Use of polysaccharide coating film in the preparation of cell culture products

Abstract

The application discloses application of a polysaccharide wrapping film in preparation of a cell culture product and belongs to the technical field of cell culture food. The application specifically forms the polysaccharide wrapping film on the periphery of a cell inoculation support after the cell inoculation support, can relieve the phenomenon that cells fall off from the support during culture, and further guarantees effective proliferation and differentiation of muscle stem cells and / or fat stem cells on the support, thereby providing a new solution approach for efficient and standardized production of the cell culture product.

Description

Technical Field

[0001] This invention belongs to the field of cell culture product preparation technology, specifically relating to the application of polysaccharide coating membranes in the preparation of cell culture products. Background Technology

[0002] In the preparation of cell-cultured meat (such as muscle and / or fat), mimicking the in vivo environment to provide suitable growth conditions for cells is crucial. Currently, seeding cells onto scaffolds is a common method. Scaffolds provide physical support for cells, mimicking the three-dimensional structure of the extracellular matrix, and guiding cell adhesion, proliferation, and differentiation. They play a key role in forming cultured products with structures and functions similar to natural muscle or fat tissue. Common scaffold materials include natural polymers such as collagen and chitosan, as well as synthetic polymers such as polylactic-co-glycolic acid copolymer (PLGA).

[0003] However, previous studies have found that during the culture process, muscle stem cells have weak adhesion ability and will detach from the scaffold during proliferation. Furthermore, because myoblasts (i.e., muscle stem cells) have the characteristic of spontaneous contraction in the later stages of differentiation, ordinary scaffolds cannot flexibly adapt to muscle contraction movements, leading to the detachment of the differentiated myotubes after contraction. Although adipose-derived mesenchymal stem cells (i.e., adipose stem cells) have strong adhesion ability, the formation of full lipid droplets during differentiation can also cause the differentiation products to detach, resulting in poor culture results.

[0004] Cell shedding (including post-differentiation shedding) leads to uneven cell distribution, preventing the formation of a tight, orderly tissue structure and consequently affecting the texture, taste, and yield of the final product. For example, in culturing muscle tissue, uneven cell distribution may result in a product lacking the fibrous structure and resilience of normal muscle; in culturing adipose tissue, cell shedding may cause fat cells to clump together or be sparsely distributed, failing to form the delicate texture similar to natural fat. Furthermore, cell shedding can trigger cell death, as cells detached from the scaffold struggle to obtain sufficient nutrients and signaling molecules to maintain survival and normal metabolism. This not only wastes cell resources but also increases production costs, requiring additional cells for culture to compensate for the loss. Moreover, the uncontrollable nature of cell shedding poses a significant challenge to large-scale industrial production, making it difficult to maintain consistent product quality and limiting the commercialization of cell-cultured muscle and fat products.

[0005] In conclusion, solving the problem of cells easily detaching from the scaffold during culture is urgent and of great significance for promoting the development of cultured meat (such as muscle and / or fat). Summary of the Invention

[0006] 1. The problem to be solved This invention addresses the problem of cells easily detaching from scaffolds during culture by providing an application of a polysaccharide-coated membrane in the preparation of cell culture products. Specifically, after cells are seeded onto a scaffold, a polysaccharide-coated membrane is formed around it. This can alleviate the phenomenon of cells detaching from the scaffold during culture, thereby ensuring the effective proliferation and differentiation of muscle stem cells and / or adipose stem cells on the scaffold. This provides a new solution for the efficient and standardized production of cell culture products.

[0007] 2. Technical Solution To solve the above problems, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides the application of a polysaccharide coating membrane in the preparation of cell culture products. This application includes: immersing a stem cell-inoculated scaffold in a polysaccharide coating membrane solution, wherein the polysaccharide coating membrane solution cross-links on the surface of the stem cell-inoculated scaffold to form a polysaccharide coating membrane; the polysaccharide coating membrane solution comprises any one of: sodium alginate, sodium alginate and chitosan, sodium alginate and sodium carboxymethyl cellulose, or sodium alginate and gellan gum. As a further illustration of the present invention, the amount of cell leakage after coating with the polysaccharide coating membrane is less than the cell leakage rate without the polysaccharide coating membrane, indicating that the coating with the polysaccharide coating membrane can effectively reduce cell leakage (shedding).

[0008] Furthermore, the above-mentioned cell culture product is cell-cultured muscle, the stem cells are muscle stem cells, and the polysaccharide coating solution includes any one of sodium alginate, sodium alginate and chitosan, sodium alginate and sodium carboxymethyl cellulose, or sodium alginate and gellan gum.

[0009] Furthermore, the aforementioned cell culture product is cell-cultured muscle, and the polysaccharide coating solution includes sodium alginate and chitosan.

[0010] Furthermore, the above-mentioned cell culture product is cell culture fat, the stem cells are adipose stem cells, and the polysaccharide coating solution includes any one of sodium alginate, sodium alginate and chitosan, sodium alginate and sodium carboxymethyl cellulose, or sodium alginate and gellan gum.

[0011] Furthermore, the aforementioned cell culture product is cell culture lipid, and the polysaccharide coating solution includes sodium alginate and chitosan. As a further illustration of the invention, the activity of adipose-derived stem cells in the sodium alginate and chitosan membrane extracts remained above 85% for three days, with a cytotoxicity grade of 1, demonstrating that it is non-toxic to the growth and proliferation of adipose-derived stem cells and can maintain their good biological activity; while sodium alginate and sodium carboxymethyl cellulose membranes, sodium alginate and gellan gum membranes, and sodium alginate membranes have potential damaging effects on adipose-derived stem cells.

[0012] Secondly, the present invention also provides a method for preparing a cell culture product, the method comprising: immersing a scaffold inoculated with stem cells into a polysaccharide coating solution, wherein the polysaccharide coating solution crosslinks on the surface of the scaffold inoculated with stem cells to form a polysaccharide coating membrane; the polysaccharide coating solution comprises any one of sodium alginate, sodium alginate and chitosan, sodium alginate and sodium carboxymethyl cellulose, or sodium alginate and gellan gum.

[0013] Furthermore, the above preparation method also includes: the scaffold after forming a polysaccharide coating is further proliferated and induced to differentiate in a culture medium to form a cell culture product.

[0014] Furthermore, the above-mentioned cell culture product is cell cultured muscle, and the method includes: immersing a scaffold inoculated with muscle stem cells into a polysaccharide encapsulation solution, wherein the polysaccharide encapsulation solution cross-links on the surface of the scaffold inoculated with muscle stem cells to form a polysaccharide encapsulation membrane; the polysaccharide encapsulation solution includes any one of sodium alginate, sodium alginate and chitosan, sodium alginate and sodium carboxymethyl cellulose, or sodium alginate and gellan gum.

[0015] Furthermore, the above-mentioned cell culture product is cell culture fat, and the method includes: immersing a scaffold inoculated with adipose stem cells into a polysaccharide coating solution, wherein the polysaccharide coating solution cross-links on the surface of the scaffold inoculated with adipose stem cells to form a polysaccharide coating membrane; the polysaccharide coating solution includes any one of sodium alginate, sodium alginate and chitosan, sodium alginate and sodium carboxymethyl cellulose, or sodium alginate and gellan gum.

[0016] Thirdly, the present invention also provides cell culture products prepared by the above-mentioned method for preparing cell culture products.

[0017] 3. Technical Effects Compared with the prior art, the advantages of this invention are as follows: (1) The application of the polysaccharide coating membrane provided by the present invention in the preparation of cell culture products: the polysaccharide coating membrane effectively reduces the shedding (overflow) of stem cells on the scaffold, and the polysaccharide coating membrane enables cells to proliferate along the pores of the scaffold, and the cells are more evenly distributed on the scaffold, improving the proliferation efficiency of stem cells on the scaffold, which is beneficial to the differentiation of stem cells in the later stage, improving the texture of the scaffold, and forming a combined scaffold for cell proliferation.

[0018] (2) The application of the polysaccharide-encapsulated membrane provided by the present invention in the preparation of cell culture products has been studied. It was found that although they are all polysaccharides, the effects of different combinations of polysaccharides on cell proliferation are not the same: For muscle stem cells, muscle stem cells can grow normally in the membrane extracts of SA-CS membrane, SA-CMC membrane, and SA-GG membrane. However, as the culture time increases, the cell activity of muscle stem cells in the membrane extracts of SA-CMC membrane and SA-GG membrane gradually decreases, while the cell activity in the SA-CS membrane extract is higher than 85% within three days; For adipose stem cells, SA-CMC membrane and SA-GG membrane are harmful to adipose stem cells, and the cell activity is extremely low after three consecutive days of culture. SA membrane has a harmful effect on adipose stem cells, but the harmful effect is less than that of SA-GG membrane and SA-CMC membrane. The cell activity in the SA-CS membrane extract is higher than 85% within three days, and the cytotoxicity reaction is grade 1, which proves that it is not toxic to the growth and proliferation of adipose stem cells and can maintain their good biological activity. Attached Figure Description

[0019] Figure 1 It is the cell spillage rate of muscle stem cells and adipose stem cells after seeding in a scaffold, with and without various polysaccharide membranes.

[0020] Figure 2 These are scanning electron microscope images of the pre- and post-support structures of the polysaccharide-coated membrane.

[0021] Figure 3 These are the observation results of muscle stem cells after the scaffold is wrapped with a polysaccharide-coated membrane (SA-CMC membrane).

[0022] Figure 4 These are the observation results of muscle stem cells after the scaffold was wrapped with a polysaccharide-coated membrane (SA-CS membrane).

[0023] Figure 5 These are the observation results of muscle stem cells after the scaffold is wrapped with a polysaccharide-coated membrane (SA-GG membrane).

[0024] Figure 6 These are the observation results of muscle stem cells after the scaffold is wrapped with a polysaccharide-coated membrane (SA membrane).

[0025] Figure 7 This is the result of observation on muscle stem cells in a scaffold that is not encapsulated with a polysaccharide membrane.

[0026] Figure 8 These are the observation results of adipose stem cells after the scaffold was wrapped with a polysaccharide-coated membrane (SA-CMC membrane).

[0027] Figure 9 These are the observation results of adipose stem cells after the scaffold was wrapped with a polysaccharide-coated membrane (SA-CS membrane).

[0028] Figure 10 These are the observation results of adipose stem cells after the scaffold was wrapped with a polysaccharide-coated membrane (SA-GG membrane).

[0029] Figure 11 These are the observation results of adipose stem cells after the scaffold is wrapped with a polysaccharide-encapsulated membrane (SA membrane).

[0030] Figure 12 This is the result of observation on adipose stem cells in scaffolds that are not encapsulated by polysaccharide membranes.

[0031] Figure 13 These are the cell viability test results of muscle stem cells and adipose stem cells cultured in various membrane extracts. Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments.

[0033] It should be noted that, unless otherwise defined, 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 pertains; the term “and / or” as used herein includes any and all combinations of one or more of the associated listed items.

[0034] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0035] As used herein, the term “about” is used to provide for the flexibility and imprecision associated with a given term, measure, or value. Those skilled in the art can readily determine the degree of flexibility for a particular variable. As used herein, the term “at least one of…” is intended to be synonymous with “one or more of…”. For example, “at least one of A, B, and C” explicitly includes only A, only B, only C, and combinations thereof.

[0036] Concentration, amount, and other numerical data may be presented in range format herein. It should be understood that such range format is used solely for convenience and brevity and should be flexibly interpreted to include not only the values ​​explicitly stated as the limits of the range, but also all individual values ​​or subranges encompassed within the range, as if each value and subrange were explicitly stated. For example, a range of values ​​from about 1 to about 4.5 should be interpreted to include not only the explicitly stated limits of 1 to 4.5, but also individual numbers (such as 2, 3, 4) and subranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges that describe only a single value, such as "less than about 4.5," which should be interpreted to include all the values ​​and ranges described above. Furthermore, this interpretation should apply regardless of the breadth of the range or characteristic described.

[0037] In this invention, unless otherwise specified, the muscle stem cell proliferation culture medium used in the proliferation stage is formulated as follows: 84 vol% DMEM / F12 basal medium, 15 vol% fetal bovine serum, 1 vol% penicillin-streptomycin antibiotics, and 5 ng / mL recombinant human fibroblast growth factor (bFGF).

[0038] In this invention, unless otherwise specified, the adipose stem cell proliferation culture medium used in the proliferation stage is formulated as follows: 89 vol% DMEM / F12 basal medium, 10 vol% fetal bovine serum, 1 vol% penicillin-streptomycin antibiotics, and 5 ng / mL recombinant human fibroblast growth factor (bFGF).

[0039] In this invention, unless otherwise specified, the culture conditions are all in a CO2 incubator at 37°C, and the CO2 concentration is 5% (v / v).

[0040] In this invention, cell detachment refers to the separation of cells from a scaffold that is not encapsulated by a polysaccharide membrane; cell spillage refers to the separation of cells from a scaffold encapsulated by a polysaccharide membrane. Both refer to the separation of cells from the scaffold and can be understood in the same way. Accordingly, cell spillage rate and cell detachment rate can be understood similarly.

[0041] In this invention, the brand of sodium alginate is Sigma-Aldrich. ® The product number is 180947; the brand of chitosan is Sigma-Aldrich. ® The product number is 448869; the brand of sodium carboxymethyl cellulose is Sigma-Aldrich. ® The product number is C5013; the brand of gellan gum is Gelrite.® The product number is G1910.

[0042] In this invention, unless otherwise specified, the muscle seed cells used are primary porcine muscle stem cells, provided by Nanjing Zhouzi Future Food Technology Co., Ltd. These cells are adherent cells, catalog number: JF-Pg-01. As a further explanation of this invention, the present invention does not impose any further limitations on the muscle seed cells. Those skilled in the art will know that adherent muscle stem cells, through proliferation and induced differentiation, can form cultured muscle tissue.

[0043] In this invention, unless otherwise specified, the adipose-seeding cells used are primary porcine adipose-derived mesenchymal stem cells, provided by Nanjing Zhouzi Future Food Technology Co., Ltd. These cells are adherent cells, catalog number: JF-Pg-03. As a further explanation of this invention, the adipose-seeding cells are not further limited in this invention. Those skilled in the art will know that adherent adipose-derived stem cells can form cultured adipose tissue through proliferation and induced differentiation.

[0044] Example 1 This embodiment provides the application of polysaccharide coating membranes in the preparation of cell culture products.

[0045] Specifically, it includes: (1) Preparation of polysaccharide coating solution Sodium alginate-chitosan (SA-CS): 2.0 g of sodium alginate (SA) was added to 100 mL of deionized water and dissolved completely at 60 °C. The solution was then degassed by sonication (40 kHz, 100 W, 25 °C) to obtain the SA membrane solution. 1.0 g of chitosan (CS) and 2.0 g of calcium chloride were dissolved in 100 mL of 2% acetic acid solution and stirred at 60 °C until completely dissolved. The solution was then degassed by sonication to obtain the CS membrane solution. The CS membrane solution was added to the SA membrane solution and mixed to obtain the polysaccharide-encapsulated membrane solution.

[0046] Sodium alginate-sodium carboxymethyl cellulose (SA-CMC): 1.0 g each of SA and sodium carboxymethyl cellulose (CMC) were weighed and mixed in a 1:1 ratio. 100 mL of deionized water was added, and the mixture was stirred at 60°C until completely dissolved. After complete degassing in an ultrasonic cleaner, a polysaccharide membrane solution was prepared. 2.0 g of anhydrous calcium chloride was weighed and added to 100 mL of deionized water to prepare a 2% (w / v) calcium chloride solution. The calcium chloride solution was added to the polysaccharide membrane solution, and the mixture was mixed to obtain the polysaccharide-coated membrane solution.

[0047] Sodium alginate-gellan gum (SA-GG): Weigh 1.5g of SA and 0.5g of gellan gum (GG) in a 3:1 ratio. First, add the gellan gum to 100 mL of deionized water and stir at 90°C until completely dissolved. Then add the SA and stir at 60°C until completely dissolved. After complete degassing in an ultrasonic cleaner, prepare a polysaccharide membrane solution. Weigh 2.0g of anhydrous calcium chloride and add it to 100 mL of deionized water to prepare a 2% (w / v) calcium chloride solution. Add the calcium chloride solution to the polysaccharide membrane solution and mix to obtain a polysaccharide-encapsulated membrane solution.

[0048] Sodium alginate (SA): Weigh 2.0 g of SA and add it to 100 mL of deionized water. Stir at 60°C until completely dissolved. After complete degassing in an ultrasonic cleaner, prepare a polysaccharide membrane solution. Weigh 2.0 g of anhydrous calcium chloride and add it to 100 mL of deionized water to prepare a 2% (w / v) calcium chloride solution. Add the calcium chloride solution to the polysaccharide membrane solution and mix to obtain a polysaccharide-coated membrane solution.

[0049] All the polysaccharide-coated membrane solutions mentioned above were prepared fresh for immediate use.

[0050] (2) Encapsulation Cell culture scaffolds (all peanut protein scaffolds in this invention, product name: Weimei, series: 46723) were soaked in ultrapure water for 5-10 min and then longitudinally cut into 6 mm × 6 mm × 1 mm scaffolds along the pores. The scaffolds were placed in a culture dish, and 75% alcohol was poured in to cover them. The scaffolds were then sterilized by UV irradiation in a clean bench for 2 hours. After sterilization, the scaffolds were washed twice with PBS buffer to remove the alcohol from the inside. Muscle stem cell proliferation medium was added, and high-purity porcine muscle stem cells (JF-Pg-01) before passage P6 were added at a ratio of 1 × 10⁻⁶. 6 Seeds were placed on the surface of a peanut protein scaffold at a density of 1 × 10⁶ cells / scaffold, or adipose-derived stem cell proliferation medium was added and high-purity porcine adipose-derived stem cells (JF-Pg-03) prior to passage P6 were seeded at a density of 1 × 10⁶ cells / scaffold. 6 Cells were seeded onto the surface of a peanut protein scaffold at a density of 100 cells / scaffold. Two hours after cell seeding, the scaffolds seeded with stem cells were immersed in a freshly prepared polysaccharide coating solution for at least 2 minutes to allow for full cross-linking, forming polysaccharide coating membranes (SA-CS membrane, SA-CMC membrane, SA-GG membrane, and SA membrane) on the surface of the peanut protein scaffolds seeded with stem cells. The scaffolds were then placed in 24-well plates and cultured in proliferation medium. Peanut protein scaffolds without polysaccharide coating membranes served as a blank control.

[0051] After culturing in 24-well plates for 20 h, the cell overflow rate (exfoliation rate) was measured.

[0052] The cell spillover rate (exfoliation rate) is calculated using formula (1): , formula (1), in: The overflow cell density was determined by aspirating 1 mL of culture medium from the well after 20 h of culture, mixing it thoroughly with 20 μL of trypan blue and 20 μL of resuspension, taking 10 μL of the mixture and counting and recording the cell count on a cell counter, and calculating the number of cells per mL. Culture medium volume is the volume of the proliferation medium used in proliferation culture, expressed in mL; The number of cells seeded per scaffold refers to the initial number of cells seeded, expressed in units of individual cells.

[0053] The results are as follows Figure 1 As shown, this is a comparison of the cell spillage rate of muscle stem cells and adipose stem cells after seeding in the scaffold with each polysaccharide membrane and the cell detachment rate of those without polysaccharide membrane. It can be seen that the cell spillage rate after 20 h of polysaccharide membrane coating is less than that of the blank control (without polysaccharide membrane), indicating that the polysaccharide membrane coating can effectively reduce cell spillage (detachment).

[0054] The initial structure of the scaffold (before encapsulation) was observed using scanning electron microscopy. Figure 2 (Middle left image) The results show that the scaffold surface is porous and has many grooves, which is not conducive to cell adhesion and proliferation. After coating with a polysaccharide-coated membrane (SA-CS membrane), the surface is smoother and more suitable for uniform cell proliferation and growth. Cells can firmly adhere to and extend on the inner wall and surface of the scaffold, which is beneficial for the overall expansion and culture of cells on the scaffold. Figure 2 (Right image in the middle)

[0055] Example 2 This embodiment provides the detection of cell growth on a scaffold after encapsulating polysaccharide membranes.

[0056] Referring to Example 1, the difference is that cell staining is performed before cell seeding.

[0057] The specific steps for live cell tracing detection are as follows: (a) Cell staining: Before collection and digestion, the cells were washed with PBS and the working solution was prepared according to the ratio of live cell tracer stain (CellTracker™, catalog number: C2925): serum-free medium (myogenic cell serum-free proliferation medium, Nanjing Zhouzi Future Food Technology Co., Ltd., catalog number: JF-MYO-A24) = 1:250. 2 mL of working solution was added to each 10 cm culture dish and the cells were cultured at 37°C for 20-40 min. The staining was observed under a microscope to check if it was complete. If the brightness was sufficient, the staining solution was washed off with PBS and the cells were digested and collected.

[0058] (2) Cell seeding: at 1×106 The density of cells / scaffold involves seeding stained cells onto the surface of a sterilized 6 mm × 6 mm × 1 mm peanut protein scaffold.

[0059] (3) Coating: After the cells are seeded on the scaffold and dried for 2 h, the scaffold with cells is immersed in the polysaccharide coating solution with tweezers for more than 2 min to ensure full cross-linking, and then placed in a 24-well plate for culture.

[0060] (4) Cell observation: During the culture period, the growth of live cells inside the scaffold, at the edge and in the culture medium was observed daily using a fluorescence microscope with an eyepiece of 10x, an objective lens of 4x and a total magnification of 40x.

[0061] The results are as follows Figures 3-12 As shown, Figures 3-7 These are the observation results of muscle stem cells on a cell scaffold. Figure 8-12 The results of observations on adipose-derived stem cells (ADSCs) on a cell scaffold show that both muscle stem cells and ADSCs can grow well in both the polysaccharide-coated scaffold and in a blank scaffold. Furthermore, a significant number of viable cells remain on the scaffold even with increasing culture time, demonstrating that cells can proliferate within both the scaffold and the polysaccharide-coated scaffold. Meanwhile, muscle stem cells on the blank scaffold tend to grow in clusters, especially in the later stages of proliferation, resulting in highly uneven cell growth across the entire scaffold. In contrast, both types of stem cells are more evenly distributed within the polysaccharide-coated scaffold and proliferate along the scaffold's pores.

[0062] Example 3 This embodiment provides a method for detecting the toxicity of polysaccharide-coated membranes to cells.

[0063] Specifically, the steps include the following: (1) Preparation of polysaccharide coating material extract Referring to Example 1, the preparation of a polysaccharide-encapsulated membrane solution and the fabrication of a polysaccharide-encapsulated membrane specifically includes: Prepare a polysaccharide-encapsulated membrane solution, pour it into a mold, and form a uniform membrane; pre-freeze at -80℃ or in a gradient until completely solid; freeze-dry under vacuum (low temperature and high vacuum); remove the membrane, seal and dry for storage.

[0064] After weighing the lyophilized polysaccharide-coated membrane material, cut it into small pieces, soaked it in alcohol, and irradiated it overnight under UV light for thorough sterilization. After rinsing twice with PBS buffer, add serum-free culture medium (myogenic cell serum-free proliferation medium, Nanjing Zhouzi Future Food Technology Co., Ltd., catalog number: JF-MYO-A24) and irradiate overnight under UV light. Add serum-free culture medium to a final polysaccharide coating concentration of 50 mg / mL and extract in a 37°C water bath for 24 h. Filter through a 0.22 μm sterile filter membrane to obtain the polysaccharide-coated membrane material extract. Store the extract at 4°C. Before use, heat in a water bath and add appropriate concentrations of fetal bovine serum and bFGF according to the cell type.

[0065] (2) Cell proliferation and cytotoxicity detection Seed cells (cell populations collected and digested after 2D expansion culture) were collected, and the cell suspension concentration was adjusted. Cells were then aliquoted into 96-well plates (100 μL per well, 4000 cells / well). Experimental groups (extraction solutions of various polysaccharide-coated membrane materials), control groups (extraction solutions of pure SA membrane materials), and a blank control group were set up, with 5 replicates per group. Cells were incubated at 37℃ in a 5% CO2 incubator. After 24 hours, the culture medium in the experimental groups was replaced with the extraction solution. Cells were analyzed on days 1, 2, and 3 after the extraction solution was replaced. Before analysis, the growth status of cells in each well was observed under 10x eyepiece, 10x objective lens, and a total magnification of 100x.

[0066] The detection procedure was as follows: First, the supernatant was aspirated, and 90 μL of serum-containing DMEM / F12 basal medium (serum brand: Sigma-Aldrich, catalog number: F0193; DMEM / F12 brand: Sigma-Aldrich, catalog number: D0697) was added, followed by 10 μL of MTT solution. The mixture was then incubated for 4 h. The supernatant was aspirated, and 110 μL of Formazan dissolving solution was added to each well. The wells were then shaken at low speed (50 rpm) for 10 min to fully dissolve the crystals. The absorbance of each well was measured at 490 nm using an ELISA reader.

[0067] Cell viability is calculated using formula (2): , formula (2).

[0068] The results are as follows Figure 13 As shown, For muscle stem cells, they can grow normally in various membrane extracts. However, with increasing culture time, the cell viability of muscle stem cells in SA-CMC and SA-GG membrane extracts gradually decreases. In contrast, the cell viability of muscle stem cells in SA-CS membrane extracts remains above 85% for three days.

[0069] For adipose-derived stem cells, the cell viability in the SA-CS membrane extract remained above 85% for three days, with a cytotoxicity grade of 1, demonstrating that it is non-toxic to the growth and proliferation of adipose-derived stem cells and can maintain their good biological activity. In contrast, the SA-CMC and SA-GG membranes damaged adipose-derived stem cells, resulting in extremely low cell viability after three consecutive days of culture. While the SA membrane has a damaging effect on adipose-derived stem cells, the effect is less than that of the SA-GG and SA-CMC membranes.

[0070] The examples disclosed above are for illustrative purposes only and should not be construed as limitations of the invention. The membrane encapsulation method mentioned herein, aimed at reducing cell leakage and thus promoting the proliferation and differentiation of muscle stem cells / adipose stem cells, provides a new pathway for cultured meat. Without departing from the scope and spirit of this invention, many different raw materials and cells can be combined to prepare different encapsulation membranes, scaffolds, and different cultured meats. Therefore, this invention is not limited to the disclosed embodiments. In fact, all modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. The application of polysaccharide-coated membrane solution in the preparation of cell culture products, characterized in that, The application includes: immersing a stem cell-inoculated scaffold in a polysaccharide coating solution, wherein the polysaccharide coating solution cross-links on the surface of the stem cell-inoculated scaffold to form a polysaccharide coating membrane; the polysaccharide coating solution includes: sodium alginate, chitosan, and calcium chloride; The cell culture product is cell-cultured muscle, and the stem cells are muscle stem cells; or the cell culture product is cell-cultured fat, and the stem cells are adipose stem cells.

2. A method for preparing a cell culture product, characterized in that, The method includes: immersing a stem cell-inoculated scaffold in a polysaccharide coating solution, wherein the polysaccharide coating solution crosslinks on the surface of the stem cell-inoculated scaffold to form a polysaccharide coating membrane; the polysaccharide coating solution includes: sodium alginate, chitosan, and calcium chloride; The cell culture product is cell-cultured muscle, and the stem cells are muscle stem cells; or the cell culture product is cell-cultured fat, and the stem cells are adipose stem cells.

3. The preparation method according to claim 2, characterized in that, The method further includes: the scaffold after forming a polysaccharide coating is further proliferated and induced to differentiate in a culture medium to form a cell culture product.

4. Cell culture products prepared by the preparation method according to claim 2 or 3.

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