Duck skeletal muscle satellite cell serum-free myoblast induced differentiation culture medium, induction method and application

Abstract

The invention discloses a duck skeletal muscle satellite cell serum-free myoblast induced differentiation culture medium and an induction method and application, and relates to the technical field of animal cell differentiation. The cell culture auxiliary factor is composed of 0.1 [mu] mol / L of insulin, 1 [mu] mol / L of dexamethasone, 1 [mu] g / mL of linoleic acid, 10 [mu] g / mL of transferrin and a 1 * GlutaMAX supplement. The duck skeletal muscle satellite cell serum-free myoblast induced differentiation culture medium 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

[0001] The invention relates to the technical field of animal cell differentiation, and in particular to a serum-free myogenic differentiation medium for duck skeletal muscle satellite cells, an induction method and an application thereof. Background Art

[0002] Cell cultured meat, also known as artificial meat or lab-grown meat, is a cultured meat product similar to traditional meat, which is obtained by extracting muscle or fat stem cells from animals (such as pigs, cattle, sheep, chickens, ducks, and pigeons), providing nutrients and growth factors in a bioreactor, and expanding the cell culture to achieve large-scale expansion of seed cells, and then using differentiation molds, bioreactors and 3D printing technology to mass-produce muscle and fat tissues, and finally using food processing technology to collect and process cells. Cell cultured meat can reduce the demand for animal agriculture, thereby reducing the impact of the meat production process on the environment, such as reducing greenhouse gas emissions and land pollution; it can also reduce the slaughter of animals, thereby improving animal welfare; and producing meat in a controlled environment can also reduce the risk of bacterial contamination and disease transmission. Cell cultured meat is similar to traditional meat in nutritional value and can be adjusted as needed to meet specific nutritional needs. In addition, cell cultured meat can also be customized according to consumer preferences, such as fat content, flavor and texture. Precisely because of the advantages mentioned above, the development prospects of cell cultured meat are optimistic and worthy of our comprehensive development. Among them, muscle production is one of our key tasks.

[0003] Skeletal muscle satellite cells are myogenic stem cells with differentiation and proliferation potential in skeletal muscle. They are usually adult stem cells that exist in a quiescent state between the sarcolemma and the basement membrane of the muscle fiber. Their position and arrangement are similar to the satellites of muscle cells, so they are called satellite cells. They are usually used as muscle seed cells in the preparation of cell cultured meat. The process of skeletal muscle satellite cells differentiating into muscle is called myogenesis, which involves a series of complex molecular and cellular events, roughly including 5 stages, namely the activation, proliferation, differentiation, fusion and maturation of satellite cells, but the myogenesis process in organisms and in vitro is slightly different.

[0004] In organisms, skeletal muscle satellite cells are activated by growth factors (such as insulin-like growth factor 1) and mechanical stimulation (such as exercise). Activated satellite cells begin to proliferate, and then when a certain cell density is reached, muscle satellite cells begin to fuse into multinucleated myotubes, which eventually form mature muscle fibers. Myotubes mature, develop sarcomeres and myofilament fibers, and thus acquire the ability to contract. Myogenesis in organisms is regulated by a variety of molecular mechanisms, including transcription factors, epigenetic regulation, and signaling pathways.

[0005] In the process of in vitro differentiation of skeletal muscle satellite cells into muscle, serum is usually added to support cell growth and differentiation. For example, the culture medium for in vitro differentiation of chicken skeletal muscle satellite cells disclosed in publication number CN116004526A needs to add 1-5% horse serum to the culture medium; Zhang Min et al. mentioned the use of chicken skeletal muscle cell differentiation medium in the article "Regulatory role of circRIPK2 in the proliferation and differentiation of chicken skeletal muscle cells": 2% horse serum + 97.5% RPMI + 0.5% penicillin and streptomycin; Liu et al. mentioned the addition of 2% horse serum to myogenic differentiation medium in the article "Construction of adenovirus vector expressing duck sclerostin and its induction effect on myogenic proliferation and differentiation in vitro"; in addition, Zhao Qian, Xu Xiaochun, Zhang Li and other researchers have also repeatedly mentioned the addition of horse serum to myogenic differentiation medium of various animals (including sheep, geese, etc.) to support cell growth and differentiation. It can be seen that the commonly used in vitro myogenic differentiation medium is to add horse serum to the basal medium. Horse serum is rich in growth factors and other nutrients that can support the proliferation and differentiation of satellite cells. It contains insulin-like growth factor 1 (IGF-1, a key growth factor known to stimulate myogenesis). However, as a complex liquid extracted from animal blood, although horse serum contains growth factors, hormones and other nutrients, the composition and quality of serum may vary from batch to batch, which may lead to variability in cell culture results. In addition, serum usually comes from animals, which implies ethical issues and the risk of infectious diseases. At the same time, serum, as an expensive culture medium component, will also increase the production cost of cell cultured meat. From the perspective of differentiation effect, proteins in animal serum may trigger immune responses, which may also affect the growth and differentiation of cultured cells. Therefore, batch-to-batch variation, concerns about animal sources, high costs and immunogenicity make it difficult for serum-containing myogenic differentiation medium to produce muscle efficiently, stably and cheaply on a large scale, hindering the industrialization of cell-cultured meat.

[0006] Therefore, it is urgent to develop a chemically defined, inexpensive, and high-differentiation-efficiency serum-free myogenic differentiation medium to overcome the disadvantages of serum use. The use of serum-free myogenic differentiation medium is crucial for the development of cell-cultured meat because it can improve product quality, reduce production costs, eliminate concerns about animal sources, and promote large-scale production. Summary of the invention

[0007] In view of the above problems in the prior art, the present invention provides a serum-free myogenic differentiation medium and an induction method for duck skeletal muscle satellite cells to solve the problem that the existing cell myogenic differentiation technology is hindered from the industrialization process of cell cultured meat due to low efficiency and poor repeatability.

[0008] The technical solution adopted by the present invention is as follows: A serum-free myogenic differentiation medium for duck skeletal muscle satellite cells, comprising a basal medium and a cell culture auxiliary factor, wherein the cell culture auxiliary factor comprises 0.1 μmol / L insulin, 1 μmol / L dexamethasone, 1 μg / mL linoleic acid, 10 μg / mL transferrin, 1×GlutaMAX ™ Supplement composition.

[0009] The object of the present invention is to provide a modified cell myogenic differentiation medium with clear chemical composition for in vitro myogenic differentiation of duck skeletal muscle satellite cells, wherein the modified cell myogenic differentiation medium does not contain serum components. The serum-free medium refers to the absence of any animal serum components, including fetal bovine serum, calf serum, chicken serum, duck serum, horse serum, etc. The serum-free medium uses synthetic growth factors and other nutrients to support cell growth and differentiation. The sources of these components can be plant-based, animal-based (non-serum) or synthetic. The modified cell myogenic differentiation medium is a skeletal muscle satellite cell differentiation medium added with cell culture auxiliary factors, and the serum components in the commonly used skeletal muscle satellite cell myogenic differentiation medium are replaced by adding cell culture auxiliary factors.

[0010] Preferably, the basal culture medium is one of DMEM and DMEM / F12 culture medium.

[0011] Preferably, the basal culture medium contains a 1× penicillin-streptomycin mixture, that is, the working concentration of penicillin G sodium salt is 100 U / mL, and the working concentration of streptomycin sulfate is 0.1 mg / mL, which can effectively avoid bacterial contamination.

[0012] The method for inducing myogenic differentiation of duck skeletal muscle satellite cells using the serum-free myogenic differentiation medium described in any one of the above items comprises the following steps: isolating duck skeletal muscle satellite cells, and performing in vitro proliferation culture to grow them to a confluence of 80-90%, discarding the proliferation medium, and then adding the serum-free myogenic differentiation medium for duck skeletal muscle satellite cells, marking the day of adding the differentiation induction medium as day 0, changing the medium every other day, and culturing for 3 days.

[0013] The duck skeletal muscle satellite cell serum-free myogenic differentiation medium is used as a myogenic differentiation medium for seed cells in the process of preparing cell culture meat.

[0014] The duck skeletal muscle satellite cell serum-free myogenic differentiation medium is used as a test reagent in the process of testing the myogenic differentiation performance of seed cells by using a cell differentiation method.

[0015] The duck skeletal muscle satellite cell serum-free myogenic differentiation medium is used as a myogenic differentiation medium in a study of muscle development mechanism through cell myogenic differentiation in vitro.

[0016] In summary, compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The serum-free myogenic differentiation medium for duck skeletal muscle satellite cells provided by the present invention does not contain any serum components and has clear chemical composition, which is convenient for promoting the industrialization process of cell cultured meat; 2. The serum-free myogenic differentiation medium for duck skeletal muscle satellite cells provided by the present invention has a good myogenic differentiation effect. Experimental studies have shown that obvious myotubes are produced after 3 days of in vitro differentiation of skeletal muscle satellite cells using the improved cell myogenic differentiation medium described in the present invention.

[0017] 3. The serum-free myogenic differentiation medium and induction method of duck skeletal muscle satellite cells provided by the present invention are of great significance for the study of the mechanism of duck skeletal muscle development and the preparation of cell-cultured meat. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a microscopic image of duck skeletal muscle satellite cells (unpurified) (scale bar: 250μm); Figure 2 This is a microscopic image of duck skeletal muscle satellite cells after 3 days of serum-free myogenic differentiation (scale bars are 1000 μm and 400 μm respectively); Figure 3 Immunofluorescence identification of duck skeletal muscle satellite cells after serum-free myogenic differentiation for 3 days (blue: cell nucleus, red: MYHC protein) and merged (MERGE) images (scale bar: 100 μm); DETAILED DESCRIPTION

[0019] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0020] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0021] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0022] The present application will be described in detail below with reference to embodiments and experimental data.

[0023] Example 1 This example explores the isolation and culture of duck skeletal muscle satellite cells, and the specific operations are as follows: (1) Under sterile conditions, obtain leg muscles from 3-day-old ducks, rinse with 75% alcohol for 1-3 min, remove bones and other non-muscle impurities, rinse with PBS three times, and then place the muscle tissue in a sterile 50 mL centrifuge tube and cut into 1 mm thick pieces with scissors. 3 Add 1-2 mg / mL collagenase I solution (2-3 times the volume of minced meat) to minced meat, place in a 37 ℃ incubator, and digest for 60-90 minutes. Shake repeatedly several times during digestion to make digestion more uniform and rapid. Centrifuge at 1000-1500 rpm for 5-10 minutes, discard the supernatant, add 0.25% trypsin solution (2-3 times the volume of minced meat), place in a 37 ℃ incubator and continue digestion for 20-30 minutes. Add an equal volume of high-glucose DMEM containing 10% fetal bovine serum to terminate digestion, and filter with a 70-150μm cell sieve.

[0024] (2) Centrifuge the filtrate at 1000-1500 rpm at room temperature for 5-10 min, discard the supernatant, and retain the cell pellet; (3) Resuspend the cell pellet in high-glucose DMEM (containing 1× penicillin-streptomycin mixture) containing 10% FBS and inoculate it into a cell culture dish. Subculture the culture medium containing duck skeletal muscle satellite cells for later use.

[0025] like Figure 1 From the cell microscopic image under the microscope shown, it is not difficult to find that the unpurified culture does contain duck skeletal muscle satellite cells with irregular long spindle shape.

[0026] Example 2 This example explores the myogenic differentiation culture of duck skeletal muscle satellite cells, and the specific operations are as follows: High-glucose DMEM (containing 1× penicillin-streptomycin mixture) containing 10% FBS was used as the complete culture medium to culture duck skeletal muscle satellite cells until the confluence of duck skeletal muscle satellite cells reached 80%~90%. The proliferation medium was removed and the serum-free myogenic induction differentiation medium for duck skeletal muscle satellite cells was added. The day of adding the induction differentiation medium was recorded as 0d, and the medium was changed every 1d. After 3d of induction differentiation, the cells were observed under a microscope and photographed.

[0027] In this embodiment, the induction differentiation medium includes a basal medium, which is a DMEM medium containing a 1× penicillin-streptomycin mixture, and then 0.1 μmol / L insulin, 1 μmol / L dexamethasone, 1 μg / mL linoleic acid, 10 μg / mL transferrin, and 1×GlutaMAX ™ Supplements, including insulin, dexamethasone, linoleic acid, transferrin, and GlutaMAX ™ Supplements are cell culture cofactors.

[0028] like Figure 2 The figures shown are microscopic images of duck skeletal muscle satellite cells at different magnifications under a microscope after 3 days of differentiation by serum-free myogenic induction. It is not difficult to see that after 3 days of differentiation, duck skeletal muscle satellite cells have obviously differentiated into multinucleated myotubes.

[0029] Example 3 This example explores the immunofluorescence identification of duck skeletal muscle satellite cells after differentiation, and the specific operation is as follows: Remove serum-free myogenic differentiation medium, wash once with PBS; add 4% PFA to fix cells for 30 minutes. Remove the fixative, add 0.5% PBSTr to permeabilize cells for 15 minutes, discard the cell permeabilization solution, wash once with PBS, add 0.2% BSA solution to block at room temperature for 45-60 minutes, discard the blocking solution, add diluted MYHC primary antibody (SC-376157, dilution ratio 1:100), incubate at 4℃ overnight, discard the primary antibody, wash 3 times with PBS, 5 minutes each time, add ABflo® 647 labeled goat anti-mouse fluorescent secondary antibody (1:100) to incubate for 1-2 hours, discard the secondary antibody, wash 3 times with PBS, 5 minutes each time. Add appropriate amount of DAPI stain (1 μg / mL), incubate for 5-10 minutes, discard the stain, wash 3 times with PBS, 5 minutes each time. Observe and take pictures under high-content laser confocal microscope.

[0030] like Figure 3The following are the immunofluorescence identification images of DAPI and MYHC (myosin heavy chain) taken under different fluorescence channels under a microscope and the merged images (DAPI and MYHC). DAPI is a specific dye for the cell nucleus and emits blue fluorescence. It is not difficult to see from the immunofluorescence staining results that most of the cells at 3d of differentiation have fused to form multinucleated myotubes, and MYHC expression is positive.

[0031] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. A serum-free myogenic differentiation medium for duck skeletal muscle satellite cells, characterized in that: The cell culture auxiliary factor comprises 0.1 μmol / L insulin, 1 μmol / L dexamethasone, 1 μg / mL linoleic acid, 10 μg / mL transferrin, 1×GlutaMAX ™ Supplement composition.

2. The serum-free myogenic differentiation medium for duck skeletal muscle satellite cells according to claim 1, characterized in that The basic culture medium is one of DMEM and DMEM / F12 culture medium.

3. The serum-free myogenic differentiation medium for duck skeletal muscle satellite cells according to claim 1, characterized in that The basic culture medium contains 1× penicillin-streptomycin mixed solution.

4. A method for cell differentiation using the duck skeletal muscle satellite cell serum-free myogenic differentiation medium as described in any one of claims 1 to 3, characterized in that: The method comprises the following steps: isolating duck skeletal muscle satellite cells, and performing in vitro proliferation culture to make them grow to a confluence of 80-90%, discarding the proliferation culture medium, and then adding a serum-free myogenic differentiation induction culture medium for duck skeletal muscle satellite cells, marking the day when the differentiation induction culture medium is added as day 0, changing the culture medium every other day, and culturing for 3 days.

5. Use of the serum-free myogenic differentiation medium for duck skeletal muscle satellite cells as claimed in any one of claims 1 to 3 as a myogenic differentiation medium for seed cells in the process of preparing cell culture meat.

6. Use of the duck skeletal muscle satellite cell serum-free myogenic differentiation medium as claimed in any one of claims 1 to 3 as a test reagent in the process of testing the myogenic differentiation performance of seed cells using a cell differentiation method.

7. Use of the serum-free myogenic differentiation medium for duck skeletal muscle satellite cells as claimed in any one of claims 1 to 3 as a myogenic differentiation medium in a study of muscle development mechanism through cell myogenic differentiation in vitro.

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