A serum-free induction adipogenic differentiation medium for fish stem cells and its application

Abstract

The present invention relates to the field of cell culture, and particularly to a serum-free induction adipogenic differentiation medium for fish stem cells and its application. It is composed of a basal medium and a combination of cell culture cofactors; the basal medium is one of DMEM and DMEM / F12 medium; the combination of cell culture cofactors consists of oleic acid, soybean lecithin, vitamin C phosphate, β-cyclodextrin, transferrin, saikosaponin A, bakuchiol, dehydroabietic acid, and phenyl lactic acid. By adding the combination of cell culture cofactors, the present invention replaces the serum in the traditional adipogenic induction differentiation medium for preadipocytes and the core component dexamethasone IBMX in the traditional adipogenic differentiation "cocktail", and obtains a medium suitable for serum-free induction adipogenic differentiation of fish stem cells, which not only solves the bottleneck problems of the prior art, but also provides reliable technical support for the large-scale production of cell-cultured fish meat.

Description

Technical Field

[0001] The present invention relates to the field of cell culture, and particularly to a serum-free induction adipogenic differentiation medium for fish stem cells and its application. Background Art

[0002] As a new biological manufacturing technology to replace traditional animal husbandry, the cell culture 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 tissues such as muscle and fat, and finally assembles them into edible meat products. Against the background of the increasingly depleted fish resources, the cell culture fish meat technology has great strategic significance for marine ecological protection.

[0003] In the field of cell culture, traditional culture media usually contain animal sera, 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 rich nutrition and growth signals for cells and support the proliferation and differentiation of cells. However, the use of animal sera has many problems: the components are complex and unstable, with significant differences due to different batches, sources and processing methods, resulting in difficult standardization of culture conditions and poor repeatability; they may carry pathogens such as viruses, bacteria and mycoplasmas, posing a biosafety risk; the extraction process involves animal slaughter, causing ethical disputes and not conforming to the concept of sustainable development; high-quality animal sera are expensive, limiting the large-scale application and industrial production of cell culture technology.

[0004] Chinese Patent CN112961825A discloses "A Serum-Free Medium and Its Preparation Method", which is composed of a basal medium and additives for serum-free medium. 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, which may cause stress responses or other adverse effects on cells; IBMX is a phosphodiesterase inhibitor, and high-concentration use may be toxic to cells. In addition, the application of traditional adipogenic differentiation media in fish cells also faces challenges, because the metabolic and differentiation mechanisms of fish cells are significantly different from those of mammalian cells, and directly following the adipogenic differentiation media of mammalian cells cannot achieve ideal differentiation effects.

[0005] Therefore, based on the above industrial pain points, there is an urgent need to develop a chemically defined, inexpensive and highly efficient serum-free fish stem cell induction adipogenic differentiation medium to solve the problems that currently hinder the industrialization process of cell culture meat due to the high cost of the 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 the core components of traditional adipogenic differentiation "cocktails", dexamethasone and 3-isobutyl-1-methylxanthine (IBMX), in fish cells faces challenges, and directly following the mammalian cell adipogenic differentiation medium cannot achieve ideal results in the adipogenic differentiation of fish cells.

[0007] To solve the above problems, the present invention obtains a serum-free induction adipogenic differentiation medium suitable for fish stem cells by adding a cell culture cofactor combination to replace the serum in the traditional adipogenic induction differentiation medium for preadipocytes and the core components of the traditional adipogenic differentiation "cocktail", dexamethasone and IBMX. This not only solves the bottleneck problems 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 object, the technical solution of the present invention is: a serum-free induction adipogenic differentiation medium for fish stem cells, which consists of a basal medium and a cell culture cofactor combination; the basal medium is one of DMEM and DMEM / F12 medium; the cell culture cofactor combination consists of 10 - 500 μg / mL oleic acid, 10 - 500 μg / mL soy lecithin, 2 - 200 μg / mL L-Ascorbic acid 2-phosphate, 10 - 500 μg / mL β-cyclodextrin, 5 - 100 μg / mL transferrin, 1 - 100 μg / mL saikosaponin A, 1 - 100 μg / mL corylin, 1 - 100 μg / mL dehydroabietic acid, and 1 - 100 μg / mL phenyl lactic acid.

[0009] The serum-free 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 soy lecithin in the medium provide essential fatty acids and phospholipids, replacing the lipid components in serum, and supporting cell membrane formation and lipid droplet accumulation; β-cyclodextrin enhances the solubility and stability of lipophilic components (such as oleic acid), ensuring the uniform distribution and efficient utilization of nutrients; L-Ascorbic acid 2-phosphate acts as an antioxidant to protect cells from oxidative damage and simultaneously promotes collagen synthesis and extracellular matrix stability. Transferrin is responsible for the transport and metabolism of iron ions, replacing the function of iron-binding proteins in serum and maintaining the iron homeostasis required for normal cell metabolic activity.

[0010] The key components for inducing adipogenic differentiation of cells in the present invention include oleic acid, soybean lecithin, vitamin C phosphate, β-cyclodextrin, transferrin, saikosaponin A, corylin, dehydroabietic acid, and phenyl lactic acid. Among them, oleic acid and soybean lecithin provide the necessary lipid precursor substances, vitamin C phosphate and transferrin promote the antioxidant capacity and iron metabolism of cells, β-cyclodextrin enhances the solubility and stability of lipophilic components, and saikosaponin A, corylin, dehydroabietic acid, and phenyl lactic acid replace the functions of dexamethasone and IBMX in the traditional adipogenic differentiation medium by activating the PPARγ / RXR heterodimer and AMPK pathways.

[0011] Furthermore, the volume-mass ratio (v / w) of the basal medium to the cell cofactors combination is 1000:0.041 - 1000:2.2. Specifically, the cell cofactors to be added per liter of the basal medium include: 10 - 500 mg of oleic acid, 10 - 500 mg of soybean lecithin, 2 - 200 mg of vitamin C phosphate (L-Ascorbic acid 2-phosphate), 10 - 500 mg of β-cyclodextrin, 5 - 100 mg of transferrin, 1 - 100 mg of saikosaponin A, 1 - 100 mg of corylin, 1 - 100 mg of dehydroabietic acid, and 1 - 100 mg of phenyl lactic acid.

[0012] An application of the above-mentioned medium in the adipogenic differentiation of fish stem cells.

[0013] Furthermore, it includes the following steps: taking fish stem cells for in vitro culture, waiting until the cell density of the fish stem cells reaches more than 90%, removing the cell proliferation medium, adding the above-mentioned adipogenic differentiation medium, and continuing the culture to induce the differentiation of adipose tissue-derived stem cells of large yellow croaker; every 1 - 2 days, replacing the culture medium of the fish stem cells in the culture with the above-mentioned adipogenic differentiation medium, and continuing the culture until the intracellular lipids of the adipose tissue-derived stem cells of large yellow croaker continuously accumulate.

[0014] Furthermore, the environmental conditions for culturing the above-mentioned fish stem cells are carried out under the conditions of 28°C and 5% carbon dioxide.

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

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

[0017] The beneficial effects of the present invention are:

[0018] (1)The serum-free adipogenic induction and differentiation medium for fish provided by the present invention contains no serum components and has a clear chemical composition, facilitating the industrialization process of cell-cultured meat.

[0019] (2)The serum-free induction and adipogenic differentiation medium for fish stem cells provided by the present invention has good adipogenic differentiation effect. Experimental studies have shown that after culturing adipose precursor cells in vitro for 6 days using the improved cell adipogenic induction and differentiation medium described in the present invention, lipid droplets appear in almost all cells, and the cell differentiation efficiency can reach 100%.

[0020] (3)The present invention provides a serum-free induction and adipogenic differentiation medium and an induction method for fish stem cells. This innovative formulation not only solves the bottleneck problems of the existing technology but also provides reliable technical support for the large-scale production of cell-cultured fish meat. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0022] Figure 1 : Microscopic image of large yellow croaker muscle satellite cells (scale bar: 500 μm);

[0023] Figure 2 : Fluorescence microscopic image of large yellow croaker muscle satellite cells differentiated and cultured in 3 induction and adipogenic differentiation media for 6 days respectively (scale bar: 100 μm);

[0024] Figure 3 : Microscopic image of large yellow croaker adipose tissue-derived stem cells (scale bar: 500 μm);

[0025] Figure 4 : Fluorescence microscopic image of large yellow croaker adipose tissue-derived stem cells differentiated and cultured in 3 induction and adipogenic differentiation media for 6 days respectively (scale bar: 100 μm). DETAILED DESCRIPTION OF THE INVENTION

[0026] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation embodiments of the present invention.

[0027] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes 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 related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0029] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are merely exemplary.

[0030] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0031] Unless otherwise specified, the test 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 commercially or prepared by existing methods.

[0032] Example 1:

[0033] This example explores the adipogenic differentiation of Pseudosciaena crocea muscle satellite cells, and the specific operations are as follows:

[0034] (1) Resuscitate the Pseudosciaena crocea muscle satellite cell line: Take out the cryopreserved Pseudosciaena crocea muscle satellite cell line from liquid nitrogen, quickly thaw it in a 37°C water bath. After the cell suspension is completely melted, centrifuge it at 300×g for 5 minutes in a centrifuge, remove the supernatant, resuspend it with DMEM / F12 medium (containing 10% fetal bovine serum), add it to a T25 culture flask, and culture it at 28°C. The microscopic morphological images of Pseudosciaena crocea muscle satellite cells are as Figure 1 shown.

[0035] (2) When the density of large yellow croaker muscle satellite cells reaches over 90%, remove the proliferation medium, and add the amplified large yellow croaker muscle satellite cells to the serum-free induction adipogenic differentiation medium for fish stem cells. Record the day when the differentiation medium is added as day 0. The differentiation medium is composed of a basal medium and a combination of cell culture cofactors, where the volume-to-mass ratio (v / w) of the basal medium to the cell cofactor combination is 1000:0.585. Specifically, the cell cofactors to be added to each liter of the basal medium include: 200 mg oleic acid, 50 mg soy lecithin, 100 mg L-Ascorbic acid 2-phosphate, 200 mg β-cyclodextrin, 10 mg transferrin, 5 mg saikosaponin A, 5 mg bakuchiol, 5 mg dehydroabietic acid, and 10 mg phenyl lactic acid. The basal medium is DMEM / F12 medium.

[0036] In addition, use the traditional induction adipogenic differentiation "cocktail" combination medium as a control experiment. The formula of control adipogenic differentiation medium ① is: using DMEM / F12 as the basal medium, containing 0.5 mM IBMX, 1 μM dexamethasone, and 10% fetal bovine serum; the formula of control adipogenic differentiation medium ② is: using DMEM / F12 as the basal medium, containing 0.5 mM IBMX, 1 μM dexamethasone, and 10% horse serum; the formula of control adipogenic differentiation medium ③ is: using DMEM / F12 as the basal medium, and containing 200 μg / mL oleic acid, 50 μg / mL soy lecithin, 100 μg / mL L-Ascorbic acid 2-phosphate, 200 μg / mL β-cyclodextrin, 10 μg / mL transferrin, 0.5 mM IBMX, and 1 μM dexamethasone. Induce differentiation for 6 days to achieve adipogenic induction and differentiation of large yellow croaker muscle satellite cells. During the induction differentiation process, change the differentiation medium every 2 days.

[0037] (3) Conduct fluorescence staining on the lipid droplets accumulated during the cell induction adipogenic differentiation to examine the differentiation situation:

[0038] ① Remove the induction adipogenic differentiation medium, and gently wash 3 times with PBS;

[0039] ② Add 4% paraformaldehyde cell fixative to fix the cells for 15 min;

[0040] ③ Add 0.1% cell permeabilization solution (0.1% Triton X-100 prepared with PBS) to permeabilize the cells for 15 min;

[0041] ④ Remove the permeabilization solution, and wash 1 time with PBS;

[0042] ⑤ Add 10 μmol / L BODIPY staining solution to stain the cell lipids for 10 min. After staining, wash once with PBS, and then add 1 μg / mL DAPI staining solution to stain the cell nuclei for 5 min;

[0043] ⑥ Discard the staining solution and rinse three times with PBS;

[0044] ⑦ Observe and take pictures using a fluorescence microscope.

[0045] As Figure 2 shown, the lipid accumulation of cells differentiated with the serum-free induction adipogenic differentiation medium for fish stem cells is significantly higher than that using the control adipogenic differentiation medium (traditional induction adipogenic differentiation "cocktail" combined medium), and the adipogenic differentiation efficiency can reach 100%.

[0046] Example 2:

[0047] The adipogenic differentiation of adipose-derived stem cells from large yellow croaker was investigated, and the specific operations were as follows:

[0048] (1) Isolation and preparation of the adipose-derived stem cell line from large yellow croaker: Obtain adipose tissue from 3-month-old large yellow croaker fry, cut it into small pieces, and then add digestive solution for digestion. The formula of the digestive solution is 1 mg / mL type I collagenase and 1 mg / mL trypsin, and the digestion time is 1 hour. Subsequently, add washing solution to the digested tissue mixture, usually wash with D-Hanks solution, and obtain a cell suspension by filtration. Then centrifuge at 300×g / min for 5 min to collect cells. After that, resuspend the cells with the complete medium for large yellow croaker stem cells (DMEM / F12 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 3 hours of culture, supplement the medium to 5 mL, and then place it in an incubator at 28°C for culture. The microscopic morphological image of the adipose-derived stem cells from large yellow croaker is as Figure 3 shown.

[0049] (2)When the density of large yellow croaker adipose-derived stem cells reaches over 90%, remove the proliferation medium, and add the amplified large yellow croaker adipose-derived stem cells to the serum-free induction adipogenic differentiation medium for fish stem cells. Denote the day of adding the differentiation medium as day 0. The differentiation medium is composed of a basal medium and a cell culture cofactor combination, where the volume-mass ratio (v / w) of the basal medium to the cell cofactor combination is 1000:0.375. Specifically, the cell cofactors 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 bakuchiol, 5 mg dehydroabietic acid, and 20 mg phenyllactic acid. The basal medium is DMEM / F12 medium.

[0050] In addition, use the traditional induction adipogenic differentiation "cocktail" combined medium as a control experiment. The formula of control adipogenic differentiation medium ① is: using DMEM / F12 as the basal medium, containing 0.5 mM IBMX, 1 μM dexamethasone, and 10% fetal bovine serum; the formula of control adipogenic differentiation medium ② is: using DMEM / F12 as the basal medium, containing 0.5 mM IBMX, 1 μM dexamethasone, and 10% horse serum; the formula of control adipogenic differentiation medium ③ is: using DMEM / F12 as the basal medium, and containing 100 μg / mL oleic acid, 20 μg / mL soy lecithin, 100 μg / mL vitamin C phosphate, 100 μg / mL β-cyclodextrin, 10 μg / mL transferrin, 0.5 mM IBMX, and 1 μM dexamethasone. Induce differentiation for 6 days to achieve adipogenic induction differentiation of large yellow croaker adipose-derived stem cells. During the induction differentiation process, change the differentiation medium every 2 days.

[0051] (3)Conduct fluorescence staining on the lipid droplets accumulated during cell induction adipogenic differentiation to examine the differentiation situation:

[0052] ① Remove the induction adipogenic differentiation medium, and gently wash 3 times with PBS;

[0053] ② Add 4% paraformaldehyde cell fixative to fix the cells for 15 min;

[0054] ③ Add 0.1% cell permeabilization solution (0.1% Triton X-100 prepared with PBS) to permeabilize the cells for 15 min;

[0055] ④ Remove the permeabilization solution, and wash 1 time with PBS;

[0056] ⑤ Add 10 μmol / L BODIPY staining solution to stain cell lipids for 10 minutes. After staining, wash once with PBS, and then add 1 μg / mL DAPI staining solution to stain cell nuclei for 5 minutes.

[0057] ⑥ Discard the staining solution and rinse three times with PBS.

[0058] ⑦ Observe and take pictures using a fluorescence microscope.

[0059] As Figure 4 shown, the lipid accumulation of cells differentiated with the serum-free induction adipogenic differentiation medium for fish stem cells is significantly higher than that using the control adipogenic differentiation medium (traditional induction adipogenic differentiation "cocktail" combined medium), and the adipogenic differentiation efficiency can reach 100%.

[0060] The above are only the preferred embodiments of the present invention, and do not limit the present invention in any way. Any simple modifications, changes, and equivalent transformations made to the above embodiments according to the technical essence of the present invention still belong to the protection scope of the technical solution of the present invention.

Claims

1. A serum-free induction adipogenic differentiation medium for fish stem cells, characterized in that: It is composed of a basal medium and a combination of cell culture cofactors; the fish stem cells are large yellow croaker muscle satellite cells or large yellow croaker adipose-derived stem cells; The basal medium is one of DMEM and DMEM / F12 medium; the combination of cell culture cofactors consists of 100-500 μg / mL oleic acid, 10-50 μg / mL soy lecithin, 100-200 μg / mL vitamin C phosphate, 100-500 μg / mL β-cyclodextrin, 5-10 μg / mL transferrin, 1-10 μg / mL saikosaponin A, 1-10 μg / mL bavachinin, 1-5 μg / mL dehydroabietic acid, and 1-20 μg / mL phenyl lactic acid.

2. The culture medium according to claim 1, characterized in that: The volume-mass ratio of the basal medium to the cell cofactor combination is 1000:0.375 to 1000:0.

585.

3. Use of the medium according to claim 1 in the adipogenic differentiation of fish stem cells; the fish stem cells are large yellow croaker muscle satellite cells or large yellow croaker adipose-derived stem cells.

4. The application according to claim 3, wherein It includes the following steps: Take large yellow croaker adipose-derived stem cells for in vitro culture. When the cell density of the stem cells reaches more than 90%, remove the cell proliferation medium, add the adipogenic differentiation medium, and continue to culture to induce the differentiation of large yellow croaker adipose-derived stem cells; every 1-2 days, change the medium with the adipogenic differentiation medium and continue to culture until the intracellular lipids of large yellow croaker adipose-derived stem cells continue to accumulate.

5. The application according to claim 4, characterized in that: The environmental conditions for culturing the large yellow croaker adipose-derived stem cells are culturing at 28°C and 5% carbon dioxide.

6. The application according to claim 4, wherein: The cell proliferation medium is DMEM medium containing 10% fetal bovine serum or DMEM / F12 medium containing 10% fetal bovine serum.

Images