Method for culturing sheep precursor fat cells in vitro and application thereof

Abstract

The invention discloses a method for culturing sheep precursor fat cells in vitro and application thereof, and belongs to the technical field of cell culture engineering. By adopting the in-vitro culture method of the sheep precursor fat cells, provided by the invention, the obtained cells are uniform in component, the growth speed of the sheep precursor fat cells is high, and a large number of sheep precursor fat cells can be obtained within 12 days; the cultured sheep precursor fat cells are high in survival rate and good in induced differentiation performance, and the phenomena of aging, slow proliferation and death can be avoided after multiple times of passage; the sheep precursor fat cell in-vitro culture method provided by the invention is simple in process, easy to operate, easy to control in culture process and environment-friendly.

Description

Technical Field

[0001] The invention relates to the technical field of cell culture engineering, in particular to a method for culturing sheep precursor fat cells in vitro and application thereof. Background Art

[0002] Adipose tissue is an important organ for energy storage and metabolism, and the fat content of livestock will affect the quality of their meat. Adipose tissue contains both adipocytes and pre-adipocytes. If adipocytes are directly separated from adipose tissue, they will die within 24 hours after separation. Therefore, the current adipocyte culture starts with pre-adipocytes.

[0003] Preadipocytes are a type of adipocyte that differentiates from pluripotent stem cells or embryonic stem cells. They are small in size, spindle-shaped, have little stroma, and do not contain lipid droplets in the cytoplasm, but have the ability to accumulate lipid droplets in the cytoplasm. They can divide and proliferate both in vivo and in vitro, and accumulate lipid droplets in the cytoplasm to differentiate into mature adipocytes.

[0004] Preadipocytes are the best model for studying animal fat deposition and metabolism. In addition, the in vitro culture technology of preadipocytes has also shown a wide range of application value in many other fields, mainly in the following aspects:

[0005] 1) Basic scientific research: By culturing adipocyte precursor cells in vitro, scientists can better understand the development process of adipocytes, including cell differentiation, proliferation, apoptosis, and the molecular mechanism of fat metabolism. This helps to reveal the pathogenesis of metabolic diseases such as obesity, diabetes, and cardiovascular disease.

[0006] 2) Drug screening and toxicity testing: Preadipocytes and differentiated adipocytes can be used as model systems to test the effects of new drugs on fat metabolism and evaluate the potential side effects of drugs. This test is particularly important when developing drugs to treat metabolic diseases.

[0007] 3) Regenerative medicine: Adipose tissue is rich in mesenchymal stem cells and fat precursor cells, which have certain pluripotency and can differentiate into various cell types under specific conditions, such as bone, cartilage, muscle cells, etc. Therefore, they have application potential in tissue engineering, wound repair and regenerative medicine, such as promoting skin wound healing and soft tissue reconstruction.

[0008] 4) Obesity and diabetes research: The adipocyte precursor cell model can be used to study the mechanism of obesity-related insulin resistance, explore the relationship between obesity and type 2 diabetes, and develop new treatment strategies.

[0009] 5) Cosmetics and beauty industry: After in vitro expansion, fat precursor cells can differentiate into mature fat cells and be used in autologous fat transplantation surgery to improve facial contours, breast reconstruction and other cosmetic medical procedures, providing a more natural filling material option.

[0010] 6) Disease model establishment: Constructing disease models of fat precursor cells and adipocytes to simulate metabolic disorders such as human obesity and fatty liver will help to further study the disease process and discover new therapeutic targets.

[0011] However, primary fat cells are difficult to adhere to the wall, grow slowly, and are easily contaminated by fibroblasts, which affects the later induction and differentiation process. The survival rate of cultured precursor cells is low, and the time to passage is long, and the number of cells obtained is small. Therefore, how to improve the survival rate of in vitro cultured fat cells, especially the adhesion rate of non-model animal fat cells such as sheep, and improve their cell proliferation and differentiation efficiency is the focus of sheep precursor adipocyte culture.

[0012] At present, the culture methods used for pre-adipocytes are mainly tissue block culture and digestion culture. The tissue block culture method is time-consuming, and the cells reach monolayer fusion in about 10-12 days, which requires a lot of manpower; the digestion culture method takes less time than the tissue block culture method, and the cell growth and fusion time also takes 7-8 days, and the cells are prone to aging, slow proliferation and death after multiple passages. Therefore, providing a simple, efficient, time-saving, and well-maintained sheep pre-adipocyte in vitro culture method has broad application prospects, and can also provide more powerful technical support for exploring the molecular mechanism of sheep body fat deposition and improving meat quality. Summary of the invention

[0013] The purpose of the present invention is to provide a method for culturing sheep precursor fat cells in vitro and its application, so as to solve the technical problems that the sheep precursor fat cells are difficult to adhere to the wall, grow slowly, are easily contaminated by fibroblasts, affect the later induction differentiation process, have a low survival rate, take a long time to be subcultured, and obtain a small number of cells.

[0014] To achieve the above object, the present invention provides a method for culturing sheep preadipocytes in vitro, comprising the following steps:

[0015] S1. Prepare type I collagenase solution, dissolve type I collagenase in 1% bovine serum albumin PBS solution, sterilize and store in aliquots;

[0016] S2. Prepare a complete culture medium, wherein the components of the complete culture medium include 87-89% DMEM / F12 basal culture medium, 10-12% fetal bovine serum, and 1% penicillin-streptomycin;

[0017] S3. Sampling and pretreatment of adipose tissue: 2×2-2×4 cm of sheep adipose tissue was collected and placed in 37°C physiological saline containing penicillin-streptomycin. The sheep tissue was immersed in 37°C ethanol between cells, and then quickly rinsed repeatedly with a PBS solution containing gradient penicillin-streptomycin, and then the blood vessels and connective tissue in the adipose tissue were removed;

[0018] S4, separating and culturing sheep pre-adipocytes in vitro, cutting the sheep pre-adipocytes into pieces, washing them, and adjusting the cell density for culturing after digestion;

[0019] S5. Subculture the sheep preadipocytes. When the sheep preadipocytes in step S4 grow to 80-90% confluence, discard the culture medium, add trypsin digestion solution for digestion, and then subculture.

[0020] Preferably, the concentration of type I collagenase in step S1 is 0.1%, and the type I collagenase solution is sterilized using a 0.22 μm filter membrane and stored at -20°C for later use.

[0021] Preferably, in step S2, after the components of the complete culture medium are mixed, they are placed in a 5% CO 2 , place in a 37℃ incubator, equilibrate for 6-8h, and store at 4℃ for later use; the mass ratio of penicillin:streptomycin in penicillin-streptomycin is 1:1.

[0022] Preferably, the concentration of penicillin and streptomycin in the physiological saline in step S3 is 1%, and the mass ratio of penicillin and streptomycin in the penicillin and streptomycin is 1:1; the mixture is immersed in ethanol for 30-45s, and the ethanol has a volume fraction of 75%.

[0023] Preferably, in step S3, PBS solution containing 3%, 2%, and 1% gradient penicillin-streptomycin is used for repeated washing 3-5 times.

[0024] Preferably, the washing in step S4 refers to washing with PBS solution for 2-3 times, and the digestion refers to adding 1-2 times the type I collagenase solution prepared in step S1, mixing and digesting in a constant temperature shaker at 37°C and 200 rpm for 1-1.5 hours, shaking once every 3-10 minutes, and the cell density during culture is 1.0×10 5 Pieces / mL.

[0025] Preferably, in step S4, the cells are cultured at 37°C and 5% CO 2 After 24 hours of culture, the original culture medium was discarded, and the cells were washed 2-3 times with PBS solution to remove blood cells and non-adherent cells. New culture medium prepared in step S2 was added, and the culture medium was changed every 2-3 days.

[0026] Preferably, the concentration of trypsin in step S5 is 0.25%, and the amount added is 2-3 mL / 5×10 6 The cells were inoculated and subcultured at a ratio of 1:2-4.

[0027] An application of the above-mentioned in vitro culture method of sheep preadipocytes in in vitro culture of preadipocytes.

[0028] Therefore, the method for culturing sheep preadipocytes in vitro and its application provided by the present invention have the following specific technical effects:

[0029] (1) The in vitro culture method of sheep preadipocytes provided by the present invention can obtain uniform cell components, and the sheep preadipocytes grow rapidly, and a large number of sheep preadipocytes can be obtained in 12 days;

[0030] (2) The sheep preadipocyte in vitro culture method provided by the present invention has a high survival rate and good differentiation induction performance, and can be cultured for multiple generations without aging, slowing proliferation and death;

[0031] (3) The in vitro culture method of sheep preadipocytes provided by the present invention is simple and easy to operate, the culture process is easy to control, and it is environmentally friendly.

[0032] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0034] Figure 1 These are photographs of the primary culture of sheep preadipocytes at 0d (A), 3d (B), 9d (C), and 12d (D) in Example 1 of the present invention under a 100× field of view;

[0035] Figure 2 These are photographs of the P1 sheep preadipocyte culture at 6h (A), 1d (B), 2d (C), 3d (D), 4d (E), and 5d (F) under a 100× field of view in Example 1 of the present invention;

[0036] Figure 3The results of cell flow cytometry in Example 2 of the present invention; wherein Part A is the result of the test at 0 h after induction of differentiation; Part B is the result of the test at 12 h after induction of differentiation; Part C is the result of the test at 24 h after induction of differentiation; Part D is the result of the test at 36 h after induction of differentiation; and Part E is the result of the test at 48 h after induction of differentiation;

[0037] Figure 4 is the cck8d detection result in Example 2 of the present invention;

[0038] Figure 5 is the expression identification result of the cell proliferation marker gene in Example 2 of the present invention;

[0039] Figure 6 is the expression detection result of the cell differentiation adipogenesis marker gene in Example 2 of the present invention;

[0040] Figure 7 are photographs of cells at 100× field of view on days 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 24 of induced differentiation in Example 2 of the present invention;

[0041] Figure 8 This is the result of fluorescence microscopy observation of lipid droplet staining in Example 3 of the present invention;

[0042] Fig. 9 This is the triglyceride detection result curve in Example 4 of the present invention. DETAILED DESCRIPTION

[0043] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0044] In order to make the purpose, technical scheme and advantages of the present application clearer, more thorough and more complete, the technical scheme of the present invention is clearly and completely described below through the accompanying drawings and examples. The following detailed descriptions are all descriptions of the embodiments, and are intended to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present application belongs.

[0045] The instruments, equipment, reagents and materials used in the examples were all obtained from commercial sources, wherein the specification of penicillin and streptomycin was 10000 μg / mL, and the mass ratio of penicillin to streptomycin was 1:1; methods not described are all commonly used methods in the art.

[0046] Embodiment 1

[0047] The sheep preadipocytes were cultured in vitro, and the specific steps were as follows:

[0048] S1. Prepare 0.1% type I collagenase solution. Accurately weigh 100 mg type I collagenase and dissolve it in 100 mL 1% bovine serum albumin PBS solution. After complete dissolution, sterilize it through a 0.22 μm filter membrane, dispense it into 1 mL / tube, and store it at -20°C for later use.

[0049] S2. Prepare complete culture medium. Accurately pipette 44.5 mL of Gibco DMEM / F-12 basal culture medium, add 5 mL of fetal bovine serum and 0.5 mL of penicillin-streptomycin, mix thoroughly, and place in a 5% CO 2 , 37 ℃ incubator, equilibrate for 8 hours and then store at 4 ℃ for use.

[0050] S3. Sampling and pretreatment of adipose tissue. Immediately after slaughtering the sheep, collect 2*2-2*4cm of sheep adipose tissue and add it to 37℃ physiological saline containing 1% concentration of penicillin-streptomycin. Take out the sheep tissue between cells and soak it in 75% ethanol at 37℃ for 30s. Then quickly rinse it with PBS solution containing 3%, 2%, and 1% gradient penicillin-streptomycin for 5 times, and then use ophthalmic scissors and tweezers to remove the blood vessels and connective tissue in the adipose tissue.

[0051] S4, separate and culture sheep prefat cells in vitro, use ophthalmic scissors to cut the sheep prefat tissue obtained in step S3 into pieces and wash it with PBS solution three times, then add 1 volume of type I collagenase solution prepared in step S1 to the sheep prefat tissue, mix well and digest in a constant temperature shaker at 37°C and 150rpm for 1h, and shake it manually every 10min. Then add an equal volume of complete medium prepared in step S2 to terminate digestion, filter with a cell sieve to remove undigested tissue blocks and fat cells, centrifuge at 1000rpm for 5min, remove the supernatant, add DMEM / F12 basal medium to wash twice, centrifuge at 1000rpm for 5min, remove the supernatant, add an appropriate amount of complete medium prepared in step S2, blow and beat to make a uniform cell suspension, count by cell technology plate, add complete medium prepared in step S2 to adjust the cell density to 1.0×10 5 / mL and inoculated into culture flasks.

[0052] After culturing for 24 hours, the original culture medium was discarded, and the cells were washed twice with PBS solution to remove blood cells and non-adherent cells. New culture medium prepared in step S2 was added, and the cell status was observed every day and the culture medium was changed every 3 days.

[0053] S5, sheep preadipocytes are subcultured, when the sheep preadipocytes in step S4 grow to 80-90% confluence, the culture fluid is discarded, washed twice with PBS, 2mL 0.25% trypsin digestion solution is added and digested at 37°C for 3min, observed under a microscope every one minute, when the cell morphology shrinks and partially floats, 6mL of complete medium prepared in step S2 is added to terminate digestion, the adherent cells are blown up and transferred to a centrifuge tube for centrifugation at 1000r for 5min, the supernatant is discarded and added to the complete medium prepared in step S2 to make a cell suspension, and subcultured and inoculated at a ratio of 1:2, marked as P1 (first generation). Change the complete culture fluid once every 3d, until the cells converge and cover the entire culture dish with 80-90%, and then repeat the above operation to continue subculture.

[0054] See the photo of sheep preadipocytes cultured in step S4. Figure 1 The photo of the P1 sheep preadipocytes cultured in step S5 is shown in Figure 2 .

[0055] Embodiment 2

[0056] The sheep preadipocytes cultured in Example 1 were induced to differentiate, and the specific steps were as follows:

[0057] The density is 1×10 5 Equal amounts of cell suspensions of 100 cells / mL were inoculated into culture dishes previously covered with sterile coverslips, and cultured with the complete culture medium prepared in step S2 of Example 1 until cell contact inhibition occurred. Differentiation culture medium was then used to induce differentiation (recorded as 0 h of differentiation induction). Cell flow cytometry was performed at 0, 12, 24, 36, and 48 h after differentiation induction. The results are shown in FIG. Figure 3 As shown, the cck8d detection results are as follows Figure 4 The expression of cell proliferation marker genes is shown in Figure 5 As shown. After that, the differentiation medium was changed every 2 days until the 12th day. On the 12th day, the cell differentiation adipogenic marker genes were detected. Figure 6 The photos of cells on days 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 24 of induction differentiation are shown in Figure 7 .

[0058] The components of the differentiation culture medium are as follows: complete culture medium with a final concentration of 10 μg / mL insulin (INS, Sigma, I5500), 1.0 μmol / L dexamethasone (DEX, Sigma, D1756), and 0.5 mmol / L 3-isobutyl-1-methylxanthine (IBMX, Sigma, 17018).

[0059] It can be seen that the induced differentiation effect is good, and the differentiated cells have the functions of proliferation, apoptosis and adipogenesis, which is consistent with the morphological changes of intracellular fat formation after adipocyte differentiation induction.

[0060] Embodiment 3

[0061] The cultured sheep preadipocytes were identified by Oil Red O staining, and the specific steps are as follows:

[0062] The coverslip covered with cells in Example 2 was taken out, washed 3 times with PBS, fixed with 4% paraformaldehyde fixative for 20 min, washed 3 times with PBS, and stained with Oil Red O staining solution: ddH 2 The working solution was prepared with a volume ratio of 3:2 and stained at room temperature for 30 minutes. The solution was washed with PBS three times and the coloration of lipid droplets was observed under a fluorescence microscope. Figure 8 As shown, with the extension of differentiation induction time, it can be clearly observed that the lipid droplets increase, and after staining on the 22nd day, relatively large lipid droplets can be clearly observed.

[0063] Embodiment 4

[0064] Triglyceride testing, the specific steps are as follows:

[0065] (1) After cell treatment, digest the cells, wash with PBS, and centrifuge to keep the cell pellet.

[0066] (2) Resuspend the cells in saline and disrupt the cells by ultrasonication or grinding.

[0067] (3) Mix 0.2 mL of the lysate and 2.3 mL of isopropanol, incubate in a 60°C water bath for 2 min, then add 0.5 g of alumina, rapidly shake for 2 min, centrifuge at 3000 rpm for 5 min, and take the supernatant (i.e., the extract) for later use.

[0068] (4) Prepare the Glycerol working solution: Glycerol standard (4 mg / mL): protein precipitation solution = 1:49, i.e. the final concentration is 0.08 mg / mL.

[0069] (5) Use the protein precipitate as the blank well, the glycerol standard as the standard well, and the extract as the measurement well. Add the solutions in the order shown in the table below to avoid generating bubbles.

[0070]

[0071]

[0072] (6) Measure absorbance at 420 nm using an ELISA reader

[0073] After induction of differentiation, triglyceride detection revealed that as the number of days increased, Fig. 9The triglyceride content shown continued to increase linearly, and the results were consistent with the morphological changes of intracellular adipogenesis after induction.

[0074] Therefore, by adopting the in vitro culture method of sheep preadipocytes provided by the present invention, the obtained cell components are uniform, the sheep preadipocytes grow fast, and a large number of sheep preadipocytes can be obtained in 12 days; the cultured sheep preadipocytes have a high survival rate and good induction differentiation performance, and can be passed through multiple generations without aging, slowed proliferation and death; the provided in vitro culture method of sheep preadipocytes has a simple process and is easy to operate, the culture process is easy to control, and it is environmentally friendly.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for culturing sheep preadipocytes in vitro, characterized in that: The steps include: S1. Prepare type I collagenase solution, dissolve type I collagenase in 1% bovine serum albumin PBS solution, sterilize and store in aliquots; S2. Prepare a complete culture medium, wherein the components of the complete culture medium include 87-89% DMEM / F12 basal culture medium, 10-12% fetal bovine serum, and 1% penicillin-streptomycin; S3. Sampling and pretreatment of adipose tissue: 2×2-2×4 cm of sheep adipose tissue was collected and placed in 37°C physiological saline containing penicillin-streptomycin. The sheep tissue was immersed in 37°C ethanol between cells, and then quickly rinsed repeatedly with a PBS solution containing gradient penicillin-streptomycin, and then the blood vessels and connective tissue in the adipose tissue were removed; S4, separating and culturing sheep pre-adipocytes in vitro, cutting the sheep pre-adipocytes into pieces, washing them, and adjusting the cell density for culturing after digestion; S5. Subculture the sheep preadipocytes. When the sheep preadipocytes in step S4 grow to 80-90% confluence, discard the culture medium, add trypsin digestion solution for digestion, and then subculture.

2. The method for culturing sheep preadipocytes in vitro according to claim 1, characterized in that: In step S1, the concentration of type I collagenase is 0.1%, and the type I collagenase solution is sterilized using a 0.22 μm filter membrane and stored at -20°C for later use.

3. The method for culturing sheep preadipocytes in vitro according to claim 1, characterized in that: In step S2, the components of the complete culture medium are mixed, placed in a 5% CO2, 37°C incubator, equilibrated for 6-8 hours, and stored at 4°C for later use; the mass ratio of penicillin to streptomycin in the penicillin-streptomycin is 1:

1.

4. The method for culturing sheep preadipocytes in vitro according to claim 1, characterized in that: In step S3, the concentration of penicillin and streptomycin in the physiological saline is 1%, and the mass ratio of penicillin to streptomycin is 1:1; the mixture is immersed in ethanol for 30-45 seconds, and the ethanol has a volume fraction of 75%.

5. The method for culturing sheep preadipocytes in vitro according to claim 1, characterized in that: In step S3, the cells are repeatedly rinsed 3-5 times with a PBS solution containing 3%, 2%, and 1% gradient penicillin-streptomycin.

6. The method for culturing sheep preadipocytes in vitro according to claim 1, characterized in that: In step S4, washing refers to washing with PBS solution for 2-3 times, and digestion refers to adding 1-2 times the type I collagenase solution prepared in step S1, mixing and digesting in a constant temperature shaker at 37°C and 200 rpm for 1-1.5 h, shaking once every 3-10 min during the period, and the cell density during culture was 1.0×10 5 Pieces / mL.

7. The method for culturing sheep preadipocytes in vitro according to claim 1, characterized in that: In step S4, the cells are cultured in an incubator at 37°C and 5% CO2. After 24 hours of culture, the original culture medium is discarded and the cells are washed 2-3 times with PBS solution to remove blood cells and non-adherent cells. New culture medium prepared in step S2 is added and the culture medium is changed every 2-3 days.

8. The method for culturing sheep preadipocytes in vitro according to claim 1, characterized in that: The concentration of trypsin in step S5 is 0.25%, and the amount added is 2-3 mL / 5×10 6 The cells were inoculated and subcultured at a ratio of 1:2-4.

9. Use of the in vitro culture method of sheep preadipocytes according to any one of claims 1 to 8 in in vitro culture of preadipocytes.

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