SPECIAL CULTURE APPARATUS FOR 3D BIOLOGICAL TISSUE, AND METHOD FOR PREPARING BLOCK-SHAPED CULTURED MEAT

Abstract

The present disclosure provides a special culture apparatus for a 3D biological tissue, and a method for preparing block-shaped cultured meat. The special culture apparatus for a 3D biological tissue includes: a 3D biological tissue culture tank for accommodating a 3D biological tissue, and a liquid storage tank for containing a culture medium; the 3D biological tissue culture tank is connected to the liquid storage tank by means of a pipeline to form a circuit for the culture medium to circularly flow; and an opening of the 3D biological tissue culture tank is provided with a sealing plug, an inner side of the sealing plug is provided with a plurality of culture medium infusion needles that penetrate the 3D biological tissue when in use.

Claims

1. A special culture apparatus for a 3D biological tissue, comprising: a 3D biological tissue culture tank for accommodating a 3D biological tissue, and a liquid storage tank for containing a culture medium; wherein the 3D biological tissue culture tank is connected to the liquid storage tank by means of a pipeline to form a circuit for the culture medium to circularly flow; and an opening of the 3D biological tissue culture tank is provided with a sealing plug, with an inner side of the sealing plug provided with a plurality of culture medium infusion needles that penetrate the 3D biological tissue when in use.

2. A method for preparing block-shaped cultured meat, comprising steps of: mixing animal skeletal muscle satellite cells with bioink for 3D bioprinting, and placing a printed 3D animal skeletal muscle satellite cell tissue in the special culture apparatus for a 3D biological tissue according to claim 1 for proliferation culture and differentiation.

3. The method for preparing block-shaped cultured meat according to claim 2, wherein the bioink is methacrylic anhydride-modified gelatin (GelMa) and/or nanocellulose.

4. The method for preparing block-shaped cultured meat according to claim 2, wherein when the animal skeletal muscle satellite cells are porcine skeletal muscle satellite cells, the printed 3D animal skeletal muscle satellite cell tissue is placed in the special culture apparatus for a 3D biological tissue according to claim 1, and the whole is cultured at 36.5-37.5° C. in 5% CO.sub.2; after 1-2 days, when cell morphology is stable, a proliferation medium is replaced with a differentiation medium; after 3-5 days, the differentiation medium is replaced with the proliferation medium; and further culture is continued until the cells differentiate and fuse to form the block-shaped cultured meat.

5. The method for preparing block-shaped cultured meat according to claim 4, wherein when the animal skeletal muscle satellite cells are porcine skeletal muscle satellite cells, the proliferation medium used comprises 8-12 ng/mL epidermal growth factor, 0.5-2 ng/mL fibroblast growth factor, 0.005-0.015 mg/L insulin, and 0.3-0.5 μg/mL dexamethasone, and the differentiation medium used comprises 0.005-0.015 mg/L insulin.

6. The method for preparing block-shaped cultured meat according to claim 2, wherein when the animal skeletal muscle satellite cells are chicken skeletal muscle satellite cells, the printed 3D animal skeletal muscle satellite cell tissue is placed in the special culture apparatus for a 3D biological tissue according to claim 1, and the whole is cultured at 40.5-41.5° C. in 5% CO.sub.2; after 1-2 days, when cell morphology is stable, a proliferation medium is replaced with a differentiation medium; after 3-5 days, the differentiation medium is replaced with the proliferation medium; and further culture is continued until the cells differentiate and fuse to form the block-shaped cultured meat.

7. The method for preparing block-shaped cultured meat according to claim 6, wherein when the animal skeletal muscle satellite cells are chicken skeletal muscle satellite cells, the proliferation medium used is a McCoy's 5A Medium supplemented with 10-20% chicken serum or fetal bovine serum (FBS), and the differentiation medium used is a McCoy's 5A Medium supplemented with 0-5% chicken serum or FBS.

8. The method for preparing block-shaped cultured meat according to claim 2, wherein the animal skeletal muscle satellite cells are obtained by extraction and in vitro culture, wherein the porcine skeletal muscle satellite cells are extracted from a skeletal muscle tissue of a neonatal animal, and the chicken skeletal muscle satellite cells are extracted from an embryo in an incubator.

9. The method for preparing block-shaped cultured meat according to claim 8, wherein the in vitro culture is implemented by an adherent culture method or a suspension culture method by means of loading on the surface of a microcarrier microsphere.

10. Block-shaped cultured meat, wherein the block-shaped cultured meat is prepared by the method for preparing block-shaped cultured meat according to claim 2.

11. The block-shaped cultured meat according to claim 10, wherein the bioink is methacrylic anhydride-modified gelatin (GelMa) and/or nanocellulose.

12. The block-shaped cultured meat according to claim 10, wherein when the animal skeletal muscle satellite cells are porcine skeletal muscle satellite cells, the printed 3D animal skeletal muscle satellite cell tissue is placed in the special culture apparatus for a 3D biological tissue according to claim 1, and the whole is cultured at 36.5-37.5° C. in 5% CO.sub.2; after 1-2 days, when cell morphology is stable, a proliferation medium is replaced with a differentiation medium; after 3-5 days, the differentiation medium is replaced with the proliferation medium; and further culture is continued until the cells differentiate and fuse to form the block-shaped cultured meat.

13. The block-shaped cultured meat according to claim 10, wherein when the animal skeletal muscle satellite cells are porcine skeletal muscle satellite cells, the proliferation medium used comprises 8-12 ng/mL epidermal growth factor, 0.5-2 ng/mL fibroblast growth factor, 0.005-0.015 mg/L insulin, and 0.3-0.5 μg/mL dexamethasone, and the differentiation medium used comprises 0.005-0.015 mg/L insulin.

14. The block-shaped cultured meat according to claim 10, wherein when the animal skeletal muscle satellite cells are chicken skeletal muscle satellite cells, the printed 3D animal skeletal muscle satellite cell tissue is placed in the special culture apparatus for a 3D biological tissue according to claim 1, and the whole is cultured at 40.5-41.5° C. in 5% CO.sub.2; after 1-2 days, when cell morphology is stable, a proliferation medium is replaced with a differentiation medium; after 3-5 days, the differentiation medium is replaced with the proliferation medium; and further culture is continued until the cells differentiate and fuse to form the block-shaped cultured meat.

15. The block-shaped cultured meat according to claim 10, wherein when the animal skeletal muscle satellite cells are chicken skeletal muscle satellite cells, the proliferation medium used is a McCoy's 5A Medium supplemented with 10-20% chicken serum or fetal calf serum (FCS), and the differentiation medium used is a McCoy's 5A Medium supplemented with 0-5% chicken serum or FCS.

16. The block-shaped cultured meat according to claim 10, wherein the animal skeletal muscle satellite cells are obtained by extraction and in vitro culture, wherein the porcine skeletal muscle satellite cells are extracted from a skeletal muscle tissue of a neonatal animal, and the chicken skeletal muscle satellite cells are extracted from an embryo in an incubator.

17. The block-shaped cultured meat according to claim 10, wherein the in vitro culture is implemented by an adherent culture method or a suspension culture method by means of loading on the surface of a microcarrier microsphere.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0045] FIG. 1 is a structural representation of a special culture apparatus for a 3D biological tissue provided in Example 1 of the present disclosure, where 1 represents a 3D biological tissue culture tank; 2 represents a sealing plug; 3 represents a liquid storage tank; 4 represents a culture medium infusion needle; 5 represents a first differential pressure gauge; 6 represents a filling pump; 7 represents a second differential pressure gauge; and 8 represents a suction pump;

[0046] FIGS. 2 and 3 illustrate the morphology of porcine skeletal muscle satellite cells cultured for 48 h and 72 h in step (2) of Example 2 of the present disclosure, respectively;

[0047] FIGS. 4 and 5 illustrate the morphology of chicken skeletal muscle satellite cells cultured for 24 h and 48 h in step (2) of Example 3 of the present disclosure, respectively;

[0048] FIG. 6 illustrates the morphology of porcine skeletal muscle satellite cells cultured for 72 h in step (2) of Example 4 of the present disclosure;

[0049] FIG. 7 illustrates the morphology of chicken skeletal muscle satellite cells cultured for 48 h in step (2) of Example 5 of the present disclosure;

[0050] FIG. 8 illustrates the morphology of porcine skeletal muscle satellite cells cultured for 5 days in step (2) of Example 2 of the present disclosure;

[0051] FIGS. 9 and 10 illustrate the morphology of a 3D porcine skeletal muscle satellite cell tissue cultured for 10 and 15 days in Example 2 of the present disclosure, respectively;

[0052] FIG. 11 illustrates the morphology of a 3D chicken skeletal muscle satellite cell tissue cultured for 15 days in Example 3 of the present disclosure;

[0053] FIG. 12 illustrates the myogenesis of porcine muscle stem cells in Example 2 of the present disclosure;

[0054] FIG. 13 illustrates the myogenesis of chicken muscle stem cells in Example 3 of the present disclosure;

[0055] FIG. 14 illustrates the morphology of porcine skeletal muscle satellite cells extracted by the digestion tissue explant adherent method in Comparative Example 1 of the present disclosure;

[0056] FIG. 15 illustrates the morphology of porcine skeletal muscle satellite cells extracted by the tissue explant adherent method in Example 2 of the present disclosure; and

[0057] FIG. 16 illustrates the morphology of porcine skeletal muscle satellite cells after static culture in Comparative Example 2 of the present disclosure.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0058] In order to make the objectives, technical solutions and advantages of the embodiments of the present disclosure clearer, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below with reference to the accompanying drawings in the examples of the present disclosure. Obviously, the described examples are a part of, not all of, embodiments of the present disclosure. All other embodiments obtained by a person of ordinary skill in the art based on the examples of the present invention without creative efforts shall fall within the protection scope of the present invention.

Example 1

[0059] This example of the present disclosure provided a special culture apparatus for a 3D biological tissue, and the structural representation thereof is shown in FIG. 1, including: [0060] a 3D biological tissue culture tank 1 for accommodating a 3D biological tissue, and [0061] a liquid storage tank 3 for containing a culture medium; [0062] where the 3D biological tissue culture tank 1 was connected to the liquid storage tank 3 by means of a pipeline to form a circuit for the culture medium to circularly flow; the pipeline flowing into the 3D biological tissue culture tank 1 was provided with a first differential pressure gauge 5 and a filling pump 6, and a second differential pressure gauge 7 and a suction pump 8 were provided on the pipeline flowing out of the 3D biological tissue culture tank 1; and [0063] an opening of the 3D biological tissue culture tank 1 was provided with a sealing plug 2, with an inner side of the sealing plug 2 provided with a plurality of culture medium infusion needles 4 that penetrate the 3D biological tissue when in use.

Example 2

[0064] This example of the present disclosure provided a method for preparing block-shaped porcine-derived cultured meat, specifically as follows:

[0065] (1) Extraction of Porcine Skeletal Muscle Satellite Cells

[0066] A newborn pig was sacrificed by CO.sub.2, put in 75% alcohol solution for immersion disinfection for 5 min, and dissected to obtain the skeletal muscle tissue of its thigh; the fascia tissues were torn off, and cut into about 1 mm.sup.3 tissue explants with ophthalmic scissors. Phosphate buffered saline (PBS) was added and the tissue explants were washed gently with a pipette. After that, the tissue explants were centrifuged at 700 rpm for 5 min, a resulting supernatant was aspirated, and the lower pellet was the porcine skeletal muscle tissue explant. The above tissue explant was resuspended in DMEM supplemented with 20% FBS, spread on a Petri dish, and placed in an incubator at 37° C. in 5% CO.sub.2 for static culture. After cultured for 3 days, the porcine skeletal muscle tissue explant was pipetted off the Petri dish, and fresh DMEM supplemented with 20% FBS was changed.

[0067] (2) Adherent and In Vitro Culture of Porcine Skeletal Muscle Satellite Cells

[0068] After the porcine skeletal muscle satellite cells extracted in step (1) covered more than 50% of the bottom area of the Petri dish, the culture medium was discarded, and the cells at the bottom of the Petri dish were rinsed once with Dulbecco's phosphate-buffered saline (DPBS). Thereafter, the cells were digested with 1 mL of 0.25% pancreatin for 4.5 min at 37° C.; then, 1 mL of 10% FBS in PBS was added to terminate the digestion, and the adherent cells were gently pipetted off, transferred to a centrifuge tube, and centrifuged at 900 rpm for 5 min; the supernatant was aspirated, and the lower pellets were primary porcine skeletal muscle satellite cells. Subsequently, the primary porcine skeletal muscle satellite cells were resuspended in 1 mL of DMEM supplemented with 20% FBS and counted. An appropriate quantity of the cells were inoculated in a cell factory (about 3×10.sup.6 cells/layer), and placed in an incubator at 37° C. in 5% CO.sub.2 for static culture. The skeletal muscle satellite cells were passaged after expansion to cover 80% of the bottom area of the cell factory, and the skeletal muscle satellite cells were continuously expanded.

[0069] (3) 3D Bioprinting of Porcine Skeletal Muscle Satellite Cells

[0070] Under aseptic conditions, an appropriate quantity of the porcine skeletal muscle satellite cells obtained in step (2) were mixed with a volume of bioink GelMa, so that the volume ratio of the bioink was between 1% and 20%. The porcine skeletal muscle satellite cells were added into a bioprinter for 3D bioprinting into a block or grid in a sterile Petri dish.

[0071] (4) Proliferation Culture and Differentiation of 3D Porcine Skeletal Muscle Satellite Cell Tissue

[0072] An animal skeletal muscle satellite cell tissue printed in step (3) was placed in the special culture apparatus for a 3D biological tissue provided in Example 1 of the present disclosure, and the whole was cultured at 37° C. in 5% CO.sub.2; after 1-2 days, when the cell morphology was stable, a proliferation medium for porcine skeletal muscle satellite cells (see Table 1 for specific components) was replaced with a differentiation medium for porcine skeletal muscle satellite cells (see Table 2 for specific components). After 3-5 days, the differentiation medium was replaced with the differentiation medium for further culture. Once the skeletal muscle satellite cells inside the 3D biological tissue differentiated and fused to form a unified whole, with the tissue elastic and the tissue surface lustrous, the 3D biological tissue culture and differentiation were completed and a cultured meat structure unit was formed.

TABLE-US-00001 TABLE 1 Components of the proliferation medium for porcine skeletal muscle satellite cells Molecular Content Component name weight (mg/L) Glycine 75 30 L-Arginine hydrochloride 211 84 L-Cystine•2HCl 313 63 L-Glutamine 146 584 L-Histidine hydrochloride•H.sub.2O 210 42 L-Isoleucine 131 105 L-Leucine 131 105 L-Lysine hydrochloride 183 146 L-Methionine 149 30 L-Phenylalanine 165 66 L-Serine 105 42 L-Threonine 119 95 L-Tryptophan 204 16 L-Tyrosine disodium. salt dihydrate 261 104 L-Valine 117 94 Choline chloride 140 4 D-Calcium pantothenate 477 4 Folic Acid 441 4 Niacinamide 122 4 Pyridoxine hydrochloride 206 4 Riboflavin 376 0.4 Thiamine hydrochloride 337 4 i-Inositol 180 7.2 Calcium Chloride (CaCl.sub.2) (anhyd.) 111 200 Ferric Nitrate (Fe(NO.sub.3).sub.3•9H.sub.2O) 404 0.1 Magnesium Sulfate (MgSO.sub.4) (anhyd.) 120 97.67 Potassium Chloride (KCl) 75 400 Sodium Bicarbonate (NaHCO.sub.3) 84 3700 Sodium Chloride (NaCl) 58 6400 Sodium Phosphate monobasic 138 125 (NaH2PO4—H2O) D-Glucose 180 4500 Fetal Calf Serum 0.05 mL/mL mL/mL Fetuin 0.05 Epidermal Growth Factor (recombinant human) 10 ng/mL Basic Fibroblast Growth Factor  1 ng/mL (recombinant human) Insulin (recombinant human) 0.01 Dexamethasone 392 0.4 μg/mL μg/mL

TABLE-US-00002 TABLE 2 Components of the differentiation medium for porcine skeletal muscle satellite cells Molecular Content Component name weight (mg/L) Glycine 75 30 L-Arginine hydrochloride 211 84 L-Cystine•2HCl 313 63 L-Glutamine 146 584 L-Histidine hydrochloride•H.sub.2O 210 42 L-Isoleucine 131 105 L-Leucine 131 105 L-Lysine hydrochloride 183 146 L-Methionine 149 30 L-Phenylalanine 165 66 L-Serine 105 42 L-Threonine 119 95 L-Tryptophan 204 16 L-Tyrosine disodium salt dihydrate 261 104 L-Valine 117 94 Choline chloride 140 4 D-Calcium pantothenate 477 4 Folic Acid 441 4 Niacinamide 122 4 Pyridoxine hydrochloride 206 4 Riboflavin 376 0.4 Thiamine hydrochloride 337 4 i-Inositol 180 7.2 Calcium Chloride (CaCl.sub.2) (anhyd.) 111 200 Ferric Nitrate (Fe(NO.sub.3).sub.3•9H.sub.2O) 404 0.1 Magnesium Sulfate (MgSO.sub.4) (anhyd.) 120 97.67 Potassium Chloride (KCl) 75 400 Sodium. Bicarbonate (NaHCO.sub.3) 84 3700 Sodium Chloride (NaCl) 58 6400 Sodium Phosphate monobasic (NaH.sub.2PO.sub.4•H.sub.2O) 138 125 D-Glucose 180 4500 Insulin 0.01

Example 3

[0073] This example of the present disclosure provided a method for preparing block-shaped chicken-origin cultured meat, specifically as follows:

[0074] (1) Extraction of Chicken Skeletal Muscle Satellite Cells

[0075] A well-developed egg without damaged egg shell and obvious unremovable dirt was selected, gently wiped and disinfected with a 75% alcohol cotton ball. The air cell was gently broken by medical tweezers and the inner membrane attached to the shell was removed. The chicken embryo was taken out with elbow tweezers and dipped in 75% alcohol for 3 seconds. Subsequently, the disinfected chicken embryo was put into a Petri dish with PBS in advance, and rinsed with PBS twice. The PBS was absorbed. The chicken embryo thigh was cut off, and the outer fascia was torn off. Muscles were immobilized with a pair of tweezers. The muscles were torn and pulled with a pair of elbow tweezers tear, and the muscles attached to the bones on one thigh of the chicken embryo were torn into minced meat. The PBS was added to the minced chicken skeletal muscle tissue until it submerged the muscle tissue. The minced chicken skeletal muscle tissue was pipetted evenly and centrifuged at 800 rpm for 5 min, and the supernatant was discarded. The lower pellet was the target chicken skeletal muscle tissue explant. Subsequently, the above-mentioned tissue explant was resuspended in DMEM supplemented with 20% FBS, spread on a Petri dish, and placed in an incubator at 37° C. in 5% CO.sub.2 for static culture. After culturing for one day, the skeletal muscle tissue explant was pipetted off the Petri dish, and fresh DMEM supplemented with 20% FBS was changed.

[0076] (2) Adherent and In Vitro Culture of Chicken Skeletal Muscle Satellite Cells

[0077] After the chicken skeletal muscle satellite cells extracted in step (1) covered more than 50% of the bottom area of the Petri dish, the culture medium was discarded, and the cells at the bottom of the Petri dish were rinsed once with DPBS. Then, the cells were digested with 1 mL of 0.25% pancreatin for 4.5 min at 37° C.; after that, 1 mL of 10% FBS in PBS was added to terminate the digestion, and the adherent cells were gently pipetted off, transferred to a centrifuge tube, and centrifuged at 900 rpm for 5 min; the supernatant was aspirated, and the lower pellets were primary chicken skeletal muscle satellite cells. Subsequently, the primary chicken skeletal muscle satellite cells were resuspended in 1 mL of DMEM supplemented with 20% FBS and counted. An appropriate quantity of the cells were inoculated in a cell factory (about 3×10.sup.6 cells/layer), and placed in an incubator at 37° C. in 5% CO.sub.2 for static culture. The skeletal muscle satellite cells were passaged after expansion to cover 80% of the bottom area of the cell factory, and the number of skeletal muscle satellite cells was continuously expanded.

[0078] (3) 3D Bioprinting of Chicken Skeletal Muscle Satellite Cells

[0079] Under aseptic conditions, an appropriate quantity of the chicken skeletal muscle satellite cells obtained in step (2) were mixed with a volume of bioink GelMa, so that the volume ratio of the bioink was between 1% and 20%. The chicken skeletal muscle satellite cells were added into a bioprinter for 3D bioprinting into a block or grid in a sterile Petri dish.

[0080] (4) Proliferation Culture and Differentiation of Chicken Skeletal Muscle Satellite Cell Tissue

[0081] A chicken skeletal muscle satellite cell tissue printed in step (3) was placed in the special culture apparatus for a 3D biological tissue provided in Example 1 of the present disclosure, and the whole was cultured at 41° C. in 5% CO.sub.2; after 1-2 days, when the cell morphology was stable, a proliferation medium for chicken skeletal muscle satellite cells (see Table 3 for specific components) was replaced with a differentiation medium for chicken skeletal muscle satellite cells (see Table 4 for specific components). After 3-5 days, the differentiation medium was replaced with the differentiation medium for further culture. Once the skeletal muscle satellite cells inside the 3D biological tissue differentiated and fused to form a unified whole, with the tissue elastic and the tissue surface lustrous, the 3D biological tissue culture and differentiation were completed, and a cultured meat structure unit was formed.

TABLE-US-00003 TABLE 3 Components of the proliferation medium for chicken skeletal muscle satellite cells Molecular Content Component name (in English) weight (mg/L) Glycine 75 30 L-Arginine hydrochloride 211 84 L-Cystine•2HCl 313 63 L-Glutamine 146 584 L-Histidine hydrochloride•H.sub.2O 210 42 L-Isoleucine 131 105 L-Leucine 131 105 L-Lysine hydrochloride 183 146 L-Methionine 149 30 L-Phenylalanine 165 66 L-Serine 105 42 L-Threonine 119 95 L-Tryptophan 204 16 L-Tyrosine disodium salt dihydrate 261 104 L-Valine 117 94 Choline chloride 140 4 D-Calcium pantothenate 477 4 Folic Acid 441 4 Niacinamide 122 4 Pyridoxine hydrochloride 206 4 Riboflavin. 376 0.4 Thiamine hydrochloride 337 4 i-Inositol 180 7.2 Calcium Chloride (CaCl.sub.2) (anhyd.) 111 200 Feme Nitrate (Fe(NO.sub.3).sub.3•9H.sub.2O) 404 0.1 Magnesium Sulfate (MgSO.sub.4) (anhyd.) 120 97.67 Potassium Chloride (KCl) 75 400 Sodium Bicarbonate (NaHCO.sub.3) 84 3700 Sodium Chloride (NaCl) 58 6400 Sodium Phosphate monobasic 138 125 D-Glucose 180 4500 Fetal Calf Serum 0.2 mL/mL

TABLE-US-00004 TABLE 4 Components of the differentiation medium for chicken skeletal muscle satellite cells Molecular Content Component name (in English) weight (mg/L) Glycine 75 30 L-Arginine hydrochloride 211 84 L-Cystine•2HCl 313 63 L-Glutamine 146 584 L-Histidine hydrochloride•H.sub.2O 210 42 L-Isoleucine 131 105 L-Leucine 131 105 L-Lysine hydrochloride 183 146 L-Methionine 149 30 L-Phenylalanine 165 66 L-Serine 105 42 L-Threonine 119 95 L-Tryptophan 204 16 L-Tyrosine disodium salt dihydrate 261 104 L-Valine 117 94 Choline chloride 140 4 D-Calcium pantothenate 477 4 Folic Acid 441 4 Niacinamide 122 4 Pyridoxine hydrochloride 206 4 Riboflavin 376 0.4 Thiamine hydrochloride 337 4 i-Inositol 180 7.2 Calcium Chloride (CaCl.sub.2) (anhyd.) 111 200 Ferric Nitrate (Fe(NO.sub.3).sub.3•9H.sub.2O) 404 0.1 Magnesium Sulfate (MgSO.sub.4) (anhyd.) 120 97.67 Potassium Chloride (KCl) 75 400 Sodium Bicarbonate (NaHCO.sub.3) 84 3700 Sodium Chloride (NaCl) 58 6400 Sodium Phosphate monobasic (NaH.sub.2PO.sub.4•H.sub.2O) 138 125 D-Glucose 180 4500

Example 4

[0082] This example provided a method for preparing block-shaped porcine-derived cultured meat the same as Example 2 with exception that the adherent culture method was replaced with the suspension culture method by means of loading on the surface of a microcarrier microsphere during the in vitro culture of porcine skeletal muscle satellite cells in step (2).

Example 5

[0083] This example provided a method for preparing block-shaped chicken-derived cultured meat the same as Example 3 with exception that the adherent culture method was replaced with the suspension culture method by means of loading on the surface of a microcarrier microsphere during the in vitro culture of chicken skeletal muscle satellite cells in step (2).

Comparative Example 1

[0084] This comparative example provided a method for preparing porcine-derived cultured meat the same as Example 2 with exception that the tissue explant adherent method was replaced with the digestion tissue explant adherent method when extracting skeletal muscle satellite cells in step (1).

Comparative Example 2

[0085] This comparative example provided a method for preparing porcine-derived cultured meat the same as Example 1 with exception that static culture in an incubator was used, instead of the special culture apparatus for a 3D biological tissue provided in Example 1, during the proliferation culture and differentiation of the 3D porcine skeletal muscle satellite cell tissue in step (4).

[0086] FIGS. 2 and 3 illustrate the morphology of porcine skeletal muscle satellite cells cultured for 48 h and 72 h in step (2) of Example 2 of the present disclosure, respectively. As can be seen from the figures, the primary porcine skeletal muscle satellite cells were fusiform. With the extension of the culture time, tentacles gradually increased, showing a satellite radial cell morphology, consistent shape, and rapid proliferation.

[0087] FIGS. 4 and 5 illustrate the morphology of chicken skeletal muscle satellite cells cultured for 24 h and 48 h in step (2) of Example 3 of the present disclosure, respectively. As can be seen from the figures, the extraction efficiency of primary chicken skeletal muscle satellite cells was high (approximately 5×10.sup.5 primary chicken skeletal muscle satellite cells were obtained per gram of chicken skeletal muscle satellite cell tissue), the cell growth rate was extremely fast, and the morphology was stable.

[0088] FIG. 6 illustrates the morphology of porcine skeletal muscle satellite cells cultured for 72 h in step (2) of Example 4 of the present disclosure; FIG. 7 illustrates the morphology of chicken skeletal muscle satellite cells cultured for 48 h in step (2) of Example 5 of the present disclosure. According to the comparison of FIGS. 6 and 7 versus FIGS. 3 and 5, during the in vitro culture, both the adherent culture method and the suspension culture method by means of loading on the surface of a microcarrier microsphere were feasible. However, the suspension culture method by means of loading on the surface of a microcarrier microsphere led to a higher cell expansion density. According to calculations, if a microcarrier microsphere are used as carriers for the growth of skeletal muscle satellite cells (MSCs) in suspension culture spinner flasks for suspension culture, 3.5×10.sup.7 chicken MSCs or 2.5×10.sup.7 porcine MSCs can be produced per milliliter of culture system, which can increase the use efficiency of the culture medium by 20-50 times; moreover, the spinner flasks were repeatable, which may effectively reduce the production cost.

[0089] FIG. 8 illustrates the morphology of porcine skeletal muscle satellite cells cultured for 5 days in step (2) of Example 2 of the present disclosure. As can be seen from the figure, the skeletal muscle satellite cells in the 3D culture state proliferated rapidly and grew substantial tentaculiform skeletons, and the mutual contact and fusion occurred, indicating that the cells grew well in the bioink matrix.

[0090] FIGS. 9 and 10 illustrate the morphology of a 3D porcine skeletal muscle satellite cell tissue cultured for 10 and 15 days in Example 2 of the present disclosure, respectively. It can be seen that the block-shaped cultured meat had been substantially formed.

[0091] FIG. 11 illustrates the morphology of a 3D chicken skeletal muscle satellite cell tissue cultured for 15 days in Example 3 of the present disclosure. It can be seen that the block-shaped cultured meat with a diameter of 1 cm had been formed.

[0092] FIG. 12 illustrates the myogenesis of porcine muscle stem cells in Example 2 of the present disclosure; FIG. 13 illustrates the myogenesis of chicken muscle stem cells in Example 3 of the present disclosure. As can be seen from the figures, the cells were in a fully extended state and differentiate into 3D fibrous myotubes in situ, and the cell proliferation and differentiation viability still existed after 3D forming.

[0093] FIG. 14 illustrates the morphology of porcine skeletal muscle satellite cells extracted by the digestion tissue explant adherent method in Comparative Example 1 of the present disclosure; FIG. 15 illustrates the morphology of porcine skeletal muscle satellite cells extracted by the tissue explant adherent method in Example 2 of the present disclosure. As can be seen from the figures, extraction by the tissue explant adherent method reduced the damage to skeletal muscle satellite cells, and the animal skeletal muscle satellite cells obtained exhibited high purity, uniform and stable morphology, stable stemness maintenance, high regenerative activity, and fast expansion rate.

[0094] FIG. 16 illustrates the morphology of porcine skeletal muscle satellite cells after static culture in Comparative Example 2 of the present disclosure. As can be seen from the figure, substantial cell deaths occurred in the middle of the tissue explant, which can be in sharp contrast with FIG. 8.

[0095] The results show that the examples of the present disclosure provide an effective method for preparing block-shaped cultured meat. The method for extracting animal skeletal muscle satellite cells provided in the examples of the present disclosure provides seed cells for cultured meat production with high efficiency. The in vitro expansion method of animal skeletal muscle satellite cells provided in the examples of the present disclosure provides substantial animal skeletal muscle satellite cells for the production of the cultured meat in a relatively short period of time at low cost with simple operations. The 3D forming method of animal skeletal muscle satellite cells provided in the examples of the present disclosure realizes rapid and automatic production. The method of proliferation culture and differentiation of the cell tissue provided in the examples of the present disclosure realizes the overall culture and differentiation of the cultured meat, which is beneficial to enhance the food properties of the cultured meat and improve the eating taste of the cultured meat.

[0096] Finally, it should be noted that the above examples are only intended to illustrate, but not to limit, the technical solutions of the present disclosure; although the present disclosure has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that: the technical solutions recorded in the foregoing examples may be still modified, or some of the technical features may be equivalently substituted; these modifications or substitutions do not cause the essence of the corresponding technical solutions to depart from the spirit and scope of the technical solutions of the examples of the present disclosure.

INDUSTRIAL APPLICABILITY

[0097] The present disclosure provides a special culture apparatus for a 3D biological tissue, and a method for preparing block-shaped cultured meat. The special culture apparatus for a 3D biological tissue includes a 3D biological tissue culture tank for accommodating a 3D biological tissue, and a liquid storage tank for containing a culture medium; the 3D biological tissue culture tank is connected to the liquid storage tank by means of a pipeline to form a circuit for the culture medium to circularly flow; and an opening of the 3D biological tissue culture tank is provided with a sealing plug, an inner side of the sealing plug is provided with a plurality of culture medium infusion needles that penetrate the 3D biological tissue when in use. The special culture apparatus for a 3D biological tissue provided by the present disclosure may effectively guarantee the uniform growth and differentiation of cells in the 3D biological tissue and is suitable to prepare the block-shaped cultured meat. The method for preparing block-shaped cultured meat provided by the present disclosure may significantly increase the preparation scale of cultured animal meat, and truly achieve the preparation of high-quality block-shaped cultured meat with a chewing sensation of meat products, which has excellent economic value and application prospect.