A serum-free culture medium with a clearly defined chemical composition for the in vitro proliferation of chicken embryo fibroblasts.

Abstract

This invention provides a serum-free culture medium with clearly defined chemical composition for the in vitro proliferation of chicken embryo fibroblasts, comprising a basal medium and cell culture supplementary factors. The cell culture supplementary factors include insulin, hydrocortisone, transferrin, basic fibroblast growth factor, epidermal growth factor, bovine serum albumin, L-ascorbic acid-2-phosphate trisodium salt, sodium selenite, ethanolamine, hypoxanthine, and thymidine. This invention also provides the application of the aforementioned serum-free culture medium in the in vitro proliferation culture of chicken embryo fibroblasts. Using the serum-free culture medium of this invention, chicken embryo fibroblasts can maintain normal adhesion and proliferation capacity. DF-1 cells maintain a 3-fold proliferation capacity per generation during at least 10 passages, with a viable cell percentage of not less than 80%. Primary chicken embryo fibroblasts maintain normal adhesion and cell morphology within 5 passages, with a greater than 3-fold increase in number per generation and a viable cell rate maintained above 80%.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, specifically to a culture medium with clearly defined chemical composition for the in vitro proliferation of chicken embryo fibroblasts and its application. Background Technology

[0002] Cell cultured meat technology produces meat by culturing animal cells in vitro. It has significant advantages such as high efficiency in resource utilization, controllable production process, and green environmental protection, providing a highly promising technological path for building a diversified food supply system and implementing the "big food concept".

[0003] The industrialization of cultured meat relies on a stable, efficient, and low-cost source of seed cells. While muscle stem cells are widely studied, their isolation process is complex, and they are prone to stemness loss, decreased proliferation and differentiation capacity during long-term in vitro culture, making them unsuitable as a sustainable source of seed cells. In contrast, fibroblasts, as one of the most widely distributed and abundant cell types in animals, offer advantages such as ease of acquisition via minimally invasive biopsy, strong in vitro proliferation potential, and passage stability. They have also been proven to participate in the formation of muscle, fat, and extracellular matrix, playing a crucial role in shaping the texture and flavor of cultured meat. Therefore, they are considered a highly promising alternative seed cell for cultured meat production.

[0004] However, current in vitro expansion of animal cells (including fibroblasts) generally relies on basal media supplemented with fetal bovine serum (FBS). While FBS provides a variety of nutrients and growth factors necessary for cell growth, its composition is complex and unclear, and it suffers from inherent drawbacks such as significant batch-to-batch variability, high cost, potential pathogen contamination risks, and animal welfare and ethical controversies. These factors severely restrict the standardization, scaling up, and cost control of cell-cultured meat production processes.

[0005] Fibroblasts are a promising alternative seed cell for cell-cultured meat production. However, current in vitro expansion of chicken embryo fibroblasts generally relies on basal culture media supplemented with fetal bovine serum, which has significant limitations in many aspects, including biochemistry, medicine, food science, and the development and industrialization of cell-cultured meat.

[0006] Currently, various serum substitutes or serum-free culture media are available for different cell types, including induced pluripotent stem cells, embryonic stem cells, and muscle stem cells. These findings provide valuable insights into the nutritional requirements of livestock stem cells and have driven the development of serum-free culture media for cell cultured meat. For example, patent application CN202311586016A describes a culture medium with a clearly defined chemical composition for the in vitro proliferation of myogenic cells. By adding cell culture supplementary factors, the function of serum in the proliferation of myogenic cells is replaced, enabling myogenic cells to maintain a proliferative capacity of approximately 3-fold per generation for at least 9 days, with a viable cell ratio maintained at around 90%. There is also some research and development on serum-free culture media for fibroblasts. For example, patent application US20210139843A1 relates to systems and methods for in vitro cell culture, which, for cell lines obtained through spontaneous immortalization, uses the addition of growth factors, hormones, and other substances to maintain a population doubling of at least 90 times in long-passaged chicken embryo fibroblast lines. However, compared to other cell cultures such as muscle stem cells, there is still relatively little research on serum-free culture media for fibroblasts. In particular, primary cells are more prone to aging, and their proliferation capacity weakens with passage number and time. The patent application with publication number US20210139843A1 has not solved the proliferation problem of primary chicken embryo fibroblasts.

[0007] Therefore, developing a fibroblast proliferation culture medium with a clearly defined chemical composition, stable performance, and no need for the addition of animal serum is of vital importance for achieving efficient, stable, and large-scale expansion of fibroblasts as seed cells, reducing the production cost of cultured meat, and promoting its industrialization. Summary of the Invention

[0008] To address the problems existing in the prior art, the purpose of this invention is to replace the serum component in traditional fibroblast proliferation culture media and provide a fibroblast proliferation culture medium with clearly defined chemical composition and its application method.

[0009] The serum-free culture medium for in vitro proliferation of chicken embryo fibroblasts described in this invention has a clearly defined chemical composition, meaning that the culture medium formulation does not contain any animal serum components, including fetal bovine serum, calf serum, horse serum, human serum, etc. By adding cell culture supplementary factors to the basal culture medium, better proliferation culture results are achieved, and the composition is clearly defined, making quality control easier and reducing the use of traditional fibroblast proliferation culture media.

[0010] The first objective of this invention is to provide a serum-free culture medium with a clearly defined chemical composition for the in vitro proliferation of chicken embryo fibroblasts, wherein the serum-free culture medium comprises a basal culture medium and a cell culture supplement factor, and neither the basal culture medium nor the cell culture supplement factor contains serum components. Cell culture supplements include insulin, hydrocortisone, transferrin, basic fibroblast growth factor, epidermal growth factor, bovine serum albumin, L-ascorbic acid-2-phosphate trisodium salt, sodium selenite, ethanolamine, hypoxanthine, and thymidine.

[0011] Furthermore, the concentrations of cell culture supplementary factors in the serum-free culture medium are shown in Table 1: Table 1. Concentration of cell culture supplementation factors insulin 10-30 μg / ml Hydrocortisone 1-5 μM Transferrin 5.5-16.5 μg / ml Basic fibroblast growth factor 20-100 ng / ml Epidermal growth factor 10-50 ng / ml Bovine serum albumin 1.5-2.5 mg / ml L-Ascorbic acid-2-trisodium phosphate 100-300 μM Sodium selenite 6.7-20.1 ng / ml ethanolamine 2-6 μg / ml Hypoxanthine (HYPO) 1-3 μg / ml Thymidine (THY) 50-150 ng / ml .

[0012] Furthermore, the basal culture medium includes an initial culture medium and a penicillin-streptomycin dual antibiotic solution.

[0013] Furthermore, the initial culture medium is selected from one or more of DMEM culture medium and DMEM / F12 culture medium.

[0014] Furthermore, the initial culture medium and penicillin-streptomycin dual antibiotic solution were in a volume ratio of 99:1.

[0015] The second objective of this invention is to provide the application of the aforementioned serum-free culture medium in the in vitro proliferation culture of chicken embryo fibroblasts, wherein after the chicken embryo fibroblasts reach the adherent state, the aforementioned serum-free culture medium is used for in vitro proliferation culture.

[0016] Chicken embryo fibroblasts can be adhered to the culture medium using either serum-containing or serum-free medium. In a specific embodiment, the serum-containing medium used is DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution.

[0017] Furthermore, the chicken embryo fibroblasts are selected from spontaneously immortalized chicken embryo fibroblast lines (e.g., UMNSAH / DF-1, or DF-1 for short) or primary chicken embryo fibroblasts.

[0018] Furthermore, the serum-free culture medium is able to maintain: a) Self-immortified chicken embryo fibroblasts exhibit normal cell morphology, normal fold increase, and viable cell rate during proliferation; maintain normal adhesion and proliferation capacity of self-immortified chicken embryo fibroblasts, and / or maintain normal cell morphology, and / or maintain a proliferation capacity greater than 5-fold per generation during at least 10 passages (40 days), and / or a viable cell ratio of not less than 80%, maintain the expression of fibroblast-specific marker proteins, maintain cell purity, and retain adipogenic transdifferentiation capacity; or b) Primary chicken embryo fibroblasts maintain normal adhesion and cell morphology within 3 generations, and the number of cells increases more than 3 times in each generation with a viable cell rate of more than 80%.

[0019] Furthermore, the application includes the following steps: (1) After the chicken embryo fibroblasts reach the adherent state, change the medium with the aforementioned serum-free proliferation medium. The same serum-free proliferation medium is used to change the medium again the day after the medium change. (2) When the cell density reaches 6×10 5 Digest the cells with 0.25% trypsin, centrifuge the cell suspension at 300 g for 5 minutes, resuspend the cells, and repeat step (1) for passage culture.

[0020] Further, step (1) involves mixing chicken embryo fibroblasts at a ratio of 3 × 10⁻⁶. 5 One cell / plate was inoculated into a serum-containing culture medium to allow chicken embryo fibroblasts to adhere to the culture medium.

[0021] Furthermore, cell cultures were maintained in a carbon dioxide incubator environment at 39 °C with a 5% CO2 concentration.

[0022] In this invention, "DF-1" refers to the chicken embryo fibroblast cell line UMNSAH / DF-1.

[0023] The beneficial effects of the technical solution in this application are as follows: The serum-free culture medium of the present invention, with its clearly defined chemical composition for the in vitro proliferation of chicken embryo fibroblasts, avoids the use of serum and enables chicken embryo fibroblasts to maintain normal adhesion and proliferation capacity. DF-1 maintains a 3-fold proliferation capacity per generation during at least 10 passages (40 days), with a viable cell ratio of not less than 80%. Primary chicken embryo fibroblasts maintain normal adhesion and cell morphology within 5 passages, and each generation can achieve a greater than 3-fold increase in number with a viable cell rate maintained above 80%. Attached Figure Description

[0024] Figure 1 Bright-field photograph of DF-1 cells after 20 generations of in vitro proliferation and passage in a culture medium with well-defined chemical composition.

[0025] Figure 2 The fold increase and survival rate of DF-1 were determined by in vitro propagation and subculturing for 20 generations in a culture medium with well-defined chemical composition.

[0026] Figure 3 The cumulative number of DF-1 cells after 20 generations of in vitro proliferation and passage in a culture medium with clearly defined chemical composition.

[0027] Figure 4 After 7 generations of in vitro proliferation and passage in a culture medium with well-defined chemical composition, DF-1 was observed. Figure A shows the immunofluorescence staining of vimentin, a fibroblast-specific marker protein, and Figure B shows the positive rate.

[0028] Figure 5After 7 generations of in vitro proliferation and subculturing in a culture medium with a clearly defined chemical composition, the lipid droplets of DF-1 were subjected to adipogenic transdifferentiation culture to obtain green fluorescence staining. DF-1 cultured under the same conditions in serum-containing medium and then undergoing adipogenic transdifferentiation culture, as well as DF-1 cultured in serum-containing medium but without adipogenic transdifferentiation culture, were used as positive and negative controls, respectively.

[0029] Figure 6 After 7 generations of in vitro proliferation and subculturing in a culture medium with a clearly defined chemical composition, DF-1 was subjected to adipogenic transdifferentiation culture. Oil Red O staining was used as positive and negative controls for DF-1 cultured under the same conditions in serum-containing medium and cultured in serum-containing medium without adipogenic transdifferentiation culture.

[0030] Figure 7 Three primary chicken embryo fibroblasts were identified. Figure A shows the immunofluorescence staining of vimentin, a fibroblast-specific marker protein, and Figure B shows the positive rate.

[0031] Figure 8 Bright field photographs of the cell state of the above three primary chicken embryo fibroblasts after five generations of in vitro proliferation and passage in a culture medium with clearly defined chemical composition.

[0032] Figure 9 The proliferation fold and survival rate of the above three primary chicken embryo fibroblasts were determined by in vitro proliferation and passage five times in a culture medium with clearly defined chemical composition.

[0033] Figure 10 The cumulative number and survival rate of primary chicken embryo fibroblasts (CEF3) were determined by in vitro proliferation and passage for 10 generations in a culture medium with clearly defined chemical composition. Detailed Implementation

[0034] The present invention will be further explained below with reference to the embodiments, but the embodiments do not limit the present invention in any way.

[0035] The present invention will be further described in detail below with reference to specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0036] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0037] The statistical analysis used in the following examples, as a preferred method, employed GraphPad Prism 10.1.2. A t-test was used to compare two experimental groups. For groups with two or more experimental groups, Dunnett's multiple comparisons analysis with one-way ANOVA was used. Statistical significance was defined as *p<0.05, *p<0.01, and ***p<0.001.

[0038] The culture conditions used in the following examples were all 5% CO2 concentration and 39°C temperature.

[0039] Example 1: In vitro proliferation and passage culture of DF-1 cells and detection of cell proliferation fold and viability. (1) Pre-coating: Dissolve type I rat tail collagen in 0.02 mol / L acetic acid solution to a final concentration of 0.5 mg / mL, spread the solution on the surface of cell culture dish, incubate at 37℃ for 4-8 hours, wash twice with PBS and air dry for later use.

[0040] (2) Inoculation and medium change: DF-1 is added at a ratio of 3×10 5 Chicken embryo fibroblasts were seeded per cell / plate into pre-coated culture dishes and cultured in serum-containing medium (DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution) for 12 h until they reached adherence. The medium was then changed to the serum-free proliferation medium, and the same serum-free proliferation medium was used again the following day.

[0041] In this embodiment, the serum-free proliferation medium consisted of the initial DMEM medium and a penicillin-streptomycin antibiotic solution, with a volume ratio of 99:1. The types and amounts of cell culture supplementary factors are shown in Table 2.

[0042] (3) Passage and counting: On the 4th day after inoculation, the cells were digested with 0.25% trypsin, the cell suspension was centrifuged at 300 g for 5 minutes, the cells were resuspended, 10 μl of cell suspension was mixed with 10 μl of trypan blue dye for counting, and then the cells were re-passaged and inoculated into fresh serum-containing culture medium.

[0043] (4) The results showed that DF-1 cells cultured in the culture medium maintained normal adhesion and cell morphology in the 7th generation. Figure 1 Furthermore, the number of cells can increase approximately tenfold per generation from 10 to 20 generations, while maintaining a viable cell rate of over 80%. Figure 2 In the 20th generation, the cumulative cell count reached 10. 26 indivual( Figure 3 ).

[0044] Table 2: Cell culture supplementary factors in culture media with clearly defined chemical compositions

[0045] Example 2: Immunofluorescence detection of specific marker proteins during DF-1 in vitro passage (1) After 7 passages of DF-1 cells in serum-free proliferation medium with well-defined chemical composition as described in Example 1, wash with PBS, fix with 4% paraformaldehyde at room temperature for 20 minutes, wash three times with PBS, then permeate with 0.5% (v / v) Triton X-100 (prepared with PBS) for 15 minutes, wash three times with PBS on a shaker for 5 minutes each time, add vimentin immunofluorescence primary antibody prepared with 1% BSA solution, and incubate overnight at 4 °C. Wash three times with PBS for 5 minutes each time. Add fluorescent secondary antibody, incubate for 2 hours, and wash twice with PBS for 5 minutes each time. Note that light protection is required from the time the secondary antibody is added. Add anti-fluorescence quenching agent containing DAPI, cover with a suitable coverslip, and observe and acquire images under a fluorescence microscope.

[0046] Meanwhile, a serum-containing culture medium was used as a control. Except for replacing the serum-free proliferation medium with a serum-containing culture medium (DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution), the operation was the same as in Example 1.

[0047] (2) Results showed that after 7 consecutive passages in the culture medium with clearly defined chemical composition of the present invention, DF-1 cells were stained with vimentin and DAPI, which are specific marker proteins of fibroblasts. After 10 consecutive passages, the proportion of vimentin remained above 95%, indicating that the cell purity of fibroblasts did not decrease during the culture process in the culture medium of the present invention, demonstrating that the culture medium of the present invention can stably maintain cell characteristics. Figure 4 ).

[0048] Example 3: DF-1 lipid transdifferentiation and lipid droplet green fluorescence detection experiment, Oil Red O detection experiment.

[0049] (1) Pre-plating: Pre-cool the PBS solution and pipette tip, mix the matrix gel (manufacturer: Corning, model: 456234) with the pre-cooled PBS solution at a ratio of 1:50, add 1 ml of the mixture to each well of the 6-well plate, incubate at 37 ℃ for 1 hour, wash twice with PBS and then air dry for use.

[0050] (2) Preparation of transdifferentiation medium: Add 200 mM oleic acid, 10 mM rosiglitazone, and 15.83 μM L-α phosphatidylcholine to serum-containing medium (same as step (2) in Example 1).

[0051] (3) DF-1 cells, after being passaged seven times in the culture medium with clearly defined chemical composition as described in Example 1, were inoculated into 6-well plates pre-coated with matrix gel for adherent culture. 5 × 10⁶ cells were inoculated into each well. 4 Cells were cultured for 12 hours with 2 mL of serum-containing medium, then the medium was changed with culture medium 2 with clearly defined chemical composition. After 3 days of culture, the medium was changed with transdifferentiation medium and cultured for 4 days.

[0052] (4) Lipid droplet green fluorescence staining: Remove the cells to be tested, wash twice with PBS, and remove the PBS. Add 4% paraformaldehyde fixative and fix at room temperature for 10-15 minutes, wash 1-2 times with PBS, add 1 ml of BODIPY staining solution to each well of a 6-well plate, incubate at room temperature in the dark for 10-20 minutes, and wash twice with PBS. All reagents should be added slowly along the sidewall to avoid cell floating and loss. Add a DAPI-containing anti-fluorescence quencher and observe using a fluorescence microscope.

[0053] (5) Oil Red O staining: Remove the cells to be tested, add 4% paraformaldehyde fixative and fix at room temperature for 10 minutes, then wash twice with PBS. Add 1 ml of staining wash buffer to each well of a 6-well plate to cover the cells for 20 seconds, remove the staining wash buffer, add 1 ml of Oil Red O working solution to each well of the 6-well plate, and stain for 20 minutes. Remove the Oil Red O working solution, add an appropriate amount of staining wash buffer and let stand for 30 seconds, remove the staining wash buffer, and wash with PBS for 20 seconds. All reagents should be applied slowly along the sidewall to avoid cell floating and loss. Add an appropriate amount of PBS to evenly cover the cells, and observe and photograph under a microscope.

[0054] (6) The results showed that in the lipid droplet green fluorescence staining experiment, there was obvious lipid droplet aggregation in the transdifferentiated cells, and the intracellular lipid droplets were stained with bright green fluorescence, while the untransdifferentiated cells showed weaker green fluorescence. Figure 5 In the Oil Red O staining experiment, the dye dissolved in intracellular lipid droplets in transdifferentiated cells, resulting in obvious red granules, while undifferentiated cells showed no red granules. Figure 6 These all indicate that after culturing in the culture medium with clearly defined chemical composition, the cells maintain the adipogenic transdifferentiation function of chicken embryo fibroblasts, demonstrating that the culture medium described in this invention can stably maintain cell characteristics.

[0055] Example 4: In vitro proliferation and passage culture of primary chicken embryo fibroblasts and detection of cell proliferation fold and survival rate (1) Isolation and immunofluorescence identification of primary chicken embryo fibroblasts. The shells of 10-day-old fertilized, specific pathogen-free eggs were disinfected with 75% alcohol. The embryos were removed, the head was quickly severed, and feathers, limbs, internal organs, and any visible bones were removed to obtain the embryonic body. The embryonic body was washed with PBS to remove blood and other residues, and then rinsed with DMEM. The embryonic body was shredded, and an appropriate amount of 0.25% trypsin was added and mixed. The mixture was digested in a 37°C water bath for 30 minutes, centrifuged at 500g for 10 minutes, and the supernatant was collected. 15 ml of primary cell culture medium (DMEM medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin solution, and 1% GlutaMAX) was added and mixed to terminate the digestion. The resulting mixture was diluted with an appropriate amount of PBS, filtered through a 100- and 40-μm filter, centrifuged at 300g for 5 minutes, and the supernatant was discarded. Add an appropriate amount of red blood cell lysis buffer to the precipitate and mix well. Incubate at room temperature for 10 min, centrifuge at 500 g for 5 min, and repeat this step until there is no red part in the precipitate. Resuspend in primary cell culture medium and inoculate in T75 culture flasks for 2 days to obtain primary chicken embryo fibroblasts.

[0056] In a 6-well plate, press 5×10 4 Primary chicken embryo fibroblasts were seeded and cultured at a density of / wells for 3 days. The culture medium was aspirated, and the cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 20 minutes. The cells were then washed three times with PBS, followed by permeabilization with 0.5% (v / v) Triton X-100 (prepared in PBS) for 15 minutes, and then washed three times with PBS on a shaker for 5 minutes each time. Vimentin immunofluorescence primary antibody prepared with 1% BSA solution was added, and the cells were incubated overnight at 4°C. The cells were washed three times with PBS for 5 minutes each time. Secondary fluorescent antibody was added, and the cells were incubated for 2 hours, followed by two PBS washes for 5 minutes each time. Note that light protection is required from the time the secondary antibody is added. A DAPI-containing anti-fluorescence quencher was added, a suitable coverslip was placed, and images were observed and acquired under a fluorescence microscope.

[0057] Immunofluorescence identification showed that the positive rate of vimentin, a fibroblast-specific marker protein, was greater than 95%, indicating that the isolated cells were primary chicken embryo fibroblasts (CEF2, 3, and 4 were primary chicken embryo fibroblasts isolated from three different embryos). Figure 7 ).

[0058] (2) Following the DF-1 in vitro proliferation and passage culture method in Example 1, the above primary chicken embryo fibroblasts were cultured in a culture medium with a clearly defined chemical composition, and the cell proliferation fold and survival rate were detected.

[0059] (3) The results showed that primary chicken embryo fibroblasts CEF2, 3, and 4 cultured in the culture medium with clearly defined chemical composition could exhibit normal adhesion and cell morphology within 5 generations. Figure 8Furthermore, it can achieve a more than 3-fold increase in number in each generation while maintaining a viable cell rate of over 80%. Figure 9 After 10 consecutive passages in the culture medium, CEF3 had a higher cumulative cell count than that of serum-containing cultures. Figure 10 ).

[0060] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A serum-free culture medium with a clearly defined chemical composition for the in vitro proliferation of chicken embryo fibroblasts, characterized in that, The serum-free culture medium includes a basal culture medium and a cell culture supplement factor, neither of which contains serum components; Cell culture supplements include insulin, hydrocortisone, transferrin, basic fibroblast growth factor, epidermal growth factor, bovine serum albumin, L-ascorbic acid-2-phosphate trisodium salt, sodium selenite, ethanolamine, hypoxanthine, and thymidine.

2. The serum-free culture medium according to claim 1, characterized in that, The serum-free culture medium contains the following cell culture supplementation factors: insulin 10-30 μg / ml, hydrocortisone 1-5 μM, transferrin 5.5-16.5 μg / ml, basic fibroblast growth factor 20-100 ng / ml, epidermal growth factor 10-50 ng / ml, bovine serum albumin 1.5-2.5 mg / ml, L-ascorbic acid-2-phosphate trisodium salt 100-300 μM, sodium selenite 6.7-20.1 ng / ml, ethanolamine 2-6 μg / ml, hypoxanthine 1-3 μg / ml, and thymidine 50-150 ng / ml.

3. The serum-free culture medium according to claim 1, characterized in that, The basal culture medium includes the initial culture medium and a penicillin-streptomycin double antibiotic solution.

4. The serum-free culture medium according to claim 3, characterized in that, The initial culture medium is selected from one or more of DMEM culture medium and DMEM / F12 culture medium.

5. The serum-free culture medium according to claim 3, characterized in that, The initial culture medium and penicillin-streptomycin antibiotic solution were in a volume ratio of 99:

1.

6. The application of the serum-free culture medium according to claim 1 in the in vitro proliferation culture of chicken embryo fibroblasts, characterized in that, After the chicken embryo fibroblasts reached the adherent state, they were cultured in vitro using the serum-free culture medium described in claim 1.

7. The application according to claim 6, characterized in that, The chicken embryo fibroblasts are selected from spontaneously immortalized chicken embryo fibroblast cell lines or primary chicken embryo fibroblasts.

8. The application according to claim 6, characterized in that, The serum-free culture medium can maintain: a) Self-immortified chicken embryo fibroblasts exhibit normal cell morphology, normal fold increase, and viable cell rate during proliferation; maintain normal adhesion and proliferation capacity of self-immortified chicken embryo fibroblasts, and / or maintain normal cell morphology, and / or maintain a proliferation capacity greater than 3-fold per generation during at least 10 passages, and / or a viable cell ratio of not less than 80%, maintain the expression of fibroblast-specific marker proteins, maintain cell purity, and retain adipogenic transdifferentiation capacity; or b) Primary chicken embryo fibroblasts maintain normal adhesion and cell morphology within 5 generations, and the number of cells increases more than 3 times in each generation while the viability rate remains above 80%.

9. The application according to claim 6, characterized in that, The application includes the following steps: (1) After the chicken embryo fibroblasts reach the adherent state, the medium is changed with the serum-free culture medium described in claim 1, and the medium is changed again with the same serum-free proliferation medium the day after the medium change; (2) When the number of cells reaches 6×10 5 At this point, digest the cells with 0.25% trypsin, centrifuge the cell suspension at 300 g for 5 minutes, resuspend the cells, take 10 μl and mix with 0.25% trypan blue, count the cells, and repeat step (1) for passage culture.

10. The application according to claim 9, characterized in that, Step (1) involves mixing chicken embryo fibroblasts at a ratio of 3 × 10⁻⁶. 5 One cell / plate was inoculated into a serum-containing culture medium to allow chicken embryo fibroblasts to adhere to the culture medium.

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