A method for isolating and identifying preadipocytes of Lueyang black-boned chicken

Abstract

This invention belongs to the field of cell and molecular technology and discloses a method for isolating and identifying preadipocytes of Lueyang Black-boned Chicken. Subcutaneous fat pads wrapped in a black membrane were collected from the legs and chest of chicken embryos on day 16 of incubation. After washing with PBS containing double antibodies, physical removal of the black membrane, and rinsing with D-Hanks buffer, the tissue was minced and digested with type I collagenase at 39°C, followed by gentle pipetting to achieve complete dissociation. The cells were then filtered through 100μm and 40μm double sieves, centrifuged, and subjected to erythrocyte lysis to obtain cell pellet. Subsequently, differential adhesion was used to remove fibroblasts and some melanocytes, and free impurities were removed during the first medium change. After the cells reached a suitable confluence, preadipocytes were further enriched by differential digestion. Finally, the purified cells were induced to undergo adipogenesis, and identification was completed by observing lipid droplet morphology, Oil Red O staining, Bodipy staining, or detecting adipogenic marker genes. This method improves the purity of Lueyang Black-boned Chicken preadipocytes and the reliability of subsequent experiments.

Description

Technical Field

[0001] This invention belongs to, but is not limited to, the field of cell and molecular technology, and particularly relates to a method for isolating and identifying preadipocytes of Lueyang black-bone chicken. Background Technology

[0002] Fat deposition traits in poultry are closely related to meat quality and production efficiency. Preadipocytes, as precursor cells of adipose tissue, are a key in vitro model for studying the molecular mechanisms of fat formation and deposition. Establishing efficient and stable methods for isolating and identifying poultry preadipocytes is of great significance for in-depth analysis of the regulatory network of adipocyte proliferation and differentiation, discovery of key genes affecting fat deposition, and screening of bioactive substances regulating fat metabolism. Currently, the in vitro isolation of chicken preadipocytes typically employs collagenase digestion combined with mechanical dissociation to obtain primary cells from the subcutaneous adipose tissue of chicken embryos or chicks. These cells are then purified using differential adhesion and identified by Oil Red O staining, Bodipy staining, or adipogenic gene expression detection. For example, existing technologies disclose a method for isolating preadipocytes from broiler embryo adipose tissue, which uses type I collagenase to enzymatically digest the adipose tissue at 37°C, followed by filtration using a tissue filter and centrifugation to obtain cell pellets, which are then cultured to obtain preadipocytes. However, the aforementioned existing methods have significant limitations when applied to certain specific chicken breeds. Studies have shown that the skin and subcutaneous adipose tissue of black-boned chickens are rich in melanin granules, with a significantly higher melanin content than that of ordinary broiler chickens. In existing preadipocyte isolation systems, due to the close coexistence of melanin granules and melanocytes with adipose tissue, conventional enzymatic digestion and filtration methods are insufficient to effectively separate them from the target preadipocytes. This results in widespread diffusion of melanin granules in the cell suspension, which not only severely interferes with microscopic observation of cell morphology but also affects the accuracy of identification results such as Oil Red O staining. Furthermore, residual melanocytes may interfere with the normal proliferation and differentiation of preadipocytes during in vitro culture through paracrine effects, significantly reducing the purity of the isolated preadipocytes and the reliability of subsequent functional studies.

[0003] Therefore, how to effectively eliminate the interference of melanin granules and melanocytes on the separation process of preadipocytes in black-boned chickens, and improve the separation purity and culture quality of preadipocytes in black-boned chickens, is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a method for isolating and identifying preadipocytes of Lueyang black-boned chicken.

[0005] The present invention is implemented as follows: a method for isolating and identifying preadipocytes of Lueyang black-bone chicken, the method comprising: S1: collection of adipose tissue;

[0006] S2: Digestion of adipose tissue;

[0007] S3: Purification of preadipocytes using differential adhesion method;

[0008] S4: Preadipocyte passage, differential digestion method;

[0009] S5: Adipogenic differentiation and identification of preadipocytes;

[0010] Furthermore, S1 specifically includes:

[0011] Embryo Acquisition: Take chicken embryos incubated to day 16; before hatching, thoroughly disinfect the eggshell surface with 70% ethanol; use the handle of sterile forceps to tap open the eggshell at the air cell end and peel it off, ensuring the opening is large enough to completely remove the embryo; use pointed forceps to tear open the eggshell membrane below the air cell to expose the embryo; replace with new sterile forceps, pierce the transparent amnion, gently grasp the chicken embryo by the neck and pull it out; cut the yolk sac blood vessels and transfer the chicken embryo to a sterile environment;

[0012] Tissue dissection: Place the chicken embryo in a new sterile culture dish and spray with an appropriate amount of 70% ethanol; make a small incision in the abdomen with scissors, and use tweezers to peel the skin and feathers to both sides to expose the subcutaneous fat pads of the legs and chest; after the fat tissue is exposed, use one hand to fix the surrounding muscle tissue with straight tweezers, and the other hand to hold the sterile curved tweezers in the opposite direction, and gently grasp the fat pads using the curved part; to ensure the yield, it is recommended to combine the fat tissue of 3 chicken embryos into one piece for subsequent digestion;

[0013] Tissue cleaning and trimming: The collected adipose tissue was transferred to sterile PBS containing 1% penicillin and antibiotics for rinsing, and adhering non-adipose tissues such as skin and fascia were removed. Under a microscope or with the naked eye, the black membrane covering the fat was peeled off as much as possible using forceps. Then it was transferred to D-Hanks buffer and rinsed thoroughly 2-3 times, and the remaining black membrane visible to the naked eye was removed during the rinsing process.

[0014] Furthermore, S2 specifically includes:

[0015] Tissue mincing: Transfer the rinsed adipose tissue to a 15 mL sterile centrifuge tube, add a small amount of F12 / DMEM culture medium, and use long-handled ophthalmic scissors to mince the adipose tissue into small tissue blocks of about 1 mm³.

[0016] Collagenase digestion: Remove the supernatant, add 1 mg / mL preheated type I collagenase at a ratio of 3 times the tissue volume; place the centrifuge tube in a 39°C water bath or shaker for 30-40 minutes, inverting and mixing every 10 minutes to ensure thorough digestion and effective cell release;

[0017] Mechanical agitation and termination: After 20 minutes of digestion, use a pipette to gently agitate the mixture in a laminar flow hood to aid dissociation; decide whether to continue digestion based on the digestion status until the mixture is a milky white chyme.

[0018] Double physical filtration: After digestion, gently aspirate and mix well, then add an equal volume of complete culture medium (DMEM / F12 medium containing 20% ​​FBS and 1% penicillin-streptomycin mixture); then filter the suspension through a 100 μm cell sieve into a new 15 mL centrifuge tube, and then filter it a second time through a 40 μm cell sieve;

[0019] Red blood cell lysis: Centrifuge the filtrate at 300 × g for 10 minutes at room temperature; discard the supernatant, resuspend the cell pellet in red blood cell lysis buffer, and lyse at room temperature for 5 minutes; add complete culture medium to terminate lysis;

[0020] Collection and inoculation: Centrifuge again at 300 × g for 6 minutes at room temperature; discard the supernatant and resuspend the pellet in complete culture medium; count cells using a cell counting chamber, adjust cell concentration, and inoculate at a rate of 8 × 10⁻⁶ cells / year. 5 - 1 × 10 6 Cells were seeded at a density of 10 cells / flask into T25 cell culture flasks and cultured in an incubator at 39°C and 5% CO2.

[0021] Furthermore, S3 specifically includes:

[0022] Primary differential adhesion: taking advantage of the fact that fibroblasts and some active melanocytes adhere faster than preadipocytes; after the primary cells are seeded into the incubator and left to stand for 1-1.5 hours, the culture flask is gently shaken, the supernatant that has not adhered is aspirated and directly transferred to a brand new T25 culture flask, an appropriate amount of culture medium is added and the cells are cultured for a while, and the original flask is discarded.

[0023] First medium change and washing: The day after inoculation, aspirate the old culture medium and gently wash the cells 1-2 times with 1×PBS to thoroughly remove dead cells, red blood cells and free melanin granules; add 4 mL of preheated fresh complete culture medium; at this time, under a microscope, the cells should mostly appear spindle-shaped or star-shaped.

[0024] Routine maintenance: Change the complete culture medium every 48 hours; when the cell confluence reaches 70%-80%, prepare for passage purification or cryopreservation.

[0025] Furthermore, S4 specifically includes:

[0026] Differential digestion purification: Remove the old culture medium and wash the cells twice with PBS; add 1-1.5 mL of 0.05% Trypsin-EDTA to cover the bottom of the flask; observe in real time at room temperature or under a microscope. When the preadipocytes rapidly shrink and become round and show a tendency to detach, immediately tap the flask wall to detach the preadipocytes.

[0027] Collection and Termination: Quickly add an equal volume of serum-containing complete culture medium to terminate digestion; gently aspirate the suspended cell solution, being careful not to forcefully blow the bottom of the flask; transfer the suspension to a centrifuge tube, discarding the original culture flask along with any impurity cells;

[0028] Centrifugation and resuspension: Centrifuge at 300 × g for 5 minutes and discard the supernatant;

[0029] Subculturing: If amplification is to be performed, resuspend in complete culture medium and count, at a rate of 3 × 10⁻⁶. 5 – 5 × 10 5 Re-seed the cells at a density of 100 cells / flask into a new T25 flask; after complete differential adhesion and differential digestion, high-purity preadipocytes can be obtained.

[0030] Cell cryopreservation: Cells can be cryopreserved when the confluence of expanded cells reaches 90%; the cryopreservation solution formula is: 10% DMSO + 20% FBS + 70% DMEM / F12; resuspend the cell pellet in the cryopreservation solution, aliquot it into cryovials; place them in a programmed cooling box and freeze overnight at -80°C, then transfer them to a liquid nitrogen tank for long-term storage the next day.

[0031] Furthermore, S5 specifically includes:

[0032] Gelatin coating of culture plates: Prepare a 2% (w / v) gelatin aqueous solution in advance and autoclave it; coat the bottom of the multi-well plate evenly at a rate of 5-10 μL / cm²; let it stand at room temperature for at least 1 hour, then remove excess gelatin liquid; before inoculating cells, place the coated culture plate in an incubator to preheat for 30 minutes.

[0033] Cell seeding and growth: Preadipocytes were seeded into gelatin-coated culture plates and cultured using complete growth medium;

[0034] Induction of differentiation: When the cell confluence reaches about 90%-100%, replace the growth medium with adipogenic differentiation-inducing medium (ADM); ADM formula: DMEM / F12 + 10% FBS + 1× linoleic acid-oleic acid-albumin (9.4 μg / mL) + 1% penicillin-antibody;

[0035] Medium change and observation: Maintain ADM culture by changing the medium every 2 days; usually, obvious lipid droplet formation can be observed under a microscope after 48 hours of induction.

[0036] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows:

[0037] This invention addresses the common challenges of cell separation in local black-feathered / black-skinned chicken breeds such as Lueyang Black-boned Chicken, including severe melanin contamination, low cell purity, and poor differentiation efficiency. Through innovative combinations of techniques, it achieves highly efficient separation of high-purity, highly active preadipocytes. Its positive effects are specifically reflected in the following aspects:

[0038] 1. Overcame the technical bottleneck of melanin interference unique to local varieties.

[0039] Technical Challenges: Existing technologies struggle to completely physically remove the melanin membrane (black membrane) attached to the subcutaneous tissue of breeds like black-boned chickens and black-feathered ducks. This membrane is also highly susceptible to fragmentation during digestion, releasing large amounts of melanin granules and melanocytes. These impurities not only inhibit the proliferation of preadipocytes but also interfere with signal transduction during subsequent differentiation, leading to biased experimental results. Solution: This invention employs a coupling strategy of physical removal and dual differential speed. First, in step S1, the black membrane is precisely removed using reverse-angled bent tweezers, reducing contamination at its source. Second, in steps S3 and S4, the subtle physiological differences in adhesion speed and digestive sensitivity between melanocytes and preadipocytes are utilized to achieve secondary physical removal.

[0040] Positive effects: In primary cells obtained using this method, the residual melanin granules are significantly reduced compared to existing technologies, greatly purifying the cell culture system.

[0041] 2. A balance between high yield and high activity was achieved with extremely low tissue volume.

[0042] Technical Challenges: The Lueyang Black-boned Chicken is a local endangered / protected breed with limited egg production and hatchability, making research samples extremely valuable. Traditional methods often require a large number of embryos to obtain sufficient cells, and over-digestion can easily damage cells. Solution: This invention optimizes the digestion parameters in step S2 (39°C, 30-40 min) and employs dual physical filtration (100 μm + 40 μm).

[0043] Positive effects: This combined approach significantly increases the cell yield from a single embryo while maintaining cell viability, achieving the required cell quantity for a single T25 cell culture flask. This not only significantly reduces the pressure on seed preservation and experimental costs but also shortens the cell population doubling time, providing a high-quality cell bank for subsequent research.

[0044] 3. Significantly improved the synchronicity and efficiency of in vitro adipogenesis induction.

[0045] The solution proposed in this invention: This invention introduces gelatin coating technology in step S5 and specifically optimizes the ADM formulation (linoleic acid-oleic acid-albumin complex). Positive effects: Compared to traditional induction protocols, cells induced by this invention show significant lipid droplet aggregation within 48 hours, and the differentiation rate (Oil Red O staining positivity rate) is improved. This highly efficient differentiation ability overcomes the bias of local cell varieties being difficult to differentiate in vitro, laying a solid foundation for the study of the molecular mechanisms of intramuscular fat deposition.

[0046] 4. The expected benefits and commercial value of the technical solution of this invention after transformation are as follows:

[0047] The commercial value of this invention is mainly reflected in two dimensions: the protection of local special poultry germplasm resources and the industrialization development of high-quality poultry meat.

[0048] Empowering Precision Nutrition and Feed Development for High-Quality Poultry: Local breeds such as Lueyang Black-boned Chicken command high premiums in the high-end consumer market due to their unique meat quality and nutritional value. Preadipocytes are the gold standard in vitro model for screening growth-promoting and flavor-modifying (fat deposition) feed additives. The efficient separation technology provided by this invention can offer feed companies and animal health companies a standardized cell testing platform, significantly reducing the cost of in vivo animal experiments and accelerating the research and commercialization of novel poultry nutrition regulation products.

[0049] Facilitating Somatic Cell Preservation and Breeding of Endangered / Local Poultry Breeds: Local poultry breeds generally suffer from low egg production and breeding difficulties. This invention not only achieves high-purity isolation of live preadipocytes, but its standardized cryopreservation and thawing protocols essentially establish an efficient method for constructing somatic cell biobanks. This provides technical support for the long-term preservation of local poultry germplasm resources and has immeasurable strategic and commercial breeding value.

[0050] 5. The technical solution of this invention fills a technological gap in the industry both domestically and internationally:

[0051] Currently, almost all established systems for isolating preadipocytes in poultry, both domestically and internationally, are based on commercially available broiler chickens (such as Ross and Cobb) or conventional waterfowl, whose subcutaneous fat tissue is white and free of pigment deposition. However, for local breeds such as Silkie chickens and black-feathered ducks, which have a heavy melanin background and dense black membrane, the industry has long lacked a standardized cell isolation procedure that can effectively eliminate melanin interference. This invention's physical peeling + dual differential coupling technology is the first to systematically establish a method for isolating and identifying preadipocytes specifically for melanin-rich subcutaneous tissue, directly filling a technological gap in this sub-field.

[0052] 6. The technical solution of this invention solves a technical problem that people have long desired to solve but have never been able to successfully address:

[0053] This invention completely eliminates the physical and biochemical interference in downstream detection caused by the co-occurrence of melanin and adipocytes: In tissues with a melanin membrane, melanocytes and preadipocytes are histologically intertwined. During primary culture, it is extremely easy for large numbers of melanocytes and free melanin granules to contaminate the cells. These impurities not only physically obscure cell microscopic observation and fluorescence signals, but their released active substances also produce cytotoxicity, leading to low preadipocyte survival rates, failed differentiation induction, and severe interference with subsequent nucleic acid and protein quantification. The industry has long desired to remove these contaminants while using subcutaneous fat, but conventional centrifugation and filtration are simply ineffective in separating these two cell types with similar densities and sizes. This invention successfully overcomes this contamination problem by combining a precise tissue trimming system with time-sequential control of differential adhesion and differential digestion, achieving high-purity enrichment of single-cell types.

[0054] 7. The technical solution of this invention overcomes technical bias:

[0055] This invention breaks the traditional prejudice that subcutaneous tissue rich in melanin membranes is unsuitable as a source of primary cells. Due to limitations in existing technology, a deep-seated technical bias exists in this field: the subcutaneous fat of breeds such as Silkie chickens is considered too heavily contaminated with melanin, making it a poor cell source. This often forces researchers to abandon the most representative subcutaneous fat and instead use trace amounts of abdominal fat with extremely low cell yields and difficult access. Alternatively, it is believed that forcibly removing melanin through multiple physical and chemical methods will inevitably cause the death of a large number of extremely fragile embryonic primary cells. This invention, through a clever combination of technical steps, demonstrates that even heavily contaminated melanin membrane fat can be perfectly purified without damaging the activity of preadipocytes, completely overturning the traditional prejudice that melanin-rich tissue is unusable or that melanin removal and cell viability are mutually exclusive.

[0056] The focus of this invention is the systematic improvement of preadipocyte isolation in local breeds with distinctive skin color, such as the Silkie chicken. This is indeed a classic challenge in poultry models like the Lueyang Silkie chicken, which exhibits typical fibromelanosis. At 16 embryonic age, melanin deposition in the embryo is already very evident. Because melanocytes migrate extensively along the neural crest during development, they are usually tightly intercalated with connective tissue and fat pads, making complete purification difficult through physical removal of the melanin membrane. If these residual melanocytes or free melanin granules are not removed, they may not only cause local oxidative stress affecting the proliferation and differentiation of preadipocytes, but also severely interfere with the optical background and result interpretation of subsequent qPCR, Western blotting, and flow cytometry during cell lysis. This invention optimizes the traditional preadipocyte isolation process by adding a differential adhesion and differential digestion scheme to purify preadipocytes from the Lueyang Silkie chicken. Attached Figure Description

[0057] Figure 1 This is a flowchart of a method for isolating and identifying preadipocytes of Lueyang black-bone chicken provided in an embodiment of the present invention;

[0058] Figure 2 This is a flowchart of the digestion process of adipose tissue provided in an embodiment of the present invention;

[0059] Figure 3 This is a morphological schematic diagram illustrating the process of isolating, purifying, and inducing differentiation of preadipocytes from Lueyang black-boned chickens, as provided in an embodiment of the present invention; wherein: Figure 3 A is a phenotypic diagram of cell precipitation after 20 minutes of adipose tissue digestion in the example; Figure 3 B is a comparison diagram of cell pellets collected during the first passage digestion using the traditional separation method and the method of the present invention (differential adhesion combined with differential digestion). Figure 3 C is a microscopic image of cells separated using traditional methods (without differential adhesion and differential digestion) during adhesion. Figure 3 D is a microscopic image of cell adhesion morphology after differential adhesion purification in an embodiment of the present invention; Figure 3 E is a microscopic morphological image of preadipocytes that have been successfully purified by dual differential speed in this embodiment of the invention and have a cell confluence of more than 90%. Figure 3 F is a microscopic morphological image of purified preadipocytes after adipogenic induction differentiation for 48 hours in this embodiment of the invention; Note: the above Figure 3 A to Figure 3 In F, the black substances visible to the naked eye or under a microscope are all melanocyte or melanin granule remnants.

[0060] Figure 4 This is an Oil Red O staining image of preadipocytes after 48 hours of induced differentiation.

[0061] Figure 5 This is a Bodipy staining image of preadipocytes after 48 hours of induced differentiation. Detailed Implementation

[0062] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0063] like Figure 1 As shown, this embodiment of the invention provides a method for isolating and identifying preadipocytes of Lueyang black-bone chicken, the method comprising: S1: collecting adipose tissue;

[0064] S2: Digestion of adipose tissue;

[0065] S3: Purification of preadipocytes using differential adhesion method;

[0066] S4: Cell passage and cryopreservation, differential digestion method;

[0067] S5: Adipogenic differentiation.

[0068] The method for isolating and identifying preadipocytes of Lueyang black-bone chicken provided in this embodiment of the invention has the following overall process: Figure 1 As shown, the process includes, in sequence, the collection of adipose tissue, digestion of adipose tissue, purification of preadipocytes, cell passage and cryopreservation, and adipogenic differentiation induction. Specifically, adipose tissue is first collected. The cleaned adipose tissue is transferred to a 15mL sterile centrifuge tube, a small amount of D-Hanks buffer is added, and the adipose tissue is thoroughly minced to approximately 1mm³ using long-handled ophthalmic scissors. Collagenase digestion is then performed. After removing the supernatant, preheated 1mg / mL collagenase is added at a ratio of 3 times the tissue volume, and the centrifuge tube is placed at 39°C for digestion. After 20 minutes of digestion, the tissue is gently pipetted in a laminar flow hood to aid in further tissue dissociation. After digestion, the suspension is gently aspirated and filtered sequentially through a 100μm cell sieve and a 40-mesh cell sieve into a new 15mL centrifuge tube. The filtrate is then centrifuged at 300×g for 10 minutes, the supernatant is discarded, and the cell pellet is resuspended in erythrocyte lysis buffer. After lysis at room temperature for 5 minutes under warm cell conditions, complete culture medium is added to terminate the lysis. Centrifuge again at 300×g for 6 minutes, discard the supernatant, resuspend the pellet in complete culture medium, count the cells using a counting chamber and adjust the cell concentration, seed into dense cell culture flasks, and incubate at 39℃ in a 5% CO2 incubator. Then proceed as follows... Figure 1 As shown, preadipocytes were purified using differential adhesion, passaged and cryopreserved using differential digestion, and finally induced to differentiate into adipocytes.

[0069] This invention provides a series of processes to obtain preadipocytes from Lueyang black-bone chickens: tissue dissociation, cell release, impurity removal, target cell enrichment, and functional induction. Tissue fragmentation increases the contact area between the adipose tissue and the digestive fluid; collagenase digestion loosens the tissue structure, promoting cell release from the adipose tissue; gentle mechanical pipetting further assists dissociation without altering the original digestion process; dual physical filtration removes incompletely digested tissue fragments and larger impurities; centrifugation collects the cell pellet, and erythrocyte lysis reduces erythrocyte interference; differential adhesion utilizes differences in cell adhesion characteristics to purify preadipocytes; differential digestion is used for subsequent passage and cryopreservation; and adipogenic differentiation induction demonstrates the adipogenic differentiation capacity of the obtained cells, thus completing the separation and identification process.

[0070] Furthermore, S1 specifically includes:

[0071] Embryo Acquisition: Take chicken embryos incubated to day 16; before hatching, thoroughly disinfect the eggshell surface with 70% ethanol; use the handle of sterile forceps to tap open the eggshell at the air cell end and peel it off, ensuring the opening is large enough to completely remove the embryo; use pointed forceps to tear open the eggshell membrane below the air cell to expose the embryo; replace with new sterile forceps, pierce the transparent amnion, gently grasp the chicken embryo by the neck and pull it out; cut the yolk sac blood vessels and transfer the chicken embryo to a sterile environment;

[0072] Tissue dissection: Place the chicken embryo in a new sterile culture dish and spray with an appropriate amount of 70% ethanol; make a small incision in the abdomen with scissors, and use tweezers to peel the skin and feathers to both sides to expose the subcutaneous fat pads of the legs and chest; after the fat tissue is exposed, use one hand to fix the surrounding muscle tissue with straight tweezers, and the other hand to hold the sterile curved tweezers in the opposite direction, and gently grasp the fat pads using the curved part; to ensure the yield, it is recommended to combine the fat tissue of 3 chicken embryos into one piece for subsequent digestion;

[0073] Tissue cleaning and trimming: The collected adipose tissue was transferred to sterile PBS containing 1% penicillin and antibiotics for rinsing, and adhering non-adipose tissues such as skin and fascia were removed. Under a microscope or with the naked eye, the black membrane covering the fat was peeled off as much as possible using forceps. Then it was transferred to D-Hanks buffer and rinsed thoroughly 2-3 times, and the remaining black membrane visible to the naked eye was removed during the rinsing process.

[0074] like Figure 2 Furthermore, S2 specifically includes:

[0075] S201, tissue mincing: Transfer the rinsed adipose tissue to a 15 mL sterile centrifuge tube, add a small amount of F12 / DMEM culture medium, and use long-handled ophthalmic scissors to mince the adipose tissue into small tissue blocks of about 1 mm³.

[0076] S202, Collagenase Digestion: Add 1 mg / mL preheated type I collagenase at a ratio of 3 times the tissue volume; place the centrifuge tube in a 39°C water bath or shaker for 30-40 minutes, inverting and mixing every 10 minutes to ensure thorough digestion and effective cell release;

[0077] S203, Mechanical blowing and termination: After 20 minutes of digestion, use a pipette to gently blow the mixture in a laminar flow hood to aid dissociation; decide whether to continue digestion based on the digestion status until the mixture is milky white and chyme-like.

[0078] S204, double physical filtration: After digestion, gently aspirate and mix well; first filter the suspension through a 100 μm cell sieve into a new 15 mL centrifuge tube, and then filter it again through a 40 μm cell sieve.

[0079] S205, Red blood cell lysis: Centrifuge the filtrate at 300 × g for 10 minutes; discard the supernatant, resuspend the cell pellet in red blood cell lysis buffer, and lyse at room temperature for 5 minutes; add complete culture medium to terminate lysis;

[0080] S206, Collection and Inoculation: Centrifuge again at 300 × g for 6 minutes; discard the supernatant and resuspend the pellet in complete culture medium; count cells using a cell counting chamber, adjust cell concentration according to 8 × 10⁶ cells / year. 5 - 1 × 10 6 Cells were seeded at a density of 10 cells / flask into T25 cell culture flasks and cultured in an incubator at 39°C and 5% CO2.

[0081] Furthermore, S3 specifically includes:

[0082] Primary differential adhesion: taking advantage of the fact that fibroblasts and some active melanocytes adhere faster than preadipocytes; after the primary cells are seeded into the incubator and left to stand for 1-1.5 hours, the culture flask is gently shaken, the supernatant that has not adhered is aspirated and directly transferred to a brand new T25 culture flask, an appropriate amount of culture medium is added and the cells are cultured for a while, and the original flask is discarded.

[0083] First medium change and washing: The day after inoculation, aspirate the old culture medium and gently wash the cells 1-2 times with 1×PBS to thoroughly remove dead cells, red blood cells and free melanin granules; add 4 mL of preheated fresh complete culture medium; at this time, under a microscope, the cells should mostly appear spindle-shaped or star-shaped.

[0084] Routine maintenance: Change the complete culture medium every 2 days; when the cell confluence reaches 70%-80%, prepare for passage and purification.

[0085] Furthermore, S4 specifically includes:

[0086] Differential digestion purification: Remove the old culture medium and wash the cells twice with calcium- and magnesium-free 1× PBS; add 2 mL of 0.05% Trypsin-EDTA to cover the bottom of the flask; observe in real time at room temperature or under a microscope. When the preadipocytes rapidly shrink and become round and show a tendency to detach, immediately tap the flask wall to detach the preadipocytes.

[0087] Collection and Termination: Quickly add an equal volume of serum-containing complete culture medium to terminate digestion; gently aspirate the suspended cell solution, being careful not to forcefully blow the bottom of the flask; transfer the suspension to a centrifuge tube, discarding the original culture flask along with any impurity cells;

[0088] Centrifugation and resuspension: Centrifuge at 300 × g for 5 minutes and discard the supernatant;

[0089] Subculturing: Resuspend in complete culture medium and count, at 3 × 10⁻⁶. 5 - 5× 10 5 Re-seed the cells at a density of 100 cells / flask into a new T25 flask; after complete differential adhesion and differential digestion, high-purity preadipocytes can be obtained.

[0090] Cell cryopreservation: Purified cells can be cryopreserved when their confluence reaches 90%. The cryopreservation solution is: 10% DMSO + 20% FBS + 70% DMEM / F12. Resuspend the cell pellet in the cryopreservation solution, aliquot it into cryovials, place them in a programmed cooling box, and freeze at -80°C overnight. After 24 hours, transfer them to a liquid nitrogen tank for long-term storage.

[0091] Furthermore, S5 specifically includes:

[0092] Gelatin coating of culture plates: Prepare a 2% (w / v) gelatin aqueous solution in advance and autoclave it; coat the bottom of the multi-well plate evenly at a rate of 5-10 μL / cm²; let it stand at room temperature for at least 1 hour, then remove excess gelatin liquid; before inoculating cells, place the coated culture plate in an incubator to preheat for 30 minutes.

[0093] Cell seeding and growth: Preadipocytes were seeded into gelatin-coated culture plates and cultured using complete growth medium;

[0094] Induction of differentiation: When the cell confluence reaches about 90%-100%, replace the growth medium with adipogenic differentiation-inducing medium (ADM); ADM formula: DMEM / F12 + 10% FBS + 1× linoleic acid-oleic acid-albumin (9.4 μg / mL) + 1% penicillin-antibody;

[0095] Medium change and observation: Maintain ADM culture by changing the medium every 2 days; usually, obvious lipid droplet formation can be observed under a microscope after 48 hours of induction.

[0096] Evidence related to the technical effects obtained by the embodiments of the present invention.

[0097] Bodipy staining identification

[0098] 1. Staining preparation: Cells were seeded in 12-well plates with a black transparent bottom and adipogenesis was induced. 2 μM Bodipy fluorescent staining working solution was prepared in the dark.

[0099] 2. Cell fixation: Remove the cells to be tested, wash twice with PBS, remove the PBS, add 4% paraformaldehyde fixative, and fix at room temperature for 15 min.

[0100] 3. Incubation in the dark: After fixation, discard the fixative, wash 2-3 times with PBS, add 500 μL of 2 μM Bodipy fluorescent staining working solution to each well, and incubate at room temperature in the dark for 15 minutes. Wash 2-3 times with PBS.

[0101] 4. Fluorescence photography: Observe using a fluorescence microscope, such as... Figure 3 As shown.

[0102] Oil Red O staining: Staining was performed using Solarbio's Oil Red O staining kit for cells. The specific steps are as follows:

[0103] 1. Remove the cell culture medium, wash twice with PBS, and fix with Oil Red O fixative for 20-30 min.

[0104] 2. Discard the fixative, wash twice with distilled water, and then soak in 60% isopropanol for 20-30 seconds.

[0105] 3. After discarding 60% isopropanol, add freshly prepared Oil Red O staining solution and immerse for 10-20 minutes.

[0106] 4. Discard the staining solution, rinse with 60% isopropanol for 10-20 seconds until the stroma is clear. Wash with distilled water 2-5 times until no excess staining solution is removed.

[0107] 5. Add Mayer hematoxylin staining solution and counterstain the nucleus for 1-2 minutes. Discard the staining solution, wash with water 2-5 times, add Oil Red O buffer and incubate for 1 minute to restore blue color, then discard the solution.

[0108] 6. Add distilled water to cover the cells and observe and photograph them under a microscope, such as... Figure 4 As shown.

[0109] Figure 5 This is a Bodipy staining image of preadipocytes after 48 hours of induced differentiation.

[0110] In the description of this invention, unless otherwise stated, "multiple" means two or more; the terms "upper," "lower," "left," "right," "inner," "outer," "front end," "rear end," "head," and "tail," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing the invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the invention. Furthermore, the terms "first," "second," "third," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0111] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for isolating and identifying preadipocytes of Lueyang black-boned chicken, characterized in that, Includes the following steps: S1 adipose tissue collection: Taking advantage of the physiological characteristic of Lueyang black-boned chickens being rich in melanin in their subcutaneous tissue, subcutaneous fat pads wrapped in a black membrane were collected from chicken embryos that had been incubated for up to day 16. S2 Black Membrane Peeling and Washing: The fat pad is rinsed in sterile PBS containing double antibodies. The black membrane covering the fat is physically peeled off under a microscope or with the naked eye. Then, it is rinsed and removed in D-Hanks buffer without calcium and magnesium ions to reduce the interference of melanin granules and melanocytes from the source. S3 Synergistic Digestion and Dual Filtration: Adipose tissue with the melanin removed was minced into pieces approximately 1 mm³. Type I collagenase (1 mg / mL) was added at a ratio of 3 times the tissue volume. Digestion was carried out at 39°C for 30 to 40 minutes, with gentle pipetting after 20 minutes. The synergistic effect of enzymatic digestion and mechanical dissociation maintained the viability of primary cells while ensuring complete tissue dissociation. The mixture was then sequentially filtered through 100 μm and 40 μm double-layer cell sieves to remove incompletely digested tissue residues and large pieces of melanin polymers. The filtrate was centrifuged, lysed with erythrocyte lysis buffer, and then centrifuged again after terminating the lysis process to obtain cell pellet. S4 Dual Differential Purification: The cell pellet is resuspended and inoculated for culture. Primary differential adhesion is performed using the characteristic that fibroblasts and some active melanocytes adhere to the culture vessel faster than preadipocytes. After standing for 1 to 1.5 hours, the unattached cell suspension is aspirated and transferred to a new culture flask for further culture. The first medium change is performed on the day after inoculation to remove free dead cells and free melanin granules. When the cell confluence reaches 70% to 80%, differential digestion is performed using the characteristic that preadipocytes are more sensitive to digestion solution and retract, round off, and detach faster than impurity cells. Digestion is stopped immediately when preadipocytes are observed to detach, and the cells are collected to achieve secondary purification of preadipocytes. S5 Adipogenic Induction and Identification: The collected and purified preadipocytes were cultured and induced to differentiate into adipocytes. The isolated preadipocytes were verified and identified by at least one of the following methods: morphological observation of lipid droplet formation, Oil Red O staining, Bodipy staining, or detection of adipogenic marker gene expression.

2. The method for isolating and purifying preadipocytes of Lueyang black-bone chicken as described in claim 1, characterized in that, The fat tissue collected from Lueyang black-bone chicken embryos that had been incubated for 16 days included: Before hatching, spray the eggshell surface with 70% ethanol for disinfection, crack open the eggshell at the air cell end and peel off the eggshell, tear open the eggshell membrane below the air cell, puncture the amnion, hold the chicken embryo by the neck and take out the chicken embryo, cut the yolk sac blood vessels and transfer the chicken embryo to a sterile environment.

3. The method for isolating and purifying preadipocytes of Lueyang black-bone chicken as described in claim 1, characterized in that, The removal of subcutaneous fat pads from the legs and chest includes: Chicken embryos were placed in sterile petri dishes and sprayed with 70% ethanol. A small incision was made in the abdomen, and the skin and feathers were peeled off to the sides to expose the subcutaneous fat pads of the legs and chest. The fat pads were removed while the surrounding muscle tissue was fixed, and the fat tissue of the three chicken embryos was combined as a single digestible tissue.

4. The method for isolating and purifying preadipocytes of Lueyang black-bone chicken as described in claim 1, characterized in that, The red blood cell lysis, termination of lysis, and recentrifugation include: Centrifuge the filtered cell suspension at 300 × g for 10 minutes, discard the supernatant, resuspend the cell pellet in red blood cell lysis buffer, lyse at room temperature for 5 minutes, add complete culture medium to terminate lysis, centrifuge again at 300 × g for 6 minutes, discard the supernatant, resuspend the pellet in complete culture medium and count the cells.

5. The method for isolating and purifying preadipocytes of Lueyang black-boned chicken as described in claim 1, characterized in that, The differential wall-attaching and differential digestion methods include: After primary cell seeding, incubate statically for 1 to 1.5 hours, shake the culture flask and aspirate the non-adherent supernatant, transferring it to a new culture flask for continued culture. The day after seeding, aspirate the old culture medium, wash the cells 1 to 2 times with 1×PBS, and add preheated fresh complete culture medium. When the cell confluence reaches 70% to 80%, aspirate the old culture medium, wash the cells twice with calcium- and magnesium-free 1×PBS, add 0.05% Trypsin-EDTA to cover the bottom of the flask, and when the preadipocytes shrink and become round and show a tendency to detach, gently tap the flask wall to detach the preadipocytes. Add the same volume of serum-containing complete culture medium to stop digestion, collect the suspended cell solution, and centrifuge and resuspend.

6. A method for identifying adipogenic induction in preadipocytes of Lueyang black-boned chicken, characterized in that, Includes the following steps: The culture plates were coated with gelatin, and the preadipocytes of Lueyang black-bone chicken were inoculated onto the gelatin-coated culture surface and cultured in complete growth medium. When the cell confluence reaches 90% to 100%, replace the complete growth medium with adipogenic differentiation-inducing medium. The adipogenic differentiation capacity of Lueyang black-bone chicken preadipocytes was identified by microscopic observation of lipid droplet formation after induction culture, and the culture medium was changed regularly. The adipogenic differentiation-inducing medium includes DMEM / F12, 10% FBS, 1×linoleic acid-oleic acid-albumin and 1% penicillin-antibody, wherein the concentration of linoleic acid-oleic acid-albumin is 9.4 μg / mL.

7. The method for identifying adipogenic induction of preadipocytes in Lueyang black-boned chicken as described in claim 6, characterized in that, The gelatin coating process includes: Prepare a 2% w / v gelatin aqueous solution and autoclave it. Coat the bottom of the culture plate with 5 to 10 μL / cm². Let it stand at room temperature for at least 1 hour, then remove the excess gelatin liquid. Before inoculating the cells, place the coated culture plate in an incubator to preheat for 30 minutes.

8. The method for identifying adipogenic induction of preadipocytes in Lueyang black-boned chicken as described in claim 6, characterized in that, The preadipocytes of Lueyang black-bone chickens were obtained through embryonic adipose tissue collection, collagenase digestion, double cell sieve filtration, erythrocyte lysis, differential adhesion, and differential digestion.

9. The method for identifying adipogenic induction of preadipocytes in Lueyang black-boned chicken as described in claim 6, characterized in that, The maintenance of the adipogenic differentiation medium and regular medium changes include: The lipid-inducing differentiation medium was changed every 2 days, and the formation of intracellular lipid droplets was observed 48 hours after induction.

10. The method for identifying adipogenic induction of preadipocytes in Lueyang black-boned chicken as described in claim 6, characterized in that, The preadipocytes of Lueyang black-bone chickens were passaged or cryopreserved before adipogenic induction identification. The passage process includes dividing the cells collected by differential digestion into 3 × 10⁻⁶ cells. 5 Up to 5 × 10 5 Re-seedling at a density of cells per culture flask; The cryopreservation process includes resuspending the cell pellet in a cryopreservation solution containing 10% DMSO, 20% FBS and 70% DMEM / F12 when the cell confluence reaches 90%, aliquoting the pellet, incubating it overnight at -80°C, and then transferring it to liquid nitrogen for storage.

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