A method for in vitro three-dimensional culture and isolation of chicken embryo fibroblasts

Abstract

This invention discloses a method for the in vitro three-dimensional culture and isolation of chicken embryonic fibroblasts, belonging to the field of cell culture technology. The culture method of this invention includes the following steps: (1) After reviving chicken embryonic fibroblasts, passage them for at least one generation to obtain a cell suspension, centrifuge, discard the supernatant, add GelMA hydrogel solution, resuspend the cells, and obtain a mixture; (2) Irradiate the mixture with an ultraviolet light source to obtain a chicken embryonic fibroblast-GelMA hydrogel complex; (3) Transfer the chicken embryonic fibroblast-GelMA hydrogel complex to a culture dish, add culture medium, and culture the cells for 7-9 days; (4) After the cell culture is completed, discard the culture medium, add collagen II enzyme for digestion for 5-10 minutes, centrifuge, and collect the cell pellet. The three-dimensional culture system of chicken embryonic fibroblasts established by this invention better simulates the in vivo survival environment of cells, and can realize the rapid proliferation of chicken embryonic fibroblasts in an in vitro three-dimensional culture medium, which has greater research value.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, specifically to a method for in vitro three-dimensional culture and isolation of chicken embryonic fibroblasts. Background Technology

[0002] Chicken embryonic fibroblasts, derived from the mesoderm of the embryo, possess advantages such as ease of isolation and culture, strong proliferative capacity, high adaptability, and stable traits, making them an excellent cell experimental model with a wide range of applications. For example, due to their ability to detect viral receptors, these cells are widely used in virology research and are an important resource for vaccine production. Vaccines for several common infectious diseases, such as infectious bursal disease, Newcastle disease, and Marek's disease, can be produced using chicken embryonic fibroblasts. Their good tolerance allows for gene transfection, microinjection, and expression of genetically engineered products. In the late 20th century, scientists domesticated a continuously passaged chicken embryonic fibroblast cell line, DF-1, which, compared to primary chicken embryonic fibroblasts, exhibits stronger growth potential and lacks tumor genes. In conclusion, due to their numerous beneficial characteristics, chicken embryonic fibroblasts are widely used in animal virus research, vaccine development, cancer research, and many other fields, making them an important biomaterial in the life sciences.

[0003] Traditional chicken embryo fibroblast culture methods involve two-dimensional, planar culture. However, this method results in cells adhering to plastic or glass petri dishes, failing to replicate the complex in vivo tissue environment. To address this issue, we have focused on the in vitro three-dimensional culture of chicken embryo fibroblasts. In recent years, with advancements in biotechnology, research on three-dimensional cell culture has increased significantly. This involves placing cells in an artificially created three-dimensional environment, enabling them to grow better within a specific microenvironment constructed in vitro, maintaining their three-dimensional morphology and function, and interacting and exchanging information with their surroundings and neighboring cell populations. Currently, three-dimensional cell culture technology has been applied in research areas such as drug screening, organoid construction, and artificial meat.

[0004] The emergence of three-dimensional culture is attributed to advancements in cell biology, biophysics, and biochemical engineering, along with the application of new methods and materials. Methacrylamide gelatin (GelMa) is a semi-synthetic hydrogel obtained by reacting gelatin with methacrylic anhydride, resulting in the replacement of numerous active amino acid groups on the gelatin side chains with methacryloyl groups from the methacrylic anhydride. The modified gelatin, due to the presence of the methacryloyl groups, acquires photocrosslinking properties. With the addition of a photoinitiator and under ultraviolet light irradiation, a crosslinking reaction occurs, leading to a stable, three-dimensional shape. GelMa hydrogels are stable, easy to prepare, and safe and non-toxic, making them widely used in regenerative medicine, tissue engineering, and drug delivery.

[0005] There are reports on the use of GelMa for three-dimensional cell culture, such as Chinese patents CN 113667147A and CN114058040A. However, due to the different sizes, nutritional and environmental requirements of different cell types, the performance requirements of scaffold materials also vary when performing three-dimensional cell culture. In addition, how to effectively separate the cells from GelMa after the three-dimensional cell culture is completed is also one of the technical challenges in three-dimensional cell culture. Summary of the Invention

[0006] In view of the above-mentioned prior art, the purpose of this invention is to provide a method for the in vitro three-dimensional culture and isolation of chicken embryo fibroblasts. This invention can achieve rapid proliferation of chicken embryo fibroblasts in an in vitro three-dimensional culture medium.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a method for in vitro three-dimensional culture and isolation of chicken embryo fibroblasts, comprising the following steps:

[0009] (1) After reviving chicken embryonic fibroblasts, passage them for at least one generation to obtain a cell suspension. Centrifuge the suspension, discard the supernatant, add GelMA hydrogel solution, resuspend the cells, and obtain a mixture with a cell density of 5 × 10⁻⁶ cells / mL. 5 / mL~5×10 6 / mL;

[0010] (2) Irradiate the mixture with an ultraviolet light source to obtain chicken embryo fibroblast-GelMA hydrogel complex;

[0011] (3) Transfer the chicken embryo fibroblast-GelMA hydrogel complex to a culture dish, add culture medium, and culture the cells for 7-9 days;

[0012] (4) After cell culture is completed, discard the culture medium, add collagen II enzyme for digestion for 5-10 min, centrifuge, and collect the cell pellet.

[0013] Preferably, in step (1), the revived chicken embryo fibroblasts are resuspended in RPMI-1640 medium containing 12% fetal bovine serum and 1% penicillin-streptomycin by volume, and cultured in an incubator at 39°C with 5% CO2 by volume. When the culture density reaches 70-80%, they are passaged.

[0014] Preferably, in step (1), the concentration of the added GelMA hydrogel solution is 4-9 wt%. Different concentrations of GelMA hydrogel have different pore sizes, with higher concentrations resulting in smaller pore sizes.

[0015] Preferably, in step (2), the UV irradiation time is 10-20s. The curing time affects the pore size of the hydrogel. Generally speaking, the longer the curing time, the smaller the pore size of the hydrogel. Since too long a UV irradiation time will increase the possibility of cell mutation, a UV irradiation time of 10-20s is appropriate.

[0016] Preferably, in step (3), the culture medium is RPMI-1640 medium containing 12% fetal bovine serum and 1% penicillin-streptomycin by volume.

[0017] Preferably, in step (3), the cell culture conditions are: 39°C in an incubator containing 5% CO2 by volume.

[0018] Preferably, in step (4), the concentration of collagen II enzyme added is 2000 U / ml.

[0019] Preferably, in step (4), the digestion conditions are: 39°C, in an incubator containing 5% CO2 by volume for 5-10 minutes.

[0020] The beneficial effects of this invention are:

[0021] (1) Based on the characteristics of chicken embryonic fibroblasts, the present invention first constructed an in vitro three-dimensional culture system using a specific concentration of GelMa; and the present invention also found that the hydrogel can be digested by collagen II enzyme, which is beneficial for separating cultured cells from the three-dimensional scaffold.

[0022] (2) Compared with two-dimensional planar culture, the three-dimensional culture system of chicken embryo fibroblasts established in this invention better simulates the cell's living environment and can realize the rapid proliferation of chicken embryo fibroblasts in in vitro three-dimensional culture medium, which is more valuable for research. Attached Figure Description

[0023] Figure 1 Diagrams illustrating the culture apparatus. Figures A and B show schematic diagrams of commercial curing light sources and curing rings, while Figures C and D show the cell hydrogel complex prepared by this method.

[0024] Figure 2 Microscopic observation of DF-1 cells cultured in GelMA hydrogels of different concentrations. DF-1 cells were cultured in three-dimensional gels at concentrations of 5 wt%, 7 wt%, and 9 wt% GelMA hydrogels for 1 hour, 1 day, 3 days, 5 days, 7 days, and 9 days, respectively. The photocuring time for all hydrogels was 15 seconds, and the initial cell number was 2.5 × 10⁻⁶ cells / day. 6 Left and right. Scale bar is 200μm.

[0025] Figure 3Cell isolation from three-dimensional culture. Figure A shows a bright-field image (20×) of DF-1 cells cultured on a 5 wt% GelMA hydrogel for 15 seconds after 7 days of culture. Figure B shows a bright-field image (20×) of the cell hydrogel complex from Figure A after digestion with 2000 U / mL collagen II enzyme for 6 minutes. Figure C shows a bright-field image (10×) of cells isolated from the cell hydrogel complex from Figure B seeded in a flat culture dish after 10 minutes. Figure D shows a bright-field image (10×) of the cells from Figure C after 4 hours of culture. Scale bar: 200 μm.

[0026] Figure 4 Tracking of DF-1 cells stained with PKH26. The 2D group consisted of DF-1 cells stained with PKH26 and cultured in a plane for 3 days. The 3D group consisted of DF-1 cells stained with PKH26 and cultured in three dimensions for 3 days on a 5wt% GelMA hydrogel cured for 15 seconds. The scale bar is 200 μm.

[0027] Figure 5 DF-1 cells cultured in GelMA hydrogel for three-dimensional culture and EDU staining. DF-1 cells were cultured in a 5 wt% GelMA hydrogel for 1, 3, and 5 days. The cell-hydrogel complex was stained with EDU, and the proliferation rate was statistically analyzed. Scale bar: 200 μm.

[0028] Figure 6 SEM characterization of GelMA hydrogels at different concentrations. The figures show surface images of GelMA hydrogels at concentrations of 3 wt%, 5 wt%, 7 wt%, and 9 wt% observed under a cold field scanning electron microscope. The photocuring time for all hydrogels was 15 s, and the magnification was 2000×. Detailed Implementation

[0029] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0030] As mentioned above, in order to obtain a large number of chicken cells in vitro for subsequent research, the purpose of this invention is to provide a method for the in vitro three-dimensional culture and isolation of chicken embryonic fibroblasts and its application. This method can achieve rapid proliferation of chicken cells in an in vitro three-dimensional culture medium, laying the foundation for subsequent research. The method is as follows: first, chicken fibroblasts are resuspended in GelMA hydrogel with a concentration of 4-9 wt%, then cultured in a culture medium, and finally isolated using collagenase II. The chicken embryonic fibroblasts used in this invention are the DF-1 cell line. Specific details are as follows:

[0031] Step 1, Cell Culture: Resuscitate DF1 cells and resuspend them in 1640 medium containing 12% fetal bovine serum and 1% penicillin-streptomycin. Incubate at 39°C in a 5% CO2 incubator. Passage the cells when the culture density reaches 70-80%. After the first resuscitation, the cells must be passaged at least once before they can be used for three-dimensional culture.

[0032] Step 2, GelMA hydrogel solution preparation: GelMA hydrogel was prepared from methacrylic anhydride and gelatin. An appropriate amount of lyophilized GelMA powder was weighed and added to 1640 basal medium, dissolved in a 70°C water bath, and filtered through a 0.22μm sieve for sterilization. One-eighth of the dissolved solution volume of LAP photoinitiator was added, resulting in a final GelMA hydrogel concentration of 4–9 wt%. After mixing, the solution was temporarily stored at 39°C in the dark. These reagents are commercially available. Under the same conditions, different concentrations of GelMA hydrogel will have different pore sizes; the higher the concentration, the smaller the pore size. Microscopic characterization of 4–9 wt% GelMA hydrogels under 15s photocuring conditions is shown below. Figure 6 For chicken fibroblasts, a concentration of 5 wt% is optimal.

[0033] Step 3, Preparation of cell and GelMA hydrogel mixture: Place the cell suspension obtained after passage in Step 1 into a centrifuge and centrifuge at 1000g for 5 minutes at room temperature. Discard the supernatant, add the GelMA hydrogel solution obtained in Step 2, resuspend the cells and mix well. The cell density should be approximately 5 × 10⁻⁶ cells / mL. 5 / mL~5×10 6 / mL. If the cell density is too low, it may lead to a lack of connection between cells and apoptosis. If the cell density is too high, some cells may not have enough space to spread out and may not be able to obtain enough nutrients and oxygen in time, thus also leading to apoptosis.

[0034] Step four, photocuring: Drop the mixture obtained in step three onto a smooth, clean, transparent anti-stick membrane, and irradiate it with a 405nm ultraviolet light source for 15 seconds to obtain the cell and GelMA hydrogel complex. It is important to note that the photocuring time affects the hydrogel pore size. Generally, under the same conditions, the longer the curing time, the smaller the hydrogel pore size. However, because prolonged ultraviolet irradiation can increase the possibility of cell mutation, an ultraviolet irradiation time of 10–20 seconds is recommended.

[0035] Step 5, add culture medium: Carefully transfer the cell hydrogel complex from step 4 to a 24-well plate using tweezers or other tools. Add 1 mL of 12% fetal bovine serum and 1% penicillin-streptomycin 1640 medium to each well and incubate at 39°C in an incubator containing 5% CO2.

[0036] Step 6, Change the culture medium: 12 hours after implementing step 5, transfer the cell hydrogel complex to a new 24-well plate culture dish, add 1 mL of 12% fetal bovine serum and 1% penicillin-streptomycin 1640 medium to each well, and continue to incubate in an incubator at 39°C with 5% CO2.

[0037] 24–48 hours after performing step six, once most cells have been observed to have spread out on the hydrogel scaffold, wash with PBS buffer when changing the medium. The steps are as follows: before changing the medium, discard the waste liquid, add 1 mL of PBS buffer to each well, gently shake the culture dish for 1 minute, then discard the waste liquid again, and repeat twice.

[0038] Step 7, Cell Isolation from 3D Culture: Discard the culture medium from the culture dish in Step 6, wash the dish three times with PBS buffer, add 2000 U / mL collagenase to cover the colloid, and incubate at 39°C with 5% CO2 for 5-10 minutes. Remove the culture dish, pipette the liquid with a 1 mL pipette tip to check if the hydrogel has been fully digested, add 1640 medium containing 12% fetal bovine serum to stop the digestion, centrifuge at 1000g for 10 minutes at room temperature, discard the waste liquid, and the cells isolated from the 3D culture can be obtained.

[0039] The proliferation capacity of the isolated cells was measured, and the results showed that the cells isolated by the above method could maintain their proliferation capacity well. Compared with the initial chicken embryo fibroblasts, their proliferation capacity was comparable or even improved.

[0040] For the isolation of three-dimensional cells, the inventors experimented with various enzymes and their combinations during the research and development process. The goal was to degrade the colloidal scaffold while ensuring it did not affect the viability of the cultured cells. Ultimately, they found that treatment with 2000 U / mL collagenase II yielded the best results.

[0041] To enable those skilled in the art to better understand the technical solutions of this application, the technical solutions of this application will be described in detail below with reference to specific embodiments. Unless otherwise specified, experimental conditions not detailed in the embodiments are generally based on conventional conditions or conditions recommended by the reagent company; reagents, consumables, etc., used in the following embodiments can be obtained commercially unless otherwise specified.

[0042] Example 1: Three-dimensional culture process of chicken fibroblasts for the purpose of preparing cultured meat

[0043] Cultured meat refers to the three-dimensional in vitro culture of cells using a porous scaffold. The system of this invention can produce cultured meat that proliferates rapidly and forms a dense structure, unlike parameters used in other experiments. For example, in preparing five DF-1 cell three-dimensional culture models, the initial cell density was approximately 2.5 × 10⁻⁶ cells / cm². 6 / mL, GelMA hydrogel concentration 5wt%, photocuring time 15s. Specific steps are as follows:

[0044] 1. Pass DF-1 cells into 6 cm culture dishes. Once the cell density reaches 95%–100%, replace the medium with fresh 1640 medium containing 12% fetal bovine serum and 1% penicillin-streptomycin. Incubate at 39°C with 5% CO2 for two hours. After incubation, discard the supernatant. Add 1 mL of PBS buffer to the dish, gently agitate to thoroughly wash the bottom, discard the supernatant, and repeat twice. Add 1 mL of 0.25% trypsin digestion solution, incubate for 1 minute to allow digestion to complete, then remove the dish and add 1 mL of 1640 medium containing 12% fetal bovine serum to terminate digestion.

[0045] 2. Transfer the cell suspension to a centrifuge tube, mix well, and then take 10 μL of the suspension to count cells using a cell counting chamber. Take cells containing 3 × 10⁻⁶ cells according to the specified ratio. 6 Transfer the cell suspension (approximately 1000g) to another clean centrifuge tube, centrifuge at 1000g for 5 minutes at room temperature, discard the supernatant, and keep the cell pellet for later use.

[0046] 3. Place 75 mg of lyophilized GelMA powder into a brown, light-proof glass bottle, add RPMI-1640 medium, and heat in a 70°C water bath for 20 minutes, shaking the bottle occasionally to ensure the powder is fully dissolved, to prepare a 5 wt% GelMA hydrogel. Filter using a 0.22 μm sieve, transfer 1 mL of the filtered liquid to a clean centrifuge tube, add 125 μL of the photoinitiator phenyl-2,4,6-trimethylbenzoyl lithium phosphite (LAP), mix well, and store at 39°C in the dark for later use.

[0047] 4. Prepare a clean bench in advance. Irradiate with UV light for 10 minutes. Disinfect the anti-adhesion membrane and plastic curing rings by immersing them in 75% (volume fraction) ethanol, and air-dry them for later use. Place the anti-adhesion membrane on the UV light source plane. Take five curing rings and place them upside down on the anti-adhesion membrane. Resuspend the cell pellet obtained in step 2 in 1 mL of the GelMA hydrogel solution obtained in step 3. Mix well and drop the mixture onto the surface of the curing rings, adding 200 μL of the mixture to each ring. Let the liquid wet the surface of the rings, turn on the UV light source, and irradiate with 405 nm UV light for 15 seconds. The curing rings and UV light source are as follows. Figure 1 As shown in A and 1B.

[0048] 5. Using forceps, transfer the cell hydrogel complex from the solidified ring scaffold to a 24-well culture plate. Add 1 mL of RPMI-1640 medium containing 12% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin. Incubate at 39°C with 5% CO2. Change the medium every 24 hours thereafter. Do not use PBS buffer for the first medium change. A macroscopic image of the cells cultured on the scaffold is shown below. Figure 1 As shown in C and 1D.

[0049] 6. After 7 days of culture, microscopic observation reveals densely packed cells within the hydrogel, such as... Figure 2 As shown.

[0050] 7. Cell isolation from hydrogel: Remove the 24-well culture plate, discard the culture medium, add 1 mL of PBS buffer, gently shake the culture dish, discard the waste liquid, repeat 3 times, add 500 μL of collagenase II (2000 U / mL) to each well, incubate at 39℃, 5% CO2 for 6 minutes, remove and mix thoroughly with a 1 mL pipette tip, check for complete digestion, centrifuge at 1000g for 10 minutes at room temperature, discard the waste liquid, terminate digestion with 12% fetal bovine serum and 1% penicillin-streptomycin in 1640 medium, resuspend the cells, mix thoroughly by pipetting, and seed into 6 cm or other size culture dishes. Change the medium after 4 hours. Figure 3 As shown.

[0051] Example 2: DF-1 three-dimensional cultured cell tracking

[0052] To observe the state of cells in three-dimensional culture, PKH26 reagent was used to track cells cultured in GelMA hydrogels. PKH26 is a lipophilic red fluorescent dye with no obvious toxic side effects on cells. It can insert into the lipid region of the cell membrane and excites red fluorescence at wavelengths of 551–567 nm, thereby marking the morphology and location of cells. The specific steps are as follows:

[0053] To observe the state of cells in three-dimensional culture, PKH26 reagent was used to track cells cultured in GelMA hydrogels. PKH26 is a lipophilic red fluorescent dye with no obvious toxic side effects on cells. It can insert into the lipid region of the cell membrane and excites red fluorescence at wavelengths of 551–567 nm, thereby marking the morphology and location of cells. The specific steps are as follows:

[0054] 1. Prepare PKH26 staining solution: 1 mL = 1 mL Diluent C + 4 μL PKH26 Dye.

[0055] 2. Passage the cells into 6 cm culture dishes, following the same steps as in Example 1. After obtaining the cell pellet, resuspend the cells in 1 mL of PKH26 staining agent and incubate in the dark for 5 minutes. Then add 1 mL of culture medium to terminate the reaction, centrifuge at 1000 g for 5 minutes at room temperature, and discard the supernatant.

[0056] 3. Two-dimensional planar culture: Resuspend the cells in 1640 medium containing 4 mL of 12% fetal bovine serum and 1% penicillin-streptomycin, transfer to a 6 cm culture dish or other size culture dish, and shake well using the "figure-eight method".

[0057] 4. Three-dimensional culture: The procedure is the same as in Example 1. The stained cell pellet is resuspended in GelMA hydrogel mixture. After photocuring, the cell-hydrogel complex is transferred to a 24-well plate, and 1 mL of culture medium is added to cover the gel surface. Observation is performed under an inverted fluorescence microscope. Generally, cell extension and growth can be observed inside the hydrogel after three days of culture. Unlike two-dimensional culture where only the cell membrane glows clearly, cells in three-dimensional culture appear entirely red. See details... Figure 4 .

[0058] Example 3: Detection of DF-1 three-dimensional culture proliferation capacity

[0059] To detect the proliferative capacity of DF-1 cells under three-dimensional culture, the EDU assay was used. EDU is a thymidine nucleoside analog that can replace thymine and infiltrate replicating DNA molecules during cell proliferation. Catalyzed by substances such as divalent copper ions, it reacts with fluorescent azide compounds, and the cells are then observed under a fluorescence microscope. Using the cell hydrogel complex prepared in Example 1 as an example, the proliferative capacity of DF-1 cells cultured in 5 wt% GelMA hydrogel for one, three, and five days was tested. The specific steps were as follows:

[0060] 1. Prepare EDU incubation solution: 5mL = 5mL of 1640 medium containing 12% fetal bovine serum + 1.25μL EDU (5mg / mL), shake to mix well and set aside.

[0061] 2. Prepare EDU staining solution: 5mL = 50μL CUSO4 + 250μL Tris-HCl + 0.085g ascorbic acid + 5μL Alexa Fluor 555 dye + 4.7mL pure water.

[0062] 3. Following Example 1, after 21 hours of three-dimensional culture with DF-1, the 24-well culture plate was removed, and 1 mL of fresh 1640 medium containing 12% fetal bovine serum and 1% penicillin-streptomycin was added to each well. The plate was then returned to an incubator at 39°C and 5% CO2 for 2 hours. The culture dishes were then removed and gently rinsed with PBS buffer, the waste liquid was discarded, and 1 mL of the EDU incubation solution prepared in step 1 was added to each well. The plates were then returned to the incubator for 1 hour.

[0063] 4. Remove the culture plate, discard the waste liquid, add 1 mL of PBS buffer, shake for 5 minutes, discard the waste liquid, repeat 3 times, then add 1 mL of 4% paraformaldehyde to fix the cells in each well, fully immersing the cell hydrogel complex, and fix at 4℃ for 24 hours. Remove the culture plate, discard the waste liquid, and wash three times with PBS. Add 1 mL of the EDU staining solution prepared in step 2 to each well, fully immersing the cells. Incubate at room temperature in the dark for 1 hour. After incubation, wash three times with PBS, and finally use 50 ng / mL DAPI staining solution, incubating at room temperature in the dark for 20 minutes. Wash three times with PBS, discard the waste liquid, add 1 mL of PBS buffer to each well, and observe under an inverted fluorescence microscope. The staining steps for three and five days of cell proliferation are the same as above, with EDU incubation time at 71 hours and 119 hours after three-dimensional culture, respectively. Results are as follows. Figure 5 As shown.

[0064] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for in vitro three-dimensional culture and isolation of chicken embryonic fibroblasts, characterized in that, The method is used to prepare cell-cultured meat and includes the following steps: (1) After reviving chicken embryonic fibroblasts, passage them for at least one generation to obtain a cell suspension. Centrifuge the cells, discard the supernatant, add GelMA hydrogel solution, resuspend the cells, and obtain a mixture with a cell density of 5 × 10⁻⁶ cells / mL. 5 / mL~5×10 6 / mL; The GelMA hydrogel solution was prepared by the following method: Weigh out the lyophilized GelMA powder and add it to RPMI-1640 medium. Dissolve it in a water bath at 70°C. Filter the solution through a 0.22μm sieve to remove bacteria. Add 1 / 8 volume of LAP photoinitiator to the solution. The final concentration of the GelMA hydrogel is 4-9wt%. Mix well and store in the dark at 39°C. (2) Irradiate the mixture with a 405nm ultraviolet light source for 15s to obtain chicken embryo fibroblast-GelMA hydrogel complex. (3) Transfer the chicken embryo fibroblast-GelMA hydrogel complex to a culture dish, add culture medium, and culture the cells for 7-9 days; The culture medium was RPMI-1640 medium containing 12% fetal bovine serum and 1% penicillin-streptomycin by volume; the cell culture conditions were: 39°C in an incubator containing 5% CO2 by volume. (4) After cell culture, discard the culture medium, add collagen II enzyme at a concentration of 2000 U / ml, digest for 6 minutes in an incubator at 39°C with a volume content of 5% CO2, centrifuge, collect the cell pellet, terminate digestion with 1640 medium containing 12% fetal bovine serum and 1% penicillin-streptomycin, resuspend the cells, mix by pipetting, and inoculate into a culture dish; obtain cell culture meat that proliferates rapidly and can form a dense structure.

2. The method for in vitro three-dimensional culture and isolation of chicken embryo fibroblasts according to claim 1, characterized in that, In step (1), the revived chicken embryo fibroblasts were resuspended in RPMI-1640 medium containing 12% fetal bovine serum and 1% penicillin-streptomycin by volume, and cultured in an incubator at 39°C with 5% CO2 by volume. When the culture density reached 70-80%, they were passaged.

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