Serum-free induced adipogenic differentiation culture medium for fish stem cells and application thereof

Abstract

The invention relates to the field of cell culture, in particular to a serum-free induced adipogenic differentiation culture medium for fish stem cells and application of the serum-free induced adipogenic differentiation culture medium. The culture medium consists of a basic culture medium and a cell culture auxiliary factor combination, the basic culture medium is one of a DMEM (Dulbecco's Modified Eagle Medium) and a DMEM / F12 culture medium; the cell culture auxiliary factor combination is prepared from oleic acid, soya bean lecithin, vitamin C phosphate, beta cyclodextrin, transferrin, saikoside A, psoralen flavanone methyl ether, dehydroabietic acid and phenyllactic acid. The culture medium suitable for serum-free induced adipogenic differentiation of fish stem cells is obtained by adding a cell culture auxiliary factor combination to replace serum in a traditional adipose precursor cell adipogenic induced differentiation culture medium and dexamethasone IBMX serving as a core component of traditional adipogenic differentiation cocktail, the bottleneck problem in the prior art is solved, and the culture medium is suitable for serum-free induced adipogenic differentiation of fish stem cells. And reliable technical support is also provided for large-scale production of cell culture fish meat.

Description

Technical Field

[0001] The invention relates to the field of cell culture, and in particular to a serum-free adipogenic differentiation culture medium for fish stem cells and an application thereof. Background Art

[0002] As a new type of biomanufacturing technology that replaces traditional animal husbandry, cell cultured meat technology has become a key research direction in the global food technology field in recent years. This technology amplifies animal stem cells in vitro and induces them to differentiate into muscle, fat and other tissues, and finally assembles them into edible meat products. In the context of the increasing depletion of fish resources, cell cultured fish meat technology has great strategic significance for the protection of marine ecology.

[0003] In the field of cell culture, traditional culture media usually contain animal serum, such as fetal bovine serum, calf serum, chicken serum, duck serum and horse serum. These sera are rich in various growth factors, hormones, vitamins and minerals, which can provide cells with rich nutrition and growth signals, and support cell proliferation and differentiation. However, there are many problems with the use of animal serum: the composition is complex and unstable, and there are significant differences due to different batches, sources and processing methods, which makes it difficult to standardize the culture conditions and has poor reproducibility; it may carry pathogens such as viruses, bacteria, mycoplasma, etc., which poses a biosafety risk; the extraction process involves animal slaughter, which causes ethical controversy and is not in line with the concept of sustainable development; high-quality animal serum is expensive, which limits the large-scale application and industrial production of cell culture technology.

[0004] Chinese patent CN112961825A discloses "A serum-free culture medium and its preparation method", which is composed of basal culture medium and serum-free culture medium additives. However, in the field of cell differentiation, especially the differentiation of adipocytes, it relies on the core components of the traditional adipogenic differentiation "cocktail", dexamethasone and 3-isobutyl-1-methylxanthine (IBMX). These small molecules can induce the differentiation of adipocytes, but there are problems such as non-specific effects, potential toxicity, and lack of sustainability. Dexamethasone is a glucocorticoid that may cause stress responses or other adverse effects on cells; IBMX is a phosphodiesterase inhibitor that may be toxic to cells when used in high concentrations. In addition, the application of traditional adipogenic differentiation culture medium in fish cells also faces challenges, because the metabolism and differentiation mechanisms of fish cells are significantly different from those of mammalian cells, and the adipogenic differentiation culture medium directly derived from mammalian cells cannot achieve the ideal differentiation effect.

[0005] Therefore, based on the above-mentioned industry pain points, there is an urgent need to develop a chemically clear, inexpensive, and efficient serum-free fish stem cell induced adipogenic differentiation culture medium to solve the current problems that hinder the industrialization of cell-cultured meat due to the high price of the culture medium, low differentiation efficiency, and poor repeatability. Summary of the invention

[0006] The technical problem to be solved by the present invention is that the application of dexamethasone and 3-isobutyl-1-methylxanthine (IBMX), the core components of the traditional adipogenic differentiation "cocktail", in fish cells faces challenges, and the adipogenic differentiation culture medium directly derived from mammalian cells cannot achieve the ideal effect in the adipogenic differentiation of fish cells.

[0007] To solve the above problems, the present invention adds a combination of cell culture auxiliary factors to replace the serum in the traditional adipogenic differentiation medium for adipogenic precursor cells and the core component of the traditional adipogenic differentiation "cocktail" dexamethasone IBMX, thereby obtaining a culture medium suitable for serum-free induction adipogenic differentiation of fish stem cells. This not only solves the bottleneck problem of the prior art, but also provides reliable technical support for the large-scale production of cell-cultured fish meat.

[0008] To achieve the above-mentioned purpose, the technical scheme of the invention is: a serum-free adipogenic differentiation medium for fish stem cells, which is composed of a basal medium and a cell culture auxiliary factor combination; the basal medium is one of DMEM and DMEM / F12 medium; the cell culture auxiliary factor combination is composed of 10~500 μg / mL oleic acid, 10~500 μg / mL soybean lecithin, 2~200 μg / mL vitamin C phosphate (L-Ascorbic acid 2-phosphate), 10~500 μg / mL β-cyclodextrin, 5~100 μg / mL transferrin, 1~100 μg / mL bupleurum saponin A, 1~100 μg / mL psoralea corylifolia methyl ether, 1~100 μg / mL dehydroabietic acid, and 1~100 μg / mL phenyllactic acid.

[0009] The serum-free culture medium of the present invention does not contain any animal serum components including fetal bovine serum, calf serum, chicken serum, duck serum, horse serum, etc. Oleic acid and soybean lecithin in the culture medium provide essential fatty acids and phospholipids, replace lipid components in serum, support cell membrane formation and lipid droplet accumulation; β-cyclodextrin enhances the solubility and stability of fat-soluble components (such as oleic acid), ensures uniform distribution and efficient utilization of nutrients; vitamin C phosphate (L-Ascorbic acid 2-phosphate) acts as an antioxidant to protect cells from oxidative damage, while promoting collagen synthesis and extracellular matrix stability. Transferrin is responsible for the transportation and metabolism of iron ions, replaces the function of iron-binding proteins in serum, and maintains the iron homeostasis required for normal metabolic activity of cells.

[0010] The key components for inducing cell adipogenic differentiation of the present invention include oleic acid, soybean lecithin, vitamin C phosphate, beta-cyclodextrin, transferrin, saikosaponin A, psoralen flavonoids methyl ether, dehydroabietic acid, and phenyllactic acid. Oleic acid and soybean lecithin provide necessary lipid precursors, vitamin C phosphate and transferrin promote the antioxidant capacity and iron metabolism of cells, beta-cyclodextrin enhances the solubility and stability of fat-soluble components, and saikosaponin A, psoralen flavonoids methyl ether, dehydroabietic acid, and phenyllactic acid replace the effects of dexamethasone and IBMX in traditional adipogenic differentiation culture medium by activating PPARγ / RXR heterodimer and AMPK pathway.

[0011] Furthermore, the volume-to-mass ratio (v / w) of the basal culture medium and the cell auxiliary factor combination is 1000: 0.041~1000: 2.2. Specifically, the cell auxiliary factors to be added to each liter of the basal culture medium include: 10~500 mg oleic acid, 10~500 mg soy lecithin, 2~200 mg vitamin C phosphate (L-Ascorbic acid 2-phosphate), 10~500 mg β-cyclodextrin, 5~100 mg transferrin, 1~100 mg saikosaponin A, 1~100 mg psoralen dihydroflavonoid methyl ether, 1~100 mg dehydroabietic acid, and 1~100 mg phenyllactic acid.

[0012] An application of the culture medium in adipogenic differentiation of fish stem cells.

[0013] Furthermore, the method comprises the following steps: taking fish stem cells for in vitro culture, and when the fish stem cells are cultured to a cell density of more than 90%, removing the cell proliferation medium, adding the above-mentioned adipogenic differentiation medium, and continuing to culture to induce the differentiation of large yellow croaker fat-derived stem cells; replacing the cultured fish stem cells with the above-mentioned adipogenic differentiation medium every 1 to 2 days, and continuing to culture until the intracellular lipids of the large yellow croaker fat-derived stem cells continue to accumulate.

[0014] Furthermore, the environmental conditions for culturing the above fish stem cells are culturing at 28° C. and 5% carbon dioxide.

[0015] Furthermore, the cell proliferation medium is a DMEM medium containing 10% fetal bovine serum or a DMEM / F12 medium containing 10% fetal bovine serum.

[0016] Furthermore, the fish stem cells are large yellow croaker muscle satellite cells or large yellow croaker fat-derived stem cells.

[0017] The beneficial effects of the present invention are: (1) The fish serum-free adipogenic differentiation medium provided by the present invention does not contain any serum components and has a clear chemical composition, which is convenient for promoting the industrialization of cell cultured meat.

[0018] (2) The serum-free adipogenic differentiation medium for fish stem cells provided by the present invention has a good adipogenic differentiation effect. Experimental studies have shown that after inducing differentiation of fat precursor cells in vitro for 6 days using the improved cell adipogenic differentiation medium described in the present invention, lipid droplets appear in almost all cells, and the cell differentiation efficiency can reach 100%.

[0019] (3) The present invention provides a serum-free culture medium and an induction method for fish stem cell adipogenic differentiation. This innovative formula not only solves the bottleneck problem of the prior art, but also provides reliable technical support for the large-scale production of cell cultured fish meat. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 : Microscope images of satellite cells in yellow croaker muscle (scale bar: 500 μm); Figure 2 : Fluorescence microscopy images of large yellow croaker muscle satellite cells differentiated and cultured in three adipogenic differentiation induction media for 6 days (scale bar: 100 μm); Figure 3 :Microscope images of large yellow croaker adipose-derived stem cells (scale bar: 500 μm); Figure 4 : Fluorescence microscopy images of large yellow croaker adipose-derived stem cells cultured in three adipogenic differentiation induction media for 6 days (scale bar: 100 μm). DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels or prepared by existing methods.

[0028] Embodiment 1: This example explores the adipogenic differentiation of large yellow croaker muscle satellite cells, and the specific operation is as follows: (1) Recovery of large yellow croaker muscle satellite cell line: Take out the frozen large yellow croaker muscle satellite cell line from liquid nitrogen, thaw it quickly in a 37°C water bath, centrifuge it in a centrifuge at 300×g for 5 minutes after the cell suspension is completely thawed, remove the supernatant, resuspend it in DMEM / F12 medium (containing 10% fetal bovine serum), add it to a T25 culture flask, and culture it at 28°C. The microscopic morphology of large yellow croaker muscle satellite cells is shown in the figure. Figure 1 shown.

[0029] (2) When the density of large yellow croaker muscle satellite cells reaches more than 90%, the proliferation medium is removed, and the large yellow croaker muscle satellite cells obtained by expansion are added to the fish stem cell serum-free adipogenic differentiation medium, and the day of adding the differentiation medium is recorded as day 0, wherein the differentiation medium is composed of a basal medium and a cell culture auxiliary factor combination, wherein the volume mass ratio (v / w) of the basal medium to the cell auxiliary factor combination is 1000: 0.585. Specifically, the cell auxiliary factors to be added to each liter of the basal medium include: 200 mg oleic acid, 50 mg soy lecithin, 100 mg vitamin C phosphate (L-Ascorbic acid 2-phosphate), 200 mg β-cyclodextrin, 10 mg transferrin, 5 mg saikosaponin A, 5 mg psoralen dihydroflavonoid methyl ether, 5 mg dehydroabietic acid, and 10 mg phenyllactic acid. The basal medium is DMEM / F12 medium.

[0030] In addition, the traditional adipogenic differentiation "cocktail" combination medium was used as a control experiment, in which the formula of the control adipogenic differentiation medium ① was: DMEM / F12 as the basic medium, containing 0.5 mM IBMX, 1 μM dexamethasone and 10% fetal bovine serum; the formula of the control adipogenic differentiation medium ② was: DMEM / F12 as the basic medium, containing 0.5 mM IBMX, 1 μM dexamethasone and 10% horse serum; the formula of the control adipogenic differentiation medium ③ was: DMEM / F12 as the basic medium, and containing 200 μg / mL oleic acid, 50 μg / mL soybean lecithin, 100 μg / mL vitamin C phosphate, 200 μg / mL β-cyclodextrin, 10 μg / mL transferrin, 0.5 mM IBMX and 1 μM dexamethasone. Adipogenic differentiation of large yellow croaker muscle satellite cells was achieved by inducing differentiation for 6 days, and the differentiation medium was replaced every 2 days during the induction of differentiation.

[0031] (3) Fluorescent staining of lipid droplets accumulated during adipogenic differentiation of cells to examine differentiation status: ① Remove the adipogenic differentiation induction medium and gently wash 3 times with PBS; ② Add 4% paraformaldehyde cell fixative to fix the cells for 15 min; ③ Add 0.1% cell permeabilization solution (0.1% Triton X-100 prepared in PBS) to permeabilize the cells for 15 min; ④ Remove the permeabilization solution and wash once with PBS; ⑤ Add 10 μmol / L BODIPY staining solution to stain the cell lipids for 10 min. After staining, wash once with PBS and add 1 μg / mL DAPI staining solution to stain the cell nuclei for 5 min. ⑥Discard the staining solution and rinse with PBS 3 times; ⑦Observe and take pictures using a fluorescence microscope.

[0032] like Figure 2 As shown, the lipid accumulation of cells differentiated using fish stem cell serum-free adipogenic differentiation medium is significantly higher than that of cells differentiated using the control adipogenic differentiation medium (traditional adipogenic differentiation "cocktail" combination medium), and the adipogenic differentiation efficiency can reach 100%.

[0033] Embodiment 2: The purpose of this study is to explore the adipogenic differentiation of large yellow croaker adipose-derived stem cells. The specific operations are as follows: (1) Isolation and preparation of large yellow croaker adipose-derived stem cell cell line: Obtain adipose tissue from 3-month-old large yellow croaker fry, cut it into pieces and add digestion solution for digestion. The digestion solution formula is 1 mg / mL type I collagenase and 1 mg / mL trypsin, and the digestion time is 1 hour. Then add washing solution to the digested tissue mixture, usually D-Hanks solution for washing, and obtain a cell suspension by filtration. Then centrifuge at 300×g / min for 5 min to collect the cells. Then resuspend the cells in large yellow croaker stem cell complete culture medium (DMEM / F12 culture medium containing 10% fetal bovine serum) to obtain a cell suspension, inoculate it into a T25 cell culture flask, transfer it to a new T25 cell culture flask after culturing for 3 hours, add culture medium to 5 mL, and then place it at 28°C for culture. The microscopic morphology image of large yellow croaker adipose-derived stem cells is shown in the figure. Figure 3 shown.

[0034] (2) When the density of the adipose-derived stem cells of large yellow croaker reaches more than 90%, the proliferation medium is removed, and the amplified adipose-derived stem cells of large yellow croaker are added to the serum-free adipogenic differentiation medium for fish stem cells. The day of adding the differentiation medium is recorded as day 0, and the differentiation medium is composed of a basal medium and a cell culture auxiliary factor combination, wherein the volume-to-mass ratio (v / w) of the basal medium to the cell auxiliary factor combination is 1000: 0.375. Specifically, the cell auxiliary factors to be added to each liter of the basal medium include: 100 mg oleic acid, 20 mg soy lecithin, 100 mg vitamin C phosphate, 100 mg β-cyclodextrin, 10 mg transferrin, 10 mg saikosaponin A, 10 mg psoralen dihydroflavonoid methyl ether, 5 mg dehydroabietic acid, and 20 mg phenyllactic acid. The basal medium is DMEM / F12 medium.

[0035] In addition, the traditional adipogenic differentiation "cocktail" combination medium was used as a control experiment, in which the formula of the control adipogenic differentiation medium ① was: DMEM / F12 as the basic medium, containing 0.5 mM IBMX, 1 μM dexamethasone and 10% fetal bovine serum; the formula of the control adipogenic differentiation medium ② was: DMEM / F12 as the basic medium, containing 0.5 mM IBMX, 1 μM dexamethasone and 10% horse serum; the formula of the control adipogenic differentiation medium ③ was: DMEM / F12 as the basic medium, and containing 100 μg / mL oleic acid, 20 μg / mL soybean lecithin, 100 μg / mL vitamin C phosphate, 100 μg / mL β-cyclodextrin, 10 μg / mL transferrin, 0.5 mM IBMX and 1 μM dexamethasone. Adipogenic differentiation of large yellow croaker adipose-derived stem cells was achieved by inducing differentiation for 6 days, and the differentiation medium was replaced every 2 days during the induction of differentiation.

[0036] (3) Fluorescent staining of lipid droplets accumulated during adipogenic differentiation of cells to examine differentiation status: ① Remove the adipogenic differentiation induction medium and gently wash 3 times with PBS; ② Add 4% paraformaldehyde cell fixative to fix the cells for 15 min; ③ Add 0.1% cell permeabilization solution (0.1% Triton X-100 prepared in PBS) to permeabilize the cells for 15 min; ④ Remove the permeabilization solution and wash once with PBS; ⑤ Add 10 μmol / L BODIPY staining solution to stain the cell lipids for 10 minutes, wash once with PBS after staining, and add 1 ug / mL DAPI staining solution to stain the cell nucleus for 5 minutes; ⑥Discard the staining solution and rinse with PBS 3 times; ⑦Observe and take pictures using a fluorescence microscope.

[0037] like Figure 4 As shown, the lipid accumulation of cells differentiated using fish stem cell serum-free adipogenic differentiation medium is significantly higher than that of cells differentiated using the control adipogenic differentiation medium (traditional adipogenic differentiation "cocktail" combination medium), and the adipogenic differentiation efficiency can reach 100%.

[0038] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A serum-free medium for inducing adipogenic differentiation of fish stem cells, characterized in that: The invention is composed of a basal culture medium and a cell culture auxiliary factor combination; the basal culture medium is one of DMEM and DMEM / F12 culture medium; the cell culture auxiliary factor combination consists of 10-500 μg / mL oleic acid, 10-500 μg / mL soybean lecithin, 2-200 μg / mL vitamin C phosphate, 10-500 μg / mL β-cyclodextrin, 5-100 μg / mL transferrin, 1-100 μg / mL saikosaponin A, 1-100 μg / mL psoralea corylifolia methyl ether, 1-100 μg / mL dehydroabietic acid, and 1-100 μg / mL phenyllactic acid.

2. The culture medium according to claim 1, characterized in that: The volume-to-mass ratio of the basal culture medium and the cell auxiliary factor combination is 1000:0.041 to 1000:2.

2.

3. Use of the culture medium according to claim 1 in adipogenic differentiation of fish stem cells.

4. The use according to claim 3, characterized in that The method comprises the following steps: taking fish stem cells for in vitro culture, and when the fish stem cells are cultured to a cell density of more than 90%, removing the cell proliferation culture medium, adding the adipogenic differentiation culture medium, and continuing the culture to induce the differentiation of large yellow croaker fat-derived stem cells; replacing the culture medium of the fish stem cells with the adipogenic differentiation culture medium every 1 to 2 days, and continuing the culture until the intracellular lipids of the large yellow croaker fat-derived stem cells are continuously accumulated.

5. The use according to claim 4, characterized in that: The environmental conditions for fish stem cell culture are 28°C and 5% carbon dioxide.

6. The use according to claim 4, characterized in that: The cell proliferation medium is DMEM medium containing 10% fetal bovine serum or DMEM / F12 medium containing 10% fetal bovine serum.

7. The use according to claim 4, characterized in that: The fish stem cells are large yellow croaker muscle satellite cells or large yellow croaker fat-derived stem cells.

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