CN117721074B - Serum-free adipogenic induction differentiation medium for poultry, induction method and application - Google Patents
Serum-free adipogenic induction differentiation medium for poultry, induction method and application Download PDFInfo
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- CN117721074B CN117721074B CN202410034653.5A CN202410034653A CN117721074B CN 117721074 B CN117721074 B CN 117721074B CN 202410034653 A CN202410034653 A CN 202410034653A CN 117721074 B CN117721074 B CN 117721074B
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a serum-free adipogenic induction differentiation medium for poultry, an induction method and application thereof, and relates to the technical field of animal cell differentiation. The serum-free adipogenic induction differentiation medium for poultry provided by the invention does not contain any serum component, has definite chemical components and good differentiation effect, and is convenient for promoting the industrialization process of cell culture meat.
Description
Technical Field
The invention relates to the technical field of animal cell differentiation, in particular to a serum-free adipogenesis induced differentiation culture medium for poultry, an induction method and application.
Background
The cell culture meat is a product obtained by extracting animal stem cells, performing proliferation and directional differentiation in vitro, and collecting and processing. Fat is one of the main components of meat and plays a key role in the texture, mouthfeel and oral sensation of cell-cultured meat. First, adipocytes can provide the fullness and mouthfeel of meat. Fat droplets in adipocytes can give the meat a juicy character, making it more palatable. In addition, the fat cells can influence the viscosity and the oral feeling of the meat, so that the meat is more similar to the taste of the traditional meat. In addition, adipocytes are also critical to the nutritional properties of cell-cultured meats.
Fat has an important impact on the health and physiological functions of birds. First, fat plays a role in energy storage and energy supply in birds. The fat also provides protection and insulation to the birds, which can help the birds maintain a stable body temperature in a cold environment. In poultry food production, fat is also very important for the quality and mouthfeel of the product. The proper amount of fat can endow the poultry meat with rich mouthfeel and flavor, and make the poultry meat more juicy and delicious.
Adipocyte differentiation refers to the process by which undifferentiated adipose precursor cells are transformed into mature adipocytes. This process is critical to the proper development and function of adipose tissue. First, adipocyte differentiation is a critical step in adipose tissue formation and growth. During adipocyte differentiation, adipose precursor cells undergo multiple stages of cell proliferation, cell cycle regulation, and cell differentiation, and eventually differentiate into mature adipocytes. These mature adipocytes can synthesize and store fat, form adipose tissue, and have an impact on energy metabolism and endocrine function.
Serum, which is often used as one of the components of cell proliferation and differentiation media in cell culture, contains various growth factors, nutrients and extracellular matrix components, which can provide the support required for cell growth and differentiation. However, serum also presents some potential risks and limitations in cell culture. First, there may be batch-to-batch differences in serum composition and quality. Different batches of serum may contain different components and concentrations, which may have an effect on cell growth and differentiation and lead to instability of the experimental results. Second, serum may contain pathogenic microorganisms, viruses and other contaminants. Although commercial serum products are processed and tested to ensure that they meet relevant quality standards, there is a potential risk of infection. Furthermore, serum may introduce unnecessary variables or limitations. For example, lipid and protein components in serum may interfere with cell signaling, metabolism, and synthesis of extracellular matrix. In adipocyte culture, the use of serum may interfere with the differentiation and functional expression of adipocytes. Serum is expensive, and the preparation of serum requires slaughtering animals, so that the cost for cultivating meat by cells is high, and animal source substances should be avoided as much as possible.
Fat of poultry is mainly synthesized in liver, and the head synthesis capacity of in vitro fat cells is poor, so that the cocktail method (insulin, dexamethasone, IBMX) used by traditional mammals has poor induction effect and almost no fat drop is generated. The in vitro differentiation of avian adipocytes requires the addition of exogenous fatty acids. The formula of the culture medium for inducing the differentiation of the fat precursor cells of the poultry, which is commonly used at present, is that 10 percent of fetal bovine serum and oleic acid are added into a basic culture medium. However, the chemical components of the culture medium are not clear due to the addition of the fetal calf serum, and the culture medium has the problems of unstable components of different batches of culture medium, easy pathogen pollution such as viruses, high cost and the like. In addition, the differentiation effect is poor and unstable. This makes it impossible to produce cell culture meat efficiently, stably and inexpensively on a large scale using the medium, and hinders the progress of industrialization of cell culture meat.
Therefore, there is an urgent need to develop a serum-free adipogenic induction differentiation medium with definite chemical composition, low cost and high efficiency, and solve the problem that the industrialization process of cell culture meat is hindered due to low differentiation efficiency and poor repeatability at present.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a serum-free adipogenesis induction differentiation medium for poultry and an induction method thereof, which are used for solving the problem that the existing cell adipogenesis differentiation technology hinders the industrialization process of cell culture meat due to low efficiency and poor repeatability.
The technical scheme adopted by the invention is as follows:
A serum-free adipogenic induction differentiation culture medium for poultry consists of a basal culture medium and a cell culture auxiliary factor, wherein the cell culture auxiliary factor consists of 5-10 mu g/mL of insulin, 100-200 mu mol/L of sodium oleate, 10-50 mu mol/L of linoleic acid, 5-10 mu g/mL of transferrin, 0.5% -1% by volume of essential amino acid additive, 0.05-0.1 mmol/L of non-essential amino acid additive and 1-2 mmol/L of GlutaMAX ™ additive.
The invention aims to provide an improved cell adipogenic induction differentiation medium with definite chemical composition for adipogenic induction differentiation of fat precursor cells in vitro, which does not contain serum components. The serum-free means that any animal serum components including fetal bovine serum, calf serum, chicken serum, duck serum, horse serum and the like are not added, the modified cell adipogenic induction differentiation culture medium is a fat precursor cell differentiation culture medium added with cell culture auxiliary factors, and the serum components in the traditional fat precursor cell adipogenic induction differentiation culture medium are replaced by adding the cell culture auxiliary factors; the basal medium also does not contain penicillin streptomycin diabody solution.
Preferably, the basal medium is one of DMEM and DMEM/F12 medium.
Preferably, the essential amino acid additive comprises L-arginine, L-cystine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, L-tyrosine, L-valine.
Preferably, the non-essential amino acid additives include L-alanine, L-glutamic acid, L-asparagine, L-aspartic acid, L-proline, L-serine and glycine.
The induction method for inducing the cell adipogenic differentiation by adopting the serum-free adipogenic induced differentiation medium for poultry, which comprises the following steps:
(1) Taking fat precursor cells of poultry, culturing in vitro, growing to a confluence of more than 80%, discarding proliferation culture medium, adding serum-free adipogenic induction differentiation culture medium of the poultry, and recording the current day of the addition of the differentiation culture medium as 0 d;
(2) And (3) inducing differentiation for 2-6 d to realize fat-forming induced differentiation of the poultry, wherein in the induced differentiation process, a differentiation culture medium is changed every 1-2 d.
The serum-free adipogenesis induced differentiation medium for poultry is applied to the preparation of the differentiation medium for seed cells in the process of cultivating meat by poultry organisms.
The serum-free adipogenic induction differentiation medium for poultry is applied as a test reagent in the process of testing the differentiation performance of the poultry seed cells by using a cell differentiation method.
In summary, compared with the prior art, the invention has the following advantages and beneficial effects:
1. the serum-free adipogenic induction differentiation culture medium for poultry provided by the invention does not contain any serum component, has definite chemical components, and is convenient for promoting the industrialization process of cell culture meat;
2. the serum-free adipogenic induced differentiation medium for poultry provided by the invention has good adipogenic differentiation effect, and experimental researches show that after the improved adipogenic induced differentiation medium for cells of the invention is adopted to conduct in vitro induced differentiation on fat precursor cells for 6 d, lipid drops appear on almost all cells, and the cell differentiation efficiency reaches 100%;
3. The invention provides a universal serum-free adipogenesis induced differentiation culture medium for poultry and an induction method, which have important significance for researching the mechanism of poultry fat development and preparing cell culture meat.
Drawings
FIG. 1 is a microscopic view of chicken fat precursor cells;
FIG. 2 is a microscopic view of chicken fat precursor cells after serum-free adipogenic induced differentiation 6 d;
FIG. 3 shows the results of repeated experiments of chicken fat precursor cells after serum-free adipogenic induced differentiation of 6 d;
FIG. 4 is a microscopic view of duck fat precursor cells;
FIG. 5 is a microscopic view of duck fat precursor cells after serum-free adipogenic induced differentiation 6 d;
FIG. 6 shows the results of repeated experiments on duck fat precursor cells after serum-free adipogenic induced differentiation of 6 d.
Detailed Description
The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.
Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification will control.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.
The present application will be described in detail with reference to examples and experimental data.
Example 1 this example explores the differentiation of chicken fat precursor cells, and is performed as follows:
(1) Extracting chicken fat precursor cells: cutting subcutaneous fat tissue of chicken into 1mm 3 pieces, adding collagenase I working solution, digesting 40 min at 37 ℃, adding proper amount of complete culture medium to stop digestion, filtering and centrifuging to obtain fat precursor cells, adding complete culture medium to resuspend cell precipitate, counting, spreading on a cell culture dish for proliferation culture, wherein the microscopic morphology of the obtained chicken fat precursor cells is shown in figure 1;
(2) Removing proliferation medium, adding the amplified chicken fat precursor cells into a fowl serum-free adipogenic induction differentiation medium, adding 0.0 d of a local diary added with the differentiation medium, wherein the differentiation medium consists of a basic medium and a cell culture auxiliary factor, the basic medium is a DMEM medium, the cell culture auxiliary factor consists of 6 mug/mL of insulin, 150 mug/L of sodium oleate, 40 mug/L of linoleic acid, 8 mug/mL of transferrin, 0.6 volume percent of essential amino acid additive, 0.08 mmol/L of non-essential amino acid additive and 1.5 mmol/L of GlutaMAX ™ additive, and the essential amino acid additive is Gibcom TM MEM amino acid solution (50X) with the commodity number of 11130051; the non-essential amino acid additive is Gibcom TM MEM non-essential amino acid solution (100X), and the product number is 11140050;
(3) Induced differentiation 6d realizes the induced differentiation of chicken fat, and in the induced differentiation process, the differentiation medium is changed every 1-2 d.
The differentiation effect is checked by the following steps:
(1) Removing the adipogenic induction differentiation medium, and gently rinsing with PBS for 3 times;
(2) Cell fixation was performed by adding 4% PFA 20 min;
(3) Cell permeabilization was performed by adding 0.1% PBSTr (0.1% triton X-100 in PBS) for 15: 15 min cell permeabilization, with replacement of permeabilization every 5: 5min;
(4) Removing the penetrating fluid and rinsing for 1 time by PBS;
(5) Adding 5 mu mol/L BODIPY dye solution and 1.5 mu g/mL DAPI dye solution, and dyeing in a dark place for 12 min;
(6) Discarding the staining solution, and rinsing with PBS for 3 times;
(7) Anti-fluorescence quenchers were added, observed using a fluorescence microscope and photographed.
As shown in FIG. 2, it can be seen that after the fat precursor cells are subjected to in vitro induced differentiation by using a specific modified cell adipogenic differentiation medium for 6 d, the cell differentiation efficiency reaches 100%, and as shown in FIG. 3, we repeat the above experimental process for 2 times, and after the in vitro induced differentiation is performed for 6 d, the cell differentiation efficiency reaches 100% and 100%, respectively, which indicates that the method has good repeatability and high cell differentiation rate.
Example 2 this example explores the differentiation of duck fat precursor cells, and is performed as follows:
(1) Extracting duck fat precursor cells: cutting subcutaneous fat tissue of duck into pieces of 1 mm 3, adding collagenase I working solution, digesting 40 min at 37deg.C, adding 9 times of complete culture medium to stop digestion, filtering, spreading onto cell culture dish, and performing proliferation culture to obtain microscopic morphology of duck fat precursor cells shown in figure 4;
(2) Removing proliferation medium, adding the amplified duck fat precursor cells into a fowl serum-free adipogenic induction differentiation medium, adding 0.0 d of a local mark into the differentiation medium, wherein the differentiation medium consists of a basic medium and a cell culture auxiliary factor, the basic medium is a DMEM medium, the cell culture auxiliary factor consists of 6 mug/mL of insulin, 150 mug/L of sodium oleate, 40 mug/L of linoleic acid, 8 mug/mL of transferrin, 0.6 volume percent of essential amino acid additive, 0.08 mmol/L of non-essential amino acid additive and 1.5 mmol/L of GlutaMAX ™ additive, and the essential amino acid additive is Gibcom TM MEM amino acid solution (50X) with the commodity number of 11130051; the non-essential amino acid additive is Gibcom TM MEM non-essential amino acid solution (100X), and the product number is 11140050;
(3) The induced differentiation is 6d to realize the induced differentiation of the duck fat, and the differentiation culture medium is changed every 1-2 d in the induced differentiation process.
The differentiation effect is checked by the following steps:
(1) Removing the differentiation medium, and gently rinsing with PBS for 3 times;
(2) Cell fixation was performed by adding 4% PFA 20 min;
(3) Adding 0.1% PBSTr for cell permeation 15 min, and changing permeation liquid every 5 min;
(4) Removing the penetrating fluid and rinsing for 1 time by PBS;
(5) Adding 5 mu mol/L BODIPY dyeing liquid and 1.5 mu g/mLDAPI dyeing liquid, and dyeing in dark for 12 min;
(6) Discarding the staining solution, and rinsing with PBS for 3 times;
(7) Anti-fluorescence quenchers were added, observed using a fluorescence microscope and photographed.
As shown in FIG. 5, it is known that the cell differentiation efficiency reaches 100% after the in vitro induced differentiation of the fat precursor cells by using the specific modified cell adipogenic differentiation medium is 6 d, and as shown in FIG. 6, we repeat the above experimental process for 2 times, and after the in vitro induced differentiation is 6 d, the cell differentiation efficiency reaches 100% and 100%, respectively, which indicates that the method has good repeatability and high cell differentiation rate.
The above examples merely illustrate specific embodiments of the application, which are described in more detail and are not to be construed as limiting the scope of the application. It should be noted that it is possible for a person skilled in the art to make several variants and modifications without departing from the technical idea of the application, which fall within the scope of protection of the application.
Claims (6)
1. A serum-free adipogenic induction differentiation culture medium for poultry is characterized by comprising a basal culture medium and a cell culture auxiliary factor, wherein the cell culture auxiliary factor comprises 5-10 mu g/mL of insulin, 100-200 mu mol/L of sodium oleate, 10-50 mu mol/L of linoleic acid, 5-10 mu g/mL of transferrin, 0.5% -1% by volume of essential amino acid additive, 0.05-0.1 mmol/L of non-essential amino acid additive and 1-2 mmol/L of GlutaMAX ™ additive.
2. The avian serum-free lipid-forming induction differentiation medium according to claim 1 wherein the basal medium is one of DMEM, DMEM/F12 medium.
3. The avian serum-free lipid-forming induction differentiation medium according to claim 1 wherein the essential amino acid additives comprise L-arginine, L-cystine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, L-tyrosine and L-valine.
4. The avian serum-free lipid-forming induction differentiation medium according to claim 1 wherein the non-essential amino acid additives comprise L-alanine, L-glutamic acid, L-asparagine, L-aspartic acid, L-proline, L-serine and glycine.
5. The induction method for cell differentiation by using the serum-free adipogenic induction differentiation medium for poultry according to any one of claims 1 to 4, which is characterized by comprising the following steps:
(1) Taking fat precursor cells of poultry, culturing in vitro, growing to a confluence of more than 80%, discarding proliferation culture medium, adding serum-free adipogenic induction differentiation culture medium of the poultry, and recording the current day of the addition of the differentiation culture medium as 0 d;
(2) And (3) inducing differentiation for 2-6 d to realize adipogenic induced differentiation of the avian fat precursor cells, wherein in the induced differentiation process, a differentiation culture medium is changed every 1-2 d.
6. The use of the serum-free adipogenic differentiation medium for poultry according to any one of claims 1 to 4 for adipogenic differentiation of avian fat precursor cells.
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| JP2000157260A (en) * | 1998-11-26 | 2000-06-13 | Toyobo Co Ltd | Differentiation induction of primary precursor adipose cell, and culture medium for its differentiation |
| CN115927172A (en) * | 2022-12-15 | 2023-04-07 | 南京周子未来食品科技有限公司 | Improved culture medium with specific chemical components for inducing in vitro adipogenic differentiation and application thereof |
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| JP2000157260A (en) * | 1998-11-26 | 2000-06-13 | Toyobo Co Ltd | Differentiation induction of primary precursor adipose cell, and culture medium for its differentiation |
| CN115927172A (en) * | 2022-12-15 | 2023-04-07 | 南京周子未来食品科技有限公司 | Improved culture medium with specific chemical components for inducing in vitro adipogenic differentiation and application thereof |
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