Method for preparing pet food by using animal-derived muscle stem cells, and product obtained therefrom

Abstract

A method for preparing pet food by using animal-derived muscle stem cells, and a product obtained therefrom. The method comprises an integrated separation and culture technique for highly active muscle and adipose progenitor cells, and a large-scale three-dimensional culture and differentiation technique combined with a cell scaffold. The method can significantly improve the efficiency of separating myogenic and adipogenic progenitor cells and reduce costs.

Description

A method for preparing pet food by animal-derived muscle stem cells and the resulting product TECHNICAL FIELD

[0001] The present application belongs to the field of stem cell application, and particularly relates to a method for preparing pet food by animal-derived muscle stem cells and the resulting product. BACKGROUND

[0002] Mouse meat is a kind of food meat that cats and other pets naturally like. Since mouse meat is rich in a large amount of protein, balanced amino acids, lipids and vitamins and other nutrients, it can make cats more healthy and lively, and the fur color is bright. Most importantly, mouse meat is rich in taurine and other ingredients necessary for cats, which can enhance the night vision of cats. A long-term lack of taurine in cats will cause night blindness. In addition, a lack of taurine will also cause a decrease in the reproductive ability of cats, mainly manifested as pregnancy failure, miscarriage and stillbirth. In addition, it will cause abnormal development of the hind legs of kittens in the growth stage, such as excessive abduction, paralysis, decrease in muscle volume and tension, etc., leading to abnormal gait; in addition, the weight of the cerebellum of kittens will also decrease. The above clinical symptoms are all manifestations of cerebellar dysfunction and cerebellar immaturity, and the reason is that the lack of taurine causes a delay in cell changes during the maturation of the cerebellum. In addition, taurine plays an important role in the development of the central nervous system of cats and the maintenance of normal function. Taurine is a relatively abundant amino acid in the central nervous system of vertebrates, which participates in maintaining the integrity of cell membrane structure, regulating calcium binding and transport, and is also a permeability regulator and inhibitory neurotransmitter.

[0003] In the case where it is inconvenient to use mice as cat food, although taurine and other ingredients can be added to cat food, the source of meat quality of pet food has always been difficult to guarantee and various problems are easy to occur. In particular, live mice are easy to carry a variety of dangerous bacteria and viruses, and are not suitable as a source of pet food. In recent years, it has attracted widespread attention from global public opinion and markets. The quality of various commodities in the pet food market is uneven, and the meat comes from farmed or wild animals, which has the risk of spreading pathogens in the process of production, sales and use. Due to the complexity of the current large-scale livestock production methods and public health problems, as well as the problems of greenhouse effect, environmental degradation, and animal welfare and protection related to meat production, the growing global demand for meat has caused more and more concerns and negative consequences. The large-scale development of livestock is related to the scale prevalence of foodborne diseases, antibiotic resistance and infectious diseases, and closely affects human health. SUMMARY

[0004] In view of the technical blank in the prior art, the present application provides a method for preparing pet food by animal-derived muscle stem cells.

[0005] Another object of the present application is to provide pet food prepared by the above method.

[0006] The application adopts the technical scheme for realizing the above-mentioned purpose:

[0007] The application provides a method for preparing pet food by animal-derived muscle stem cells, comprising the following steps:

[0008] (1) Integrated separation and culture technology of high-activity muscle and fat precursor cells: muscle and fat tissues are chopped and mixed, and then primary cells are obtained by enzymolysis, and then two groups of cells are quickly obtained by differential adhesion method, and cells with adipogenic differentiation ability are obtained as fat precursor cells; cells with myogenic differentiation ability with high expression of ALDH1 / VCAM1 are screened out according to the expression levels of ALDH1 and VCAM1 by using a flow cytometer, and the cells are used as muscle precursor cells;

[0009] (2) Large-scale three-dimensional culture and differentiation technology combined with cell scaffolds: muscle precursor cells and fat precursor cells in the logarithmic growth phase (the logarithmic growth phase refers to a period of time after the primary cells are cultured into a rapid growth stage, and the application is the 4th to 6th generation) are seeded into a cell growth scaffold prepared from the cell extracellular matrix of the de-cellularized plant leaves at a certain ratio, a culture medium is added, and then expansion culture is carried out, and then differentiation culture is carried out to obtain pet food.

[0010] Further, in step (1), the specific process of enzymolysis is that: the chopped and mixed muscle and fat tissues are added with digestive enzymes and trypsin for enzymolysis; the digestive enzymes are a mixed enzyme of type I collagenase and neutral protease II, the final concentration of type I collagenase is 0.1%-0.5% (w / v), the final concentration of neutral protease II is 0.2-0.5% (w / v), and the digestion time is 1-2 hours; the final concentration of trypsin is 1%-5% (w / v), and the digestion time is 0.5-1 hour.

[0011] Further, in step (1), the two groups of cells are specifically: (1) a mixed cell group of fat mesenchymal stem cells and muscle mesenchymal stem cells adhering within 2-4 hours; (2) muscle precursor cells adhering after 48-60 hours.

[0012] Further, in step (1), the muscle precursor cells with high expression of ALDH1 / VCAM1 screened and separated account for 10%-40% of the total amount.

[0013] Further, in step (2), the ratio of the muscle precursor cells and the fat precursor cells is 4:1-10:1.

[0014] Further, in step (2), the preparation method of the cell growth scaffold is: the plant leaf is treated with 0.5-2.5% SDS and 2-4% triton-x100 in sequence for 2-6 hours, so that the acellular extracellular matrix thin layer is obtained, and the acellular extracellular matrix thin layer is washed with water and sterilized.

[0015] Further, in step (2), the culture medium is DMEM containing a Jagged1 polypeptide (20 mg / L), NMN (300 mg / L) and 10 mg / L FBS, and the expansion culture time is 10-15 days.

[0016] Further, in step (2), the differentiation is specifically: after expansion culture, the culture medium is replaced with myogenic differentiation culture solution, and myogenic differentiation is carried out for 7-10 days; then the myogenic differentiation culture solution is replaced with adipogenic differentiation culture solution again, and adipogenic differentiation is carried out for 3-6 days.

[0017] Further, the composition of the myogenic differentiation culture solution is: 95% (v / v) DMEM culture medium, 3% (v / v) fetal bovine serum, 2% (v / v) 100X glutamine, 35 mg / mL glucose and 300 mg / L NMN; and the composition of the adipogenic differentiation culture solution is: 83% (v / v) DMEM culture medium, 15% (v / v) fetal bovine serum, 2% (v / v) 100X glutamine, 35 mg / mL glucose, 300 mg / L NMN, 10 μM Y-27632, 1.5 μg / mL insulin and 1.5 mg / mL phosphatidylcholine.

[0018] The application further provides a pet food prepared by the above method.

[0019] The muscle and fat tissue used in the application is extracted from healthy young mice; but the source is not limited to mice. The application can also be extended to artificial rabbit meat or beef as dog food and the like, so as to realize new selection of various pet food raw materials which are environmentally friendly, safe and free of animal ethics problems.

[0020] The cell growth scaffold used in the application can adopt any plant leaf; and the application adopts a mint leaf.

[0021] The application has the following beneficial effects:

[0022] (1) The cell culture meat technology provided by the application can alleviate the harm related to the livestock industry to a certain extent. The cell culture meat (CBM) technology based on muscle stem cells can realize the direct production of mouse meat and other animal meat, and no longer needs wild or farmed animals with the risk of pathogen hazards such as infectious diseases. Artificial meat is not only nutritious, but also does not need to add various ingredients, and can improve nutrition and taste by combining a certain ratio of adipose tissue; at the same time, the method can also significantly improve the efficiency of the separation of myogenic and lipogenic precursor cells, and reduce the cost.

[0023] (2) The method provided by the application is based on the successful extraction of high-activity muscle and fat precursor cells from mice and the in-vitro myofiber and adipogenic differentiation, and is based on the high-activity muscle and fat precursor cell acquisition method, the efficient 3D cell growth and differentiation system, and the efficient cell culture solution preparation method. BRIEF DESCRIPTION OF DRAWINGS

[0024] Fig. 1 is a technical route schematic diagram of the preparation method provided by the application;

[0025] Fig. 2 is a cell culture meat tissue prepared by the embodiment 1 of the application;

[0026] Fig. 3 is a comparison diagram of the number of cells in different culture modes. DETAILED DESCRIPTION

[0027] The technical solutions of the application will be further explained and described below through specific embodiments.

[0028] The preparation method provided by the application is shown in the schematic diagram of Fig. 1, and the specific operation steps include:

[0029] (1) Rapid and efficient acquisition of two groups of precursor cells for forming muscle and fat:

[0030] Muscle and fat tissues from healthy young mouse individuals are extracted, mixed and chopped at a certain ratio (5:1 ~ 1:1), and digestive enzymes and trypsin are added for enzymolysis to release individual cells. The digestive enzyme is a mixed enzyme of type I collagenase and neutral protease II, the final concentration of type I collagenase is 0.1%~0.5% (w / v), the final concentration of neutral protease II is 0.2~0.5% (w / v), and the digestion time is about 1-2 hours. The final concentration of trypsin is 1%~5% (w / v), and the digestion time is about 0.5-1 hour.

[0031] Then the primary cells are divided into two groups using differential adherence method, i.e. the different adherence speed of different cells on the surface of collagen type 1 embedded cell culture plate, which are (1) the mixed cell group of adipose-derived mesenchymal stromal / stem cells (ADSC) and muscle-derived mesenchymal stromal / stem cells (MD-MSCs) adhered within 2-4 hours, and (2) muscle progenitor cells (MPCs) adhered after 48-60 hours. The culture condition used in this stage is DMEM medium containing 5-15% FBS, 37 o C, 5% CO2.

[0032] The mixed cells of adipose-derived mesenchymal stromal / stem cells and muscle-derived mesenchymal stromal / stem cells are used for mass expansion and adipogenic differentiation to produce adipose tissue. The muscle progenitor cells are then screened for high-activity muscle progenitor cells with high expression of ALDH1 and VCAM1 using flow cytometry according to the expression levels of ALDH1 and VCAM1. The high-activity muscle progenitor cells (10-40%) sorted using flow cytometry are further cultured and myogenic differentiated to produce muscle tissue.

[0033] (2) Obtain the acellular extracellular matrix sheet: the plant leaves (such as mint) are treated with 0.5-2.5% SDS and 2-4% triton-x100 by shaking for 2-6 hours in sequence to obtain the acellular extracellular matrix sheet, which is then washed with water and sterilized for use as a cell culture substrate.

[0034] (3) 3D cell culture combined with extracellular matrix: the muscle progenitor cells and adipose precursor cells in logarithmic growth phase are mixed at a certain ratio and introduced into the acellular extracellular matrix, and large-scale cell culture expansion is carried out using this 3D extracellular matrix. The culture solution is DMEM containing Jagged1 polypeptide (10-30 mg / L), NMN (200-400 mg / L) and 10-15% FBS, 37 o C, and cultured for 10-15 days.

[0035] (4) Myogenic and adipogenic differentiation:

[0036] The culture medium is then changed to myogenic differentiation medium: 95-98.5% (v / v) DMEM medium, 1-3% (v / v) fetal bovine serum, 0.5-2% (v / v) 100X glutamine, 35-60 mg / mL glucose, 200-400 mg / L NMN. The myogenic differentiation is performed for 7-10 days, during which the muscle precursor cells will differentiate into mature muscle fibrocytes.

[0037] The culture medium is then changed to adipogenic differentiation medium: 83-89.5% (v / v) DMEM medium, 10-15% (v / v) fetal bovine serum, 0.5-2% (v / v) 100X glutamine, 35-60 mg / mL glucose, 200-400 mg / L NMN, 5-10 mM Y-27632, 1.5-3 mg / mL insulin, and 1.5-3 mg / mL phosphatidylcholine. The adipogenic differentiation is performed for 3-6 days, with the specific time arranged according to the designed ratio of muscle / fat in the target product design.

[0038] Example 1

[0039] (1) Obtain two groups of mouse precursor cells for forming muscle and fat:

[0040] Muscle and fat tissues from healthy young mice are extracted, mixed at a ratio of 1:1 (2 grams each), and chopped, and digestive enzymes and trypsin are added for enzymolysis to release individual cells. The digestive enzymes are a mixture of type I collagenase and neutral protease II, with a final concentration of 0.2% (w / v) for type I collagenase and a final concentration of 0.2% (w / v) for neutral protease II, and the digestion time is about 1 hour. The final concentration of trypsin is 1% (w / v), and the digestion time is about 0.5 hours.

[0041] The primary cells are then divided into two groups using the differential adhesion method, i.e., the characteristics of different cells adhering to the surface of a type I collagen-embedded cell culture plate at different speeds: (1) a mixed cell population of adipose-derived mesenchymal stromal / stem cells (ADSCs) and muscle-derived mesenchymal stromal / stem cells (MD-MSCs) that adhere within 2 hours (used together as precursor cells for adipogenic differentiation), and (2) muscle precursor cells (MPCs) that adhere after 48 hours. The conditions used for this stage of culture are 10% FBS DMEM medium, 37 o C, 5% CO2.

[0042] The muscle precursor cells are then screened for high expression of ALDH1 / VCAM1 using flow cytometry according to the expression levels of ALDH1 and VCAM1, respectively. The high-activity muscle precursor cells with high expression of ALDH1 / VCAM1 screened using flow cytometry (20%) are then cultured in large quantities and subjected to myogenic differentiation to produce muscle tissue.

[0043] (2) Obtain a thin layer of acellular extracellular matrix: the mint leaves are sequentially treated with 1% SDS and 3% triton-x100 by shaking for 4 hours to obtain a thin layer of acellular extracellular matrix, which is then washed with water and sterilized to obtain a cell growth scaffold, which is used as a cell culture substrate;

[0044] (3) 3D cell culture combined with extracellular matrix: muscle precursor cells and fat precursor cells in the logarithmic growth phase are mixed at a certain ratio (8:1) and introduced into the acellular mint leaf cell culture substrate. This 3D extracellular matrix is used for large-scale cell culture expansion. The culture medium is DMEM containing Jagged1 polypeptide (20 mg / L), NMN (300 mg / L), and 10 mg / L FBS, and the culture is carried out for 14 days.

[0045] (4) Myogenic and adipogenic differentiation:

[0046] The culture medium is replaced with myogenic differentiation culture medium: 95% (v / v) DMEM medium, 3% (v / v) fetal bovine serum, 2% (v / v) 100X glutamine, 35 mg / mL glucose, and 300 mg / L NMN. Myogenic differentiation is carried out for 7 days, during which the muscle precursor cells will differentiate into mature muscle fiber cells.

[0047] Then replace it with adipogenic differentiation culture medium: 83% (v / v) DMEM medium, 15% (v / v) fetal bovine serum, 2% (v / v) 100X glutamine, 35 mg / mL glucose, 300 mg / L NMN, 10 μM Y-27632, 1.5 μg / mL insulin, and 1.5 mg / mL phosphatidylcholine. Adipogenic differentiation is carried out for 4 days, and the specific time can also be arranged according to the design ratio of muscle / fat in the target product design.

[0048] Ester precursor cells and myogenic precursor cells are added to the extracellular matrix for cell culture as shown in Figure 2. Using the method provided by the present application, mature muscle fibers can be obtained to prepare pet food.

[0049] Comparative Example 1

[0050] Step (1) Obtain two groups of mouse precursor cells for forming muscle and fat as in Example 1;

[0051] Step (3) uses 2D cell culture: muscle precursor cells and fat precursor cells in logarithmic growth phase are mixed at a certain ratio (8:1) for cell culture expansion, using culture solution DMEM containing Jagged1 polypeptide (20 mg / L), NMN (300 mg / L) and 10 mg / L FBS, and cultured for 14 days.

[0052] Other same as Example 1.

[0053] By comparing the difference in cell number between 2D (flat culture) and cell scaffold culture cells after 5 days, it is shown that the number of cells cultured using the cell scaffold is significantly increased (Figure 3). After continuing to complete the entire growth and differentiation process, the final cell number difference between the two is more than 4 times. Therefore, the use of cell scaffold significantly improves efficiency and yield.

Claims

1. A method for preparing a pet food from animal-derived muscle stem cells, characterized by, The method comprises the following steps: (1) integrated separation and culture technology of high-activity muscle and fat precursor cells: primary cells are obtained by chopping and mixing muscle and fat tissues and then enzymatic digestion, and two groups of cells are quickly obtained by differential adhesion method, and cells with adipogenic differentiation ability are obtained as fat precursor cells; ALDH1 / VCAM1 high expression cells with myogenic differentiation ability are screened according to the expression levels of ALDH1 and VCAM1 by using a flow cytometer, and the cells are used as muscle precursor cells; (2) large-scale three-dimensional culture differentiation technology combined with cell scaffolds: muscle precursor cells and fat precursor cells in logarithmic growth phase are seeded into a cell growth scaffold prepared from a decellularized extracellular matrix of a plant leaf at a certain ratio, a culture medium is added, and then expansion culture is carried out, and then differentiation culture is carried out to obtain pet food.

2. The method of claim 1, wherein, In step (1), the specific process of enzymatic digestion is: adding digestive enzymes and trypsin to the chopped and mixed muscle and fat tissues for enzymatic digestion; the digestive enzymes are a mixture of type I collagenase and neutral protease II, the final concentration of type I collagenase is 0.1%-0.5% (w / v), the final concentration of neutral protease II is 0.2-0.5% (w / v), and the digestion time is 1-2 hours; the final concentration of trypsin is 1%-5% (w / v), and the digestion time is 0.5-1 hour.

3. The method according to claim 1 or 2, characterized in that, In step (1), the two groups of cells are specifically: (1) a mixed cell group of fat mesenchymal stem cells and muscle mesenchymal stem cells adhering within 2-4 hours; (2) muscle precursor cells adhering after 48-60 hours.

4. The method according to claim 1 or 3, characterized in that, In step (1), the muscle precursor cells with high expression of ALDH1 / VCAM1 screened and separated account for 10-40% of the total amount.

5. The method according to any one of claims 1 to 4, characterized in that, In step (2), the ratio of muscle precursor cells to fat precursor cells is 4:1-10:

1.

6. The method of claim 1, wherein, In step (2), the preparation method of the cell growth scaffold is: treating a plant leaf with 0.5-2.5% SDS and 2-4% triton-x100 in sequence for 2-6 hours by shaking, thereby obtaining a thin layer of decellularized extracellular matrix, and then washing with water and sterilizing to obtain.

7. The method according to any one of claims 1 to 6, characterized in that, In step (2), the culture medium is DMEM containing Jagged1 polypeptide (20 mg / L), NMN (300 mg / L), and 10 mg / L FBS; the expansion culture time is 10-15 days.

8. The method according to any one of claims 1 to 7, characterized in that, In step (2), the differentiation is specifically: after expansion culture, the culture medium is replaced with myogenic differentiation culture solution for myogenic differentiation for 7-10 days; then it is replaced with adipogenic differentiation culture solution again for adipogenic differentiation for 3-6 days.

9. The method of claim 8, wherein, The myogenic differentiation medium comprises 95% (v / v) DMEM medium, 3% (v / v) fetal bovine serum, 2% (v / v) 100X glutamine, 35 mg / mL glucose, and 300 mg / L NMN; and the adipogenic differentiation medium comprises 83% (v / v) DMEM medium, 15% (v / v) fetal bovine serum, 2% (v / v) 100X glutamine, 35 mg / mL glucose, 300 mg / L NMN, 10 μM Y-27632, 1.5 μg / mL insulin, and 1.5 mg / mL phosphatidylcholine.

10. A pet food prepared by the method of any one of claims 1 to 9.

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