CN115584341A - Fish myoblast differentiation culture medium and artificial fish tissue method - Google Patents

Abstract

The method screens Notch and TGF-beta signal paths from a large number of signal paths by a transcriptome sequencing means, provides a fish myoblast differentiation medium capable of simultaneously inhibiting the Notch and TGF-beta signal paths, and promotes the efficient differentiation of myoblasts into myotubes. The invention also provides a three-dimensional culture method of fish myoblasts based on the culture medium, so as to obtain fish muscle tissues, and further obtain artificial fish meat with a texture highly similar to that of natural fish meat by filling fat cells. Therefore, the invention can realize the preparation of high-quality artificial fish meat through two steps of cell culture and cell filling, is simple, convenient and efficient, and is suitable for large-scale implementation and application.

Description

A kind of fish myoblast differentiation medium and artificial fish tissue method

technical field

The invention belongs to the field of food processing, and in particular relates to a fish myoblast differentiation medium and a method for artificial fish tissue.

Background technique

In recent years, cell-cultured meat technology has emerged as a partial alternative to traditional livestock meat production based on concerns about efficiency, sustainability, environmental burden, and animal welfare. Tissue-like cell-cultured meat, which is similar in composition and structure to real muscle tissue and consists mainly of fat cells and lined muscle cells, has been successfully developed in poultry cattle and pigs. In vitro three-dimensional (3D) cell culture conditions, including media (e.g., fetal bovine serum) and support materials, have been extensively studied in human or other mammalian cell cultures for regenerative medicine and organoid purposes. However, the cost and scale of food constructs, as well as other key aspects such as target species, nutrients, and sensory characteristics, are quite different from those of biomedical organizations. When it comes to cell-cultured meat, there are still many challenges to establishing your own methods.

The production of cell culture meat is that isolated cells (seed cells) form multinucleated myotubes through proliferation and differentiation in a suitable culture environment (medium), such as temperature, oxygen, nutrients and growth factors, and the myotubes mature to form muscle fibers , eventually becoming a product that mimics meat. The differentiation of myoblasts is highly susceptible to a variety of regulatory factors and signaling pathways, resulting in unintended cell fate differentiation, such as apoptosis and fibrous necrosis, resulting in the inability of cells to form myotubes and further fusion into muscle bundles. However, it is now known that there are tens of thousands of animal genes, and the interaction between different genes and signaling pathways is extremely complex. It is extremely important to accurately find the combination of signaling pathways that regulate cell differentiation behavior, and then make myoblasts move towards the correct differentiation fate. challenge.

As a key part of three-dimensional tissue culture in vitro, cell scaffolds provide the necessary attachments and carriers for the proliferation and differentiation of myoblasts and adipocytes. The selection of scaffold materials in cell culture meat should not only meet the green, safe and edible production conditions, but also meet the biological characteristics of cells (supporting cell adhesion, proliferation, migration, differentiation) and rheological transformation characteristics (lower than cell growth temperature). Freezing point, mild cross-linking mode, necessary viscosity to protect cells). At the same time, as the final part of edible products, it must also have a certain mechanical strength to meet consumers' needs for chewiness, viscoelasticity and other textures. This is a difficult technical challenge across the fields of food, biology, materials, machinery and so on.

Seafood is a favorite food for many and is rich in protein, omega-3 fatty acids and micronutrients. As human population increases, coupled with environmental pressures and climate change, have led to the overexploitation of marine food resources in recent decades, which has had a huge impact on ecosystems, innovative technologies (e.g. cell cultured fish) are a viable option for seafood production. The urgent need for continuity. At present, although there are some researches on tissue cultured meat in mammals (pigs, cattle, rabbits), there is still a big gap between its structure and natural meat, and most of them adopt some simple stacking molding methods. Elasticity, etc. are still not comparable to natural meat. Producing cell-cultured fish fillets with tissue-like texture remains an unfinished task due to the lack of fish muscle tissue texture properties and realistic 3D scaffolds for mechanical support.

Contents of the invention

On the one hand, the present invention screens out a combination of Notch and TGF-β signaling pathways from a large number of signaling pathways by means of transcriptome sequencing. And found that by simultaneously inhibiting Notch and TGF-β signaling pathways, it can significantly improve myoblast differentiation.

In some preferred embodiments of the present invention, growth factors LY411575 and RepSox are used to inhibit the aforementioned Notch and TGF-β signaling pathways. The data show that the combination of LY411575 and RepSox can effectively inhibit the above-mentioned Notch and TGF-β signaling pathways without the assistance of other growth factors, and improve the differentiation efficiency of myoblasts.

In some preferred embodiments of the present invention, the concentration of LY411575 is 1-20 nM, and the concentration of RepSox is 1-10 μM.

In some preferred embodiments of the present invention, the medium is F12 medium. F12 medium provides energy supplement for the cell differentiation process, and cooperates with LY411575 and RepSox to promote cell differentiation efficiency and increase myotube fusion.

As another aspect of the present invention, the present invention provides a three-dimensional culture method of myoblasts. In this method, myoblasts and scaffold precursors are composed of bio-ink, and 3D simulation printing is performed. After curing, the above-mentioned medium is used for culture to obtain a three-dimensional muscle tissue. In a three-dimensional system, myoblasts grow three-dimensionally in a gelatin-based scaffold, and the above medium that inhibits Notch and TGF-β signaling pathways promotes the myogenic differentiation of fish myoblasts, and exerts its unique advantages in a 3D system . It has been proved by experiments that the differentiation promotion rate of the same medium in the three-dimensional system is increased by 9.79% compared with that in the two-dimensional system.

In some preferred embodiments of the present invention, the scaffold precursor includes gelatin, and the concentration of gelatin in the bio-ink is 5-10%. Gelatin can provide biological sites for cell adhesion, increase cell viability and provide living space for its growth.

In some preferred embodiments of the present invention, the cell concentration in the bio-ink is 1×10 ⁶ -1×10 ⁷ .

In some preferred embodiments of the present invention, the bio-ink also has a curing agent for shaping the gelatin; the hardness of the cured gelatin is 9-14 kPa. Appropriate scaffold stiffness helps to promote cell attachment, increase cell viability and provide bearing capacity for its differentiation.

In some preferred embodiments of the present invention, described curing agent is sodium alginate, chitosan, and the curing of curing agent is realized by curing initiator, and described curing initiator comprises calcium chloride, calcium chloride, lemon Sodium Chloride, Magnesium Chloride and Zinc Chloride etc.

In some preferred embodiments of the present invention, the curing concentration is 0.1-5%, and the curing time is 1-10 minutes.

In some preferred embodiments of the present invention, the culture conditions are 27°C and 5% carbon dioxide concentration.

In some preferred embodiments of the present invention, the 3D simulation printing is based on a fish muscle tissue model to obtain a three-dimensional muscle tissue with a fish muscle tissue structure. Specifically, the soft tissue is stained, and then micro-CT scans are used to obtain two-dimensional slice images of real meat to generate a three-dimensional model.

Finally, the present invention also relates to a method for forming artificial fish flesh tissue, filling fish flesh fat cells into the aforementioned cultured muscle tissue. Compared with the existing stacking method, the method is simpler and more efficient, and its hardness, viscosity, elasticity, elasticity, etc. are more similar to natural fish muscle tissue.

The beneficial effects of the present invention are:

(1) Using the medium containing LY411575 and RepSox in the present invention, the two-dimensional differentiation efficiency of fish myoblasts increased from 1% to 32%, creating a good foundation for the subsequent formation of muscle tissue;

(2) The gelatin-based cell scaffold in the present invention has excellent biocompatibility to fish cells, supports fish cell attachment, proliferation, and differentiation, and is used in combination with the differentiation medium containing LY411575 and RepSox in this study to make fish myoblasts The differentiation efficiency reached 41.79%;

(3) The artificial fish meat tissue method of the present invention can obtain cell culture meat products similar to natural fish meat in hardness, viscosity, elasticity, elasticity, etc.

Description of drawings

Figure 1a is the bright field and immunofluorescence images during the myogenic differentiation process (the upper is the bright field image, the lower is the fluorescence microscope observation image after Dsemin staining), b is the muscle fusion index image during the myogenic differentiation process (0 day, 3 days, 6 days);

Figure 2a is a heat map showing all differentially expressed genes during myogenic differentiation on day 0, day 3 and day 6, b is a volcano map showing genes differentially expressed between day 0 and day 3, and c is Go analysis of the top 10 enrichments of up-regulated (red bars) and down-regulated (blue bars) on day 3 compared to day 0, d is a heatmap of gene expression patterns related to muscle formation, e is cell cycle related Heat map of genes, f is the heat map of Notch-related gene expression, g is the heat map of TGF-β-related gene expression, h is the heat map of myofibroblast-related gene expression, i is the heat map of extracellular matrix-related gene expression , j is the differential gene enrichment analysis diagram in the process of myogenic differentiation;

Figure 3a is the image of LY411575 and RepSox alone and in combination (upper is the bright field image of the microscope, and the lower is the image of fluorescence microscope observation after Dsemin staining), b is the muscle fusion index image of LY411575 and RepSox alone and in combination;

Figure 4 is a graph showing the effect of LY411575 and RepSox alone and in combination on the myogenic effect of three-dimensional growing cells;

Figure 5 is a comparison of the texture of cell cultured fish meat and natural fish meat.

detailed description

In order to further explain the technical means and effects of the present invention to achieve the intended purpose of the invention, the specific implementation, structure, features and effects of the present invention will be described in detail below in conjunction with the accompanying drawings and preferred embodiments.

Example 1

After the yellow croaker myoblasts were cultured to 80% confluence on the well plate, the cells containing 8% HS (horse serum), 10 ng/mL IGF-1, 50 nM necrotic sulfonamide, 200 μM ascorbic acid (Phytotech, A106), 1 × PS F12 medium for differentiation culture. Differentiation efficiency is shown in Figure 1. As can be seen from Figure 1, on day 3 of culture in this differentiation medium, elongated myotubes began to appear, and on day 6, streaks resembling skeletal muscle were observed in some myotubes (Figure 1f). Immunofluorescent staining for Desmin, a muscle-specific protein, revealed that Desmin-positive myotubes with multiple nuclei were detected on both day 3 and day 6 (Fig. 1a). Whereas the fusion index (proportion of nuclei in multinucleated myotubes) was higher at day 3 (6.39%) than at day 6 (1.30%) (Fig. 1b). Although the average number of nuclei in individual myotubes was higher on day 6, the total number of myotubes was much less on day 6 than on day 3, suggesting that myogenic differentiation was inhibited on days 3–6.

Based on the above experimental results, the present invention analyzes the differentially expressed genes of cells cultured at different times (day 0, day 3, and day 6), and further uses KEGG analysis to select the signal pathway that inhibits myogenic differentiation. details as follows:

(1) By performing transcriptomic analysis on the RNA sequences of the 0th day, the 3rd day, and the 6th day. Cluster analysis showed that the expression profile changed more drastically on day 3 than on day 6 compared to day 0 (Fig. 2a), which is consistent with the fusion index. Differentially expressed gene (DEG) analysis showed that from day 0 to day 3, 1650 genes were significantly up-regulated (log2 FC>1, FDR<0.05) and 1063 genes were significantly down-regulated (log2 FC<-1 , FDR<0.05) (Fig. 2b).

(2) After annotating the above differentially expressed genes with KEGG pathways, it can be seen that many important signaling pathways, including MAPK, TGF-β, Wnt, Notch, hippo, HIF-1, etc., are enriched in differentially expressed genes (Fig. 2d~i).

Among them, on day 3, the expression of some members of TGF-β signaling (dcn, tgffbr3, tmoda, and smad3) was increased, while the expression of some members (smad6, smad2, tgfb1a, tgfb2, and tgfbr1b) was decreased; on day 6, the expression of DCN, TGFBR2 and Smad7 increased, while the expression of Smad6 and Smad2 decreased. Therefore, the low fusion index is most likely due to TGF-β signaling-induced conversion of myoblasts to myofibroblasts in serum-starved cultures.

Example 2

In this example, the purpose is to improve the efficiency of myogenesis and prevent myofibrosis by inhibiting the TGF-β signaling pathway and the Notch signaling pathway related to myogenic differentiation.

In this example, two small molecule drugs: LY411575 (a Notch inhibitor) and RepSox (a TGFβR-1/ALK5 inhibitor) were used to achieve simultaneous inhibition of the above two signaling pathways.

Experimental group (1): Add 10nMLY411575 and 5 μM RepSox to the basal medium (F12 medium containing 8% HS, 1×PS) for the cultivation of fish myoblasts for the first two days, and then transfer to DMEM medium (containing 8% FBS, 1×PS in DMEM) for routine culture in the later stage.

In addition, a control group was set up as follows:

Control 1: 10 nMLY411575 was added to the basal medium (F12 medium containing 8% HS, 1 × PS) for the first two days of culture of fish myoblasts, and then transferred to DMEM medium (containing 8% FBS, 1 ×PS DMEM) for the later conventional culture.

Control 2: 5 μM RepSox was added to the basal medium (F12 medium containing 8% HS, 1 × PS) for the cultivation of fish myoblasts for the first two days, and then transferred to DMEM medium (containing 8% FBS, 1 × PS) ×PS DMEM) for the later conventional culture.

The test results after 4 days of culture in DMEM showed a slight increase in the number of myotubes in control 1 and control 2. In the experimental group (LY411575 and RepSox added at the same time), compared with the control group 1 and control group 2, the number of myotubes increased significantly (Figure 3). The fusion index of the blank group was about 1%, which was increased to 8% and 3.5% by RepSox or LY411575 treatment, respectively, and the experimental group was synergistically increased to 32% by the two chemicals. The results showed that the myogenic efficiency of PSCs was significantly enhanced by synchronous inhibition of Notch and TGF-β signaling. It can be seen that the synergistic effect of LY411575 and RepSox, in addition to their respective inhibition of Notch and TGF-β signaling pathways, also has unexpected technical effects.

Experimental group (2): 20nM LY411575 and 2 μM RepSox were added to the basal medium (F12 medium containing 8% HS, 1×PS) for the cultivation of fish myoblasts for the first two days, and then transferred to DMEM medium ( The later routine culture was carried out in DMEM containing 8% FBS and 1×PS.

Experimental group (3): Add 1nM LY411575 and 10μM RepSox to the basal medium (F12 medium containing 10% HS, 1×PS) for the cultivation of fish myoblasts for the first two days, and then transfer to DMEM medium ( The later conventional culture was carried out in DMEM containing 10% FBS and 1×PS.

Experimental group (4): 5nM LY411575 and 1 μM RepSox were added to the basal medium (F12 medium containing 6% HS, 1×PS) for the cultivation of fish myoblasts for the first two days, and then transferred to the DMEM medium ( The later conventional culture was carried out in DMEM containing 6% FBS and 1×PS.

After cultured in DMEM for 4 days, the muscle cell fusion index of experimental group 2 was 16%; the muscle cell fusion index of experimental group 3 was 21%. The myocyte fusion index of experimental group 4 was 14%. Compared with the separate superposition of the blank group and the control group, there is a significant improvement.

Example 3

This example aims to study the conditions of gel solidification to screen out a three-dimensional framework suitable for cell printing and growth.

(1) Mix 10wt% fish gelatin, 1wt% sodium alginate and fish myoblasts at a cell concentration of 1×10 ⁶ /mL, cool at 4°C for 5 minutes, and then soak in 1% CaCl ₂ for 10 minutes to solidify. Under this condition, the hardness of the hydrogel is 9kPa. After culturing for 5 days, the cell adhesion rate was 80.69%. It shows that the cell viability is high, which provides favorable growth conditions for the subsequent cell differentiation and formation.

(2) Mix 10wt% fish gelatin, 1wt% sodium alginate and fish myoblasts at a cell concentration of 5×10 ⁶ /mL, cool at 4°C for 5 minutes, and then soak in 5% CaCl ₂ for 10 minutes to solidify. Under this condition, the hardness of the hydrogel is 21kPa. After 5 days of culture, the cell adhesion rate was only 9%. Most of the cells are dead, making it impossible to grow tissue later.

(3) Mix 5wt% fish gelatin, 1wt% sodium alginate and fish myoblasts, cool at 4°C for 5 minutes, the cell concentration is 1×10 ⁷ /mL, and then soak in 5% CaCl ₂ for 5 minutes to solidify. Under this condition, the hardness of the hydrogel is 14kPa. After culturing for 5 days, the cell adhesion rate was 51%. It shows that the cell viability is acceptable, and the basic conditions for cell growth are still available.

It can be seen that the cured gel needs to be kept at 9-14kPa to ensure the growth of fish myoblasts. Those skilled in the art can adjust the concentration of gel and curing agent to obtain the mechanical environment for cell growth in the above matters.

Example 4

(1) The yellow croaker meat samples were fixed in tissue fixative for 24 hours. After the fixation, the samples were taken out and washed with saline, weighed and stained with staining solution. After the dyeing is completed, use 75% alcohol to wash off the staining solution attached to the surface, and perform CT scanning; wherein, the staining solution is: dissolve 10g of potassium iodide in a small amount of distilled water, add 5g of iodine and stir to completely dissolve the iodine, continue to add distilled water to make the volume of the distilled water For 100ml, a 3.75% IKI solution can be obtained.

(2) Scan the above-mentioned test sample with a resolution of 3 μm/pixel by using SkyScan1272 micro-CT equipment to obtain continuous two-dimensional slice images of salmon muscle microstructure.

(3) Use Gaussian Filter to filter the scanned two-dimensional data; use VolumeRendering to perform preliminary three-dimensional rendering of the data set and use Crop Editor to cut the data to an appropriate size; use Interactive Thresholding to perform dynamic threshold segmentation on the data to determine each Threshold distribution of components for extraction of muscle fibers.

(4) Under the premise of not affecting the main body of the fiber, use Image Segmentation to remove small-scale noise and fill small-scale cavities; use Separate Objects to segment adjacent muscle fibers; use GenerateSurface to smooth the surface of the 3D model to Export the 3D model in .stl format.

(5) After mixing 10wt% fish gelatin, 1wt% sodium alginate and fish myoblasts, the cell concentration is 5×10 ⁶ /mL, use the .stl format model generated above for 3D printing, and obtain the yellow croaker simulation muscle scaffold .

(6) Place the scaffold in the culture medium (F12 medium containing 8% HS, 1×PS) supplemented with 10 nMLY411575 and 5 μM RepSox for two days, and then transfer to DMEM medium (containing 8% FBS, 1×PS DMEM) for the later stage of culture, and obtained muscle tissue after 5 days of culture. Observing its differentiation effect, the fusion index reached 41.79%. Compared with the differentiation effect in the 2-dimensional environment in Example 2, it has increased by 9.79%.

In addition, the scaffold obtained in step 5 was placed in the culture medium (F12 medium containing 8% HS, 1 × PS) supplemented with LY411575 alone and cultured for two days, and then transferred to DMEM medium (containing 8% FBS, 1 × PS). PS in DMEM) for subsequent conventional culture. The differentiation effect was observed after culturing for 7 days. Under the synergistic effect of materials and differentiation medium, the fusion index was 13.6%.

It can be seen that in the three-dimensional system, the synergistic effect of LY411575 and RepSox is particularly significant.

Example 5

The differentiated fish adipocytes were filled into the muscle tissue cultured in Step 6 of Example 4. The difference in texture between the cell cultured fish meat obtained by this method and the natural yellow croaker was compared, and the results are shown in Figure 5. The hardness of cultured fish meat was 5.37±0.42N, which was in the same range as that of natural yellow croaker fillets (5.72±0.80N). There was also no noticeable difference in stickiness, elasticity, and elasticity between cultured meat and natural muscle tissue. It shows that the cell cultured fish meat constructed by this method has high molding efficiency, and the texture is closer to real fish meat, which is better than the current method of assembling and splicing to construct tissue-like cell cultured meat.

The above description is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art , without departing from the scope of the technical solution of the present invention, when the technical content disclosed above can be used to make some changes or be modified into equivalent embodiments with equivalent changes, but as long as it does not depart from the technical solution of the present invention, the technical content of the present invention In essence, any brief modifications, equivalent changes and modifications made to the above embodiments still fall within the scope of the technical solution of the present invention.

Claims (10)

1. A fish myoblast differentiation medium that inhibits both Notch and TGF- β signaling pathways.

2. The culture medium according to claim 1, wherein the growth factors of the culture medium are LY411575 and RepSox.

3. The culture medium according to claim 2, wherein the concentration of LY411575 is 1 to 20nM, and the concentration of RepSox is 1 to 10. Mu.M; the culture medium is an F12 culture medium.

4. A three-dimensional culture method of fish myoblasts is characterized in that biological ink consisting of the fish myoblasts and a scaffold precursor is subjected to 3D simulation printing, and after solidification, the culture medium according to any one of claims 1 to 4 is adopted for culture to obtain three-dimensional muscle tissues.

5. The three-dimensional culture method according to claim 4, wherein the scaffold precursor comprises gelatin, and the concentration of gelatin in the bio-ink is 5 to 10%.

6. The three-dimensional culture method according to claim 4, wherein the cell concentration in the bio-ink is 1 x 10 ⁶ ～1×10 ⁷ one/mL.

7. The three-dimensional culture method according to claim 4, wherein the bio-ink further comprises a curing agent for curing gelatin; the hardness of the solidified gelatin is 9-14 kPa.

8. The three-dimensional culture method according to claim 7, wherein the curing agent is sodium alginate or chitosan, and the curing of the curing agent is realized by a curing initiator which comprises calcium chloride, sodium citrate, magnesium chloride, zinc chloride and the like.

9. The three-dimensional culture method according to claim 4, wherein the 3D simulation printing is based on a fish muscle tissue model to obtain the three-dimensional muscle tissue with the fish muscle tissue structure.

10. A method for organizing an artificial fish meat, characterized in that the muscle tissue cultured by the method of claim 5 is filled with fish fat cells.

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