CN117247897A

CN117247897A - Cell culture product and preparation method thereof

Info

Publication number: CN117247897A
Application number: CN202210649911.1A
Authority: CN
Inventors: 周光宏; 丁世杰; 孟子晴; 陈虹竹; 洪婉琪; 朱浩哲; 陶天艺; 史义凡
Original assignee: Nanjing Zhouzi Future Food Technology Co ltd
Current assignee: Nanjing Zhouzi Future Food Technology Co ltd
Priority date: 2022-06-10
Filing date: 2022-06-10
Publication date: 2023-12-19

Abstract

The invention discloses a cell culture product and a preparation method thereof, and belongs to the technical field of foods. The product comprises integrated cell culture muscle tissue, cell culture adipose tissue and a hydrogel biological scaffold, and the specific preparation method comprises the steps of respectively inoculating muscle stem cells and interstitial stem cells on the color-adjustable biological scaffold, wherein the surface of a colored part of the muscle stem cells is inoculated, the color of the prepared cell culture muscle tissue is more similar to that of natural pig muscle, and the cell culture muscle tissue and the cell culture adipose tissue are included, so that the structure composition of natural meat can be simulated, and the cell culture meat has good texture and color, and in addition, the cell culture product can be well adhered, proliferated and differentiated in the edible biological scaffold.

Description

Cell culture product and preparation method thereof

Technical Field

The invention belongs to the technical field of foods, and particularly relates to a cell culture product and a preparation method thereof.

Background

The cell culture meat technology is a technology with subversion meat production, not only can reduce the waste of various natural resources such as environment, land and the like caused by the development of the traditional animal husbandry, but also can effectively relieve the pressure of effectively supplying meat products and proteins. The production of cell culture meat mainly utilizes an in-vitro culture technology of animal cells, which comprises the steps of collecting muscle cell samples from animals, separating stem cells in the muscle samples, culturing on a biological scaffold, inducing differentiation to generate muscle fibers, and finally preparing the cell culture meat. Likewise, mesenchymal stem cells may also be collected to prepare cell culture adipose tissue.

The Chinese diet has good color, smell and taste, the natural pork products are products with interphase muscle tissues (namely 'lean meat') and fat tissues (namely 'fat meat'), muscle cells are filled with protein and nutrients, fat cells are filled with fat, and during heating (such as boiling, frying and the like), fat molecules are heated or combined with oxygen and are converted into molecules with fruit, flowers, nuts, green grass taste or the like, and the two types of cells play an important role in the sensory experience when meat animals chew meat. However, current cell culture meat products include only muscle tissue, and the formation of complete fat-interphase meat for eating still requires later processing and assembly due to the lack of fat cells and their lack of aroma after cooking; moreover, the bioscaffold used to culture muscle stem cells is generally colorless, thereby resulting in the lack of natural "lean" color of the existing cell culture meat products, which severely affects the development of cell culture meat.

Therefore, the development of a product which is close to the color of natural pork and is alternately fat and thin is more beneficial to meeting the requirements of meat animals and promoting the development of future food technology.

Disclosure of Invention

1. Object of the invention

Aiming at the problems that the current cell culture meat biological scaffold only provides proliferation and differentiation culture of single cells and the cell culture meat prepared by the method cannot form a complete fat-lean interphase product, the invention provides the biological scaffold with an edible polyphenol proteoglycan bionic structure, the scaffold comprises edible polyphenol, edible protein and edible polysaccharide, the brightness and the redness value of the scaffold can be adjusted, and muscle stem cells and mesenchymal stem cells are respectively inoculated on the surfaces with different brightness and redness values in the biological scaffold, so that the prepared cell culture product comprises cell culture muscle tissue and cell culture adipose tissue, and the color and the composition of the cell culture product are closer to those of natural pork so as to meet the requirements of social consumption.

2. Technical proposal

In order to solve the problems, the technical scheme adopted by the invention is as follows:

the invention provides a cell culture product, which comprises integrated cell culture muscle tissue, cell culture adipose tissue and a hydrogel biological scaffold, wherein the hydrogel biological scaffold comprises a colored part and a white part, the colored part is reddish brown to mauve, the brightness value is 5-70, the redness value is 0-40, the cell culture muscle tissue is combined with the colored part on the biological scaffold, the cell culture adipose tissue is combined with the white part on the biological scaffold, and the combination is formed by proliferation and differentiation of muscle stem cells and mesenchymal stem cells respectively in the colored part and the white part

Preferably, the hydrogel biological scaffold is a hydrogel biological scaffold comprising polyphenol, plant protein and polysaccharide, wherein the polyphenol is crosslinked with the plant protein, so that the brightness and the redness value of the biological scaffold can be adjusted; in addition, polyphenols crosslink with proteins, the porosities of the red part and the white part of the gel are significantly different, and a microenvironment basis is provided for co-culture of different types of cells.

Preferably, the polyphenols, vegetable proteins and polysaccharides are food grade, and the bioscaffold thus prepared can be consumed directly without isolation from the cell culture product.

Preferably, the content of the polyphenol in the hydrogel biological scaffold is 0.05-1% (w/v).

Preferably, the polyphenols are selected from tannins, flavonoids or other types of polyphenols.

Preferably, the polyphenol is selected from one or more of procyanidins, gallic acid, tea polyphenols, mung bean polyphenols, grape polyphenols, grapefruit polyphenols, catechins, etc.

Preferably, the polyphenols are selected from procyanidins, and the color of the biological scaffold ranges from reddish brown to mauve, and the biological scaffold has bionic color and structure.

Preferably, the content of the procyanidine in the hydrogel biological scaffold is 0.05-0.5% (w/v).

Preferably, the content of the polysaccharide in the hydrogel biological scaffold is 1-10% (w/v).

Preferably, the polysaccharide is selected from one or more of alginate, carrageenan, konjac gum and other plant gel.

Preferably, the polysaccharide is selected from sodium alginate.

Preferably, the content of the sodium alginate in the hydrogel biological scaffold is 1-3% (w/v).

Preferably, the content of the plant protein in the hydrogel biological scaffold is 5-50% (w/v).

Preferably, the vegetable protein is selected from any one or a combination of several of soybean protein, soybean protein isolate, peanut protein, wheat gluten, corn protein, pea protein and the like.

The invention also provides a preparation method of the hydrogel biological scaffold of the polyphenol, the plant protein and the polysaccharide, which comprises the following steps:

s1: mixing polyphenol, vegetable protein, polysaccharide and water thoroughly, and adjusting pH to 6.5-7;

s2: transferring the mixed protein solution into a bracket mold, heating at a high temperature of not lower than 95deg.C for more than 30min to obtain biological bracket solution, layering the components in the heated solution due to different densities, and mixing with CaCl ₂ After cross-linking, white and red colors similar to those of natural pork (such as streaky pork) can be formed, and after the mesenchymal stem cells and the muscle stem cells are respectively inoculated, cell culture adipose tissue and cell culture muscle tissue are formed, and the fat and thin parts corresponding to the natural pork (such as streaky pork) are formed;

s3: caCl is added into the unfixed biological stent solution ₂ And (3) standing the solution overnight in a refrigerator at the temperature of 4 ℃ to obtain the biological scaffold with the bionic structure.

Preferably, the mass and the dosage ratio of the vegetable protein to the polysaccharide are (1-50): 1.

preferably, the mass and the dosage ratio of the vegetable protein to the polysaccharide are 16:1,8:1, or 4:1.

preferably, the vegetable protein is soy protein isolate.

Preferably, sodium alginate is used as the polysaccharide.

Preferably, the polyphenol is selected from procyanidins.

The invention also provides an application of the hydrogel biological scaffold in preparing cell culture muscle tissue or cell culture adipose tissue.

The invention also provides a preparation method of the cell culture product, which comprises the following steps:

s1: sterilizing the hydrogel biological scaffold (75% alcohol+ultraviolet);

s2: respectively inoculating the solution containing muscle stem cells and adipose-derived mesenchymal stem cells on the colored (red, similar to lean meat) part and the white (similar to fat) part of the surface of the hydrogel biological scaffold, wherein the inoculation density is 10-50×10 ⁶ cells/cm ² Culturing in a carbon dioxide incubator at 37 ℃ for 2 hours to form hydrogel tissues, and then adding a culture medium for co-culturing to perform proliferation and differentiation culture to obtain cell culture meat and cell culture adipose tissue interphase cell culture products.

Preferably, the proliferation and differentiation culture comprises adding proliferation medium to culture and proliferate for 1-2 days, and replacing differentiation medium to culture for 4-20 days.

Preferably, the CO in the incubator of the carbon dioxide ₂ The concentration of (C) was 5% (v/v).

Preferably, the proliferation medium is formulated as follows: 5% -20% (v/v) fetal bovine serum and 1% (v/v) diabody were added to DMEM F12 complete medium, and 5ng/mL growth factor (human fibroblast growth factor, bFGF) was added thereto.

Preferably, the proliferation medium is formulated as follows: 10% -15% (v/v) fetal bovine serum and 1% (v/v) diabody were added to DMEM F12 complete medium, and 5ng/mL growth factor (human fibroblast growth factor, bFGF) was added thereto.

Preferably, the proliferation medium is formulated as follows: 12.5% (v/v) fetal bovine serum and 1% (v/v) diabody were added to DMEM F12 complete medium, and 5ng/mL growth factor (human fibroblast growth factor, bFGF) was added thereto.

Preferably, the differentiation medium comprises a muscle stem cell differentiation medium and a mesenchymal stem cell differentiation medium, wherein the volume of the muscle stem cell differentiation medium accounts for 30% -70%. Further, the volume ratio of the muscle stem cell differentiation medium is 30%, 40%, 50%, 60% or 70%, i.e., the ratio of the muscle stem cell differentiation medium to the mesenchymal stem cell differentiation medium is 1:1,2:3,3:7,7:3 or 3:2.

Preferably, the above-mentioned muscle stem cell differentiation medium is formulated as follows: 2% horse serum, 1% diabody, DMEM or F10 or F12 basal medium;

preferably, the mesenchymal stem cell differentiation medium is formulated as follows: 10% of fetal bovine serum, 1% of diabody, a basic culture medium such as DMEM or F10 or F12 and the like, and a certain amount of dexamethasone, insulin, 3-isobutyl-1-methylxanthine, indomethacin and rosiglitazone are added.

Preferably, in the above differentiation medium, the final concentration ratios are respectively: 0-5 uM of dexamethasone, 0-1mM of 3-isobutyl-1-methylxanthine, 0-100ug/mL of insulin, 0-1mM of indomethacin and 0-10uM of rosiglitazone.

3. Advantageous effects

Compared with the prior art, the invention has the beneficial effects that:

(1) The biological stent provided by the invention comprises a hydrogel system of polyphenol, plant protein and polysaccharide, wherein the polyphenol is crosslinked with the plant protein, so that the brightness and the redness value of the biological stent can be adjusted; in addition, the polyphenols and the proteins are crosslinked, the porosities of the red part and the white part of the gel are obviously different, a microenvironment basis is provided for the co-culture of different types of cells, and the co-culture of the muscle stem cells and the mesenchymal stem cells can be carried out. Still further, the present invention provides a biological stent, wherein the raw materials involved are proteins of non-animal origin, and edible polysaccharides, polyphenols. The selected material and the biological scaffold prepared by the material not only have good cell compatibility and biological safety and support cell adhesion and differentiation, but also belong to food-grade materials, and the biological scaffold is used for preparing cell culture meat and can be directly eaten.

(2) The biological scaffold provided by the invention comprises a hydrogel system of polyphenol, plant protein and polysaccharide, wherein the polyphenol takes procyanidine as an example, the brightness and the redness value of the biological scaffold can be adjusted, and the cell culture products formed after the muscle stem cells and the interstitial stem cells are respectively inoculated in different color parts for culture, wherein the color of the cell culture muscle tissue is more similar to that of natural pig muscle, and the cell culture muscle tissue and the cell culture adipose tissue are included simultaneously, so that the structure composition of natural meat can be simulated, and good taste is brought to the cell culture meat.

(3) The cell culture product provided by the invention simultaneously comprises muscle tissues and fat tissues, fat cells are filled with fat, and after being heated or combined with oxygen in the heating process, fat molecules can emit meat flavor and fat flavor, so that the sensory experience of natural pork on meat animals when chewing meat can be simulated.

Drawings

FIG. 1 is a graph of the macroscopic morphology of the hydrogel bioscaffold of example 1.

FIG. 2 is a graph of the living cell tracing results of a hydrogel bioscaffold.

FIG. 3 shows the results of the differentiation test of cells on a hydrogel bioscaffold.

FIG. 4 is a schematic diagram of a cell culture food according to the present invention.

FIG. 5 is a graph showing the cooking effect of the cell culture food of the present invention.

Detailed Description

The invention is further described below in connection with specific embodiments.

The terms such as "upper", "lower", "left", "right", "middle" and the like are also used in the present specification for convenience of description, and are not intended to limit the scope of the present invention, but rather to change or adjust the relative relationship thereof, and are also considered to be within the scope of the present invention without substantial change of technical content.

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; the term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

As used herein, the term "about" is used to provide the flexibility and inaccuracy associated with a given term, metric or value. The degree of flexibility of a particular variable can be readily determined by one skilled in the art.

As used herein, the term "is intended to be synonymous with" one or more of ". For example, "at least one of A, B and C" expressly includes a only, B only, C only, and respective combinations thereof.

Concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a numerical range of about 1 to about 4.5 should be interpreted to include not only the explicitly recited limits of 1 to about 4.5, but also include individual numbers (such as 2, 3, 4) and subranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges reciting only one numerical value, such as "less than about 4.5," which should be construed to include all such values and ranges. Moreover, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.

Any steps recited in any method or process claims may be performed in any order and are not limited to the order set forth in the claims.

The cells used in the following examples were piglet muscle stem cells, mesenchymal stem cells, further adherent cells, and further, after sorting by a cell flow sorter, muscle stem cells with Pax7 surface antibody positive rate of 90% or more, and mesenchymal stem cells with CD x 8 of more than 90%.

The isolated soy proteins in the following examples were purchased from Xin Er Rui Biotechnology, procyanidins were purchased from Shanghai Seiyaku Biotechnology Co., ltd, and sodium alginate was purchased from Shanghai Ala Latin Biotechnology Co., ltd.

The culture conditions used in the examples below were all CO ₂ Culturing at 37deg.C in incubator, CO ₂ The concentration of (C) was 5% (v/v).

The detection methods employed in the examples below are experimental methods, detection methods and preparation methods disclosed in the art unless otherwise specified. See in particular Shijie Ding et al document "Maintaining bovine satellite cells stemness through p path" (DOI: 10.1038/s 41598-018-28746-7)

Example 1

The embodiment provides a hydrogel biological scaffold with an edible polyphenol proteoglycan bionic structure, which comprises polyphenol, protein and polysaccharide, wherein the polyphenol is selected from procyanidine, and the content of the polyphenol in the hydrogel biological scaffold is 0.05-1% (w/v); the protein is selected from soybean protein isolate, and the content of the protein in the hydrogel biological scaffold is 5-50% (w/v); the polysaccharide is selected from sodium alginate, and the content of the polysaccharide in the hydrogel biological scaffold is 1-10% (w/v).

Taking soybean protein isolate with 16% (w/v), procyanidine with 0.4 (w/v) and sodium alginate with 1% (w/v) as an example, the preparation method of the hydrogel biological scaffold comprises the following steps:

s1: preparing 16% (w/v) soybean protein isolate solution with distilled water, stirring to dissolve, standing at 4deg.C, and refrigerating for storage; adding 0.4% (w/v) of procyanidine powder into the soybean protein isolate solution in S1, and stirring at 800rpm until procyanidine is completely dissolved; stirring the mixed solution on a magnetic stirrer at 800rpm for 10-20 min at room temperature, weighing 1% (w/v) sodium alginate, adding the sodium alginate into the stirring solution, adjusting the stirring speed to 1000rpm, and adjusting the pH of the mixed solution to 6.5-7 after uniform stirring; s2: heating in a water bath kettle at 95 ℃ for more than 30min, taking out and standing for 30min after obvious delamination of the solution is observed;

s2: preparing 2% (w/v) CaCl ₂ The solution is added into the mixed solution after standing, and the mixed solution is placed at the temperature of 4 ℃ for standing reaction for 6 to 24 hours, so that complete gelation is ensured.

Analysis of results:

cutting the prepared polyphenol proteoglycan gel into cuboid with length of 6cm, width of 3cm and height of 0.5cm, photographing macroscopic appearance under pure black background,as shown in FIG. 1, the gel is obviously divided into red and white layers, wherein the colored part is reddish brown to mauve, the brightness value is 5-70, the redness value is 0-40, after the procyanidine, the protein and the sodium alginate are crosslinked, each component in the solution is divided into two layers due to different densities after heating, namely, in the sodium alginate solution, the procyanidine and the soybean protein isolate are crosslinked and float on the upper layer, and all the solution is mixed with CaCl ₂ After cross-linking, white (mainly sodium alginate) and red (mainly procyanidine and soybean protein isolate) similar to natural pork (such as streaky pork) can be formed, and after the mesenchymal stem cells and the muscle stem cells are respectively inoculated, cell culture adipose tissue and cell culture muscle tissue can be formed, so that the fat and thin parts of the natural pork (such as streaky pork) can be correspondingly formed.

Accordingly, the present embodiment provides a hydrogel bioscaffold of an edible polyphenol proteoglycan biomimetic structure, the bioscaffold comprising polyphenol, protein and polysaccharide, wherein the polyphenol is selected from one or more of procyanidins, gallic acid, tea polyphenols, mung bean polyphenols, grape polyphenols, grapefruit polyphenols, catechins, etc.; the polysaccharide is selected from one or more of alginate, carrageenan, konjac gum and other plant gel; the plant protein is selected from any one or a combination of a plurality of proteins such as soybean protein, soybean isolated protein, peanut protein, wheat gluten, corn protein, pea protein and the like, and can achieve the same effect.

Example 2

The present example provides a cell culture product and a preparation method thereof, wherein the red part and the white part of the biological scaffold in the example 1 are respectively inoculated with muscle stem cells and mesenchymal stem cells, and a culture medium is added for proliferation and differentiation co-culture, so as to obtain a cell culture product with interphase cell culture meat and cell culture adipose tissue. The method specifically comprises the following steps:

s1: sterilizing the hydrogel biological stent, soaking for 1-3 times by adopting 75% alcohol, and then irradiating for 0.5-12 hours by ultraviolet; or selecting ethylene oxide sterilization, irradiation sterilization or other sterilization modes;

s2: will contain muscle stem cells and adipose-derived mesenchymal stemThe cell solution is respectively connected to the red part and the white part of the biological stent, and can be inoculated by adopting a liquid-transfering gun for inoculation, a liquid-discharging gun for inoculation or special equipment for inoculation, and the inoculation density is 10-50 multiplied by 10 ⁶ cells/cm ² Culturing in a carbon dioxide incubator at 37 ℃ for 2 hours to form hydrogel tissues, then adding a proliferation culture medium to culture and proliferate for 1-2 days, and replacing a differentiation culture medium to culture for 4-20 days, wherein:

proliferation medium was formulated as follows:

1) 10% (v/v) fetal bovine serum and 1% (v/v) diabody were added to DMEM F12 complete medium, and 5ng/mL growth factor (human fibroblast growth factor, bFGF) was added thereto

2) 15% (v/v) fetal bovine serum and 1% (v/v) diabody were added to DMEM F12 complete medium, and 5ng/mL growth factor (human fibroblast growth factor, bFGF) was added thereto

3) 1) and 2) were mixed 1:1 for co-cultivation.

The differentiation medium was formulated as follows:

the muscle stem cell differentiation medium comprises a muscle stem cell differentiation medium and a mesenchymal stem cell differentiation medium, wherein the volume ratio of the muscle stem cell differentiation medium is 30% -70%, and the muscle stem cell differentiation medium comprises 2% horse serum, 1% diabody or DMEM or F10 or F12; the mesenchymal stem cell differentiation culture medium is 10% fetal bovine serum, 1% diabody and DMEM or F10 or F12, and the final concentration after mixing is 0-5 uM dexamethasone, 0-1mM 3-isobutyl-1-methylxanthine, 0-100ug/mL insulin, 0-1mM indomethacin and 0-10uM rosiglitazone.

The gene and protein levels after the adhesion and induced differentiation of the hydrogel biological scaffold support cells were detected, and the results were as follows:

(1) Live cell tracer detection

The estimation is carried out according to the growth state of the previous generation, and the staining can be carried out on the day of passage if the total number of cells is enough. Cells were washed once with sterile PBS buffer, live cell tracer: serum-free medium was used at 1:250, preparing a dyeing working solution; adding 2mL of working solution into each dish of 10cm culture dish; dyeing in an incubator at 37 ℃ for 30 min. After that, the mixture was washed out with PBS and observed under a microscope, and the dyeing was completed with sufficient brightness. Cells were digested, counted, and scaffolds were prepared. Throughout this process, it should be noted that the living cell tracer needs to be protected from light. Taking a photo: turning off the bright field lamp under a fluorescence microscope, striking blue light on the bracket, focusing, taking cell photographs at 4 times and 10 times respectively, and analyzing the proliferation condition of cells. The results are shown in FIG. 2, where both cells can adhere efficiently.

(2) Gene level detection

The qPCR samples were collected before and after differentiation, and the gene expression levels of Myog, MHC, cav-3, plin1, fabp4, PPARgamma and C/EBPbeta were detected by real-time fluorescent quantitative PCR, and the results are shown in FIG. 3, which shows that the gene expression levels of the relevant genes related to muscle stem cell differentiation and fat differentiation are up-regulated on the scaffold according to the present invention, including MHC, cav-3, plin1 and Fabp4 4 terminal differentiation stages, compared with the pre-differentiation.

(3) Protein level detection

The cultured bioscaffold was added with 200. Mu.L of RIPA (containing final concentration 1mM PMSF), and the mixture was lysed on ice for 30min and collected at-20℃for further use. The tissue disrupter then disrupts 3 times, each for 90s. Then 12000g was centrifuged for 5 minutes, the supernatant was collected, the BCA kit for the Siemens flight was used for measurement and protein concentration, 5X loading buffer was added at a ratio of 4:1 (V: V), and the mixture was stirred uniformly and heated at 95℃for 5 minutes to denature the protein, and the mixture was stored at-80 ℃.

SDS-page gel electrophoresis, namely preparing electrophoresis buffer solution and transfer solution in advance (10% methanol is needed to be added in the transfer solution), taking 20ug denatured protein to be respectively added into the sample holes under the condition that the electrophoresis buffer solution is over 12% denatured agarose precast gel plates, setting two procedures of voltage 80V electrophoresis for 30min and 120 electrophoresis for 90min and more, and observing whether the protein sample solution reaches the bottom of the precast plates.

Transferring: placing PVDF film into methanol for activation for about 10s, placing into transfer printing liquid for standby, placing according to sponge, 2 layers of filter paper, gel, activated PVDF film, 2 layers of filter paper and sponge, clamping by a transfer printing clamp, placing into an electrophoresis tank, adding prepared transfer printing liquid for 90V, and running for 90min.

Closing: the PVDF film after transfer was put into a blocking solution (5% skimmed milk powder prepared with TBST), and after blocking for 2 hours in a shaker at room temperature, the blocking solution was sucked off.

Primary and secondary antibody incubation: MHC, plin1, fabp4 primary antibody was diluted according to the specific condition of the antibody and incubated at 4℃for 14-16h. The primary antibody is recovered after the incubation of the primary antibody is finished, and TBST is washed three times for 5min each time. The diluted secondary antibody was added and incubated for 2h, and after the completion, TBST was washed three times for 5min each time.

Developing: the PVDF film is covered in a dark place by using the developing solution, the developing solution is sucked after incubation for 5min, and the film is photographed under a gel imager. Gray scale analysis was performed using quality One analysis software, and the reference protein used in this experiment was Gapdh.

The results show that: the hydrogel scaffold can support cell adhesion growth, can effectively differentiate, and has obviously up-regulated MHC, plin1 and Fabp4 expression.

(4) The cell culture product after cell co-culture, which includes red cell culture muscle tissue and white cell culture adipose tissue, is visually similar to natural pork, as shown in fig. 4.

Accordingly, if only one of the cells, such as muscle stem cells or mesenchymal stem cells, is inoculated, the cells may be cultured alone to prepare a cultured meat product or a cell cultured adipose tissue product.

Example 3

Preparation of scaffolds and production of cell culture products and co-cultivation of cells were performed as in example 1 and example 2, and then the differentiated products were cooked and color analysis was performed before and after cooking.

S1, cutting the product into strips with the width of 1cm and the length of 3-5 cm, scalding for 3-10 min, frying the oil and the side dish in a pot, wherein the power used in the electromagnetic equipment processing is 300-5000W, and frying for 3-15min, and the result is shown in figure 4.

S2, measuring the color of the product, measuring Lab values of 5 brackets by using a color difference meter, wherein the Lab values represent chromaticity values of object colors, L is bright and dark, a is red and green, and b is yellow and blue, and analyzing and comparing the color change condition of the cell culture streaky pork before and after cooking by the Lab values. Firstly, zeroing the color difference meter before measurement; the lens was wiped before measurement. And then the lens of the color difference meter is vertically arranged on the gel bracket for measurement, the small opening of the lens needs to be tightly fastened on the surface of the sample, light cannot leak, the stability is kept for about 3-5 s, the lens can be removed after the instrument displays the reading, and data are recorded. The lens was wiped clean after each measurement. Each set of samples was measured at least 3 times.

The results are shown in table 2, with decreased L and increased a of the muscle (lean) fraction of the cell culture product before and after cooking; fat (fat meat) fraction, L reduced, a increased, b increased in cell culture streaky pork. Namely, after cooking, the lean meat in the bracket becomes dark in brightness, the color is biased towards red and yellow, and the color change rule of the lean meat before and after pork cooking is met.

TABLE 2 color analysis before and after cooking

The above description of the present invention is further illustrated in detail and should not be taken as limiting the practice of the present invention. It is within the scope of the present invention for those skilled in the art to make simple deductions or substitutions without departing from the concept of the present invention.

Claims

1. A cell culture product comprising an integrated cell culture muscle tissue, cell culture adipose tissue, and a hydrogel bioscaffold.

2. The cell culture product of claim 1, wherein the bioscaffold is a hydrogel bioscaffold comprising polyphenols, vegetable proteins, and polysaccharides, wherein: the content of polyphenol in the hydrogel biological scaffold is 0.05-1% (w/v); the content of the vegetable protein in the hydrogel biological scaffold is 5-50% (w/v); the content of polysaccharide in the hydrogel biological scaffold is 1-10% (w/v).

3. A cell culture product according to claim 2, wherein the polyphenols are selected from one or more of procyanidins, gallic acid, tea polyphenols, mung bean polyphenols, grape polyphenols, grapefruit polyphenols, catechins, etc.; the polysaccharide is selected from one or more of alginate, carrageenan, konjac gum and other plant gel; the vegetable protein is selected from any one or a combination of a plurality of proteins such as soybean protein, soybean isolated protein, peanut protein, wheat gluten, corn protein, pea protein and the like.

4. A cell culture product according to claim 3, wherein the polyphenol is selected from procyanidins; the polysaccharide is selected from sodium alginate; the vegetable protein is selected from soy protein isolate.

5. The cell culture product of claim 4, wherein the procyanidin is present in the hydrogel bioscaffold in an amount of 0.05-0.5% (w/v); the content of the sodium alginate in the hydrogel biological scaffold is 1-3% (w/v).

6. A cell culture product according to any one of claims 2-5, wherein the method of preparing the bioscaffold comprises the steps of:

s2: transferring the mixed protein solution into a bracket mold, and heating at a high temperature of not lower than 95 ℃ for more than 30min to obtain a biological bracket solution;

s3: caCl is added into the unfixed biological stent solution ₂ And (3) standing the solution overnight in a refrigerator at the temperature of 4 ℃ to obtain the biological scaffold.

7. A method of preparing a cell culture product according to any one of claims 1 to 6, comprising the steps of:

s1: sterilizing the hydrogel biological scaffold;

s2: inoculating solutions containing muscle stem cells and adipose-derived mesenchymal stem cells to the colored part and white part of the surface of the hydrogel biological scaffold respectively, and inoculatingThe seed density is 10-50 multiplied by 10 ⁶ cells/cm ² Culturing in a carbon dioxide incubator at 37 ℃ for 2 hours to form hydrogel tissues, and then adding a culture medium for co-culturing to perform proliferation and differentiation culture to obtain cell culture meat and cell culture adipose tissue interphase cell culture products.

8. The method according to claim 7, wherein the proliferation and differentiation culture comprises adding proliferation medium to culture and proliferate for 1-2 days, and replacing differentiation medium to culture for 4-20 days.

9. The method of claim 8, wherein the proliferation medium is formulated as follows:

5% -20% (v/v) fetal bovine serum and 1% (v/v) diabody were added to DMEM F12 complete medium, and 5ng/mL growth factor (human fibroblast growth factor, bFGF) was added thereto.

10. The method for preparing a cell culture product according to claim 8 or 9, wherein the differentiation medium comprises a muscle stem cell differentiation medium and a mesenchymal stem cell differentiation medium, wherein the volume of the muscle stem cell differentiation medium is 30% -70%.

11. The method of claim 10, wherein the muscle stem cell differentiation medium is formulated as follows: 2% horse serum, 1% diabody, DMEM or F10 or F12 basal medium; the mesenchymal stem cell differentiation medium was formulated as follows: 10% fetal bovine serum, 1% diabody and DMEM or F10 or F12 basal medium, and 0-5 uM dexamethasone, 0-1mM 3-isobutyl-1-methylxanthine, 0-100ug/mL insulin, 0-1mM indomethacin, 0-10uM rosiglitazone in the final concentration in the differentiation medium.

12. A hydrogel bioscaffold comprising polyphenols, vegetable proteins, and polysaccharides, wherein: the content of polyphenol in the hydrogel biological scaffold is 0.05-1% (w/v); the content of the vegetable protein in the hydrogel biological scaffold is 5-50% (w/v); the content of polysaccharide in the hydrogel biological scaffold is 1-10% (w/v).

13. The hydrogel bioscaffold of claim 12, wherein said polyphenol is selected from procyanidins; the polysaccharide is selected from sodium alginate; the vegetable protein is selected from soy protein isolate.

14. The hydrogel biological scaffold of claim 13, wherein the procyanidins are present in the hydrogel biological scaffold in an amount of 0.05-0.5% (w/v); the content of the sodium alginate in the hydrogel biological scaffold is 1-3% (w/v).

15. A method for preparing a hydrogel biological scaffold according to any one of claims 12-14, comprising the steps of:

16. Use of the hydrogel bioscaffold according to any one of claims 12-14 or the method for producing a hydrogel bioscaffold according to claim 15 for producing cell culture muscle tissue or cell culture adipose tissue.