Adipogenic induced differentiation culture medium suitable for natural immortalized cells of chick embryos, adipogenic differentiation method and application of adipogenic induced differentiation culture medium and adipogenic differentiation method

Abstract

The invention discloses an adipogenic induced differentiation culture medium suitable for natural immortalized cells of chick embryos, an adipogenic differentiation method and application of the adipogenic induced differentiation culture medium and the adipogenic differentiation method, and belongs to the field of poultry cell differentiation. The chick embryo naturally immortalized cells are obtained by naturally immortalizing and screening 9-11-day-old SPF chick embryos, and auxiliary factors of the induced differentiation medium comprise dexamethasone, insulin, IBMX, rosiglitazone, oleic acid, glutamine, transferrin and BSA. The culture medium suitable for the natural immortalized cells of the chick embryos has a good adipogenic differentiation effect, almost all cells have lipid droplets after the natural immortalized cells of the chick embryos are subjected to in-vitro induced differentiation for 48 hours, the cell differentiation efficiency reaches about 95% or above, the differentiation efficiency of the natural immortalized cells of the chick embryos to an adipocyte lineage is remarkably improved, and the differentiation rate of the natural immortalized cells of the chick embryos to the adipocyte lineage is greatly improved. And the differentiation time is greatly shortened, so that the method has important significance on the research on the lipogenic differentiation mechanism of the fiber-derived cells and the preparation of cell culture meat.

Description

Technical Field

[0001] This invention relates to the field of avian cell differentiation, specifically to a lipid-inducing differentiation culture medium, a lipid-differentiation method, and its application suitable for naturally immortalized chicken embryo cells. Background Technology

[0002] Cell-cultured meat is a novel product similar to traditional animal meat, produced by extracting muscle and adipose-derived stem cells from livestock and poultry, conducting stem cell proliferation and directed myoblastic / adipogenic differentiation in a bioreactor, and then collecting and processing the cells. Fat provides tenderness and juiciness to meat, affecting its texture and taste, but primary adipose-derived stem cells have limited expansion capacity (apoptosis rate >80% after P8), and significant batch-to-batch variations (lipid composition fluctuation ±35%). While universal immortalized cells (such as 3T3-L1) have solved the expansion bottleneck, they lack sufficient simulation of species flavor—mammalian cells cannot synthesize characteristic flavor substances of poultry / fish meat (such as 2,4-decadienal content <30% of natural content).

[0003] Traditional adipogenic differentiation media used in mammals consist of a basal medium supplemented with 10% FBS and a cocktail of insulin, dexamethasone, and IBMX. This results in poor induction, with few and unevenly distributed lipid droplets, and an induction time exceeding two days. This makes it impossible to use this traditional adipogenic differentiation medium for large-scale, efficient, stable, and inexpensive fat production, hindering the industrialization of cell-cultured meat.

[0004] Traditional 3D culture techniques suffer from poor lipid droplet uniformity (CV value > 40%) and insufficient thermal stability (lipid droplet fusion rate > 60% at 60℃), failing to meet the requirements of cooking and processing (US11499163B2). Furthermore, traditional 3D culture has systemic defects in high-throughput screening, data reliability, and industrial scale-up.

[0005] Therefore, there is an urgent need for new cell line lipid-derived differentiation media and 3D culture technology for precise regulation of lipid industrialization, in order to solve the systemic defects of lipid cultivation in terms of cell source, differentiation efficiency and traditional 3D culture. Summary of the Invention

[0006] One of the objectives of this invention is to provide a lipid-inducing differentiation medium, a lipid-inducing differentiation method, and its application suitable for naturally immortalized chicken embryo cells, thereby solving the problems of poor induction effect, low number of lipid droplets, and uneven distribution of traditional lipid-inducing differentiation mediums.

[0007] This invention provides a lipid-inducing differentiation culture medium suitable for naturally immortalized chicken embryo cells. The naturally immortalized chicken embryo cells are obtained from 9-11 day old SPF chicken embryos through natural immortalization screening. The lipid-inducing differentiation culture medium consists of a first basic supply component, a first cell culture cofactor, and a first auxiliary reagent. The first basic supply components include: 10% FBS, 1% Pen-strep, and basal culture medium; The first cell culture cofactor includes: dexamethasone at a concentration of 0.01 μmol / L to 1 μmol / L, insulin at a concentration of 33 μmol / mL to 66 μmol / mL, IBMX at a concentration of 0.1 mmol / L to 0.3 mmol / L, rosiglitazone at a concentration of 0.5 μmol / L to 1 μmol / L, oleic acid at a concentration of 120 μmol / L to 150 μmol / L, glutamine at a concentration of 2 mM to 4 mM, transferrin at a concentration of 10 μg / mL to 20 μg / mL, and BSA at a concentration of 0.2% to 0.4%. The first auxiliary reagent includes: a concentration of 5% to 10% HEPES at a concentration of 10 mM, β-ME at a concentration of 10 μM to 50 μM, and Ascorbic Acid at a concentration of 10 μM to 20 μM.

[0008] Furthermore, the natural immortalization screening employs a natural immortalization screening medium, which comprises a second basic supply component, a second cell culture cofactor, and a second auxiliary reagent. The second basic supply components include: 10% FBS, 1% penicillin and streptomycin, and basal culture medium; The second cell culture cofactor consisted of FGF-2 at a concentration of 20 ng / mL, EGF at a concentration of 20 ng / mL, and IGF-1 at a concentration of 10 ng / mL. The second auxiliary reagent: 1×ITS-X, 0.1% F68 and 0.2% BSA.

[0009] This invention provides a lipid-maintaining differentiation culture medium suitable for naturally immortalized chicken embryo cells. This lipid-maintaining differentiation culture medium is used in combination with the aforementioned lipid-inducing differentiation culture medium. The lipid-maintaining differentiation culture medium comprises a third basic supply component, a third cell culture cofactor, and a third auxiliary reagent. The third basal culture medium consists of 10% FBS, 1% Pen-strep, and basal culture medium. The third cell culture cofactors are: transferrin at a concentration of 8 μg / mL to 12 μg / mL and glutamine at a concentration of 2 mM; The third auxiliary reagent: has a concentration of 5% to 10%. HEPES at a concentration of 10 mM, β-ME at a concentration of 50 μM, and Ascorbic Acid at a concentration of 10 μM.

[0010] The present invention provides a lipid differentiation culture medium suitable for naturally immortalized chicken embryo cells, wherein the lipid differentiation culture medium is composed of the above-mentioned lipid induction differentiation culture medium and the above-mentioned lipid maintenance differentiation culture medium.

[0011] The second objective of this invention is to provide a method for adipogenic differentiation of naturally immortalized cells in chicken embryos, in order to solve the problems of poor uniformity of lipid droplet distribution and insufficient thermal stability in traditional 3D construction technology, which cannot meet the needs of cooking and processing, and can meet the requirements of both two-dimensional and three-dimensional industrial culture.

[0012] This invention provides a method for adipogenic differentiation of naturally immortalized chicken embryo cells under 2D culture conditions, comprising the following steps: Chicken embryos were naturally immortalized cells and cultured in vitro until they reached 100% confluence. The proliferation medium was discarded and the above-mentioned adipogenic differentiation induction medium was added. The day the adipogenic differentiation induction medium was added was recorded as day 0. The cells were cultured for 1-2 days. Remove the lipogenic induction differentiation medium and add the lipogenic maintenance differentiation medium described above. Continue differentiation for 2-4 days, changing the medium every 1 day.

[0013] This invention provides a method for adipogenic differentiation of naturally immortalized chicken embryo cells under 3D culture conditions, comprising the following steps: Chicken embryos were naturally immortalized cells and cultured in vitro until they reached 100% confluence. After digestion, centrifugation, and resuspension, the cells were cultured under 3D conditions until they proliferated stably and reached a certain density. Then, the above-mentioned adipogenic differentiation-inducing medium was added. The day the adipogenic differentiation-inducing medium was added was recorded as day 0. The cells were cultured for 1-2 days. Remove the lipogenic induction differentiation medium and add the lipogenic maintenance differentiation medium described above. Continue differentiation for 2-4 days, changing the medium every day.

[0014] Furthermore, in the method for adipogenic differentiation of naturally immortalized chicken embryo cells under 3D culture conditions, the cell proliferation density is required to be 3*102. ^ More than 6 cells / mL; preferably, the 3D culture conditions are provided by a 3D parallel bioreactor.

[0015] The present invention provides the application of the above-mentioned adipogenic induction differentiation medium, the above-mentioned adipogenic maintenance differentiation medium, or the above-mentioned adipogenic differentiation medium as a differentiation medium for seed cells in the process of preparing cultured meat.

[0016] The present invention provides the application of the above-mentioned adipogenic induction differentiation medium, the above-mentioned adipogenic maintenance differentiation medium, or the above-mentioned adipogenic differentiation medium as a test reagent in the process of testing the differentiation performance of seed cells using the cell differentiation method.

[0017] The present invention provides the application of the above-mentioned adipogenic induction differentiation medium, the above-mentioned adipogenic maintenance differentiation medium, or the above-mentioned adipogenic differentiation medium in the study of adipogenic development mechanism using in vitro cell differentiation.

[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The adipogenic differentiation culture medium provided by this invention is suitable for chicken embryo naturally immortalized cells. It has a good adipogenic differentiation effect. After in vitro induction differentiation of chicken embryo naturally immortalized cells for 48 hours, almost all cells showed lipid droplets, and the cell differentiation efficiency reached more than 95%. It significantly improved the differentiation efficiency of chicken embryo naturally immortalized cells into adipocyte lineage and greatly shortened the differentiation time, achieving rapid and efficient differentiation in 48 hours. This is of great significance for the study of the lipid differentiation mechanism of fibroblasts and the preparation of cell-cultured meat.

[0019] 2. The adipogenic differentiation culture medium for naturally immortalized chicken embryo cells provided by this invention has lower efficiency and longer time (about 7 days) compared to traditional induction of adipogenic differentiation. This invention uses oleic acid + high alkaline environment to achieve superactivation of PPARγ / δ dual pathways. By using a low dose of rosiglitazone (1 μmol / L), the differentiation time is greatly shortened, achieving an adipogenic efficiency of >95% in 48 hours, which greatly shortens the time for cell culture to produce meat fat.

[0020] 3. The adipogenic differentiation medium provided by this invention is suitable for the natural immortalization of chicken embryo cells. Compared with the primary cells, which have poor expansion capacity (apoptosis >80% after P8), chicken embryos can obtain immortalized cells (stable karyotype) that can be passaged >60 times through natural immortalization screening with triple synergy of growth factors (FGF-2 / EGF / IGF-1). In addition, these cells have adipogenic differentiation capacity, providing stable seed cells for cell cultured meat and facilitating the industrialization of cell cultured meat.

[0021] 4. This invention provides a method for adipogenic differentiation of naturally immortalized chicken embryo cells under 3D culture conditions. Under 3D culture conditions, the adipogenic differentiation medium provided by this invention and the naturally immortalized chicken embryo cells can achieve an adipogenic efficiency of >95% for 48 hours, reaching up to 100%. Under the integrated approach of "naturally immortalized chicken embryo cells + culture medium suitable for naturally immortalized chicken embryo cells + 3D industrial culture technology," large-scale preparation of cell-cultured meat fat can be achieved. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be considered as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort. In the drawings: Figure 1 This is a microscopic image of primary chicken embryo cells provided in Example 1 of the present invention; Figure 2 This is a microscopic image of spontaneously immortalized P50 generation chicken embryo cells provided in Example 2 of the present invention. Figure 3 The diagram provided in Example 3 of this invention shows the cell proliferation curves of chicken embryo naturally immortalized cells and chicken fat precursor cells after culture using the CCK-8 assay. Figure 4 The EdU experiment provided in Example 4 of this invention was used to detect the effects of the adipogenic differentiation medium provided in this invention and the classic adipogenic differentiation medium on the proliferation of naturally immortalized chicken embryo cells. Figure 5 Bright field and Oil Red O staining micrographs of chicken embryo naturally immortalized cells provided in Example 5 of the present invention after 48 hours of induction into differentiation on the adipogenic differentiation medium provided in the present invention. Figure 6 This is a Bodipy staining micrograph of chicken embryo naturally immortalized cells provided in Example 5 of the present invention after 48 hours of induction differentiation in the adipogenic differentiation medium provided in the present invention; Figure 7 Bright field and Oil Red O staining micrographs of chicken embryo naturally immortalized cells and chicken adipocyte precursor cells provided in Example 6 of the present invention after 48 hours of induced differentiation in the adipogenic differentiation medium provided in the present invention. Figure 8 Bright field and Oil Red O staining micrographs of chicken embryo naturally immortalized cells provided in Example 7 of the present invention after 48 hours of induction differentiation in traditional classic adipogenic differentiation medium and adipogenic differentiation medium provided in the present invention, respectively. Figure 9 The stability curves of naturally immortalized cells from P10, P30, and P60 generation chicken embryos obtained by CCK-8 assay in Example 9 of this invention are shown. Figure 10 This is a Bodipy staining micrograph of chicken embryo naturally immortalized cells 3D cultured under the adipogenic differentiation medium provided in this invention after 48 hours of induction differentiation under the adipogenic differentiation medium provided in this invention. (Example 10 of this invention) Detailed Implementation

[0023] 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 and accompanying drawings. The illustrative embodiments and descriptions of this invention are for explanation only and are not intended to limit the invention. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0024] Traditional lipid differentiation media have poor induction effects, with few lipid droplets and uneven distribution. To address this, the present invention proposes the following solution: A lipid-inducing differentiation culture medium suitable for naturally immortalized chicken embryo cells, wherein the naturally immortalized chicken embryo cells are obtained from 9-11 day old SPF chicken embryos through natural immortalization screening, and the lipid-inducing differentiation culture medium is composed of a first basic supply component, a first cell culture cofactor, and a first auxiliary reagent. The first basic supply components include: 10% FBS (fetal bovine serum), 1% Pen-strep (penicillin-streptomycin solution), and basal culture medium; The first cell culture cofactors include: dexamethasone at a concentration of 0.01 μmol / L to 1 μmol / L, insulin at a concentration of 33 μmol / L to 66 μmol / mL, IBMX (3-isobutyl-1-methylxanthine) at a concentration of 0.1 μmol / L to 0.3 mmol / L, rosiglitazone at a concentration of 0.5 μmol / L to 1 μmol / L, oleic acid at a concentration of 120 μmol / L to 150 μmol / L, glutamine at a concentration of 2 mM to 4 mM, transferrin at a concentration of 10 μg / mL to 20 μg / mL, and BSA (bovine serum albumin) at a concentration of 0.2% to 0.4%. The first auxiliary reagents include: NaHCO3 at a concentration of 5% to 10%, HEPES (4-hydroxyethylpiperazine ethanesulfonic acid) at a concentration of 10 mM, β-ME (β-mercaptoethanol) at a concentration of 10 μM to 50 μM, and Ascorbic Acid (L-ascorbic acid) at a concentration of 10 μM to 20 μM.

[0025] The adipogenic differentiation-inducing medium provided by this invention initiates the transdifferentiation of cells from fibroblast-adipocyte precursor cells to adipocytes by containing a high concentration of differentiation inducers, and supports the continuous synthesis and accumulation of lipid droplets by an adipogenic maintenance differentiation medium containing only basal nutrients, without involving cell fate transition. Induction phase (0-48 hours): Activates adipogenic transcription factors (such as PPARγ, C / EBPα) to initiate the differentiation program. Maintenance phase (48-144 hours): Provides lipid synthesis substrates (such as oleic acid) to promote lipid droplet maturation.

[0026] This invention involves natural immortalization screening of 9-11 day old SPF chicken embryos to obtain naturally immortalized chicken embryo cells. These cells exhibit typical morphological characteristics of fibroblast-adipocyte precursor cells: spindle-shaped, adherent growth, and stable expression of fibroblast marker genes such as COL1A1, Vimentin, and PDGFRα. Furthermore, these cells demonstrate basal expression of the FABP4 adipocyte marker gene, which is highly expressed in adipocytes (qPCR Ct value = 28 ± 0.5, compared to a Ct value > 35 in conventional adipocytes), indicating they are in a "prepared adipogenic state." These cells possess strong adipogenic capacity and, under the induction of adipogenic differentiation medium provided in this invention, can efficiently and stably differentiate into adipocyte lineages, characterized by a large accumulation of lipid droplets, achieving highly efficient adipogenesis with a fat generation rate reaching 100%.

[0027] The adipogenic differentiation-inducing medium provided by this invention targets the phenotypic characteristic of low FABP4 expression in naturally immortalized chicken embryo cells (pre-existing adipogenic potential but low differentiation threshold). The cofactors in the adipogenic differentiation-inducing medium achieve efficient adipogenic differentiation through a dual principle of temporal pathway activation and metabolic adaptation, as explained in detail below: 1) Temporal pathway activation: Phase I (0-24h): IBMX increases intracellular cAMP levels by inhibiting phosphodiesterase (PDE), activates the PKA / CREB axis, and initiates the expression of early adipogenic transcription factor C / EBPβ (expression increases 8-fold in 24h).

[0028] Phase II (24-48h): Rosiglitazone as a high-affinity agonist of PPARγ ( PPARγ forms a positive feedback loop with C / EBPβ, directly inducing the expression of FABP4 and ADIPOQ genes (PPARγ binding to the promoter is 12-fold more efficient). At low concentrations, dexamethasone activates glucocorticoid receptor (GR), inhibits the NF-κB inflammatory pathway, and relieves its transcriptional inhibition of PPARγ (flow cytometry showed that the percentage of PPARγ+ cells was 98.7% ± 0.8%).

[0029] Temporal pathway activation is achieved through the synergistic action of core inducers: IBMX activates the PKA / CREB signaling axis by increasing intracellular cAMP levels. This is the "first driving force" initiating adipogenic differentiation, responsible for inducing the expression of early adipogenic transcription factors (such as C / EBPβ).

[0030] Dexamethasone activates glucocorticoid receptors (GR) at low concentrations, and its synergistic effect lies in inhibiting NF-κB. NF-κB inflammatory pathway, thereby relieving the transcriptional inhibition of the key adipogenic factor PPARγ by NF-κB.

[0031] In addition, IBMX and dexamethasone together create a favorable transcription initiation environment for adipogenic differentiation.

[0032] Rosiglitazone: As a high-affinity exogenous agonist of PPARγ, it forms a positive feedback loop with C / EBPβ expressed in the first stage, strongly and continuously activating PPARγ.

[0033] Oleic acid, as a fatty acid, is also a natural agonist of PPARγ. It synergistically amplifies the activation signal of PPARγ with rosiglitazone, achieving "hyperactivation" of PPARγ. - The most important synergistic effect.

[0034] 2) Precise regulation of metabolic substrates: Oleic acid functions as both an endogenous PPARγ ligand and a precursor for lipid droplet synthesis. It is converted to triglycerides via the SCD1 enzyme (lipomomics analysis shows a C18:1 ratio of 58% ± 3%) and can also optimize membrane fluidity to reduce lipid droplet fusion resistance (electron microscopy shows lipid droplet diameter distribution of 5-20 μm, accounting for >85%). The oleic acid concentration needs to be controlled within a suitable range. If the concentration exceeds 200 μmol / L, it easily induces lipotoxicity (LDH release increases by 30%); if the concentration is below 100 μmol / L, the number of lipid droplets decreases by 40%. Insulin activates the IRS-1 / PI3K / Akt pathway, promoting GLUT4-mediated glucose transport (2-NBDG fluorescence intensity increased by 35%), providing a carbon source for acyl-CoA synthesis; Transferrin provides iron ions to activate fatty acid desaturase (FADS2), thereby improving the bioavailability of oleic acid (SCD1 activity decreases by 70% when iron is deficient).

[0035] 3) Steady-state protection components: Glutamine generates α-ketoglutarate via the TCA cycle, supporting ATP-dependent lipid acylation reactions; BSA binds to free fatty acids to prevent aggregation toxicity (>0.4% adsorbs the inducer, <0.1% increases cell death rate by 30%). The antioxidant system (β-ME + Ascorbic Acid) in the synergistic auxiliary reagent resists the ROS burst during differentiation (MDA content is reduced by 40%) and maintains the integrity of the lipid droplet membrane.

[0036] In this invention, the adipogenic differentiation maintenance culture medium consists of a first basic supply component, a first cell culture cofactor, and a first auxiliary reagent: The first basic supply components include: 10% FBS, 1% penicillin and streptomycin and basal culture medium.

[0037] The first cell culture cofactor includes dexamethasone at a concentration of 0.01 μmol / L to 1 μmol / L, which can be 0.01 μmol / L, 0.05 μmol / L, 0.1 μmol / L, 0.2 μmol / L, 0.3 μmol / L, 0.4 μmol / L, 0.5 μmol / L, 0.6 μmol / L, 0.7 μmol / L, 0.8 μmol / L, 0.9 μmol / L, 1 μmol / L, etc., but is not limited to the listed values. Other unlisted values ​​within this range are also applicable. Insulin concentrations of 33 μmol / mL to 66 μmol / mL can be 33 μmol / mL, 35 μmol / mL, 38 μmol / mL, 40 μmol / mL, 42 μmol / mL, 45 μmol / mL, 48 μmol / mL, 50 μmol / mL, 52 μmol / mL, 55 μmol / mL, 57 μmol / mL, 60 μmol / mL, 63 μmol / mL, 66 μmol / mL, etc., but are not limited to the listed values. Other unlisted values ​​within this range are also applicable. IBMX concentrations of 0.1 mmol / L to 0.3 mmol / L can be 0.1 mmol / L, 0.13 mmol / L, 0.15 mmol / L, 0.18 mmol / L, 0.2 mmol / L, 0.22 mmol / L, 0.25 mmol / L, 0.28 mmol / L, 0.3 mmol / L, etc., but are not limited to the listed values. Other unlisted values ​​within this range are also applicable. Rosiglitazone with a concentration of 0.5 μmol / L to 1 μmol / L can be 0.5 μmol / L, 0.6 μmol / L, 0.7 μmol / L, 0.8 μmol / L, 0.9 μmol / L, 1 μmol / L, etc., but is not limited to the listed values. Other unlisted values ​​within this range are also applicable. Oleic acid with a concentration of 120 μmol / L to 150 μmol / L can be 120 μmol / L, 123 μmol / L, 125 μmol / L, 127 μmol / L, 130 μmol / L, 132 μmol / L, 135 μmol / L, 138 μmol / L, 140 μmol / L, 142 μmol / L, 145 μmol / L, 147 μmol / L, 150 μmol / L, etc., but is not limited to the listed values. Other unlisted values ​​within this range are also applicable. Glutamine concentrations of 2mM to 4mM can be 2mM, 2.2mM, 2.5mM, 2.7mM, 3mM, 3.2mM, 3.5mM, 3.8mM, 4mM, etc., but are not limited to the listed values. Other unlisted values ​​within this range are also applicable. Transferrin at concentrations of 10 μg / mL to 20 μg / mL can be 10 μg / mL, 11 μg / mL, 12 μg / mL, 13 μg / mL, 14 μg / mL, 15 μg / mL, 16 μg / mL, 17 μg / mL, 18 μg / mL, 19 μg / mL, 20 μg / mL, etc., but is not limited to the listed values. Other unlisted values ​​within this range are also applicable. The concentration of BSA is 0.2% to 0.4%, which can be 0.2%, 0.22%, 0.24%, 0.26%, 0.28%, 0.3%, 0.32%, 0.34%, 0.36%, 0.38%, 0.4%, etc., but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0038] The first auxiliary reagent includes: NaHCO3 at a concentration of 5% to 10%, which can be 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, etc., but is not limited to the listed values; other unlisted values ​​within this range are also applicable; HEPES at a concentration of 10 mM; β-ME at a concentration of 10 μM to 50 μM, which can be 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, etc., but is not limited to the listed values; other unlisted values ​​within this range are also applicable; and Ascorbic acid at a concentration of 10 μM to 20 μM. Acid can be 10μM, 11μM, 12μg / mL, 13μM, 14μM, 15μM, 16μM, 17μM, 18μM, 19μM, 20μM, etc., but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0039] Furthermore, the adipogenic differentiation induction medium suitable for naturally immortalized chicken embryo cells comprises a first basic supply component, a first cell culture cofactor, and a first auxiliary reagent: the first basic supply component includes: 10% FBS, 1% Penstrep, and basal medium. The first cell culture cofactor includes: 0.2 μmol / L dexamethasone, 66 nmol / mL insulin, 0.25 mmol / L IBMX, 1 μmol / L rosiglitazone, 150 μmol / L oleic acid, 2 mM glutamine, 10 μg / mL transferrin, and 0.2% BSA; the first auxiliary reagent includes: 8%... HEPES at a concentration of 10 mM, β-ME at a concentration of 50 μM, and Ascorbic Acid at a concentration of 10 μM.

[0040] Furthermore, the natural immortalization screening employs a natural immortalization screening medium, which comprises a second basic supply component, a second cell culture cofactor, and a second auxiliary reagent. The second basic supply components include: 10% FBS, 1% penicillin and streptomycin, and basal culture medium; The second cell culture cofactors are: FGF-2 (fibroblast growth factor-2) at a concentration of 20 ng / mL, EGF (epidermal growth factor) at a concentration of 20 ng / mL, and IGF-1 (insulin-like growth factor-1) at a concentration of 10 ng / mL. The second auxiliary reagents are: 1×ITS-X (insulin-transferrin-selenium-ethanolamine and trace element complex), F68 (poloxamer 188) at a concentration of 0.1%, and BSA (bovine serum albumin) at a concentration of 0.2%.

[0041] The 9-11 day old chicken embryos selected in this invention represent the core window period, during which embryonic adipose tissue development begins and preadipocyte progenitor cells (with low FABP4 expression) are present. After trypsin washing, homogenization, digestion, dispersion, and natural immortalization screening in an immortalization screening medium (containing FGF-2 / EGF / IGF-1), the MAPK / PI3K pathway is activated, maintaining cell stemness and coupling basal FABP4 expression.

[0042] The immortalized chicken embryo cells screened for immortalization included fibroblasts expressing COL1A1+Vimentin+PDGFRα and adipocytes expressing low levels of FABP4. Normal fibroblasts all express COL1A1+Vimentin+PDGFRα, with very few expressing the FABP4 gene. FABP4, as a marker of adipogenic differentiation, indicates that the cells screened in this invention possess endogenous adipogenic potential, enabling a faster response after induction.

[0043] The naturally immortalized chicken embryo cells selected through immortalization screening exhibit typical morphological characteristics of fibroblast and adipocyte precursor cells: they are spindle-shaped, adherent to the wall, and stably express fibroblast and adipocyte marker genes such as COL1A1+Vimentin+PDGFRα+FABP4. The adipogenesis rate can reach 100% within 48 hours, and the cells still proliferate stably at generation P60, which is different from known fibroblasts or mesenchymal stem cells.

[0044] Furthermore, the basal culture medium is DMEM medium.

[0045] A lipid-maintaining differentiation culture medium suitable for naturally immortalized chicken embryo cells, wherein the lipid-maintaining differentiation culture medium is used in combination with the aforementioned induction differentiation culture medium, and the lipid-maintaining differentiation culture medium is composed of a third basic supply component, a third cell culture cofactor, and a third auxiliary reagent: The third basal culture medium includes: 10% FBS, 1% Pen-strep, and basal culture medium. The third cell culture cofactor includes: transferrin at a concentration of 8 μg / mL to 12 μg / mL, where the concentration can be 8 μg / mL, 9 μg / mL, 10 μg / mL, 11 μg / mL, 12 μg / mL, etc., but is not limited to the listed values; other unlisted values ​​within this range are also applicable; and glutamine at a concentration of 2 mM. The third auxiliary reagent includes: a concentration of 5% to 10%. The concentration can be 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, etc., but is not limited to the listed values. Other unlisted values ​​within this range are also applicable; the concentrations are 10 mM HEPES, 50 μM β-ME, and 10 μM Ascorbic Acid.

[0046] The chicken embryo immortalized cells provided by this invention are characterized by "high basal lipid synthesis activity" and "low differentiation threshold." RNA-seq showed that these cells highly express the mitochondrial β-oxidation gene (CPT1A, ACADL), requiring continuous energy supply to maintain lipid droplet expansion. Lipomics confirmed that they depend on exogenous lipid precursors (oleic acid is pre-stored during the induction phase). After differentiation initiation (48 h), the cells expressed PPARγ / C / EBPα, requiring only basal metabolic support rather than strong induction stimulation to complete lipid droplet accumulation (compared to traditional methods that require continuous addition of inducing agents).

[0047] To achieve stable and efficient lipid differentiation, the design of the differentiation culture medium is explained in detail below: After differentiation was initiated (48h), the cells expressed PPARγ / C / EBPα. Compared with the traditional method that requires continuous addition of inducing agents, the differentiation maintenance medium of the present invention only requires basal metabolic support rather than strong inducing stimulation to complete lipid droplet accumulation.

[0048] Transferrin is key to lipid droplet structure stability. Iron ions act as a cofactor for fatty acid desaturase (SCD1), promoting the conversion of oleic acid to triglycerides (GC-MS showed a 15% increase in the C18:1 ratio). It also binds to the cell surface transferrin receptor (TfR1) to activate the PI3K / Akt pathway, thereby inhibiting apoptosis during differentiation (apoptosis rate <5%). The iron ion concentration needs to be controlled within an appropriate range. If the concentration is below 5 μg / mL, lipid droplet fusion and rupture occur (electron microscopy shows membrane structure damage). If the concentration is above 15 μg / mL, iron overload triggers the Fenton reaction, leading to increased lipid peroxidation.

[0049] It works synergistically with glutamine to achieve a balance between energy supply and antioxidant activity. Glutamine can promote the activity of acyl-CoA synthase (ACS); synthesize glutathione, and protect the integrity of lipid droplet membranes (MDA content is reduced by 40%).

[0050] Furthermore, the adipogenic maintenance differentiation medium suitable for naturally immortalized chicken embryo cells consists of a third basic supply component, a third cell culture cofactor, and a third auxiliary reagent: the third basic medium comprises 10% FBS, 1% penicillin and streptomycin, and basal medium; the third cell culture cofactor comprises 10 μg / mL transferrin and 2 mM glutamine; the third auxiliary reagent comprises 8%... HEPES at a concentration of 10 mM, β-ME at a concentration of 50 μM, and Ascorbic Acid at a concentration of 10 μM.

[0051] To address the problems of poor uniformity of lipid droplet distribution and insufficient thermal stability in traditional 3D fabrication techniques, which fail to meet the requirements of cooking and processing, this invention proposes the following solution: A method for adipogenic differentiation of naturally immortalized chicken embryo cells under 2D culture conditions includes the following steps: Chicken embryos were naturally immortalized cells and cultured in vitro until they reached 100% confluence. The proliferation medium was discarded and the above-mentioned adipogenic differentiation induction medium was added. The day the adipogenic differentiation induction medium was added was recorded as day 0. The cells were cultured for 1-2 days. Remove the above-mentioned adipogenic induction differentiation medium and add the above-mentioned adipogenic maintenance differentiation medium. Continue differentiation for 2-4 days, changing the medium every 1 day.

[0052] A method for adipogenic differentiation of naturally immortalized chicken embryo cells under 3D culture conditions is disclosed. The 3D culture is conducted in a 3D parallel bioreactor. This 3D parallel bioreactor overcomes the industrialization barriers of missing naturally immortalized chicken embryo cells and cross-module synergistic failure through three major innovations: synchronized control, integrated data flow, and miniaturized process model. Specifically, the method includes the following steps: Chicken embryos were naturally immortalized cells and cultured in vitro until they reached 100% confluence. After digestion, centrifugation, and resuspension, the cells were cultured under 3D conditions until they proliferated stably and reached a certain density. Then, the above-mentioned adipogenic differentiation-inducing medium was added. The day the adipogenic differentiation-inducing medium was added was recorded as day 0. The cells were cultured for 1-2 days. Remove the above-mentioned adipogenic induction differentiation medium and add the above-mentioned adipogenic maintenance differentiation medium. Continue differentiation for 2-4 days, changing the medium every day.

[0053] Further restrictions are imposed, requiring a cell proliferation density of 3*102 ^ More than 6 per mL.

[0054] To further illustrate the present invention, the following description, in conjunction with embodiments, illustrates an adipogenic differentiation induction culture medium, adipogenic differentiation method, and application of the present invention for naturally immortalized chicken embryo cells. However, it should be understood that these embodiments are implemented under the premise of the technical solution of the present invention, providing detailed implementation methods and specific operating procedures, and are only for further illustrating the features and advantages of the present invention, and are not intended to limit the scope of the claims of the present invention. The scope of protection of the present invention is not limited to the following embodiments.

[0055] It should be noted that the basal culture medium used in the following embodiments of the present invention is DMEN medium.

[0056] Example 1: To obtain primary chicken embryo cells, the specific procedures in this embodiment are as follows: (1) Obtain SPF chicken embryos aged 9-11 days, remove the head, limbs and internal organs, rinse repeatedly with PBS to remove blood, retain only the trunk muscle and connective tissue, cut into 1-2 mm³ tissue blocks, add 1-3 mg / mL collagenase solution, transfer to tissue dissociation tube, dissociate using a tissue dissociator and place in a 37 ℃ water bath for digestion for 60-90 min, dissociate using a tissue dissociator every 15-20 min; then add an equal volume of high glucose DMEM containing 10% fetal bovine serum to terminate digestion, filter with a 70-150 μm cell sieve, wherein the volume ratio of the minced meat to the collagenase solution is 1:5; (2) Centrifuge the digestion solution at 800-1500 rpm at room temperature for 5-10 min, discard the supernatant and retain the cell pellet; (3) Add 3-5 times the volume of erythrocyte lysis buffer to the cell pellet for resuspending, lyse at room temperature for 5-10 min, centrifuge, discard the supernatant, and retain the cell pellet; (4) After resuspending the cell pellet in high-glucose DMEM containing 20% ​​FBS, the cells were seeded into cell culture dishes. After passage, high-glucose DMEM containing 10% FBS was used as the complete culture medium.

[0057] like Figure 1 As shown, microscopic observation revealed that the obtained primary chicken embryo cells were spindle-shaped and adhered to the wall.

[0058] Example 2: The primary chicken embryo cells obtained in Example 1 were subjected to spontaneous immortalization screening to obtain naturally immortalized chicken embryo cells. The specific operation is as follows: (1) When the cell confluence reaches about 80%, remove the proliferation medium and add spontaneous immortalization induction medium. Record the day the induction medium is added as 0d. Change the medium every 1 day and culture continuously.

[0059] The natural immortalization screening uses a natural immortalization screening medium, which consists of a second basic supply component, a second cell culture cofactor, and a second auxiliary reagent. The second basic supply component includes 10% FBS, 1% penicillin and streptomycin, and basal medium. The second cell culture cofactor consists of 20 ng / mL FGF-2, 20 ng / mL EGF, and 10 ng / mL IGF-1. The second auxiliary reagent consists of 1×ITS-X, 0.1% F68, and 0.2% BSA.

[0060] (2) When the cell confluence reaches about 90%, digest, centrifuge, resuspend, and passage at a ratio of 1:3 for 50 consecutive generations.

[0061] like Figure 2 As shown, microscopic observation revealed that the naturally immortalized chicken embryo cells obtained after 50 generations of continuous passage still exhibited a spindle shape and adhered growth, consistent with the typical characteristics of fibrous cells.

[0062] Example 3: The adipogenic differentiation medium for naturally immortalized chicken embryo cells provided in this embodiment consists of an adipogenic induction differentiation medium and a maintenance differentiation medium for naturally immortalized chicken embryo cells.

[0063] A culture medium suitable for inducing adipogenic differentiation of naturally immortalized chicken embryo cells comprises a first basic supply component, a first cell culture cofactor, and a first auxiliary reagent. The first basic supply component includes: 10% FBS, 1% Penstrep, and basal medium. The first cell culture cofactor includes: 0.2 μmol / L dexamethasone, 66 nmol / mL insulin, 0.25 mmol / L IBMX, 1 μmol / L rosiglitazone, 150 μmol / L oleic acid, 2 mM glutamine, 10 μg / mL transferrin, and 0.2% BSA. The first auxiliary reagent includes: 8%... HEPES at a concentration of 10 mM, β-ME at a concentration of 50 μM, and Ascorbic Acid at a concentration of 10 μM.

[0064] This adipogenic maintenance differentiation medium for naturally immortalized chicken embryo cells consists of a third basic supply component, a third cell culture cofactor, and a third auxiliary reagent: The third basic medium comprises 10% FBS, 1% penicillin / streptomycin, and basal medium; the third cell culture cofactor comprises 10 μg / mL transferrin and 2 mM glutamine; and the third auxiliary reagent comprises 8%... HEPES at a concentration of 10 mM, β-ME at a concentration of 50 μM, and Ascorbic Acid at a concentration of 10 μM.

[0065] The effect of culturing chicken embryo naturally immortalized cells and chicken fat precursor cells in a medium supplemented with induction reagents on cell proliferation was investigated as follows: Chicken embryo naturally immortalized cells and chicken adipocyte precursor cells obtained in Example 2 were resuspended and counted in the adipogenic differentiation medium provided in this example, and then divided into groups of 1×10⁻⁶ cells per well. 4 Cells were seeded into 96-well plates. The diary entry was recorded as day 0. The OD values ​​of naturally immortalized chicken embryo cells and chicken fat precursor cells were measured every 2 days using the CCK-8 method to plot growth curves.

[0066] like Figure 3 As shown, the results indicate that after 6 days of culture in the adipogenic differentiation medium provided in this embodiment, there was a difference in the growth of chicken embryo naturally immortalized cells and chicken adipocyte precursor cells. The naturally immortalized cells of chicken embryos had a stronger proliferative capacity. After 4 more days of culture, there was a significant difference in the growth of chicken embryo naturally immortalized cells and chicken adipocyte precursor cells (P<0.05), indicating that under the same treatment process, the naturally immortalized cells of chicken embryos provided by this invention showed a stronger proliferative capacity.

[0067] Example 4: The adipogenic differentiation medium for naturally immortalized chicken embryo cells provided in this embodiment consists of an adipogenic induction differentiation medium and a maintenance differentiation medium. The adipogenic induction differentiation medium for naturally immortalized chicken embryo cells comprises a first basic supply component, a first cell culture cofactor, and a first auxiliary reagent: the first basic supply component includes: 10% FBS, 1% Penstrep, and basal medium. The first cell culture cofactor includes: 0.2 μmol / L dexamethasone, 66 nmol / mL insulin, 0.25 mmol / L IBMX, 1 μmol / L rosiglitazone, 150 μmol / L oleic acid, 2 mM glutamine, 10 μg / mL transferrin, and 0.2% BSA; the first auxiliary reagent includes: 8%... HEPES at a concentration of 10 mM, β-ME at a concentration of 50 μM, and Ascorbic Acid at a concentration of 10 μM.

[0068] This adipogenic maintenance differentiation medium for naturally immortalized chicken embryo cells consists of a third basic supply component, a third cell culture cofactor, and a third auxiliary reagent: The third basic medium comprises 10% FBS, 1% penicillin / streptomycin, and basal medium; the third cell culture cofactor comprises 10 μg / mL transferrin and 2 mM glutamine; and the third auxiliary reagent comprises 8%... HEPES at a concentration of 10 mM, β-ME at a concentration of 50 μM, and Ascorbic Acid at a concentration of 10 μM.

[0069] To investigate the effects of the adipogenic differentiation medium provided in this embodiment as the experimental group and the classic adipogenic differentiation medium as the control group on the proliferation capacity of naturally immortalized chicken embryo cells, the specific procedures are as follows: (1) Take the naturally immortalized chicken embryo cells obtained in Example 2, digest, centrifuge, resuspend, and then divide into 1×10 cells per well. 5One cell was seeded into a 12-well plate. After the chicken embryos naturally immortalized and the cells adhered to the plate, the proliferation medium was removed, and traditional classic adipogenic differentiation induction medium and the adipogenic differentiation induction medium provided in Example 4 were added respectively. The day the adipogenic differentiation induction medium was added was recorded as day 0. Differentiation was induced for 2 days, and then the adipogenic differentiation induction medium was replaced with the adipogenic maintenance differentiation medium provided in Example 4. The medium was changed every 1 day to maintain differentiation for 10 days.

[0070] (2) After 10 days of cultivation, the EdU experiment was carried out.

[0071] like Figure 4 As shown, observation under a fluorescence inverted microscope revealed that after 10 days of culture in a medium with added induction reagent, the fluorescence intensity of chicken embryo immortalized cells cultured in the experimental group using the adipogenic differentiation medium provided by this invention was significantly higher than that of chicken embryo immortalized cells cultured in the control group using the classic adipogenic differentiation medium. This indicates that the adipogenic differentiation medium provided by this invention has a significant promoting effect on cell proliferation during the adipogenic differentiation process of chicken embryo immortalized cells (P<0.05).

[0072] Example 5: This embodiment investigates the differentiation culture of naturally immortalized chicken embryo cells, as well as Oil Red O staining and Bodipy staining after differentiation. The specific procedures are as follows: (1) When the confluence of naturally immortalized cells in chicken embryos reaches 100%, remove the proliferation medium and add the adipogenic differentiation induction medium provided in Example 4. The day the adipogenic differentiation induction medium is added is recorded as 0h. After 48h of induction differentiation, replace it with the adipogenic maintenance differentiation medium provided in Example 4. Change the medium every 24h and maintain differentiation for 96h.

[0073] like Figure 5 As shown in the left image, under a microscope, the naturally immortalized cells of the chicken embryo, differentiated for 48 hours, were found to be filled with spherical lipid droplets.

[0074] (2) Remove the adipogenic differentiation medium and wash once with PBS; add 4% PFA to fix the cells for 30 min. Remove the fixative and add staining wash for 30 s. Remove the wash and add Oil Red O staining solution for 15-20 min. Discard the Oil Red O staining solution and wash 1-3 times with PBS; observe and photograph under a microscope.

[0075] like Figure 5 (Right) Microscopic observation shows that Oil Red O staining results indicate that lipid droplets in naturally immortalized chicken embryo cells differentiated for 48 hours were stained red.

[0076] (3) Remove the adipogenic differentiation medium, wash once with PBS; add 4% PFA to fix cells for 30 min. Remove the fixative, wash three times with PBS, add Bodipy staining for 15-20 min. Discard the Bodipy staining solution, add PBS to wash 1-3 times; add DAPI staining for 10 min, add PBS to wash 1-3 times; observe and photograph under a microscope.

[0077] like Figure 6 As shown, microscopic observation revealed that Bodipy staining results showed that lipid droplets in cells differentiated for 48 hours were stained green, indicating that the adipogenic differentiation medium provided by this invention can induce adipogenic differentiation of naturally immortalized chicken embryo cells.

[0078] Example 6: This embodiment investigates the differentiation culture of naturally immortalized chicken embryo cells and chicken fat precursor cells, and the Oil Red O staining after differentiation. The specific procedures are as follows: (1) When the confluence of the two types of cells reaches 100%, remove the proliferation medium and add the adipogenic differentiation induction medium provided in Example 4. The day the adipogenic differentiation induction medium is added is recorded as 0h. After 48h of induction differentiation, replace it with the adipogenic maintenance differentiation medium provided in Example 4. Change the medium every 24h and maintain differentiation for 96h.

[0079] Microscopic observation revealed that the naturally immortalized cells of chicken embryos differentiated for 48 hours were filled with spherical lipid droplets. Figure 7 As shown in the upper right image, chicken fat precursor cells differentiated for 48 hours contain only a small number of spherical lipid droplets, such as... Figure 7 As shown in the top left.

[0080] (2) Remove the adipogenic differentiation medium and wash once with PBS; add 4% PFA to fix the cells for 30 min. Remove the fixative and add staining wash for 30 s. Remove the wash and add Oil Red O staining solution for 15-20 min. Discard the Oil Red O staining solution and wash 1-3 times with PBS; observe and photograph under a microscope.

[0081] Microscopic observation revealed that Oil Red O staining showed a large number of lipid droplets stained red in naturally immortalized chicken embryo cells differentiated for 48 hours. Figure 7 As shown in the lower right, only a small number of lipid droplets in chicken adipocyte precursor cells differentiated for 48 hours were stained red, as... Figure 7 As shown in the lower left, the lipid differentiation culture medium provided by this invention is specifically for chicken embryo naturally immortalized cells.

[0082] Example 7: This embodiment explores the Bodipy staining after differentiation of naturally immortalized chicken embryo cells using the adipogenic differentiation medium provided by this invention and the traditional classic adipogenic differentiation induction medium. The specific operation is as follows: (1) When the confluence of naturally immortalized chicken embryo cells reaches 100%, remove the proliferation medium and add the traditional classic adipogenic differentiation induction medium and the induction differentiation medium provided in Example 4 respectively. The day the induction differentiation medium is added is recorded as 0h. After 48h of induction differentiation, replace it with the maintenance differentiation medium provided in Example 4 (the two methods are the same). Change the medium every 24h and maintain differentiation for 96h.

[0083] Microscopic observation revealed that the naturally immortalized chicken embryo cells induced by the adipogenic differentiation medium provided in this invention, 48 hours after differentiation, were filled with spherical lipid droplets, such as... Figure 8 As shown in the upper right image, chicken embryos that have undergone natural immortalization 48 hours after induction of differentiation in traditional classic adipogenic differentiation medium contain only a small number of spherical lipid droplets, such as... Figure 8 As shown in the top left.

[0084] (2) Remove the adipogenic differentiation medium, wash once with PBS; add 4% PFA to fix cells for 30 min. Remove the fixative, wash three times with PBS, add Bodipy staining for 15-20 min. Discard the Bodipy staining solution, add PBS to wash 1-3 times; add DAPI staining for 10 min, add PBS to wash 1-3 times; observe and photograph under a microscope.

[0085] Microscopic observation revealed that Bodipy staining showed a large number of lipid droplets stained green in naturally immortalized chicken embryo cells differentiated for 48 hours. Figure 8 As shown in the lower right, only a small number of lipid droplets in chicken fat precursor cells differentiated for 48 hours were stained green, as... Figure 8 As shown in the lower left, the adipogenic differentiation culture medium for naturally immortalized chicken embryo cells provided by this invention is specifically designed for the differentiation of naturally immortalized chicken embryo cells into adipocyte lineages.

[0086] Example 8: This embodiment investigates the stability assessment of primary chicken embryo cells after spontaneous immortalization and long-term passage. The specific procedures are as follows: Primary chicken embryo cells from generations P10, P30, and P60 were resuspended in spontaneous immortalization induction medium, counted, and seeded into 96-well plates at a density of 1 × 10⁴ cells per well. Day 0 was recorded. OD values ​​of cells from generations P10, P30, and P60 were measured every two days using the CCK-8 assay to plot growth curves.

[0087] like Figure 9As shown, the results indicate that there was no significant difference in cell growth among P10, P30, and P60 generations (P>0.5), and cells proliferated stably.

[0088] Example 9: This embodiment explores a method for adipogenic differentiation of naturally immortalized chicken embryo cells under 2D culture conditions, including the following steps: Chicken embryos were naturally immortalized cells and cultured in vitro until they reached 100% confluence. The proliferation medium was discarded and the induction differentiation medium provided in Example 4 was added. The day the induction differentiation medium was added was recorded as day 0. The cells were cultured for 1-2 days. Remove the induction differentiation medium and add the maintenance differentiation medium provided in Example 4. Continue differentiation for 2-4 days, changing the medium every 1 day.

[0089] Example 10: This embodiment explores the lipid-derived differentiation of naturally immortalized chicken embryo cells in a 3D parallel bioreactor. The specific operation is as follows: (1) Chicken embryos were naturally immortalized cells and cultured in vitro until they reached 100% confluence. After digestion, centrifugation, and resuspending, the cells were added to a 3D parallel bioreactor. Once the cells had proliferated stably and reached a certain density, the proliferation medium was removed, and the induction differentiation medium provided in Example 4 was added. The day the induction differentiation medium was added was recorded as day 0. The cells were cultured for 2 days. The induction differentiation medium was then removed, and the maintenance differentiation medium provided in Example 4 was added. Differentiation continued for 4 days, with the medium changed every other day.

[0090] (2) Remove the adipogenic differentiation medium, wash once with PBS; add 4% PFA for cell fixation for 30 min. Remove the fixative, wash three times with PBS, and add Bodipy staining for 15-20 min. Discard the Bodipy staining solution, wash 1-3 times with PBS; add DAPI staining for 10 min, wash 1-3 times with PBS; observe and photograph under a microscope. Figure 10 As shown, microscopic observation revealed that Bodipy staining results showed that a large number of lipid droplets in the naturally immortalized chicken embryo cells differentiated for 48 hours in the 3D parallel bioreactor were stained green, indicating that the adipogenic differentiation medium provided by the present invention can induce the differentiation of naturally immortalized chicken embryo cells into adipocyte lineages under 3D culture conditions.

[0091] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A lipidogenic induction differentiation medium suitable for chicken embryo natural immortalized cells, characterized by, The chicken embryo natural immortalization cell is obtained by natural immortalization screening of 9-11 day old SPF chicken embryos, and the adipogenic induction differentiation medium is composed of a first basic supply component, a first cell culture auxiliary factor and a first auxiliary reagent: The first basic supply component includes: FBS with a concentration of 10%, Pen-strep with a concentration of 1% and a basic culture medium; The first cell culture auxiliary factor includes: dexamethasone with a concentration of 0.01 μmol / L-1 μmol / L, insulin with a concentration of 33 μmol / mL-66 μmol / mL, IBMX with a concentration of 0.1 mmol / L-0.3 mmol / L, rosiglitazone with a concentration of 0.5 μmol / L-1 μmol / L, oleic acid with a concentration of 120 μmol / L-150 μmol / L, glutamine with a concentration of 2 mM-4 mM, transferrin with a concentration of 10 μg / mL-20 μg / mL and BSA with a concentration of 0.2%-0.4%; The first auxiliary reagent comprises: 5%~10% of HEPES with a concentration of 10 mM, β-ME with a concentration of 10 μM ~50 μM and Ascorbic Acid with a concentration of 10 μM~20 μM.

2. The adipogenic induction differentiation medium suitable for chicken embryo natural immortalized cells according to claim 1, characterized in that, The natural immortalization screening adopts a natural immortalization screening medium, and the natural immortalization screening medium is composed of a second basic supply component, a second cell culture auxiliary factor and a second auxiliary reagent: The second basic supply component includes: FBS with a concentration of 10%, Pen-strep with a concentration of 1% and a basic culture medium; The second cell culture auxiliary factor includes: FGF-2 with a concentration of 20 ng / mL, EGF with a concentration of 20 ng / mL and IGF-1 with a concentration of 10 ng / mL; The second auxiliary reagent includes: 1×ITS-X, F68 with a concentration of 0.1% and BSA with a concentration of 0.2%.

3. A lipidogenic maintenance differentiation medium suitable for chicken embryonic natural immortalized cells, characterized in that, The adipogenic maintenance differentiation medium is used in combination with the adipogenic induction differentiation medium of claim 1 or 2, and the adipogenic maintenance differentiation medium is composed of a third basic supply component, a third cell culture auxiliary factor and a third auxiliary reagent: The third basic culture medium includes: FBS with a concentration of 10%, Pen-strep with a concentration of 1% and a basic culture medium; The third cell culture auxiliary factor includes: transferrin with a concentration of 8 μg / mL-12 μg / mL and glutamine with a concentration of 2 mM; The third auxiliary reagent: HEPES with a concentration of 10 mM, β-ME with a concentration of 50 μM, and Ascorbic Acid with a concentration of 10 μM. , HEPES with a concentration of 10 mM, β-ME with a concentration of 50 μM, and Ascorbic Acid with a concentration of 10 μM.

4. A differentiation medium for adipogenic differentiation of chicken embryonic naturally immortalized cells, characterized in that, The adipogenic differentiation medium is composed of the adipogenic induction differentiation medium of claim 1 or 2 and the adipogenic maintenance differentiation medium of claim 3.

5. A method for adipogenic differentiation of chicken embryo naturally immortalized cells under 2D culture conditions, characterized in that, The method comprises the following steps: The chicken embryo natural immortalization cell is taken and cultured in vitro for proliferation, and is grown to a confluence degree of 100%. The proliferation culture medium is discarded, and the adipogenic induction differentiation medium of claim 1 or 2 is added. The day when the adipogenic induction differentiation medium is added is recorded as 0d, and the culture is continued for 1-2d; The adipogenic induction differentiation medium is removed, and the adipogenic maintenance differentiation medium of claim 3 is added, and the differentiation is continued for 2-4d, and the medium is changed every 1d.

6. A method for adipogenic differentiation of chicken embryo naturally immortalized cells under 3D culture conditions, characterized in that, The method comprises the following steps: Take chicken embryo natural immortalized cells and proliferate in vitro, grow to 100% confluence, digest, centrifuge, resuspend, and then add the adipogenic induction differentiation medium of claim 1 or 2 under 3D culture conditions. The day when the adipogenic induction differentiation medium is added is recorded as 0d, and the cells are cultured for 1-2d; Remove the adipogenic induction differentiation medium and add the adipogenic maintenance differentiation medium of claim 3, and continue differentiation for 2-4d, with medium replacement every 1d.

7. The method of claim 6, wherein the chicken embryo naturally immortalized cells are subjected to adipogenic differentiation under 3D culture conditions. Cell proliferation density requirement 3*10 ^ 6 cells / mL or more; preferably, the 3D culture conditions provide a culture space by 3D parallel bioreactors.

8. Use of the adipogenic induction differentiation medium of claim 1-2, the adipogenic maintenance differentiation medium of claim 3, or the adipogenic differentiation medium of claim 4 in the preparation of a differentiation medium for seed cells in bio-cultured meat.

9. Use of the adipogenic induction differentiation medium of claim 1-2, the adipogenic maintenance differentiation medium of claim 3, or the adipogenic differentiation medium of claim 4 as a testing reagent in the testing of the differentiation performance of seed cells using a cell differentiation method.

10. Use of the adipogenic induction differentiation medium of claim 1-2, the adipogenic maintenance differentiation medium of claim 3, or the adipogenic differentiation medium of claim 4 in the study of fat development mechanisms using in vitro cell differentiation.

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