POROUS CELL SUPPORT CONTAINING PLANT PROTEIN AND CULTURED MEAT PREPARED USING THE SAME

Abstract

Provided are a scaffold including a plant protein and in-vitro meat produced by using the scaffold. Considering that the scaffold consists of a plant protein, cultured muscles or adipose tissues may be ingested together with the scaffold without being separated therefrom. By adjusting a ratio of a muscle cell and an adipocyte, in-vitro meat having desirable texture may be produced, and since various types of cells may adhere, proliferate, and differentiate on the scaffold, such a scaffold may be effectively utilized for mass production of in-vitro meat.

Claims

1. A porous scaffold comprising a plant-derived protein isolate, a porogen, and an emulsifier.

2. The porous scaffold of claim 1, further comprising a coagulant.

3. The porous scaffold of claim 1, wherein the porous scaffold is obtained by adding the plant-derived protein isolate, the porogen, and the emulsifier to a solvent to prepare a mixture, stirring the mixture, and adding a coagulant to the stirred mixture.

4. The porous scaffold of claim 1, wherein the porous scaffold has any one or more properties selected from (a) to (c): (a) compressive strength in a range of 80 kPa to 150 kPa; (b) tensile strength in a range of 40 gf to 70 gf, yield strength in a range of 18 kPA to 32 kPA, and yield elongation in a range of 10 % to 20 %; and (c) hardness in a range of 0.4 kgf to 1.0 kgf, cohesiveness in a range of 0.6 to 1.0, springiness in a range of 0.8 to 1.2, gumminess in a range of 0.3 to 0.7, and chewiness of 0.3 to 0.7.

5. The porous scaffold of claim 1, wherein the porous scaffold has any one or more properties selected from (d), (e), and (o): (d) open porosity in a range of 40 % to 80 %; (e) total porosity in a range of 40 % to 80 %; and (o) pore interconnectivity in a range of 80 % to 99.9 %.

6. The porous scaffold of claim 1, wherein the porous scaffold has a pore size in a range of 50 .Math.m to 400 .Math.m.

7. The porous scaffold of claim 1, wherein the plant-derived protein isolate is a protein isolated from at least one selected from the group consisting of nut, bean, soybean, mung bean, kidney bean, pea, mushroom, vegetable, grain, and a combination thereof.

8. The porous scaffold of claim 1, wherein the porogen includes at least one selected from the group consisting of agar, salt, calcium chloride, sodium carbonate, paraffin, polyethylene glycol, gelatin, sucrose, and a combination thereof.

9. The porous scaffold of claim 1, wherein the emulsifier or the solvent includes at least one selected from the group consisting of glycerin, propylene glycol, monoglyceride, diglyceride, lecithin, soybean phospholipid, and a combination thereof.

10. The porous scaffold of claim 1, wherein the coagulant includes at least one selected from the group consisting of glucono-delta-lactone, calcium chloride, calcium sulfate, magnesium chloride, and a combination thereof.

11. The porous scaffold of claim 1, wherein cells are dispensed in the porous scaffold.

12. The porous scaffold of claim 1, wherein the porous scaffold is for the production of in-vitro meat.

13. A method of producing a porous scaffold, the method comprising: adding a plant-derived protein isolate, a porogen, and an emulsifier to a solvent to prepare a mixture and stirring the mixture to produce a stirred mixture; and adding a coagulant to the stirred mixture to form a scaffold.

14. In-vitro meat comprising: the porous scaffold of claim 1; and a cell dispersed in the porous scaffold.

15. A method of producing in-vitro meat, the method comprising dispensing a cell in the porous scaffold of claim 1 and culturing the cell three-dimensionally to form a three-dimensional cell aggregate.

16. The method of claim 15, wherein the cell is at least one selected from the group consisting of a stem cell, a muscle cell, an adipocyte, and a combination thereof.

17. An edible product comprising the in-vitro meat of claim 14.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0068] FIG. 1 is a photograph of a porous scaffold produced according to an embodiment, the porous scaffold including a plant-derived protein isolate.

[0069] FIG. 2 is a stress-strain curve shown in the process of measuring compressive strength of the porous scaffold produced according to an embodiment.

[0070] FIG. 3 is a stress-strain curve shown in the process of measuring tensile strength of the porous scaffold produced according to an embodiment.

[0071] FIG. 4 is a curve showing the results of texture profile analysis (TPA) performed on the porous scaffold produced according to an embodiment.

[0072] FIGS. 5 to 8 show the shapes of scaffolds that are previously known for cell culture, wherein FIG. 5 shows the shape of a concave microwell, FIG. 6 shows the shape of a porous scaffold, FIG. 7 shows the shape of a non-porous microbead, and FIG. 8 shows the shape of a porous microbead.

[0073] FIG. 9 is a photograph obtained by staining adipose-derived stem cells on the 9th day and the 15th day of cell culture after the cells are dispensed in the porous scaffold produced according to an embodiment.

[0074] FIG. 10 is a schematic diagram illustrating a process of producing in-vitro meat in a bioreactor by using the porous scaffold produced according to an embodiment.

[0075] FIG. 11 shows the state of culturing in-vitro meat, in-vitro chicken, and in-vitro pork by using the porous scaffold produced according to an embodiment.

[0076] FIG. 12 is a photograph showing the results of live/dead assay performed on the 13th day and the 20th day in the process of culturing bovine muscle cells by using the porous scaffold produced according to an embodiment.

[0077] FIG. 13 is a photograph of the scaffold before the cell culture (left) and the scaffold at week 4 of the cell culture (right) in the process of culturing bovine muscle cells by using the porous scaffold produced according to an embodiment.

[0078] FIG. 14 is a photograph obtained by staining bovine muscle with Calcein-AM/Desmin after the cells were cultured for 7 days by using the porous scaffold produced according to an embodiment.

[0079] FIG. 15 is a photograph obtained by staining bovine muscle cells with Phalloidin/Desmin after the cells were cultured for 7 days by using the porous scaffold produced according to an embodiment.

[0080] FIG. 16 is a photograph showing the results of live/dead assay performed on chicken cardiomyocytes, wherein the cells were first cultured by using the porous scaffold produced according to an embodiment in a 10 % DMEM culture medium for 7 days, and then, cultured for another 7 days after the culture medium was replaced with a 2 % DMEM culture medium.

[0081] FIG. 17 is a photograph showing the results of live/dead assay performed on chicken cardiomyocytes after the cells were cultured by using the porous scaffold produced according to an embodiment in a 10 % DMEM culture medium for 14 days.

[0082] FIG. 18 is a photograph showing the results of live/dead assay performed on chicken cardiomyocytes after the cells were cultured by using the porous scaffold produced according to an embodiment in a 2 % DMEM culture medium for 14 days.

[0083] FIG. 19 is a photograph showing the results of live/dead assay performed on the 13th day, the 17th day, and the 38th day in the process of culturing silki muscle cells by using the porous scaffold produced according to an embodiment.

[0084] FIG. 20 is a photograph showing the results of live/dead assay performed on the 14th day in the process of culturing bovine muscles cells and on the 8th day in the process of culturing chicken muscle cells, wherein the culturing was performed by using the porous scaffold produced according to an embodiment.

DETAILED DESCRIPTION

[0085] Regarding the production cost of in-vitro meat, $ 325,000 was required to produce a single burger patty (100 g) at the beginning of the in-vitro meat development in 2013. Although the cost was reduced to $1,986/100 g as of 2017, it was still very high. In addition, due to the limitations of the production technology, the currently produced in-vitro meat has been criticized for relatively inferior taste compared to real meat. In detail, in-vitro meat is not used as an ingredient in foods of which natural taste of meat is important as in steak, but is rather used as an ingredient for a burger patty. Thus, to eliminate the difference in taste with real meat, research is actively under way to make a real meat taste by mixing in-vitro meat with muscle, oil, bones, and the like. Furthermore, considering that a cell culture medium for the production of in-vitro meat requires horse or bovine fetal serum, there is a contradiction that as the production of in-vitro meat increases, livestock slaughter also increases. Therefore, regarding the production method for in-vitro meat, technical research to solve high cost issues, enable mass production of in-vitro meat, and give a taste similar to real meat is required.

[0086] Hereinafter, preferable examples are provided to help understanding of the present disclosure. However, the following examples are only provided for easy understanding of the present disclosure, and the contents of the present disclosure are not limited by the following examples.

Example 1. Production of Porous Scaffold Including Plant-Derived Protein Isolate

[0087] A porous scaffold including a plant-derived protein isolate was produced as follows.

[0088] 10 g of powdered isolated soy protein (ISP) and 2 g of agar were added to 10 ml of edible glycerin (0409, ES food ingredient) and mixed in a steam-stirring machine at a speed of 60 rpm for 10 minutes (D-201, Daeduck Machinery) to produce a mixture. Then, 30 ml of sterile deionized water was added to the mixture, and stirred at a speed of 5,000 rpm for 10 minutes by using a high-speed stirrer (MaXtir™ 500S, DAIHAN-Brand.sup.®). Afterwards, the resultant mixture was cast to a mold (100 mm X 100 mm X 2 mm) to form a desired shape, and then frozen at -80° C. for 1 hour to prepare a frozen sample. Baking soda was sufficiently applied to all surfaces of the frozen sample, allowed for adsorption at room temperature for 4 hours, and dried at 60° C. for 4 hours. After the dried sample was added to 1 L of sterile deionized water, 2 g of glucono-delta-lactone (E575, JUNGBUNZLAUER S.A) was added thereto for coagulation at 80° C. for 2 hours. Afterwards, the coagulated sample was washed 5 times, each with 100 % ethanol and sterile deionized water at room temperature, placed in 1 L of sterile deionized water, and subjected to high-pressure sterilization twice to remove impurities other than proteins, thereby producing a porous scaffold including plant protein components. FIG. 1 is a photograph of the porous scaffold produced in this example.

[0089] All materials for producing the porous scaffold are resources approved as food, and thus the porous scaffold can be ingested.

Example 2. Characterization of Porous Scaffold

Example 2.1 Morphological Characterization of Porous Scaffold

[0090] The morphological properties of the porous scaffold of Example 1 were evaluated by using SKYSCAN1272 ex-vivo micro-CT (Bruker microCT, Belgium) under the following conditions: [0091] X-ray source : 40 kV, 200 uA, No-filter, rotation step 0.15°; [0092] Resolution: 1 um pixel resolution; and [0093] Staining with 0.1 % iodine-potassium iodide (IKI) overnight.

[0094] Section creation was analyzed by using a NRecon software, section rotation was analyzed by using a DataViewer software, analysis was performed by using a CTAn software, volume rendering creation was analyzed by using a CTVox software, and surface rendering was analyzed by using a CTAn+CTVol software, and the results are shown in Table 2.

TABLE-US-00002 Abbreviation Measured value Unit Percent objective volume Obj.V/TV 40.91257477 % Structure thickness St.Th 0.02461021 mm Structure separation St.Sp 0.09660629 mm Open porosity Po(op) 58.55148608 % Total porosity Po(tot) 59.08742523 % Pore interconnectivity 58.55/59.09 = 99.1 % Percent objective volume: volume ratio occupied by the scaffold. Structure thickness: average spacing between pores. Structure separation: average size of pores. Open porosity: ratio of pores connected to the outside to total volume of the scaffold. Total porosity: ratio of pores to total volume of the scaffold. Pore interconnectivity: ratio of pores connected to the outside to the total pores.

[0095] Since the scaffold of Example 1 had the pore interconnectivity of about 99 %, the movement of the cells and the flow of the culture medium were smooth, and thus it was confirmed that the scaffold of Example 1 was suitable for the growth and differentiation of various cells.

Example 2.2 Physical Characterization of Porous Scaffold

[0096] To analyze physical properties of the scaffold of Example 1, the compressive strength and the tensile strength were measured by using a TXA™ texture analyzer (manufactured by Yeonjin S-Tech Corp.) as follows, and Texture profile analysis (TPA) was also performed.

[0097] The compressive strength was measured according to the Compression Test protocol. In detail, a sample was placed on a plate of a texture analyzer equipped with a compression fixation device, and compressed with a square probe (10 mm x 10 mm). Then, the initial strain rate was set to be 0.1 [1/s] and 0.033[1/s] for measurement. The measurement results are shown in Table 3 and FIG. 2.

TABLE-US-00003 Testing Method Compression Test Temperature 21.5 degree Celsius Humidity Ambient Load Cell 30 kgf Width mm Length mm Thickness mm speed mm/s Max Load gf Max Stress kPa 0.1 strain rate 7.765 7.260 1.778 0.178 724.368 126.009 0.033 strain rate 7.726 7.650 1.689 0.051 637.388 105.756 Average 7.746 7.455 1.734 0.114 680.878 115.882 Standard 0.028 0.276 0.063 0.090 61.504 14.320 deviation Standard deviation (%) 0.4 3.7 3.6 78.7 9.0 12.4

[0098] Referring to Table 3 and FIG. 2 and as being measured in a way that the sample was placed on the plate of the texture analyzer and compressed with the square probe (10 mm x 10 mm) with the initial strain rate set to be 0.1 [1/s] and 0.033[1/s] for the measurement, the porous scaffold according to an embodiment had the compression strength with the average width of 7.746 mm, the average length of 7.455 mm, the average thickness of 1.734 mm, the seismic wave speed of 0.114 mm/s , and the maximum stress of 115.882 kPa, in the case of the maximum load of 680.878 gf.

[0099] Seismic wave speed: speed at which an objective deformed by an external force attempts to return to the original shape when the force is removed.

[0100] Maximum stress: (also referred to as compression strength) maximum compressive stress that an objective can withstand without breaking.

[0101] Here, the tensile strength was measured by gripping the sample to tensile grips of the texture analyzer and setting the initial strain rates to be 0.1 [1/s] and 0.033[1/s] for measurement. The measurement results are shown in Table 4 and FIG. 3.

TABLE-US-00004 Testing Method Tensile Test Temperature 21.5 degree Celsius Humidity Ambient Load Cell 30 kgf Width mm Thickness mm Length mm speed mm/s Yield Load gf Yield Stress kPa Yield Elongation % Max Load gf 0.1 strain rate 14.000 1.500 20.000 2.033 60.209 28.117 16.000 120.820 0.033 strain rate 14.000 1.500 20.000 0.617 52.489 24.511 14.297 116.659 Average 14.000 1.500 20.000 1.325 56.349 26.314 15.148 118.739 Standard deviation 0.000 0.000 0.000 1.001 5.459 2.549 1.204 2.942 Standard deviation (%) 0 0.0 0.0 75.5 9.7 9.7 7.9 2.5

[0102] Referring to Table 4 and FIG. 3 and as being measured in a way that the sample was gripped to the tensile grips of the texture analyzer and the initial strain rate was set to be 0.1 [1/s] and 0.033[1/s] for the measurement, the porous scaffold according to an embodiment had the tensile strength with the average width of 14.000 mm, the average length of 20.000 mm, the average thickness of 1.500 mm, the seismic speed of 1.325 mm/s, the yield load of 56.348 gf, the yield stress of 26.314 kPA, and the yield elongation of 15.148 %, in the case of the maximum load of 118.739 gf.

[0103] Yield load: (also referred to as tensile strength) force immediately before breaking an objective .

[0104] Yield strength (or yield strength): when an external force is applied to an objective to pull it in opposite directions, the length of the objective increases. To some extent of the force, the lengthened objective returns to the original size when the external force is removed. When force greater than some extent of the force is applied, the objective cannot return to the original state and the length of the objective further increases. Here, the maximum force at which the objective can return to the original state is called yield stress or yield strength.

[0105] Yield elongation: Change in the length of an objective until deformation increases under almost constant stress state and the stress subsequently begins to increase smoothly, as expressed in a percentage with respect to the original length of objective .

[0106] Here, the TPA was measured in a way that a sample was placed on a plate of a texture analyzer and compressed by 50 % twice with a cylinder probe having a diameter of 25.4 mm at a speed of 60 mm/min. The measurement was repeated three times, and the results are shown in Table 5 and FIG. 7.

TABLE-US-00005 Width Thickness Crosssectional are a S. A Point distance G. L Test speed Hardness Adhesiveness Cohesiveness Springiness Gumminess Chewiness Resilience Brittleness mm mm mm.sup.2 mm mm/min kgf kgf*sec - - - - - kgf TPA_1 14.70 12.70 186.69 7.60 300.00 0.6729 0.0027 0.81 0.99 0.55 0.54 0.55 0.6729 TPA_2 14.20 13.70 194.54 7.64 300.00 0.6418 0.0013 0.82 1.02 0.53 0.54 0.55 0.6417 TPA_3 14.00 14.50 203.00 7.50 300.00 0.6922 0.0029 0.81 0.98 0.56 0.55 0.54 0.6921 Average 14.30 13.63 194.74 7.58 0.00 0.6689 0.0023 0.82 1.00 0.55 0.54 0.55 0.6689 Stand.Devi 0.36 0.90 8.16 0.07 0.00 1.6388 0.0057 2.00 2.44 1.34 1.33 1.34 1.6387

[0107] Referring to Table 5 and FIG. 4 and as being measured in a way that a sample was placed on a plate of the texture analyzer, compressed by 50% with a cylinder probe having a diameter of 25.4 mm at a speed of 60 mm/min, the physical properties of the porous scaffold according to an embodiment include, in average, the hardness of 0.6689 kgf, the adhesiveness of 0.0023 kgf*sec, the cohesiveness of 0.82, the springiness of 1.00, the gumminess of 0.55, the chewiness of 0.54, the resilience of 0.55, and the brittleness of 0.6689 kgf.

[0108] Hardness: Force required to reach desired deformation.

[0109] Adhesiveness: Force required to separate an objective from a probe.

[0110] Cohesiveness: Force to sustain the shape of an objective as it is. In case of cohesiveness greater than adhesiveness, a sample does not stick to a probe.

[0111] Springiness (also referred to as elasticity): Property in which a sample deformed by compression attempts to return to the original state after the force is removed.

[0112] Gumminess: Force required to disintegrate a sample in a semi-solid state until it becomes a swallowable state that can be swallowed.

[0113] Chewiness: Energy required to transfer a solid state of a sample into a swallowable state.

[0114] Brittleness: Breaking or brittle property upon compression, which is represented as an intermediate peak that appears before reaching the maximum force of first compression, but may not appear depending on a sample.

[0115] Resilience: Property in which a sample attempts to recover the original height.

[0116] As such, it was confirmed that the scaffold produced according to Example 1 had suitable compressive strength and tensile strength for the growth and differentiation of muscle cells, and as an edible scaffold used for the production of in-vitro meat, also had good texture with suitable elasticity and resilience properties and soft swallowability with suitable hardness and gumminess.

Example 2.3 Property Comparison with Existing Scaffolds

[0117] The results of comparing the properties of the previously known scaffolds (e.g., concave microwell, porous scaffold, non-porous microbead, porous microbead, etc) and the porous scaffold of Example 1 are shown in Table 6. For the properties of the previously known scaffolds, the prior art document [Materials Science & Engineering C 103 (2019) 109782] was cited. The shapes of the previously known scaffolds are shown in FIGS. 5 to 8.

TABLE-US-00006 Concave Microwell (FIG. 5) Porous Scaffold (FIG. 6) Non-porous microbead (FIG. 7) Porous Microbead (FIG. 8) Example 1 Cell adhesion N/A *** *** *** ***** Media diffusion to the cells * *** ***** ***** ***** Enduranc e in bioreactor s * * ***** *** ***** Cell density when fully attached N/A * *** ***** ***** Price ***** * *** * ***** Material Scaffold-free Plastic Plastic/Protein Plastic/Protein Protein Suitability for in-vitro meat productio n Research use products only Research use products only Not enough yield, aggregation occurs Not strong enough to be used in bioreactors Great for in-vitro meat application

[0118] As such, it was confirmed that, compared to the existing scaffolds for cell culture, the scaffold of Example 1 had high cell adhesion, excellent permeability of the culture medium, and high durability, and was made of materials suitable for the production of in-vitro meat.

Example 3. Three-Dimensional Culture of Cells Using Porous Scaffold

[0119] Cells were cultured three-dimensionally by using the porous scaffold according to an embodiment.

[0120] In detail, the porous scaffold of Example 1 having a diameter of 10 mm and a thickness of 2 mm was placed on a culture dish, and 10% mesenchymal stem cell growth medium 2 (Promocell C-28009, C-39809) was added thereto. Then, 5.0 x 10.sup.5 adipose-derived stem cells (ATCC PCS-500-011) were dispensed and cultured therein for 15 days for proliferation and histogenesis of the cells.

[0121] As shown in FIG. 9, it was confirmed that the growth of cells continued as the culture period elapsed; the scaffold contracted as the cells adhered to the scaffold and grew thereon; and tissues were formed as the number of the cells increased and the void volume was minimized.

Example 4. Production of In-Vitro Meat by Using Porous Scaffold

[0122] In-vitro meat was produced by using the porous scaffold of Example 1. In detail, as shown in FIG. 10, bovine muscle cells, chicken muscle cells, and pig muscle cells were respectively introduced into a bioreactor along with the scaffold of Example 1, and were collectively cultured. The results are shown in FIG. 11.

[0123] As such, it was confirmed that, when the cells and the porous scaffold were simultaneously introduced into a single incubator, the one-step mass culture system in which the adhesion, attachment, proliferation, and differentiation of the cells occur at once can be established.

Example 4.1. Production of In-Vitro Beef by Using Porous Scaffold

[0124] In a culture dish, the porous scaffold of Example 1 having a size of 25 mm X 25 mm X 2 mm, 1.7 x 10.sup.6 Bovine #1 cells (i.e., primary skeletal muscle cells extracted from 150 g of rump muscles of a 36-month-old female Hanwoo (Korean native cattle, dressed weight: 339 kg, quality grade 2) slaughtered by Bunong Livestock Inc. on the same day as the experiment), and each of culture media (10 % DMEM of DMEM biowest L0103-500 and 2 % DMEM of FBS biowest S1480-500) were added, and the cells were cultured for 24 hours in a shaking incubator. As a result of the culture, it was confirmed that all cells were dispensed in the porous scaffold, except for 2 % to 3 % of the residual cells.

[0125] In addition, in the process of 4-week culture, the live&dead assay was performed on the 13th day and the 20th day, and the results are shown in FIG. 12. As shown in FIG. 12, it was confirmed that the cell adhesion was well achieved since dead cells were hardly observed on the scaffold, and that the cell proliferation was also well achieved on the scaffold through the increase of live cells within a week. Also, as shown in FIG. 13, as a results of visually observing the scaffold before and after the culturing, it was found that the scaffold after the culturing was changed to a pale pink color unlike the scaffold color before the culturing. Such a color change refers that proliferation of the muscle cells occurred on the scaffold.

[0126] Subsequently, in a culture dish, the porous scaffold of Example 1 having a size of 25 mm X 25 mm X 2 mm and culture media (10 % DMEM of DMEM biowest L0103-500 and 2 % DMEM of FBS biowest S1480-500) were each added. Then, 1.0 x 10.sup.6 Bovine #2 cells (i.e., primary skeletal muscle cells extracted from 150 g of rump muscles of a 23-month-old female Hanwoo (Korean native cattle, dressed weight: 261 kg, quality grade 2) slaughtered by Samho Livestock on the same day as the experiment) were dispensed thereon and cultured for 7 days.

[0127] After the culture was complete, the cells were stained with each of Calcein-AM/Desmin Ab and Phalloidin/Desmin Ab, and the results are shown in FIGS. 14 and 15. Live cells and morphology of the differentiated cells were observed using calcein-AM thereof, differentiated muscle cells were observed using desmin Ab which is a muscle cell marker, and morphology of the differentiated muscle cells was observed using phalloidin. As shown in FIGS. 14 and 15, it was confirmed that the differentiation into muscle cells was well performed within the scaffold.

[0128] Accordingly, it was confirmed that in-vitro beef could be produced by culturing bovine muscle cells on the porous scaffold of Example 1.

Example 4.2. Production of In-Vitro Chicken by Using Porous Scaffold

[0129] In a culture dish, the porous scaffold of Example 1 having a size of 25 mm X 25 mm X 2 mm and a culture media (10 % DMEM) were added, and 5.0 x 10.sup.5 chicken Cardiomyocyte #1 (i.e., primary cardiomyocytes extracted from the heart by taking embryonic body from a fertilized egg of a general layer chicken at day 9 to day 11 of hatching) were dispensed thereon and cultured for 7 days to proliferate the cells. After 7 days, the culture medium was replaced with 2% DMEM, and the cells were cultured for another 7 days to differentiate into cardiomyocytes. The live&dead assay was performed on the 14th day of the culture, and the results are shown in FIG. 16. As shown in FIG. 16, it was confirmed that the cells were generally well cultured enough to produce in-vitro meat.

[0130] Also, in a culture dish, the porous scaffold of Example 1 having a size of 25 mm X 25 mm X 2 mm and culture media (10 % DMEM and 2 % DMEM) were each added.

[0131] Then, 1.0 × 10.sup.6 chicken Cardiomyocyte #1 (i.e., primary cardiomyocytes extracted from the heart by taking embryonic body from a fertilized egg of a general layer chicken on the 9th to 11th day of hatching by DaNagreen) were dispensed on each culture medium and cultured for 14 days. The live&dead assay was performed on the 14th day of the culture, and the results are shown in FIGS. 17 and 18. As shown in FIGS. 17 and 18, it was confirmed that the cells were generally well cultured enough to produce in-vitro meat.

[0132] Accordingly, it was confirmed that in-vitro chicken could be produced by culturing chicken muscle cells on the porous scaffold of Example 1.

[0133] Subsequently, in a culture dish, the porous scaffold of Example 1 having a size of 25 mm X 25 mm X 2 mm and culture media (10 % DMEM of DMEM biowest L0103-500 and 2 % DMEM of FBS biowest S1480-500) were added. Then, 1.0 × 10.sup.7 Silkie #1 cells (i.e., primary skeletal muscle cells extracted from thigh by taking embryonic body from a fertilized egg of silkie on the 9th to 11th day of hatching) were dispensed on each media and cultured for 5 weeks.

[0134] The live&dead assay was performed on the 13th day, 17th day, and 38th day of the culture, and the results are shown in FIG. 19. As shown in FIG. 19, it was confirmed that the proliferation and differentiation of the silkie muscle cells were well achieved within the scaffold. In detail, live cells were full on the scaffold at the 2nd week of the culture, and the viability of the cells was confirmed even at the 5th week of the culture.

Example 4.3. Comparison of In-Vitro Beef and In-Vitro Chicken Using Porous Scaffold

[0135] In a culture dish, the porous scaffold of Example 1 having a size of 25 mm X 25 mm X 2 mm and culture media (10 % DMEM of DMEM biowest L0103-500 and 2 % DMEM of FBS biowest S1480-500) were added. Then, 1.0 × 10.sup.6 Bovine #1 cells and 2.0 × 10.sup.6 Chicken #1 cells were dispensed on each cell media and cultured for 4 weeks.

[0136] In the process of culturing, the live&dead assay was performed for the bovine cells on the 14th day and for chicken cells on the 8th day, and the results are shown in FIG. 20. As shown in FIG. 20, the chicken cells had a smaller size and a faster doubling time than the bovine cells. Thus, when cultured for the same period, it was confirmed that the scaffold for the chicken cells was filled in a much shorter period of time. It was also confirmed that, in both in-vitro meat products, the cell properties were not degraded by the scaffold, and that the in-vitro beef had different muscle fiber morphology from the in-vitro chicken.

[0137] Subsequently, as a results of tasting the in-vitro beef and the in-vitro chicken in the 3rd week and the 4th week of the in-vitro process, it was confirmed that, in the case of the in-vitro chicken, the taste and flavor of real chicken were made by only 3-week culturing of the chicken cells, and in the case of the in-vitro beef, the taste and flavor of real beef were made after 4 weeks of the bovine cell culturing was complete.

[0138] Accordingly, it was confirmed that, by using the porous scaffold of Example 1, the taste and flavor suitable for the properties of the corresponding in-vitro meat products may be exhibited.