CN117757111A

CN117757111A - Efficient low-cholesterol fat cell 3D culture material and preparation method and application thereof

Info

Publication number: CN117757111A
Application number: CN202311793089.7A
Authority: CN
Inventors: 单体中; 顾馨; 黄钰琴; 刘事奇
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2023-12-25
Filing date: 2023-12-25
Publication date: 2024-03-26

Abstract

The invention provides a high-efficiency low-cholesterol fat cell 3D culture material, and belongs to the technical fields of biological materials and food science. The preparation method of the efficient and low-cholesterol fat cell 3D culture material provided by the invention comprises the following steps: (1) Mixing konjak glucomannan and sodium alginate, and dissolving the mixture in deionized water to obtain a gel solution, wherein the total gel concentration in the gel solution is 1-2% (w/v); (2) And (3) crosslinking the gel solution to obtain the solidified fat cell 3D culture material. The 3D culture material provided by the invention has a porous structure and good cell compatibility, and can support 3D culture of various types of fat cells such as pig subcutaneous fat cells, pig fibroblast-lipid progenitor cells, mouse 3T3 cells and the like. The 3D differentiated polyester effect of the fat cells is obviously promoted, the fat has lower cholesterol content, and the in-vitro production of the high-efficiency and low-cholesterol cell culture meat can be realized.

Description

Efficient low-cholesterol fat cell 3D culture material and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological materials and food science, in particular to a high-efficiency low-cholesterol fat cell 3D culture material and a preparation method and application thereof.

Background

In recent years, with the rapid increase of global population and the improvement of living standard of people, the demand for meat foods has increased. The consumption concept of people is changed from full eating to good eating and healthy eating. However, the problems of resource environment, public health and the like caused by the large-scale aquaculture are increasingly prominent, so that new technology for driving the development of more efficient, sustainable and safer food production is urgent. In the age background of green development of agriculture, cell culture meat is used as an emerging cell agriculture technology or one of important means for solving the problem of protein shortage of human beings in the future and meeting the demands of people for healthy and high-quality meat products.

Traditional meats mainly consist of muscle, fat and the like, wherein the fat is a key source of flavor substances in the meats, and has close relation with the mouthfeel, tenderness, flavor and the like of the meats. In addition, the fat is used as an important nutrient component in meat products, can provide fatty acid necessary for human bodies, and is used as a carrier to assist the absorption of nutrient components such as fat-soluble vitamins. However, the research of cell culture meat has been focused mainly on the culture of muscle tissue, for example, CN 116804184a cell culture meat scaffold prepared from konjak polysaccharide-fibrin composite gel performs the culture of muscle cells and the repair of alveolar bone defect by preparing konjak polysaccharide-fibrin composite hydrogel. However, the different cells have different requirements on the texture properties of the scaffold material, for example, differentiation with stem cells may also lead to differentiation into different cells (myogenic cells or adipocytes) due to the different scaffold materials used. Thus, studies on 3D culture of adipocytes are urgently needed.

In addition, excessive cholesterol content in meat products is an important cause of cardiovascular diseases such as atherosclerosis and coronary heart disease in humans. Therefore, by combining with advanced cell culture meat biosynthesis technology, accurate nutrition customization of the end product is realized, and the method has important significance for promoting product iteration of low-cholesterol meat products. There is no study of reducing cholesterol content of cell culture meat using gel scaffolds.

The 3D scaffold is an important raw material for manufacturing tissue cell culture meat products, can simulate extracellular matrix components in natural tissues, and has an internal porous structure meeting the requirement of O ₂ Transport of nutrients and metabolic waste, and provides physiological environment and medium for the processes of cell growth, proliferation, differentiation and the like. The hydrogel scaffold has good hydrophilicity, is similar to the water content of extracellular matrix and the mechanical property of natural tissues, and has wide application prospect in the fields of adipose tissue engineering and cell culture meat. At present, various biological hydrogels such as alginate, collagen, gelatin, hyaluronic acid and the like have been applied to the preparation of adipocyte scaffold materials to support cell proliferation and differentiation, however, most scaffold materials contain animal-derived components such as collagen, gelatin, hyaluronic acid and the like, and extraction processes are complicated and high in cost, contrary to the concept of sustainable development. Some polymeric materials such as polylactic-co-glycolic acid (PLGA) and porcine gelatin have also been shown to support proliferation of adipose stem cells, but their edibility is controversial. Therefore, development of an edible scaffold which is simple in preparation method, controllable in cost and free of animal-derived components is needed, and technical support is provided for efficient differentiation and nutrition-controllable fat culture strategies.

Disclosure of Invention

The invention aims to solve the problems that the existing adipose cell culture scaffold has certain toxicity, is not edible, and has low efficiency and high cholesterol content of adipose tissues obtained by culture.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a preparation method of a high-efficiency low-cholesterol fat cell 3D culture material, which comprises the following steps:

(1) Mixing konjak glucomannan and sodium alginate, and dissolving the mixture in deionized water to obtain a gel solution, wherein the total gel concentration in the gel solution is 1-2% (w/v);

(2) And (3) crosslinking the gel solution to obtain the solidified fat cell 3D culture material.

Preferably, the mass ratio of the konjac glucomannan to the sodium alginate is 1:4-5.

Preferably, the temperature of the dissolution in step (1) is 80 to 95 ℃.

Preferably, the crosslinking in the step (2) is ionic crosslinking, and the crosslinking agent is calcium chloride solution with the concentration of 1-2% (w/v); the temperature of the crosslinking is 20-25 ℃. The time is 8-12 min.

The invention also provides a high-efficiency low-cholesterol fat cell 3D culture material obtained by the preparation method.

The invention also provides an application of the high-efficiency low-cholesterol fat cell 3D culture material in culturing fat cells.

Preferably, the adipocytes include porcine subcutaneous adipocytes, porcine FAPs cells, and mouse 3T3 cells.

Preferably, the method for culturing the fat cells comprises the following steps:

(1) Mixing konjak glucomannan and sodium alginate according to a mass ratio, and dissolving the mixture in deionized water to obtain a gel solution;

(2) Mixing fat cells with the gel solution and then crosslinking to obtain a cell/scaffold complex;

(3) The cell/scaffold complex is sequentially cultured in a growth medium, a adipogenic differentiation medium and a maintenance medium, and after the culture is finished, the adipose tissue with low cholesterol characteristic is obtained.

Preferably, the volume ratio of the fat cells to the gel solution is 1:2; the concentration of the adipocytes is (1-5). Times.10 ⁷ /mL。

Preferably, the cell/scaffold complex is cultured in DMEM growth medium containing 15% fetal bovine serum for 20-28 hours, in the adipogenic differentiation medium for 4-5 days, and in the maintenance medium for 9-10 days.

The konjac glucomannan (konjac glucomannan, KGM, also called konjac glucomannan) used in the present invention is a natural plant polysaccharide extracted from konjac tubers, and can form a gel having similar texture characteristics to natural meat products under the condition of heat alkali treatment, and is widely used in plant meat production. However, single KGM gel has the problems of poor structural stability, poor biocompatibility and the like, and limits the application of the single KGM gel as a cell scaffold material. Sodium alginate (Alg) is a naturally derived polymer with good biocompatibility and is widely used in the manufacture of various 3D scaffolds.

The KGM/Alg (KA) composite plant hydrogel with excellent mechanical properties is prepared by compounding KGM and Alg through the formation of a double gel network structure and the crosslinking of exogenous metal ions, does not use any animal-derived component and toxic crosslinking agent, has a porous structure and good cell compatibility, and can support the 3D culture of various types of fat cells such as pig subcutaneous fat cells, pig fibroblast fat progenitor cells (FAPs), mouse 3T3 cells and the like. Compared with the traditional Alg hydrogel, the 3D polyester differentiation effect of the subcutaneous fat cells of the pigs is obviously promoted, the cholesterol content of the subcutaneous fat cells is not influenced, and the technical support can be provided for the in vitro production of high-efficiency and low-cholesterol cell culture meat.

Drawings

FIG. 1 is a scanning electron microscope image of the KA bracket of example 1;

FIG. 2 is a comparison of adipogenic differentiation effect of subcutaneous adipocytes in 4 KA scaffolds (Nile red stained image) in example 2;

FIG. 3 is a comparison of adipogenic differentiation effect (adipogenic differentiation-related gene expression) of subcutaneous adipocytes in 4 KA scaffolds in example 2;

FIG. 4 is a graph showing the differentiation effect of KA and Alg 3D culture systems (image of dark field, oil red O staining, and Nile red staining) in example 3;

FIG. 5 is a comparison of cholesterol deposition at equivalent triglyceride levels in example 3;

FIG. 6 shows the results of analysis of the gray scale values of the bands and expression of the adipogenic differentiation-related proteins in example 3;

FIG. 7 is a photograph of Nile red stain of FAPs cells after 3D lipogenesis induction in example 4;

FIG. 8 is a BODIPY staining image of 3D adipogenic induction of 3T3 cells in example 5.

Detailed Description

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Preparation and morphology observation of scaffolds

(1) And weighing KGM powder and Alg powder with the mass ratio of 1:4 and 1:5 at normal temperature, adding deionized water, and stirring uniformly to prepare the konjac glucomannan/sodium alginate (KGM/Alg, KA) mixed solution with the total gum concentration of 1% and 2% (w/v). And (3) placing the mixed solution into a water bath kettle at the temperature of 95 ℃ for water bath dissolution, and fully stirring and uniformly mixing to obtain a mixed gel solution.

(2) Slowly adding the mixed gel solution into an equal volume of calcium chloride solution with the concentration of 2% (w/v) by using a pipette, standing for 10min, and sucking the residual calcium chloride solution to obtain the completely gelled stent.

Four KA gels (1% KA1:4, 1% KA 1:5, 2% KA1:4 and 2% KA 1:5) are put into a vacuum freeze dryer to be freeze-dried for 24 hours, so that a bracket material with directional arranged pore channels is formed, the bracket is broken by liquid nitrogen, the cross section is processed by spraying metal, and the pore structure of the bracket material is observed by a scanning electron microscope. And (3) scanning images under the conditions of continuous scanning mode and high voltage of 5kV, photographing the microscopic surface of the sample and collecting data. As shown in fig. 1. The scaffold has a loose and porous three-dimensional structure after freeze drying. The pore diameters are distributed between 200 and 800 mu m, and the large pores and the small pores are alternately distributed, so that the double-crosslinking KGM-Alg interpenetrating polymer network is formed. The porous structure can provide sufficient nutrient substances, proper growth environment and moderate contact and inhibition space for cells.

Example 2

3D differentiation and identification of pig subcutaneous adipocytes in KA scaffold

Subcutaneous adipocytes of pig at 37℃with 5% CO ₂ Culturing in an incubator until the cell confluency reaches about 70%, adding 1mL trypsin for digestion until the cell morphology tends to ellipse and floats from the bottom of the culture dish, adding an equal volume of growth medium (Sieimer's fly), and uniformly mixing to stop digestion. The cells were transferred to a 5mL centrifuge tube,centrifuging at 800rpm/min for 4min, discarding supernatant to obtain cell precipitate, and adding growth medium to resuspend.

2 mL/well of sterile 2% (w/v) calcium chloride solution was added to the 12-well cell culture plate. Cell suspension (1X 10) ⁷ cell/mL) and sterile mixed gel solution (same as step (1) of example 1) in a volume ratio of 1:2, and then transferring the mixture into a calcium chloride solution (the gun head is inserted below the liquid level) by using a 1mL liquid-transferring gun, and crosslinking the mixture for 10min to form a cell/bracket compound by 2.5 mL/hole. The remaining calcium chloride solution was then discarded, washed once with PBS, and then immersed in DMEM growth medium containing 15% fetal calf serum at 37℃with 5% CO ₂ Culturing for 24 hours under the condition. Sucking the growth medium, adding the adipogenic differentiation medium for induced differentiation, wherein the components are as follows: 10. Mu.g/mL insulin, 500. Mu.M 3-isobutyl-1-methylxanthine, 1. Mu.M dexamethasone and 10. Mu.M rosiglitazone were added to DMEM complete medium containing 10% FBS at 37℃in 5% CO ₂ Is cultured in a cell culture box for 5 days, and the differentiation medium is replaced every 48 hours. After 5 days of induced differentiation, the differentiation medium was aspirated and replaced with maintenance medium, which consisted of: adding 10 μg/mL insulin and 10 μM rosiglitazone to DMEM complete medium containing 10% FBS at 37deg.C, 5% CO ₂ Is cultured in the cell culture incubator for 9 days, and the maintenance medium is replaced every 48 hours.

Nile red staining identification

After 3D culture differentiation, the maintenance cell culture solution in each well was discarded, the cells were fixed in 4% paraformaldehyde for 20min, the fixed solution was discarded, and the cells were washed 1 time with PBS buffer. 1mg/ml nile red stock solution (prepared by absolute ethanol) was diluted with PBS to working solution (1:200), incubated with cells/scaffolds for 4h in the dark for labeling lipid droplets; the nile red working solution was discarded, and the nuclei were labeled by incubation with 5. Mu.g/ml DAPI dye at room temperature for 2h in the dark. As shown in fig. 2, it is evident that most subcutaneous fat precursor cells were differentiated under a fluorescence microscope and exhibited a pellet morphology, demonstrating the feasibility of 3D culture and induced differentiation of fat cells based on KA hydrogel scaffolds. Wherein culturing differentiated cells in a 1% KA1:4 scaffold is capable of accumulating more and larger lipid droplets.

Adipogenic differentiation related gene expression assay

After the completion of the differentiation in 3D culture, the maintenance cell culture solution in each well was discarded, and 2ml of a cell recovery solution (a mixed solution of 100mM sodium citrate and 30mM ethylenediamine tetraacetic acid) was added to each well. Gently blow with a pipette until the gel is completely dissolved and transfer to a 1.5mL centrifuge tube. Centrifuge at 1300rpm for 5min at room temperature and discard the supernatant. The cell pellet was resuspended by adding 500. Mu.LPBS, centrifuged at 1300rpm for 5min and the supernatant discarded. Each tube of cell pellet was added with 500. Mu. LTRIzol reagent, and cellular RNA was extracted and the RNA quality and concentration were measured using a NanoDrop 2000 instrument. cDNA was synthesized using a reverse transcription kit (Thermo Fisher). Real-time quantitative PCR was performed using a AppliedBiosystems StepOnePlusTM real-time PCR system and SYBR Green Master Mix (Saint, shanghai, china). Through 2 ^-ΔΔCT The relative expression level of the gene was calculated by the method. As shown in FIG. 3, the subcutaneous adipocytes induced by 3D adipogenic in 1% KA1:4 scaffolds had higher differentiation efficiency after 14 days of adipogenic induction, which was shown by higher expression levels of adipogenic differentiation-related genes Ppar gamma, C/EBP alpha, fabp4 and PLIN 1.

Example 3

Preparation of Alg gel scaffold and 3D culture

The Alg powder with certain mass is weighed by an electronic balance and is dissolved in deionized water to prepare Alg solutions with the concentration of 1 percent and 2 percent (w/v) respectively. And (3) placing the Alg solution into a water bath kettle at 95 ℃ for water bath dissolution, and fully stirring to obtain a well-mixed solution. Sterilizing the gel solution with high temperature and high pressure steam at 121deg.C for 15min to reach the sterility standard. A certain mass of calcium chloride powder is weighed and dissolved in deionized water to prepare 2% (w/v) calcium chloride solution, the calcium chloride solution is filtered by a 0.22 mu m filter membrane to ensure sterility, and the calcium chloride solution is transferred into a cell culture plate for 2 mL/hole. The cell suspension and the sterile gel solution are mixed according to the volume ratio of 1:2, transferring the mixture into a calcium chloride solution (the gun head is inserted below the liquid level) by using a 1mL liquid-transferring gun, and crosslinking the mixture for 10min at 2 mL/hole to form the cell/bracket compound. Discarding the remaining calcium chloride solution, washing with PBS once, and immersing in DMEM growth medium containing 15% fetal calf serum at 37deg.C and 5% CO ₂ Culturing under the condition. Wherein adipogenic differentiation protocol, lipid drop staining and quantitative determinationThe procedure was identical to that of KA treatment in example 2. This was compared to a 1% KA1:4 stent.

Oil red O staining and nile red staining

After 3D culture differentiation, the maintenance cell culture solution in each well was discarded, the cells were fixed in 4% paraformaldehyde for 20min, the fixed solution was discarded, and the cells were washed 1 time with PBS buffer. Oil red O stock solution (formulated with isopropyl alcohol) at 5mg/mL was mixed with deionized water at 3:2 and filtered through filter paper to prepare an oil red O working solution. Incubating the working solution and the cells/stent for 30min in a dark place, discarding the dye solution, cleaning with 60% isopropanol for 2 times, observing the size and distribution of lipid droplets in an inverted microscope, and adjusting the visual field to select a proper area for imaging. The nile red staining method was the same as in example 2. As shown in FIG. 4, compared with the Alg scaffold, the 1% KA1:4 scaffold provided by the invention can remarkably improve the adipogenic differentiation efficiency of subcutaneous fat cells of pigs, and more large cell clusters can be observed by bright field photographing. The results of oil red O staining and nile red staining show that after induced differentiation for 14 days under the same conditions, the KA bracket can enable more and larger lipid drops to be formed in the cell culture meat, so that the production efficiency of the meat is improved.

Triglyceride and cholesterol content detection

After 3D culture differentiation, the maintenance cell culture solution in each well is discarded, 200 mu L of lysate is added for full homogenization, centrifugation is carried out at 4 ℃ for 10min, and the supernatant is transferred to a new centrifuge tube for subsequent measurement. The content of triglycerides in the cell/scaffold complex was determined using a triglyceride detection kit (beijing plaril) and a cholesterol detection kit (beijing plaril) according to the manufacturer's instructions. In addition, protein concentration in the homogenate was detected using BCA protein assay kit (thermosusher), and triglyceride/cholesterol concentrations were normalized to total protein concentration. Cholesterol deposition in each set of scaffolds was analyzed under the same triglyceride deposition conditions using Graphpad Prism software. As shown in FIG. 5, compared with the traditional Alg scaffold, the 1% KA1:4 scaffold can remarkably reduce cholesterol deposition in cultured fat under the condition of depositing triglyceride at the same level, thereby improving the production efficiency and quality of cell culture meat.

Protein level detection

After 14 days of adipogenic differentiation, the maintenance cell culture broth was discarded from each well, and 2mL of cell recovery broth (100 mM sodium citrate/30 mM ethylenediamine tetraacetic acid mixed solution) was added to each well. Gently blow with a pipette until the gel is completely dissolved and transfer to a 1.5mL centrifuge tube. Centrifuge at 1300rpm for 5min at room temperature and discard the supernatant. The cell pellet was resuspended by adding 500. Mu.LPBS, centrifuged at 1300rpm for 5min and the supernatant discarded. mu.L of protease inhibitor-containing (final concentration 1 mM) protein lysis buffer was added to each tube of cell pellet, and the pellet was homogenized well or sonicated for 30min on ice, centrifuged at 12000rpm for 10min at 4℃and the supernatant was collected. After protein concentration was determined by BCA kit (Thermo Fisher), 5 Xloading buffer was added at 4:1 (V: V), and after mixing, the protein was denatured by heating at 95℃for 10min and stored at-80 ℃. 10% polyacrylamide gel is prepared, protein electrophoresis is carried out, 90V is carried out for 30min, and 130V is carried out for 1h. Through the processes of membrane transfer, 2h of sealing, primary antibody incubation overnight, TBST elution, secondary antibody incubation at room temperature and the like, protein bands are analyzed by using a gel imaging analysis system, and grey values are analyzed by using imageJ software. As shown in FIG. 6, compared with the traditional Alg scaffold, the 1% KA1:4 scaffold provided by the invention can obviously up-regulate the expression levels of adipogenic related protein PPARgamma and mature fat key marker proteins FABP4 and Adipoq after 14 days of induced differentiation, and improves the cell differentiation efficiency.

Example 4

3D differentiation and nile red staining of porcine FAPs cells in KA scaffold

Pig FAPs cells were incubated at 37℃with 5% CO ₂ Culturing in an incubator until the cell confluency reaches about 70%, adding 1mL trypsin for digestion until the cell morphology tends to ellipse and floats from the bottom of the culture dish, adding an equal volume of growth medium for uniform mixing, and stopping digestion. Cells were transferred to a 5mL centrifuge tube and centrifuged at 800rpm/min for 4min to obtain a cell pellet. The supernatant was discarded and the growth medium was added for resuspension. FAPs/KA scaffold complexes (example 21% KA 1:4) were prepared in the same manner as subcutaneous adipocytes and adipogenic induced differentiation for 14 days.

After completion of differentiation, the cell culture solution was discarded, and the cells were fixed in 4% paraformaldehyde for 20min, after which the fixed solution was discarded, the cells were washed 1 time with PBS buffer, and Nile red staining was performed (staining method was the same as that of subcutaneous adipocytes). The fluorescent staining reagent was blotted and washed 2 times with PBS buffer, red excitation light waves (Ex/em=495/635 nm) and DAPI laser/emission filters (Ex/em=364/454 nm) were selected and the appropriate areas were selected under a fluorescent microscope for imaging. As shown in FIG. 7, FAPs cells were grown at 1% KA1:4 has good mature fat morphological characteristics after differentiation for 14 days, and proves that the KA hydrogel scaffold can support 3D differentiation of different types of fat precursor cells, thereby providing technical support for in vitro production of subcutaneous fat and intramuscular fat.

Example 5

3D differentiation and BODIPY staining of mouse 3T3 cells in KA scaffold

A3T 3 cell/KA scaffold complex was prepared in the same manner as in example 4, and adipogenic differentiation was induced under the same conditions for 14 days.

After 3D culture differentiation, the maintenance cell culture solution in each well was discarded, the cells were fixed in 4% paraformaldehyde for 20min, the fixed solution was discarded, and the cells were washed 1 time with PBS buffer. 1mg of BODIPY 493/503 was dissolved in 382. Mu.L of DMSO to obtain a 10mM stock solution, and the stock solution was diluted with PBS to prepare a working solution having a concentration of 10. Mu.M. The working fluid was incubated with the cells/scaffolds for 4h in the dark to label lipid droplets. The fluorescent staining reagent was blotted and washed 2 times with PBS buffer and the appropriate area was selected for imaging under green excitation light waves of a fluorescence microscope. The results are shown in FIG. 8, 1% KA1: the 4 bracket can provide a proper growth environment for the adipogenic differentiation of 3T3 cells, supports the 3D culture of a plurality of fat cells from different sources, and has important application value in the fields of tissue engineering and cell culture meat.

According to the embodiment, the 3D bracket with a stable structure is obtained through intermolecular interaction between KGM and Alg and further ion crosslinking, the problem of poor stability of a single gel structure is solved, and the preparation method is simple and environment-friendly, does not contain any animal-derived component or toxic crosslinking agent, and has good food safety. The crosslinked stent has higher porosity and good cell compatibility, and can obviously promote adipogenic differentiation of subcutaneous fat cells of pigs compared with single Alg hydrogel; can significantly reduce cholesterol deposition under the condition of equivalent lipid accumulation. In addition, the KA bracket provided by the invention can support 3D culture of various types of fat cells including pig subcutaneous fat cells, pig FAPs cells and mouse 3T3 cells, thereby providing technical support for production of high-efficiency low-cholesterol cell culture fat and related research in the field of tissue engineering.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. The preparation method of the efficient and low-cholesterol fat cell 3D culture material is characterized by comprising the following steps of:

2. The preparation method of claim 1, wherein the mass ratio of konjac glucomannan to sodium alginate is 1:4-5.

3. The process according to claim 2, wherein the temperature of dissolution in step (1) is 80 to 95 ℃.

4. The method according to claim 3, wherein the crosslinking in the step (2) is ionic crosslinking, and the crosslinking agent is a calcium chloride solution with a concentration of 1-2% (w/v); the temperature of the crosslinking is 20-25 ℃. The time is 8-12 min.

5. A high-efficiency, low-cholesterol fat cell 3D culture material obtained by the method according to any one of claims 1 to 4.

6. Use of the high-efficiency, low-cholesterol adipocyte 3D culture material of claim 5 for culturing adipocytes.

7. The use of claim 6, wherein the adipocytes comprise porcine subcutaneous adipocytes, porcine FAPs cells, and mouse 3T3 cells.

8. The use according to claim 6 or 7, wherein the method of culturing adipocytes comprises:

9. The use according to claim 8, wherein the volume ratio of adipocytes to gel solution is 1:2; the concentration of the adipocytes is (1-5). Times.10 ⁷ /mL。

10. The use according to claim 9, wherein the cell/scaffold complex is cultured in DMEM growth medium containing 15% fetal bovine serum for a period of 20-28 hours, in the adipogenic differentiation medium for a period of 4-5 days, and in the maintenance medium for a period of 9-10 days.