A serum-free medium and culture method for fat organoids

Abstract

This invention provides a serum-free culture medium and method for adipose organoids, relating to the field of biotechnology. The medium includes an expansion medium and an induction medium. The expansion medium comprises a serum-free insulin B27 additive, transferrin, ascorbic acid, basic fibroblast growth factor, GlutaMax 1×, and N-acetylcysteine. The induction medium comprises Forskolin, insulin-like growth factor, and hydrocortisone, with the expansion medium as the solvent. This method induces adipose-derived mesenchymal stem cells to form adipose organoids with a spatial structure similar to in vivo fat. Compared to traditional media using insulin, IBMX, indomethacin, and dexamethasone, this method reduces cytotoxicity, especially by using insulin-like growth factor instead of insulin, resulting in fewer metabolic side effects. Simultaneously, it improves adipogenesis induction efficiency and cell survival rate, while making the culture medium system more defined and stable, suitable for culturing adipose organoids from different parts of the human body.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a serum-free culture medium and culture method for adipose organs. Background Technology

[0002] Obesity and type 2 diabetes have become prevalent metabolic diseases worldwide. Traditional drugs have limitations such as unstable efficacy, significant side effects, and insufficient long-term metabolic regulation. In the field of aesthetic medicine, allogeneic fat transplantation suffers from low survival rates, uneven shaping effects, and easy absorption, making it difficult to achieve precise and lasting soft tissue regeneration. Current adipose tissue models are mostly two-dimensional cultures or simple cell aggregates, which cannot simulate the in vivo adipose microenvironment, vascularized structure, and endocrine function, thus limiting the research and translation of metabolic disease mechanisms.

[0003] Adipose-derived organoids, through three-dimensional reconstruction of white / brown adipocytes, stromal vascular components, and vascular networks, can highly mimic the metabolic regulation, lipolysis-induced thermogenesis, and endocrine functions of physiological adipose tissue. In the metabolic field, they can precisely improve insulin resistance and regulate glucose and lipid homeostasis, providing a long-term, low-invasive treatment option for obesity and diabetes. In the medical aesthetics field, they can also achieve high survival rates and high stability of autologous fat regeneration for facial rejuvenation, breast repair, and soft tissue filling, improving the durability and safety of results. However, current three-dimensional culture methods for adipose-derived organoids rely on 10% FBS or human serum (such as HS), leading to the risk of exogenous pathogen contamination and large batch-to-batch variations. Traditional insulin, IBMX, indomethacin, and dexamethasone induction systems have high toxicity and poor physiological compatibility. Simply embedding ADSCs in Matrigel and directly inducing them ignores the unique physical-biochemical-gas microenvironmental synergistic regulation of adipose tissue, resulting in adipose-derived organoids that cannot meet the requirements of clinical application and industrial transformation.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] One of the objectives of this invention is to provide a serum-free culture medium for adipose organs, in order to solve the technical problems in the prior art of three-dimensional culture of adipose organs that rely on 10% FBS or human serum, which leads to the risk of exogenous pathogen contamination, large batch-to-batch fluctuations, and the high toxicity and poor physiological compatibility of traditional induction systems.

[0006] The second objective of this invention is to provide the application of the above-mentioned serum-free culture medium in culturing adipose organs or in preparing products for culturing adipose organs.

[0007] A third objective of this invention is to provide a reagent or kit.

[0008] The fourth objective of this invention is to provide a method for culturing adipose organs.

[0009] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a serum-free culture medium for adipose organs, comprising an amplification medium and an induction medium; The amplification medium comprises the following components at the following concentrations: insulin-free B27 and serum-free additive 0.5-2×, transferrin 5-20 mg / L, ascorbic acid 50-100 mg / mL, basic fibroblast growth factor 5-25 ng / mL, GlutaMax 1×, and N-acetylcysteine ​​1-2 mM. The induction medium comprises the following components at the following concentrations: Forskolin 10-30 μM, insulin-like growth factor 50-150 mg / mL, and hydrocortisone 0.1-1 μM, with the amplification medium as the solvent.

[0010] Furthermore, the induction medium uses amplification medium as a solvent and adds the following components at concentrations: Forskolin 20 μM, insulin-like growth factor 100 mg / mL and hydrocortisone 0.5 μM.

[0011] Furthermore, the amplification medium comprises the following components at the following concentrations: insulin-free B27 serum-free additive 1×, transferrin 10 mg / L, ascorbic acid 60 mg / mL, basic fibroblast growth factor 15 ng / mL, and N-acetylcysteine ​​1 mM.

[0012] Secondly, the present invention provides the application of the above-mentioned serum-free culture medium in culturing adipose organs or in preparing products for culturing adipose organs.

[0013] Thirdly, the present invention provides a reagent or kit comprising the above-described culture medium.

[0014] Fourthly, the present invention provides a method for culturing adipose-derived organs, comprising the following steps: A. Adipose-derived mesenchymal stem cells were cultured in an amplification medium until the cell culture fusion rate reached 80-90%. Bio-oil was added to the surface of the medium to cover the cells, simulating a low-oxygen environment, and pre-aggregating to form organoid precursors. B. Mix the organoid precursor with the matrix gel mixture, using induction medium supplemented with 20–50 μM deferoxamine as a diluent, to dilute until the matrix gel concentration in the mixture is 60–80% w / v, with each matrix gel bead containing 10 4 ~10 5 Cells with a matrix gel ball volume of 50 μL were placed on a collagen membrane for adipogenesis induction. The matrix gel mixture consists of matrix gel with 0.2-0.5% w / v agarose added; The amplification medium is the amplification medium described above; The induction medium is the induction medium described above.

[0015] Furthermore, the duration of the simulated low-oxygen environment is at least 12 hours.

[0016] Furthermore, the concentration of collagen in the collagen membrane is 0.5~2.0 mg / mL; Collagen includes type III collagen.

[0017] Furthermore, bio-oils include at least one of cell-grade soybean oil, cell-grade corn oil, or cell-grade coconut oil; The culture time for adipogenic induction is 5 to 9 days.

[0018] Furthermore, the preparation method of collagen membrane includes placing the collagen solution in a container for at least 1 hour, discarding the liquid, and then allowing it to dry naturally.

[0019] This invention provides a serum-free culture medium for adipose-derived organs, using small molecule and protein components to replace serum. This induction medium, based on an expansion medium, adds Forskolin, insulin-like growth factor, and hydrocortisone. Data shows that this serum-free medium can specifically culture human adipose-derived mesenchymal stem cells in vitro. During the culture process, the expansion and induction media are combined in the expansion and induction phases, respectively, to induce adipose-derived mesenchymal stem cells to form adipose-derived organs with a spatial structure similar to in vivo fat. Compared with traditional culture media using insulin, IBMX, indomethacin, and dexamethasone, this method reduces cytotoxicity, especially by using insulin-like growth factor instead of insulin, resulting in fewer metabolic side effects. Simultaneously, it improves adipogenesis induction efficiency and cell survival rate, and makes the culture medium system more defined and stable, suitable for culturing adipose-derived organs from different parts of the human body.

[0020] Another method for culturing adipose organoids involves covering the organoids with bio-oil before embedding to simulate physiological hypoxia, thus preventing mass cell death after direct embedding. Deferoxamine is added to the induction medium to inhibit hypoxia-induced iron-dependent oxidative stress damage, enhancing cell tolerance. This allows for direct mixing with the matrix gel mixture for embedding, accelerating the induction process. Agarose is used to regulate the stiffness of the matrix gel, mimicking the microenvironment of adipose tissue. A collagen membrane is used to simulate the extracellular matrix of adipose lobules, biomimetically reconstructing the matrix microenvironment. The expansion medium promotes proliferation and antioxidant activity, and its combination with the induction medium during the induction phase promotes adipogenesis, improving adipogenesis efficiency and cell survival rate. The method also shortens the culture cycle and significantly increases the uniformity of organoids. Attached Figure Description

[0021] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0022] Figure 1 The images shown are Oil Red O staining images after 7 days of planar induction provided in Example 1 of this invention, where A is a 4x magnification image, B is a 10x magnification image, and C is a 20x magnification image. Figure 2 The images shown are Oil Red O staining images after 7 days of planar induction provided in Example 2 of the present invention, where A is a 4x magnification image, B is a 10x magnification image, and C is a 20x magnification image. Figure 3 The images shown are Oil Red O staining images after 7 days of planar induction provided in Example 3 of the present invention, where A is a 4x magnification image, B is a 10x magnification image, and C is a 20x magnification image. Figure 4 The images shown are Oil Red O staining images after 7 days of planar induction provided in Comparative Example 1 of this invention, where A is a 4x magnification image, B is a 10x magnification image, and C is a 20x magnification image. Figure 5 The images shown are Oil Red O staining images after 7 days of planar induction provided in Comparative Example 2 of this invention, where A is a 4x magnification image, B is a 10x magnification image, and C is a 20x magnification image. Figure 6 The images shown are Oil Red O staining images after 7 days of planar induction provided in Comparative Example 3 of this invention, where A is a 4x magnification image, B is a 10x magnification image, and C is a 20x magnification image. Figure 7 The images shown are Oil Red O staining images after 7 days of planar induction provided in Comparative Example 4 of this invention, where A is a 4x magnification image, B is a 10x magnification image, and C is a 20x magnification image. Figure 8 The images shown are Oil Red O staining images after 7 days of planar induction provided in Comparative Example 5 of this invention, where A is a 4x magnification image, B is a 10x magnification image, and C is a 20x magnification image. Figure 9 The image shown is a Bodipy fluorescence image of an adipose organ provided in Embodiment 4 of the present invention, wherein A is a 4x magnification image and B is a 10x magnification image. Figure 10 The image shown is a Bodipy fluorescence image of an adipose organ provided in Comparative Example 6 of the present invention, wherein A is a 4x magnification image and B is a 10x magnification image. Figure 11The image shown is a Bodipy fluorescence image of an adipose organ provided in Comparative Example 7 of the present invention, wherein A is a 4x magnification image and B is a 10x magnification image. Figure 12 The image shown is a Bodipy fluorescence image of an adipose organ provided in Comparative Example 8 of the present invention, wherein A is a 4x magnification image and B is a 10x magnification image. Figure 13 The image shown is a Bodipy fluorescence image of an adipose organ provided in Comparative Example 9 of this invention, wherein A is a 4x magnification image and B is a 10x magnification image. Detailed Implementation

[0023] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0024] Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well known in the art and as described in various general and more specific references, which are cited and discussed throughout this specification.

[0025] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] In one aspect, the present invention provides a serum-free culture medium for adipose organs, comprising an amplification medium and an induction medium; The amplification medium comprises the following components at the following concentrations: insulin-free B27 and serum-free additive 0.5-2×, transferrin 5-20 mg / L, ascorbic acid 50-100 mg / mL, basic fibroblast growth factor 5-25 ng / mL, GlutaMax 1×, and N-acetylcysteine ​​1-2 mM. The induction medium comprises the following components at the following concentrations: Forskolin 10-30 μM, insulin-like growth factor 50-150 mg / mL, and hydrocortisone 0.1-1 μM, with the amplification medium as the solvent.

[0027] This invention provides a serum-free culture medium that uses small molecule and protein components to replace serum. This induction medium, based on an expansion medium, adds Forskolin, insulin-like growth factor, and hydrocortisone. Data shows that this serum-free medium can specifically culture human adipose-derived mesenchymal stem cells in vitro. During the culture process, the expansion and induction media are combined in the expansion and induction phases, respectively, to induce adipose-derived mesenchymal stem cells to form adipose-derived organelles with a spatial structure similar to in vivo fat. Compared with traditional media using insulin, IBMX, indomethacin, and dexamethasone, this invention reduces cytotoxicity, especially by using insulin-like growth factor instead of insulin, resulting in fewer metabolic side effects. Simultaneously, it improves adipogenesis induction efficiency and cell survival rate, and makes the culture medium system more defined and stable, suitable for culturing adipose-derived organelles from different parts of the human body.

[0028] The concentration of the insulin-free B27 serum-free additive can be, but is not limited to, 0.5×, 0.6×, 0.8×, 1.0×, 1.2×, 1.4×, 1.6×, 1.8× or 2.0×, or any concentration between 0.5 and 2×, preferably 1.0×.

[0029] The concentration of transferrin can be, but is not limited to, 5 mg / L, 6 mg / L, 8 mg / L, 10 mg / L, 12 mg / L, 14 mg / L, 16 mg / L, 18 mg / L or 20 mg / L, or any concentration between 5 and 20 mg / L, preferably 10 mg / L.

[0030] The concentration of ascorbic acid can be, but is not limited to, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, 95 mg / mL or 100 mg / mL, or any concentration between 50 and 100 mg / mL, preferably 60 mg / mL.

[0031] The concentration of basic fibroblast growth factor (bFGF) can be, but is not limited to, 5 ng / mL, 6 ng / mL, 8 ng / mL, 10 ng / mL, 12 ng / mL, 14 ng / mL, 15 ng / mL, 18 ng / mL, 20 ng / mL, 22 ng / mL, 24 ng / mL or 25 ng / mL, or any concentration between 5 and 25 ng / mL, preferably 15 ng / mL.

[0032] The concentration of N-acetylcysteine ​​can be, but is not limited to, 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM or 2 mM, or any concentration between 1 and 2 mM, preferably 1 mM.

[0033] The concentration of Forskolin can be, but is not limited to, 10 μM, 12 μM, 14 μM, 16 μM, 18 μM, 20 μM, 22 μM, 24 μM, 26 μM, 28 μM or 30 μM, or any concentration between 10 and 30 μM, preferably 20 μM.

[0034] The concentration of insulin-like growth factor (IGF-1) can be, but is not limited to, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL or 150 mg / mL, or any concentration between 50 and 150 mg / mL, preferably 100 mg / mL.

[0035] The concentration of hydrocortisone can be, but is not limited to, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM or 1 μM, or any concentration between 0.1 and 1 μM, preferably 0.5 μM.

[0036] In some specific embodiments, the induction medium uses an amplification medium as a solvent and adds the following components at concentrations: Forskolin 20 μM, insulin-like growth factor 100 mg / mL, and hydrocortisone 0.5 μM.

[0037] In some specific embodiments, the amplification medium comprises the following components at the following concentrations: insulin-free B27 serum-free additive 1×, transferrin 10 mg / L, ascorbic acid 60 mg / mL, basic fibroblast growth factor 15 ng / mL, and N-acetylcysteine ​​1 mM.

[0038] According to another aspect of the invention, the use of the serum-free culture medium described above in culturing adipose organs or in preparing products for culturing adipose organs is also provided.

[0039] According to another aspect of the invention, a reagent or kit comprising the above-described culture medium is also provided.

[0040] According to another aspect of the invention, a method for culturing adipose organs is also provided, comprising the following steps: A. Adipose-derived mesenchymal stem cells were cultured in an amplification medium until the cell culture fusion rate reached 80-90%. Bio-oil was added to the surface of the medium to cover the cells, simulating a low-oxygen environment, and pre-aggregating to form organoid precursors. B. Mix the organoid precursor with the matrix gel mixture, using induction medium supplemented with 20–50 μM deferoxamine as a diluent, to dilute until the matrix gel concentration in the mixture is 60–80% w / v, with each matrix gel bead containing 10 4 ~10 5 Cells with a matrix gel ball volume of 50 μL were placed on a collagen membrane for adipogenesis induction. The matrix gel mixture consists of matrix gel with 0.2-0.5% w / v agarose added; The amplification medium is the amplification medium described above; the induction medium is the induction medium described above.

[0041] This method employs bio-oil covering to simulate physiological hypoxia before organoid embedding, preventing mass cell death after direct embedding. Deferoxamine is added to the induction medium to inhibit hypoxia-induced iron-dependent oxidative stress damage, enhancing cell tolerance and allowing direct mixing with the matrix gel mixture for embedding, thus accelerating the induction process. Agarose is used to regulate matrix gel stiffness, mimicking the microenvironment of adipose tissue, while a collagen membrane simulates the extracellular matrix of adipose lobules, biomimetically reconstructing the matrix microenvironment. The expansion medium promotes proliferation and antioxidant activity, and its combination with the induction medium during the induction phase promotes adipogenesis, improving adipogenesis induction efficiency and cell survival rate. The culture cycle is shortened, and the uniformity of organoids is significantly improved.

[0042] The amount of deferoxamine can be, but is not limited to, 20 μM, 23 μM, 26 μM, 28 μM, 30 μM, 33 μM, 36 μM, 38 μM or 50 μM, or any amount between 20 and 50 μM, preferably 50 μM.

[0043] In some specific implementations, the culture time for adipogenic induction is 5 to 9 days.

[0044] In some specific embodiments, the matrix gel is a basement membrane matrix derived from mouse EHS sarcoma. Specifically, matrix gel products from Matrigel, Moji (Xiamen Moji Biotechnology Co., Ltd.), or Bozhen (Bozhen Biotechnology (Suzhou) Co., Ltd.) can be used.

[0045] In some specific implementations, the duration of the simulated hypoxic environment is at least 12 hours.

[0046] In some specific embodiments, the concentration of collagen in the collagen membrane is 0.5~2.0 mg / mL; in some specific embodiments, the collagen includes type III collagen. In some specific embodiments, the collagen membrane is prepared by placing a collagen solution in a container for at least 1 hour, discarding the solution, and then allowing it to air dry.

[0047] In some specific embodiments, bio-oil includes at least one of cell-grade soybean oil, cell-grade corn oil, or cell-grade coconut oil.

[0048] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0049] I. Serum-free culture medium for adipose organs The serum-free culture medium for adipose organs includes an amplification medium and an induction medium. The amplification medium includes serum-free insulin B27 additive, transferrin, ascorbic acid, basic fibroblast growth factor, and N-acetylcysteine. The induction medium is the amplification medium supplemented with Forskolin, IGF-1, and hydrocortisone. The specific component contents of Examples 1-3 are shown in Table 1.

[0050] Table 1

[0051] Comparative Example 1 The difference from Example 1 is that insulin was used instead of IGF-1, and the concentration of insulin was 1 μg / L.

[0052] Comparative Example 2 The difference from Example 1 is that insulin, IBMX, indomethacin, and dexamethasone were used as culture components, replacing Forskolin, IGF-1, and hydrocortisone. The concentration of insulin was 1 μg / L, the concentration of IBMX was 0.5 mM, the concentration of indomethacin was 200 μM, and the concentration of dexamethasone was 1 μM.

[0053] Comparative Example 3 The difference from Example 1 is that IGF-1 was not added to the induction medium.

[0054] Comparative Example 4 The difference from Example 1 is that Forskolin was not added to the induction medium.

[0055] Comparative Example 5 The difference from Example 1 is that hydrocortisone was not added to the induction medium.

[0056] II. Cultivating Lipid Organs Methods for extracting primary adipose-derived mesenchymal stem cells: 1. Place the adipose tissue in a 50 mL centrifuge tube containing 6% penicillin-antibody PBS solution and rinse 2-3 times under aseptic conditions; 2. Use curved forceps and ophthalmic scissors to remove the blood vessels / connective tissue around the adipose tissue, then transfer it to a 50mL centrifuge tube and repeatedly cut it with ophthalmic scissors until it becomes milky. 3. Add an equal volume of mixed digestion solution of type I and type IV collagenase to the centrifuge tube, and digest in a water bath at 37°C for 30 minutes until the tissue is completely dissociated; 4. Add an equal volume of DMEM / F12 culture medium to the centrifuge tube to stop digestion, and filter the solution through a 75μm sieve to obtain the filtrate.

[0057] 5. Centrifuge 300 g of the filtrate at 4℃ for 5 min, discard the supernatant and keep the precipitate; 6. Add 1 mL of red blood cell lysis buffer, let stand at room temperature for 5 min, centrifuge at 300 g for 5 min, discard the supernatant and keep the precipitate, which is adipose-derived mesenchymal stem cells.

[0058] 7. Resuspend the obtained adipose-derived mesenchymal stem cells in amplification culture medium, at a concentration of 1×10⁻⁶. 4 cells / cm 2 Inoculate at a density of 6 cm plates, incubate at 37°C and 5% CO2 for 24 h, then change the medium for the first time to remove non-adherent impurities; 8. When the cell fusion reaches 80-90%, discard the culture medium, wash with PBS, add 0.25% trypsin for 5 min, add an equal volume of complete culture medium to stop, centrifuge at 300 g for 5 min, discard the supernatant and keep the precipitate to obtain primary adipose mesenchymal stem cells.

[0059] The culture method for adipose organoids is performed according to the following steps: Step 1: Planar induction culture of organoid precursors Primary adipose-derived mesenchymal stem cells were extracted and passaged in an amplification medium at a ratio of 1:3 until the cell confluence reached 80-90%. Bio-oil was then added to the surface of the medium to cover the cells and simulate a hypoxic environment for 12 hours, allowing them to pre-aggregate and form organoid precursors.

[0060] Step 2: Construction of adipose-derived organs Type III collagen at a concentration of 1 mg / mL was plated and solidified to form a collagen film.

[0061] Corning (Matrigel) combined with 0.3% agarose adjusts the stiffness of the matrix gel, forming a matrix gel mixture.

[0062] The organoid precursor cells obtained in step one were digested with 0.25% trypsin and mixed with the Matrigel mixture. Induction medium supplemented with 50 μM Deferoxamine was used as the diluent for the Matrigel mixture, and the mixture was diluted to a Matrigel concentration of 70%. Each Matrigel sphere had a volume of 50 μL and contained 10 cells. 5 The sample was placed on a collagen membrane for 7 days to induce lipidation.

[0063] Experiment 1: Validation of Organ Progenitor Culture Effect The amplification and induction media provided in Examples 1-3 and Comparative Examples 1-5 were used respectively. Following step one of the fat organoid culture method, the organoid precursors were induced and cultured in a plane. After 7 days of induction, the formation status of the organoid precursors was observed by Oil Red O staining.

[0064] The results are as follows Figures 1-8 As shown, Examples 1-3 can all form organoid precursors. In Example 1, the red lipid droplets are uniform, large, and round, the cells are plump, and the boundaries are clear, indicating the best effect. Compared to Example 1, in Comparative Example 1, the number of lipid droplets is significantly reduced, and their size is small and irregular, indicating that IGF-1 can promote planar induction of adipogenesis by replacing insulin. In Comparative Example 2, a large number of cells died, and the remaining lipid droplets were sparse and unevenly distributed, indicating that the traditional induction system has high toxicity and poor physiological adaptability. In Comparative Example 3, the total amount of lipid droplets formed is significantly reduced, with only a few areas showing tiny, diffuse red staining spots, lacking typical lipid droplet aggregates; the cells adhere loosely, have blurred boundaries, and some areas show vacuolization and detachment. In Comparative Example 4, a small number of lipid droplets are generated, but their distribution is extremely uneven, alternating between clustered aggregations and large blank areas; the lipid droplets are small and irregular in shape, the Oil Red O staining is relatively light, and apoptotic shrinkage is visible in some areas. In Comparative Example 5, the number of lipid droplets is moderate, but their maturity is poor. This indicates that IGF-1, Forskolin, and hydrocortisone are essential core growth factors for maintaining adipogenesis induction, and none of them can be omitted.

[0065] The amplification medium and induction medium provided in Example 1 were used for subsequent verification.

[0066] Example 4 The amplification medium and induction medium provided in Example 1 were used to culture adipose organs according to the culture method for adipose organs.

[0067] Comparative Example 6 The difference from Example 4 is that step one, omitting the bio-oil simulation and the pre-mixing of Deferoxamine in the anaerobic pre-culture, is as follows: Primary adipose-derived mesenchymal stem cells were extracted and passaged in an amplification medium at a ratio of 1:3 until the cell confluence reached 80%–90%. Without adding bio-oil for covering, the cells were cultured normally for another 12 hours.

[0068] Comparative Example 7 The difference from Example 4 is that in step two, pre-collagen plating and curing is not performed. Induction culture is carried out directly in the culture container, as follows: The stiffness of the matrix gel is adjusted by adding 0.3% agarose, thus forming a matrix gel mixture.

[0069] The organoid precursor cells obtained in step one were digested with 0.25% trypsin and mixed with the Matrigel mixture. Induction medium supplemented with 50 μM Deferoxamine was used as the diluent for the Matrigel mixture, and the mixture was diluted to a Matrigel concentration of 70%. Each Matrigel sphere had a volume of 50 μL and contained 10 cells. 5 The cells were placed on a culture container for lipid-induced induction for 7 days.

[0070] Comparative Example 8 The difference from Example 4 is that in step two, agarose was not pre-mixed into the matrix gel; instead, the matrix gel was used directly instead of the matrix gel mixture, as detailed below: Type III collagen at a concentration of 1 mg / mL was plated and solidified to form a collagen film.

[0071] The organoid precursor cells obtained in step one were digested with 0.25% trypsin and mixed with Matrigel. Induction medium supplemented with 50 μM Deferoxamine was used as the Matrigel dilution buffer to a Matrigel concentration of 70%. Each Matrigel sphere had a volume of 50 μL and contained 10 cells. 5 The cells were placed on a collagen membrane for 7 days to induce lipidation.

[0072] Comparative Example 9 The difference from Example 4 is that step two does not use an induction culture medium. The specific steps are as follows: Type III collagen at a concentration of 1 mg / mL was plated and solidified to form a collagen film.

[0073] The stiffness of the matrix gel is adjusted by adding 0.3% agarose, thus forming a matrix gel mixture.

[0074] The organoid precursor cells obtained in step one were digested with 0.25% trypsin and mixed with the Matrigel mixture. Amplification medium supplemented with 50 μM Deferoxamine was used as the diluent for the Matrigel mixture, and the mixture was diluted to a Matrigel concentration of 70%. Each Matrigel sphere had a volume of 50 μL and contained 10 cells. 5 The cells were placed on a collagen membrane for 7 days to induce lipidation.

[0075] Experiment 2: Identification of Mature Lipid Organs Bodipy staining was performed on the adipose organs obtained in Example 4 and Comparative Examples 6-9, respectively.

[0076] Bodipy staining results for fatty organs are as follows Figures 9-13 As shown, in Example 4, the spherical / oval organoids were structurally intact, with smooth surfaces and sharp boundaries. The cells inside the organoids were densely arranged, without large areas of necrotic core regions. Compared with Example 1, Comparative Example 6 showed loose cells with a large number of fragments, indicating that bio-oil covering and Deferoxamine pretreatment successfully simulated physiological hypoxia and blocked ferroptosis; Comparative Example 7 did not form obvious organoids and showed a large number of fragments, indicating that the collagen membrane is not only an adhesion matrix but also promotes the formation of adipose organoids; Comparative Example 8 did not form obvious organoids, lacked agarose-regulated stiffness, and showed a large number of fragments, indicating that excessively high matrix stiffness inhibits adipogenesis, while too low (no agarose) cannot maintain the structure. Precisely regulating the matrix stiffness to the physiological range of adipose tissue promotes the formation of adipose organoids; Comparative Example 9 lacked typical lipid droplet cluster structures, indicating that the amplification medium and the induction medium are not interchangeable.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A serum-free culture medium for adipose organs, characterized in that, Includes amplification medium and induction medium; The amplification medium consists of the following components at the following concentrations: insulin-free B27 and serum-free additive 0.5-2×, transferrin 5-20 mg / L, ascorbic acid 50-100 mg / mL, basic fibroblast growth factor 5-25 ng / mL, GlutaMax 1×, and N-acetylcysteine ​​1-2 mM. The induction medium consists of the following components at the following concentrations: Forskolin 10-30 μM, insulin-like growth factor 50-150 mg / mL, and hydrocortisone 0.1-1 μM, with the amplification medium as the solvent.

2. The serum-free culture medium according to claim 1, characterized in that, The induction medium uses amplification medium as a solvent and adds the following components at the following concentrations: Forskolin 20 μM, insulin-like growth factor 100 mg / mL and hydrocortisone 0.5 μM.

3. The serum-free culture medium according to claim 2, characterized in that, The amplification medium consists of the following components at the following concentrations: insulin-free B27 serum-free additive 1×, transferrin 10 mg / L, ascorbic acid 60 mg / mL, basic fibroblast growth factor 15 ng / mL, and N-acetylcysteine ​​1 mM.

4. The use of the serum-free culture medium according to any one of claims 1 to 3 in culturing adipose organs or in preparing products for culturing adipose organs.

5. A reagent or kit, characterized in that, It comprises the culture medium as described in any one of claims 1 to 3.

6. A method for culturing adipose-derived organs, characterized in that, Includes the following steps: A. Adipose-derived mesenchymal stem cells were cultured in an amplification medium until the cell culture fusion rate reached 80-90%. Bio-oil was added to the surface of the medium to cover the cells, simulating a low-oxygen environment, and pre-aggregating to form organoid precursors. B. Mix the organoid precursor with the matrix gel mixture, using induction medium supplemented with 20–50 μM deferoxamine as a diluent, to dilute until the matrix gel concentration in the mixture is 60–80% w / v, with each matrix gel bead containing 10 4 ~10 5 Cells with a matrix gel ball volume of 50 μL were placed on a collagen membrane for adipogenesis induction. The matrix gel mixture consists of matrix gel with 0.2-0.5% w / v agarose added; The amplification medium is the amplification medium according to any one of claims 1 to 3; The induction medium is the induction medium according to any one of claims 1 to 3.

7. The cultivation method according to claim 6, characterized in that, The simulated low-oxygen environment lasts for at least 12 hours.

8. The cultivation method according to claim 6, characterized in that, The concentration of collagen in the collagen membrane is 0.5~2.0 mg / mL; Collagen includes type III collagen.

9. The cultivation method according to claim 6, characterized in that, Bio-oils include at least one of cell-grade soybean oil, cell-grade corn oil, or cell-grade coconut oil; The culture time for adipogenic induction is 5 to 9 days.

10. The cultivation method according to claim 9, characterized in that, The preparation method of collagen membrane includes placing the collagen solution in a container for at least 1 hour, discarding the solution, and then allowing it to dry naturally.

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