and fibroblast-specific protein 1 (FSP-1) of the porcine fibroblast cell line that can be passaged for a long period of time.

[0026] Figure 4 This is a picture showing the assay of the proliferation capacity of long-term passaged porcine fibroblasts obtained by the present invention, wherein: Figure 4 A in the figure is a long-term passage fibroblast derived from pig ears; Figure 4 B in the figure is a long-term passage fibroblast derived from pig hind legs.

[0027] Figure 5 This is a picture showing the extracellular matrix secretion ability of the porcine fibroblasts that can be passaged for a long time obtained by the present invention, wherein: Figure 5A in the figure is a long-term passage fibroblast derived from pig ears; Figure 5 B in the figure is a long-term passage fibroblast derived from pig hind legs.

[0028] Biomaterial deposit information

[0029] The pig ear fibroblast (FAP Sus scrofa) strain of the present invention was deposited in the China Center for Type Culture Collection (CCTCC) on February 26, 2025. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code: 430072, and the deposit number is: CCTCC NO: C202511. The classification name is pig ear fibroblast FAP Sus scrofa.

[0030] The pig hind leg fibroblast (FHLP Sus scrofa) strain of the present invention was deposited in the China Center for Type Culture Collection (CCTCC) on February 26, 2025. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code: 430072, the deposit number is: CCTCC NO: C202512, and the classification name is pig hind leg fibroblast FHLP Sus scrofa. DETAILED DESCRIPTION

[0031] The present invention is further explained below with reference to the following examples, but the examples do not limit the present invention in any form. Example

[0032] This embodiment provides a porcine fibroblast cell line capable of long-term passage and a method for constructing the same, which specifically includes the following steps.

[0033] S1, cell separation and sorting: After the piglets were drowned, their whole bodies were immersed in 75% alcohol for 3 minutes, and then they were taken out and placed in a clean bench; they were disinfected three times with iodine cotton balls, and then deiodinated with 75% alcohol. The fibroblast tissues of different parts of the pigs, such as the connective tissue of the ear edge (E), front leg (F), longissimus dorsi muscle area (B) and hind limb area (H), were removed with a surgical blade. After the removal of the hair, they were cleaned with 75% alcohol and 3 cm 2Fibroblast tissue blocks of different sizes were prepared; the tissue was quickly placed in 75% alcohol for 3 minutes for disinfection, and then rinsed with PBS containing double-antibody to remove the hair on the surface of the tissue block; the wound site, cartilage tissue, and epidermal skin were removed, and the remaining tissue was cut into small pieces (at least less than 2-3 mm) with ophthalmic scissors and placed in a centrifuge tube, and 200 μL of serum was added; the tissue homogenate was evenly spread on a 10 cm culture dish, transferred to an incubator, and incubated at 37°C, 5% CO2 for 5-8 hours; after drying, 5 mL of complete culture medium was carefully added, and 5 mL of complete culture medium was added after 24 hours for continued incubation. The culture medium was changed every 2-3 days. When cells migrated out after 4-5 days, the medium was changed according to the cell growth status and culture medium color.

[0034] After primary culture, a small number of cells may be observed attaching and migrating along the periphery of the tissue block on the fifth day. A few cells may adhere to the bottom of the dish in the empty space. Cells primarily cluster at the edge of the dish, often forming short, slender, triangular structures. With increasing culture time, the number of cells increases, extending outward from the tissue block, forming radial or swirling outgrowths. The cells are three-dimensional and plump, exhibiting a typical spindle-shaped morphology. In addition to fibroblasts, densely packed epithelial cells are also present around the tissue block, forming polygonal structures with distinct cell boundaries between the epithelial and fibroblast cells. After 10-11 days of culture, cells can be passaged or cryopreserved.

[0035] Typically, fibroblasts of high purity can be obtained after 2-3 passages. The morphology of passaged fibroblasts is identical to that of the original culture, with long, spindle-shaped cells with plump cytoplasm, good growth, slight intercellular spacing, and visible retracted cells in the dividing phase. The cells appear to have a swirling or flame-like appearance. Gently shaking the culture dish reveals a small number of dead cells suspended in the culture medium. Passaged cells grow faster than the original culture and can generally be passaged again after 2-3 days.

[0036] S2, cell culture and passaging: The high-purity porcine fibroblasts screened in S1 were cultured at a rate of 1.5 ± 0.5 × 10 cells / well per 10 cm dish. 5 The number of cells was inoculated into fibroblast growth medium and cultured for long-term medium replacement. The doubling time of cells from different batches and different parts of each generation was collected. The fibroblast growth medium composition included: 10 vol% fetal bovine serum, 89 vol% DMEM / F12 culture medium, and 1 vol% penicillin-streptomycin double antibody solution. The content of penicillin in the penicillin-streptomycin double antibody solution was 10,000 U / ml and the content of streptomycin was 10 mg / ml.

[0037] S3, cell monoclonal screening: After continuous culture for 24 generations, fibroblasts with fast proliferation rate and good vitality are screened, and monoclonal cell lines are obtained by limiting dilution method.

[0038] Due to genetic differences and cellular diversity among individual pigs, porcine fibroblasts were obtained from different parts of three piglets in this study, resulting in differences in their proliferation capacity during in vitro culture. Fibroblasts from connective tissue of the ear margin (E), foreleg region (F), longissimus dorsi (B), and hind limb (H) were cultured for long periods in vitro. Cells from different piglets were designated 1, 2, and 3, respectively, and further screened for long-term cell survival. Approximately 100 days after culture, the cell doubling time reached 170 hours, and the cell culture reached passage 24. At this point, cells from the foreleg region (Q1, Q2, Q3) and longissimus dorsi (B1, B2, B3), as well as the ear margin (E2, E3), and hind limb (H2, H3) reached a senescent state and were unable to break through this senescence bottleneck, significantly limiting their growth capacity. In stark contrast, cells from the ear edge (E1) and hind limb region (H1) of one of the piglets exhibited unique growth characteristics, able to overcome the limitations of cell aging, proliferate rapidly, and exhibit good vitality. After long-term subculture for up to 180 days, the doubling time of cells from these two locations was still maintained at 20-30 hours, demonstrating good sustained proliferation capacity ( Figure 1 ).

[0039] The fibroblasts from the ear edge (E1) and hind limb region (H1) obtained by the above screening were named pig ear fibroblasts (Ep) and pig hind leg fibroblasts (Hp). 1±0.2×10 5 Cell gradients were diluted to 100 ± 10 cells / ml in fibroblast growth medium. Single cells were seeded into 96-well plates at a concentration of 10 μL per well. After 3-5 days of culture, densely growing single cell clones were selected and digested with 0.25% trypsin to obtain single-cell-expanded porcine fibroblast stem cell lines. These porcine fibroblasts were designated porcine ear fibroblasts (FAP) and porcine hind leg fibroblasts (FHLP) and deposited with the China Center for Type Culture Collection (CCTCC) at Wuhan University, China, under the accession numbers CCTCC NO: C202511 and C202512, respectively. These two porcine fibroblast cell lines were completely isolated from porcine subcutaneous fibroblast tissue, lacking the introduction of exogenous genes, capable of exceeding the Hayflick limit and long-term passage.

[0040] The porcine ear fibroblasts (Ep) and porcine hind leg fibroblasts (Hp) obtained from the screening were observed under optical microscopy at passages 2 to 42. During the cell culture process, the cells of the first passage grew well, exhibiting a typical spindle-shaped morphology and a rapid proliferation rate. At passage 18, some cells began to show signs of senescence. Senescent cells underwent significant morphological changes, increasing in size and gradually spreading out, and their proliferation rate slowed. However, it is noteworthy that even when some cells exhibited senescent features, some retained a relatively good morphology. Further culture of these cells revealed that they were able to aggregate into cell clusters. These clusters, leveraging their growth advantages, gradually eliminated senescent cells during subsequent culture. By passage 32, the cells had regained their spindle-shaped morphology, their proliferation rate accelerated again, and their population doubling index (PDI) reached 100, meeting the current definition of a long-term cell line in this research field (i.e., a PDI of 100 is considered to indicate that the cells have exceeded the Hayflick limit and are suitable for long-term cell culture). So far, the cells have been successfully passaged to the 42nd generation and still maintain a good growth state and rapid proliferation ability, which can be used as a good seed cell resource for cell culture meat. Based on the above-mentioned 32nd generation, which meets the definition of a long-term passage cell line, we selected the previous generation cells (2nd generation), senescent cells (18th generation), cells that have broken through senescence (24th generation) and cells that can be long-term passaged (32nd generation) as our research objects according to the doubling time of pig ear fibroblasts (Ep) and pig hind leg fibroblasts (Hp) to carry out subsequent experiments ( Figure 2 ). Example

[0041] This example provides protein identification of the porcine fibroblast cell line of the present invention, specifically comprising the following steps: pig ear fibroblasts (Ep) and pig hind leg fibroblasts (Hp) of different passages screened in Example 1 were digested and transferred to 3.5 cm culture dishes and cultured in fibroblast growth medium; after culturing for 3 days, the culture medium was removed, the cells were washed once with phosphate buffer, 4% paraformaldehyde was added, and the cells were fixed at room temperature at 4°C overnight; after aspirating the supernatant, the cells were washed three times with phosphate buffer, 5 min each time; then 0.5% Triton X-100 was added, permeabilized at room temperature for 15-20 min, and washed three times with phosphate buffer on a shaker, 5 min each time; Vim and FSP-1 (purchased from Abcam, ab8978, ab124805) diluted 1:1000 were added dropwise to each glass-bottomed culture dish, and the cells were placed in a humidified box and incubated overnight at 4°C; the primary antibody was recovered, the cells were washed three times with phosphate buffer, 5 min each time, a fluorescent secondary antibody diluted 1:1000 was added dropwise, and the cells were incubated in a humidified box at room temperature for 1 minute. h, washed twice with phosphate buffer, 5 min each time; added anti-quencher containing DAPI and sealed the slides.

[0042] Results show that vimentin (Vim) and fibroblast-specific protein 1 (FSP-1) are often used as key markers for identifying fibroblasts in cell identification. Immunofluorescence analysis of long-term subcultured porcine ear fibroblasts (Ep) and porcine hind leg fibroblasts (Hp) revealed that both cells expressed vimentin, with a 99% positive rate. This strongly suggests that the essential characteristics of porcine fibroblasts remain unchanged after long-term subculture, maintaining a high level of cell purity and typical fibroblast properties. Furthermore, the expression of fibroblast-specific protein 1 (FSP-1) varied with increasing passage number. Specifically, in porcine ear fibroblasts (Ep), FSP-1 expression initially increased and then decreased, whereas in porcine hind leg fibroblasts (Hp), FSP-1 expression continued to increase. Although there are differences in the expression of FSP-1 in cells of different origins or passages, all passages of cells express FSP-1. Based on the expression of vimentin and FSP-1, these cells are all fibroblasts ( Figure 3 ). Example

[0043] This example provides an identification of the proliferation ability of the porcine fibroblast cell line of the present invention, which specifically includes the following steps: the porcine ear fibroblast cell line (Ep) and the porcine hind leg fibroblast cell line (Hp) screened in Example 1 are plated at 5×10 41 mL of EdU working solution was added to each well and the 6-well plate was gently shaken to mix. The wells were incubated on a shaker for 2 h. The culture medium and EdU working solution were aspirated and 1 mL of fixative solution was added to each well and fixed at room temperature for 15 min. The fixative solution was aspirated and 1 mL of wash solution was added to each well and the wells were allowed to stand for 5 min. This step was repeated three times. The wash solution was completely aspirated and 1 mL of 0.3% TritonX-100 was added to each well and permeabilized at room temperature for 10 min. The permeabilization solution was completely aspirated and 1 mL of wash solution was added to each well and the wells were allowed to stand for 5 min. This step was repeated three times. The Click working solution was prepared according to the kit instructions. The wash solution in the 6-well plate was discarded and 0.5 mL of the wash solution was added to each well. Incubate the cells in the dark at room temperature with Click working solution for 30 minutes; discard the Click working solution, add 1 mL of washing solution to each well, let it stand for 5 minutes, and repeat this step three times; discard the washing solution, add 1 mL of Hoechst33342 working solution to each well, and incubate it in the dark at room temperature for 10 minutes; discard the Hoechst 33342 solution, add 1 mL of washing solution to each well, and wash three times, each for 5 minutes; take pictures with an inverted fluorescence microscope, and randomly select at least three fields of view for each well; use ImageJ software to count the number of EdU-positive cells and the total number of cell nuclei in each field of view, and calculate the ratio of the two.

[0044] This study measured the proliferation capacity of porcine ear fibroblasts (Ep) and porcine hind leg fibroblasts (Hp). The results showed that EdU detection in both cell types exhibited an initial upward and then downward trend. Initially, primary cells exhibited robust proliferation, with approximately 25% EdU-positive. As the culture progressed, the cells gradually entered a senescent phase, with a corresponding decrease in proliferation capacity. However, once the cells broke through the senescence bottleneck, their proliferation capacity increased again.

[0045] Notably, the proliferation capacity of the two long-term subcultured cell lines was relatively stable, with the EdU-positive percentage remaining at approximately 30%. Overall, after long-term subculture, the proliferation capacity of these two cell lines has not declined, but has actually increased.

[0046] Based on the above experimental results, it can be clearly concluded that the long-term passaged pig ear fibroblast cell line and pig hind leg fibroblast cell line screened by the present invention have strong proliferation ability ( Figure 4 ). Example

[0047] This example provides an identification of the ability of the porcine fibroblast cell line of the present invention to secrete extracellular matrix so as to be used in the production of cell-cultured meat, which specifically includes the following steps.

[0048] Total cell protein extraction and protein concentration determination: Cell proteins from the pig ear fibroblast cell line (Ep) and the pig hind leg fibroblast cell line (Hp) screened in Example 1 were collected. An ice box was prepared in advance, and all operations during the protein collection process were performed on ice. The cell culture medium was first aspirated, and the cells were washed once with 4 mL of PBS and then washed a second time with 4 mL of ice-cold PBS. After washing, the culture dish was tilted and placed for 2-3 minutes, and the residual PBS in the dish was aspirated. 100 μL of RIPA lysis buffer (containing 1% PMSF) was then added to lyse the cells, and the RIPA lysis buffer was evenly spread over the entire culture dish using a cell scraper. After lysis on ice for 30 minutes, the cell lysate was collected into a 1.5 mL centrifuge tube and temporarily stored at -20°C for use. Before determining the protein concentration, the pre-collected cell protein lysate was centrifuged at 12,000 rpm for 15 min at 4°C. After centrifugation, the supernatant was aspirated into a new 1.5 mL centrifuge tube and the supernatant volume was recorded for total protein concentration determination. The bovine serum albumin (BSA) standard was diluted into different concentration gradients. The sample was diluted as needed. 10 μL of BSA standard, the protein sample to be tested, and 200 μL of BCA working solution were added to each well. The cells were incubated in a 37°C incubator in the dark for 30 min. The absorbance was measured at a wavelength of 562 nm using a microplate reader. The protein sample after the concentration was measured was diluted to the same concentration with ddH2O and mixed thoroughly. The loading buffer was added at a ratio of 4:1, mixed thoroughly, and boiled at 95°C for 5 min.

[0049] Western blotting: Prepare a 15-well precast gel, insert it into an electrophoresis tank, and add running buffer. Sample loading: Load 20 μg of denatured protein onto each well. Add 4 μL of protein marker to each side of the sample. Set the voltage to 130 V for 75 min. Activate the PVDF membrane in methanol. Place the sponge, PVDF membrane, gel, and sponge in this order, from bottom to top. Secure with a transfer chuck and place in a rapid wet transfer apparatus. Place the transferred PVDF membrane in blocking solution (5% skim milk powder in TBST) and block on a shaker at room temperature for 2 h. Discard the blocking solution. Incubate with the primary antibody diluted according to the reagents used (Collagen I 1:100, Elastin 1:1000, Fibronectin 1:500) for 14-16 h. After incubation with the primary antibody, wash three times and incubate with the diluted secondary antibody for 2 h. Develop the membrane with developer solution for 5 min and image using a molecular imaging system. GAPDH was used as the internal control protein in all experiments.

[0050] Results show that in cultured meat production, the connective tissue structure is primarily dominated by collagen networks synthesized by fibroblasts. Tenderness and water retention can be controlled by adjusting cross-link density, making it crucial for the seed cells to possess ECM secretion capacity. Collagen I, elastin, and fibronectin, as the primary extracellular matrix (ECM) components secreted by fibroblasts, are commonly used to assess fibroblast ECM secretion capacity. The data in the figure show that in porcine ear fibroblasts (Ep), collagen I secretion generally increases, indicating that the cell's ability to secrete collagen I improves with increasing passage number. In porcine hind leg fibroblasts (Hp), although collagen I secretion decreased at passage 24, it remained elevated at passage 32, indicating that the ability of fibroblasts to secrete collagen I is not lost with increasing passage number. Elastin secretion exhibited distinct characteristics between the two cell types. In porcine ear fibroblasts (Ep), elastin secretion initially increased and then decreased, but by passage 32, secretion levels were not significantly different from those in primary cells. In porcine hind leg fibroblasts (Hp), elastin secretion continued to increase with increasing passage number. The secretion patterns of fibronectin also differed significantly between the two cell types. In porcine ear fibroblasts (Ep), fibronectin secretion increased more than fourfold during passage compared to primary cells, demonstrating a significant enhancement in secretory capacity. However, in porcine hind leg fibroblasts (Hp), fibronectin secretion was extremely low by passage 32, presumably due to weakened cell-cell interactions with increasing passage number, leading to decreased fibronectin secretion. In summary, after long-term passage, the long-term passage fibroblast cell line provided by the present invention still has the ability to secrete ECM, which is the core element of cell culture meat technology to achieve "structural biomimetic" and "functional biomimetic". It not only provides physical support and nutrient matrix for cultured meat, but also promotes tissue engineering construction by regulating the cell microenvironment, while reducing the cost threshold for industrial production ( Figure 5 ).

[0051] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A porcine fibroblast cell line capable of long-term passage, characterized in that: The porcine fibroblast cell line can be passaged for a long time in vitro and maintain a proliferation state. The porcine fibroblast cell line is derived from the pig ear edge or the pig hind leg. The long-term passaged porcine fibroblast cell line derived from the pig ear edge has a preservation number of CCTCC NO: C202511, and the spontaneously immortalized porcine fibroblast cell line derived from the pig hind leg has a preservation number of CCTCC NO: C202512.

2. Use of the long-term passaged porcine fibroblast cell line according to claim 1 in the preparation of cell-cultured meat.

3. The use according to claim 2, characterized in that The application is to use the long-term passageable pig fibroblast cell line described in claim 1 as a raw material for cell cultured meat.

4. The use according to claim 2, characterized in that The application is to increase the tenderness of cell cultured meat by using the extracellular matrix secreted by the long-term passaged pig fibroblast cell line according to claim 1.

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