A multi-enzyme preparation for industrial cell dissociation, its method of use and application

Abstract

This invention discloses a multi-enzyme preparation for industrial cell dissociation, its usage method, and applications, belonging to the field of seed cell extraction technology for industrial meat culture. The invention employs a multi-enzyme preparation composed of component 1 and component 2; component 1 includes neutral protease, hyaluronidase, and elastase; component 2 includes matrix metalloproteinase, papain, and plasmin. The invention also discloses the preparation method and applications of the multi-enzyme preparation. The rational enzyme ratio and optimized treatment conditions of this invention help protect cell membrane integrity, maintain high cell activity, and significantly improve dissociation efficiency and cell recovery. The compound enzyme preparation is highly adaptable, applicable to various tissue types and dissociation requirements, and facilitates standardized and large-scale production. Combined with automated dissociation equipment, the compound enzyme preparation enables efficient and stable industrial operation, providing important technical support and broad application prospects for the cultured meat industry, tissue engineering, and cell therapy.

Description

Technical Field

[0001] This invention relates to the field of seed cell extraction technology for industrially cultured meat, specifically to a multi-enzyme preparation for industrial cell dissociation and its usage and application. Background Technology

[0002] Cell dissociation is a crucial step in cell culture, tissue engineering, and cultured meat production, aiming to efficiently and gently isolate highly viable single cells from tissue samples. Currently, commonly used enzymes for cell dissociation include matrix metalloproteinases (MMPs), trypsin, hyaluronidase, and elastase. These enzymes achieve cell-to-cell or cell-to-matrix separation by degrading specific components of the extracellular matrix (such as collagen, hyaluronic acid, and elastin). However, traditional enzyme preparations suffer from critical bottlenecks such as high requirements for enzymatic hydrolysis, cumbersome operation, high cost, limited dissociation effects, low efficiency, and extremely low total cell count. Furthermore, the industrial applications of technologies like biomanufacturing, cell agriculture, and cultured meat place extremely high demands on cell viability and total cell count. Traditional methods are limited to laboratory-scale research and cannot address the key issues highlighted in industrialization.

[0003] Existing single-enzyme or simple enzyme combination methods are suitable for small-scale, laboratory-scale cell extraction, but they have significant limitations in high-density, high-volume industrial dissociation scenarios. Single-enzyme methods often have low dissociation efficiency, requiring longer processing times in complex tissues, which may lead to decreased cell viability. Furthermore, due to the significant differences in extracellular matrix composition among different tissue types, single enzymes cannot achieve comprehensive effects and have poor adaptability. More importantly, these methods fail to meet the demands of efficient, stable, and large-scale operations in industrial production, and cannot simultaneously achieve both cell yield and quality.

[0004] The development of combined enzyme formulations offers a novel solution for industrialized cell dissociation. Through the synergistic action of multiple enzymes, combined enzyme formulations can comprehensively degrade diverse extracellular matrix components in complex tissues, thereby significantly improving dissociation efficiency. Summary of the Invention

[0005] The purpose of this invention is to provide a multi-enzyme preparation for industrial cell dissociation, along with its usage and applications. Addressing the problems of low efficiency, insufficient cell recovery, and difficulty in ensuring cell viability in existing enzymatic hydrolysis technologies for industrial cell dissociation, this invention innovatively proposes a multi-enzyme preparation and its usage method to meet the needs of large-scale cell culture.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a multi-enzyme preparation for industrial cell dissociation, wherein the multi-enzyme preparation comprises component 1 and component 2; component 1 includes neutral protease, hyaluronidase, and elastase; and component 2 includes matrix metalloproteinase, papain, and plasmin.

[0007] Preferably, in component 1: the concentration of the neutral protease is 0.7 mg / ml, the concentration of the hyaluronidase is 0.4 mg / ml, and the concentration of the elastase is 0.3 mg / ml;

[0008] In component 2: the concentration of the matrix metalloproteinase is 0.3 mg / ml, the concentration of the papain is 0.5 mg / ml, and the concentration of the plasmin is 0.5 mg / ml.

[0009] Preferably, the matrix metalloprotein has a number-average molecular weight of 68,000 to 125,000 and an optimal pH of 6.3 to 8.8; the matrix metalloproteinase is matrix metalloproteinase II.

[0010] Preferably, the neutral protease is neutral protease II, with an optimal pH of 7.5.

[0011] Preferably, the matrix metalloproteinase solution, the papain solution, and the plasmin solution can all be prepared using any of the following reagents: TESCA buffer, calcium-magnesium PBS, calcium-magnesium Hanks solution, or serum-free culture medium.

[0012] Preferably, the matrix metalloproteinase II powder is pre-dissolved using DMSO reagent as a co-solvent.

[0013] Preferably, the neutral protease, the hyaluronidase, and the elastase are all prepared from calcium- and magnesium-free PBS, calcium- and magnesium-free Hanks solution, or serum-free culture medium.

[0014] A method for using a multi-enzyme preparation for industrial cell dissociation, wherein the multi-enzyme preparation is prepared as follows:

[0015] S1: Prepare the solution of component 1 and the solution of component 2:

[0016] The solution of component 1 is prepared by dissolving the neutral protease, the hyaluronidase, and the elastase in a calcium- and magnesium-free PBS solution.

[0017] The solution of component 2 is prepared by dissolving the matrix metalloproteinase, the papain, and the plasmin in a calcium- and magnesium-containing Hanks solution.

[0018] S2: Preparation of target enzymatic hydrolysate: Dissect the muscle tissue of 12-day-old chicken embryos; pre-treat the muscle tissue to a thickness of 0.5–1.0 mm. 3 Small pieces of meat;

[0019] S3: Preliminary tissue digestion and dissociation: Using the solution of component 1, the tissue fragments are enzymatically digested and then dissociated using a tissue dissociation apparatus to obtain a preliminary tissue digestion and dissociation solution;

[0020] S4: Secondary digestion and dissociation of tissue: Add component 2 solution to the initial digestion and dissociation solution of the tissue, perform secondary enzymatic digestion, and dissociate the tissue using a tissue dissociation apparatus;

[0021] S5: Obtain a single-cell suspension: Filter using a filter, collect the supernatant and centrifuge, then resuspend using cell culture medium.

[0022] Preferably, in step S2: the muscle tissue mass is 5g; the pre-treated muscle tissue diced meat particle size is 0.5mm;

[0023] The specific steps of S3 are as follows: using the solution of component 1, the tissue fragments are enzymatically digested at 37°C for 10 min, and then dissociated in a tissue dissociator for 30 s to obtain a preliminary tissue digestion and dissociation solution;

[0024] The specific steps of S4 are as follows: Add component 2 solution to the initial digestion and dissociation solution of the tissue, perform a second digestion at 37°C for 5 minutes, and dissociate the tissue using a tissue dissociator for 30 seconds;

[0025] The specific steps of S5 are as follows: filter using a 100μm filter, collect the supernatant and centrifuge at 1000rpm for 3min, and resuspend in 20% FBSDMEM cell culture medium.

[0026] An application of a multi-enzyme preparation for industrial cell dissociation or a method of using a multi-enzyme preparation for industrial cell dissociation, characterized in that: the multi-enzyme preparation is applied to a single-cell suspension of muscle tissue for preparing chicken embryos; the multi-enzyme preparation is applied to dissociate and obtain the required number of seed cells for the number of muscle cells in industrial-grade chicken embryos required for the meat industry.

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] (1) This invention significantly improves desorption efficiency and cell activity.

[0029] (2) The present invention can efficiently obtain seed cells for meat industry cultivation, and the cell acquisition volume is more than 10,000 times that of traditional dissociation methods, which is suitable for large-scale cell culture.

[0030] (3) The enzymes used in this invention have excellent stability and biocompatibility, ensuring the quality and purity of the cells.

[0031] (4) The present invention automates and streamlines the separation process, which facilitates industrialized mass production and process-oriented quality control.

[0032] (5) This invention not only solves the key problems in the prior art, but also broadens the application prospects of cell dissociation technology in food science, cell agriculture and other fields, and provides reliable technical support for the efficient development of the meat industry. Attached Figure Description

[0033] Figure 1 These are microscopic images of tissue and cell suspensions after trypsin digestion and dissociation in Experiment 1 of this invention.

[0034] Figure 2 These are microscopic images of tissue and cell suspensions after digestion and dissociation of matrix metalloproteinases, Comparative Example 1 of this invention.

[0035] Figure 3 These are microscopic images of tissue and cell suspensions after hyaluronidase digestion and dissociation, Comparative Example 2 of this invention.

[0036] Figure 4 These are microscopic images of tissue and cell suspensions after digestion and dissociation of neutral protease in Comparative Example 3 of this invention.

[0037] Figure 5 These are microscopic images of tissue and cell suspensions after digestion and dissociation of elastase, Comparative Example 4 of this invention.

[0038] Figure 6 These are microscopic images of tissue and cell suspensions after digestion and dissociation of a mixture of papain and plasmin in Comparative Example 5 of this invention.

[0039] Figure 7 These are microscopic images of tissue and cell suspensions after digestion and dissociation of the complex enzyme I in Experiment Example 2 of this invention.

[0040] Figure 8 These are microscopic images of tissue and cell suspensions after digestion and dissociation of complex enzyme II in Comparative Example 6 of this invention.

[0041] Figure 9 These are microscopic images of tissue and cell suspensions after digestion and dissociation of complex enzyme III in Comparative Example 7 of this invention.

[0042] Figure 10 These are microscopic images of tissue and cell suspensions after digestion and dissociation of complex enzyme IV in Comparative Example 8 of this invention.

[0043] Figure 11 These are microscopic images of the tissue and cell suspensions after digestion and dissociation of the complex enzyme V in Comparative Example 9 of this invention.

[0044] Figure 12These are microscopic images of tissue and cell suspensions after digestion and dissociation of compound enzyme IV (multi-enzyme preparation) in Example 1 of the present invention, and microscopic images of cells cultured for 1 day and 2 days after dissociation.

[0045] Figure 13 The cell count results are as follows: after the cell suspension of the compound enzyme IV (multi-enzyme preparation) in Example 1 of this invention was digested and dissociated, and then diluted 100 times. Detailed Implementation

[0046] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials can be obtained commercially.

[0047] The neutral protease, hyaluronidase, elastase, matrix metalloproteinase, papain, and other raw materials used below were all purchased from Beijing Solarbio.

[0048] Muscles consist of myofibrils, muscle fibers, and muscle bundles, which are respectively wrapped by the perimysium, endomysium, and epimysium. The connective tissue of each membrane is continuous, and blood vessels and nerves are distributed to all parts of the muscle along the connective tissue membranes. Muscle tissue is a complex structure, and obtaining muscle cells requires layer-by-layer enzymatic digestion of each part. To efficiently obtain muscle cells, based on the characteristics of muscle tissue and the enzymatic digestion range of commercially available digestive enzymes, trypsin, hyaluronidase, neutral protease, matrix metalloproteinases, elastase, and papain were selected to digest fragments of chicken embryo muscle tissue, and the enzymatic digestion effect was recorded.

[0049] Trypsin is currently the most widely used digestive reagent. It removes intercellular mucoproteins and glycoproteins by acting on peptide bonds linked to lysine or arginine, thereby affecting the cytoskeleton and thus separating cells.

[0050] Experimental Example 1

[0051] The specific steps are as follows:

[0052] S1: Enzyme solution preparation: Select 0.25% commercially available enzyme solution without EDTA for pancreatic enzyme solution.

[0053] S2: Tissue block pretreatment: Select 12-day-old chicken embryos, dissect to obtain muscle tissue, and cut 5g of muscle tissue block into small meat cubes with a medical scalpel with a particle size of 0.5-1mm. Preferably, the meat cubes have a particle size of about 0.5mm.

[0054] S3: Preliminary tissue digestion: Tissue fragments were digested using a 0.125% EDTA-free trypsin solution at 37°C for 15, 30, 45, and 60 minutes. 5g of muscle tissue was digested using 5ml of enzyme solution.

[0055] S4: Dissociation: After enzymatic digestion at 15-minute intervals, the tissue is dissociated once by a tissue dissociation instrument;

[0056] S5: Obtain single-cell suspensions: After enzymatic hydrolysis and dissociation at 15 min, 30 min, 45 min, and 60 min, tissue and cell suspensions with different degrees of dissociation were obtained. The state of tissues and cells was observed, and the digestive effect of pancreatic enzymes was evaluated.

[0057] In Experiment 1, 0.125% EDTA-free trypsin was used for digestion. Figure 1 As can be seen, no tissue fragments were observed under the microscope after 30 minutes of digestion. From 45 to 60 minutes, the muscle fibers were further digested and shortened. After 60 minutes, the digestive fluid consisted entirely of fiber fragments. Therefore, 0.125% EDTA-free trypsin digestion is very effective, and the digestion time should be controlled between 15 and 30 minutes.

[0058] Comparative Example 1

[0059] The only difference between Comparative Example 1 and Experimental Example 1 is the preparation of the S1 enzyme solution:

[0060] S1-1 Reagent preparation: MMP-2 protein powder, Tris-HCl buffer (20mM, pH 7.5, containing 10mM CaCl2), DMSO (for pre-dissolution).

[0061] Step S1-2:

[0062] 1. Prepare Tris-HCl buffer (1L) on ice: Dissolve 2.422g Tris and 0.147g CaCl2 in 900mL distilled water, adjust the pH to 7.5 and then bring the total volume to 1L.

[0063] 2. Take 1 mg MMP-2 into a sterile 1.5 mL centrifuge tube, add 10 μL DMSO, and let stand for 5 minutes to pre-dissolve.

[0064] 3. Add 490 μL of Tris-HCl buffer to the tube and vortex to mix. After dissolution, stabilize at 4°C for 30 minutes to allow the enzyme to return to its native conformation.

[0065] 4. Sterilize the solution by filtering it through a 0.22μm sterile filter membrane, then aliquot and store at -80℃.

[0066] 5g of muscle tissue was digested using 5ml of enzyme solution.

[0067] Matrix metalloproteinases (MMPs) disperse cells by hydrolyzing proline in the extracellular matrix. MMPs have a strong digestive effect on the extracellular matrix, and this effect is limited to the extracellular matrix, resulting in minimal cell damage. They are suitable for fibrous and relatively hard tissues.

[0068] according to Figure 2 Microscopic observation at scale 1 shows that after 15 minutes of digestion, no tissue fragments were observed under the microscope, and the fibers were relatively long; after 45 minutes of digestion, the fibers shortened further.

[0069] Comparative Example 2

[0070] The only difference between Comparative Example 2 and Experimental Example 1 is the preparation of the S1 enzyme solution: the hyaluronidase powder was prepared into a 0.4 mg / ml hyaluronidase solution using a Hanks solution containing calcium and magnesium ions.

[0071] S1-1 Reagent preparation: Hyaluronidase powder, Hanks solution (pH 7.0, containing 1mM CaCl2), ultrasonic water bath.

[0072] Step S1-2:

[0073] 1. Prepare a Hanks solution containing calcium and magnesium ions and adjust the pH to 7.0.

[0074] 2. Take 10 mg of hyaluronidase powder into a sterile centrifuge tube and add 1 mL of the above buffer solution.

[0075] 3. Place the centrifuge tubes in an ice bath, then place them in an ultrasonic water bath and sonicate at low power for 5 minutes, gently shaking to mix every minute.

[0076] 4. After sonication, slowly aspirate the solution at the bottom with a pipette to ensure that all enzyme particles are completely dissolved.

[0077] 5. Transfer the solution to a 1.5 mL sterile centrifuge tube and use immediately or store at 4°C (for no more than 24 hours).

[0078] 5g of muscle tissue was digested using 5ml of enzyme solution.

[0079] Hyaluronidase reduces the activity of hyaluronic acid by randomly degrading the β-N-acetylglucosamine-[1-4] glycosidic bonds of hyaluronic acid, and is used for the separation of connective tissue.

[0080] according to Figure 3The results of Comparative Example 2 showed that: after 15-45 minutes of digestion, there were no significant changes in fiber length and tissue state under the microscope; after 60 minutes of digestion, most muscle bundles were opened, but the length of muscle fibers was further shortened. Digestion with 0.4 mg / ml hyaluronidase for more than 60 minutes yielded better results.

[0081] Comparative Example 3

[0082] The only difference between Comparative Example 3 and Experimental Example 1 is the preparation of the S1 enzyme solution: the neutral protease powder was prepared into a 0.7 mg / ml neutral protease solution using PBS solution without calcium and magnesium ions.

[0083] S1-1 Reagent Preparation: Neutral protease powder, sterile PBS (pH 7.4), magnetic stirrer, and sterile beaker.

[0084] Step S1-2:

[0085] 1. Weigh 35mg of neutral protease powder, ensuring accuracy using an electronic analytical balance.

[0086] 2. Preheat 50 mL of sterile PBS to 37°C and pour it into a sterile beaker.

[0087] 3. Stir at medium speed on a magnetic stirrer and gradually sprinkle the neutral protease powder into the PBS, avoiding direct addition of powder to prevent clumping.

[0088] 4. Continue stirring for 15 minutes to ensure the enzyme is completely dissolved.

[0089] 5. After dissolution, filter the solution (using a 0.22μm sterile filter membrane) to remove undissolved particles and potential impurities.

[0090] 6. Aliquot the solution into sterile centrifuge tubes (5 mL per tube) and store at -20°C for later use.

[0091] 7. Before each use, melt the enzyme at 4°C, then gently shake to distribute it evenly.

[0092] 5g of muscle tissue was digested using 5ml of enzyme solution.

[0093] Neutral proteases have mild proteolytic activity and do not damage the integrity of cell membranes.

[0094] according to Figure 4 The results of the comparative example 3 show that the neutral protease has a mild enzymatic hydrolysis effect, and the tissue block fiber length is shortened after 45 minutes of digestion.

[0095] Comparative Example 4

[0096] The only difference between Comparative Example 4 and Experimental Example 1 is the preparation of the S1 enzyme solution: the elastase powder was prepared into a 0.3 mg / ml elastase solution using PBS solution without calcium and magnesium ions.

[0097] S1-1 Reagent Preparation: Elastase, calcium and magnesium-free PBS (pH 8.0), sterile test tubes.

[0098] Step S1-2:

[0099] 1. Prepare PBS buffer: Dissolve 0.681g of disodium glycerol-phosphate (Na2HPO4) and 0.522g of monosodium glycerol-phosphate (NaH2PO4) in 100mL of distilled water and adjust the pH to 8.0.

[0100] 2. Add 15 mg of elastase powder to a sterile test tube, and gradually add 1 ml of buffer solution. Vortex at high speed for 3 minutes using a micro vortex mixer to ensure no lumps form.

[0101] 3. Check the solubility. If there are undissolved particles, dissolve them by repeatedly blowing and agitating with a sterile pipette.

[0102] 4. Add another 49ml of buffer solution and stir with a magnetic stirrer for 5 minutes until completely transparent.

[0103] 5. Use immediately after dissolving to avoid deactivation due to prolonged exposure to air.

[0104] 5g of muscle tissue was digested using 5ml of enzyme solution.

[0105] Elastase hydrolyzes elastin by acting on its peptide, amide, and ester bonds, and is used to separate connective tissue and tissues containing a large number of cellular reticular fibers.

[0106] according to Figure 5 Comparative Example 4 showed that after digestion with 0.3 mg / ml elastase for 15 minutes, the tissue was essentially digested and separated under a microscope, with no long fibrous segments. After digestion for 30-60 minutes, no fibrous segments were observed under the microscope. Elastase digestion was highly effective; the longer the digestion time, the more cell debris was generated, resulting in significant cell damage. The digestion time with 0.3 mg / ml elastase should be controlled within 15 minutes or the elastase concentration should be reduced to avoid cell damage.

[0107] Comparative Example 5

[0108] The only difference between Comparative Example 5 and Experimental Example 1 is the preparation of the S1 enzyme solution: Papain powder and plasmin powder were prepared using a weakly acidic PBS solution or Hanks solution containing calcium and magnesium ions. The specific concentration of each enzyme was: a mixed solution of 0.5 mg / ml papain and 0.5 mg / ml plasmin.

[0109] S1-1 Reagent Preparation: Fibrinolytic enzyme powder, papain powder, weakly acidic PBS (CaCl2, pH 6.5).

[0110] Step S1-2:

[0111] 1. Dissolve 0.8g NaCl, 0.144g Na2HPO4, 0.024g KH2PO4, and 0.02g KCl in 100mL of distilled water. After completely dissolving with an appropriate amount of distilled water, add 0.0184g calcium chloride dihydrate (CaCl2·2H2O) and 0.01g magnesium chloride hexahydrate (MgCl2·6H2O). Continue stirring until dissolved. Adjust the pH to 6.5 to prepare weakly acidic PBS.

[0112] 2. Take 25 mg of fibrinolytic enzyme and 25 mg of papain powder into a sterile beaker, and add 50 mL of the above buffer solution.

[0113] 3. Stir manually with a stirring stick for 3 minutes, then stir with a magnetic stirrer at low speed for 15 minutes.

[0114] 4. If the solubility is insufficient, place the beaker in a 37°C constant temperature incubator and let it stand for 10 minutes.

[0115] 5. Transfer the solution to a sterile centrifuge tube and centrifuge at low speed (1000g, 5 minutes) to remove residual particles.

[0116] 6. After filtration through a 0.22μm filter membrane, store on ice. The solution must be used on the same day.

[0117] 5g of muscle tissue was digested using 5ml of enzyme solution.

[0118] Papain is a thiol-containing (-SH) endopeptidase that possesses both protease and esterase activities. It can break down proteins in acidic, neutral, and alkaline environments. Papain is a food additive, characterized by its low cost and easy availability. Fibrinolytic enzymes can break down muscle fibers, but have a weak effect on fibrin.

[0119] according to Figure 6The results of Comparative Example 5 showed that after 15 minutes of digestion, all cells were short fibrous segments under the microscope; as the digestion time increased, the fibrous segments became shorter, but cell debris increased. The mixed solution of 0.5 mg / ml papain and 0.5 mg / ml plasmin had a strong digestive capacity, and the effect was better when the digestion time was controlled within 15 minutes.

[0120] Based on the final results of the single-enzyme solution enzymatic hydrolysis of tissues in Experiment 1 and Comparative Examples 1-5, and according to the main modes of action and principles of various digestive enzymes in the tissue hydrolysis process, they were formulated and used in combination. Experiment 2 and Comparative Examples 6-9 show the experimental steps and final digestion results of different types of compound enzymes.

[0121] Experiment Example 2

[0122] Complex Enzyme I Components: Pancreatic enzyme, neutral protease, hyaluronidase, elastase

[0123] The experimental steps are as follows:

[0124] S1 Complex Enzyme I Preparation: The specific concentrations of each enzyme are: 0.125% pancreatin, 0.7 mg / ml neutral protease, 0.4 mg / ml hyaluronidase, and 0.3 mg / ml elastase.

[0125] S2 Tissue Block Pretreatment: Select 12-day-old chicken embryos, dissect to obtain muscle tissue, and cut 5g of muscle tissue block into small meat cubes with a medical scalpel with a particle size of 0.5-1mm. Preferably, the particle size of the meat cubes is about 0.5mm.

[0126] S3 tissue preliminary digestion: The tissue fragments were digested using compound enzyme I solution at 37°C for 30 min.

[0127] S4 Dissociation: The tissue dissociation apparatus performs dissociation once;

[0128] S5 Obtain single-cell suspensions: Obtain tissue and cell suspensions with different degrees of dissociation, observe the state of tissues and cells, and evaluate the digestion effect of complex enzyme I.

[0129] 5g of muscle tissue was digested using 5ml of enzyme solution.

[0130] according to Figure 7 Experimental Example 2 showed that the cell suspension was poorly digested by the complex enzyme I, which could not completely digest the tight junctions between tissues. Even after 30 minutes of digestion, a large number of tissue fragments remained.

[0131] Comparative Example 6

[0132] The only difference between Comparative Example 6 and Experimental Example 2 is the preparation of S1 complex enzyme II: the specific concentrations of each enzyme are: 0.125% pancreatin, 0.7 mg / ml neutral protease, 0.4 mg / ml hyaluronidase, 0.3 mg / ml elastase, 0.3 mg / ml matrix metalloproteinase, 0.5 mg / ml papain, and 0.5 mg / ml plasmin.

[0133] According to the appendix Figure 8 The observation of the cell suspension in the comparative example 6 showed that the enzymatic digestion effect of compound enzyme II was generally poor. After 30 minutes of digestion and dissociation, there were a small number of myofilaments and some completely digested single cells in the suspension.

[0134] Comparative Example 7

[0135] The difference between Comparative Example 7 and Experimental Example 2 is that

[0136] S1 Complex Enzyme III Preparation: The specific concentrations of each enzyme are: 0.7 mg / ml neutral protease, 0.4 mg / ml hyaluronidase, and 0.3 mg / ml elastase.

[0137] Preliminary digestion of S3 tissue: Digested for 15 min at 37°C using compound enzyme III.

[0138] S4 tissue secondary digestion: Add 0.125% trypsin and digest for 15 min at 37°C.

[0139] 5g of muscle tissue was digested using 5ml of enzyme solution.

[0140] according to Figure 9 The observation of the cell suspension in Comparative Example 7 showed that the enzymatic hydrolysis effect of Complex Enzyme III was too strong. After digestion and dissociation, the suspension contained only fully digested single cells, without tissue fragments or myofilaments, indicating severe cell damage.

[0141] Comparative Example 8

[0142] The difference between Comparative Example 8 and Experimental Example 2 is that

[0143] Preparation of S1 complex enzyme IV:

[0144] Component 1: The specific concentrations of each enzyme are: 0.7 mg / ml neutral protease, 0.4 mg / ml hyaluronidase, and 0.3 mg / ml elastase.

[0145] Component 2: 0.3 mg / ml matrix metalloproteinase, 0.5 mg / ml papain, 0.5 mg / ml plasmin.

[0146] S3 tissue preliminary digestion: Digest with component 1 for 15 min;

[0147] S4 tissue secondary digestion: Add component 2 and digest for 15 min;

[0148] according to Figure 10 The observation of the control group 8 cell suspension showed that the complex enzyme IV had a strong digestive effect. After digestion and dissociation, there were no tissue fragments in the suspension, but a large number of completely digested single cells and a small number of myofilaments, with minimal cell damage. The results indicate that the complex enzyme IV has a highly efficient enzymatic digestive ability to separate cells.

[0149] Comparative Example 9

[0150] The only difference between Comparative Example 9 and Experimental Example 2 is the preparation of S1 complex enzyme V: the specific concentrations of each enzyme are: 0.7 mg / ml neutral protease, 0.4 mg / ml hyaluronidase, and 0.3 mg / ml elastase.

[0151] according to Figure 11 Comparative Example 9: Observation of cell suspension showed that the enzymatic digestion effect of compound enzyme V was poor. After digestion and dissociation, the suspension contained a large number of tissue fragments, a small amount of myofilaments, and a small number of cells. The results indicate that compound enzyme V cannot effectively digest and separate cells.

[0152] Example 1

[0153] The experimental steps are as follows:

[0154] Preparation of S1 complex enzyme IV (i.e., the multi-enzyme preparation mentioned in this invention):

[0155] Component 1: Neutral protease, hyaluronidase, and elastase are dissolved in calcium- and magnesium-free PBS solution. The specific concentrations of each enzyme are: 0.7 mg / ml neutral protease, 0.4 mg / ml hyaluronidase, and 0.3 mg / ml elastase.

[0156] The specific preparation steps for component 1 are as follows:

[0157] Reagent and material preparation:

[0158] Enzyme powder: 70mg neutral protease; 40mg hyaluronidase; 30mg elastase; PBS without calcium and magnesium: pre-prepared (or purchased ready-made), pH 7.4.

[0159] Sterile materials: sterile centrifuge tubes; micropipette and sterile pipette tips; electronic balance; magnetic stirrer and stir bar.

[0160] Preparation steps (taking 100mL solution as an example (volume can be adjusted as needed)):

[0161] 1. Prepare calcium- and magnesium-free PBS: Pour 100 mL of sterile calcium- and magnesium-free PBS solution into a sterile beaker or container. Gently stir on a magnetic stirrer to maintain homogeneity.

[0162] 2. Dissolve neutral protease: Weigh 70 mg of neutral protease powder and carefully transfer it to PBS solution using a sterile pipette tip; stir at medium speed on a magnetic stirrer for 10 minutes to ensure complete dissolution; use a pipette to aspirate the liquid at the bottom to check for undissolved particles; if necessary, filter the solution using a 0.22 μm sterile filter membrane.

[0163] 3. Dissolve hyaluronidase: Weigh 40 mg of hyaluronidase powder and slowly sprinkle it into the PBS solution containing dissolved neutral protease; stir at medium speed with a magnetic stirrer for 15 minutes and observe the dissolution of the enzyme; after ensuring that the enzyme is completely dissolved, gently mix with a pipette.

[0164] 4. Dissolve elastase: Weigh 30 mg of elastase and add it to the above mixed solution in portions to avoid clumping caused by adding it all at once; stir continuously for 10 minutes and observe whether it is completely dissolved; check the transparency of the solution to ensure that there are no particles; if necessary, filter using a sterile filter membrane.

[0165] 5. Final mixing and storage: Transfer the dissolved enzyme solution to sterile centrifuge tubes or storage bottles; store on ice and label with usage information. Note: This mixture should be used within a short period of time (it is recommended to store at 4°C for no more than 24 hours).

[0166] Component 2: Dissolve matrix metalloproteinase, papain, and plasmin in a calcium- and magnesium-containing Hanks solution. The specific concentrations are: 0.3 mg / ml matrix metalloproteinase, 0.5 mg / ml papain, and 0.5 mg / ml plasmin.

[0167] The specific preparation steps for component 2 are as follows:

[0168] Reagents: Matrix metalloproteinase: 30 mg; Papain: 50 mg; Fibrinolytic enzyme: 50 mg; DMSO: 1 mL (Matrix metalloproteinase adjuvant, final solution concentration 1%)

[0169] CaCl2·2H2O (calcium chloride): 0.1g (used to prepare a 10mM stock solution, with a final concentration of 1mM)

[0170] Solutions and equipment: Hanks' solution (HBSS): 100 mL (for dissolving enzyme); sterile pipettes and tips; electronic balance (for accurate enzyme weighing); magnetic stirrer and stir bar; sterile filter membrane (optional, for filtering solution); sterile centrifuge tubes or reagent bottles for storing solution.

[0171] Preparation steps (taking 100mL solution as an example):

[0172] 1. Preparation of Hanks' solution containing calcium and magnesium: To obtain a 1 mM CaCl2 concentration, first prepare a 10 mM CaCl2 stock solution; weigh 0.1 g of calcium chloride (CaCl2·2H2O), dissolve it in a small amount of water, add PBS or Hanks' solution, and adjust the volume to 10 mL to obtain a 10 mM CaCl2 stock solution; add the 10 mM CaCl2 stock solution to 100 mL of Hanks' solution, ensuring that the final CaCl2 concentration is 1 mM; continue stirring to ensure that the CaCl2 is completely dissolved.

[0173] 2. Dissolving matrix metalloproteinases: Weigh the required amount of matrix metalloproteinase (30 mg), add 1 ml of DMSO and stir to pre-dissolve. Slowly add the matrix metalloproteinase to the calcium-magnesium Hanks solution and stir with a magnetic stirrer to ensure complete dissolution. If the enzyme is difficult to dissolve, it can be slightly warmed, but the temperature should not exceed 37°C to avoid loss of enzyme activity.

[0174] 3. Dissolve papain: Weigh the required amount of papain (50 mg); slowly add the papain to the solution containing dissolved matrix metalloproteinases and continue stirring; stir for about 10-15 minutes to ensure the solution is completely clear and free of particles or precipitates; if necessary, filter the solution using a sterile 0.22 μm filter membrane to remove any possible precipitates or particles.

[0175] 4. Dissolve the plasmin: Weigh out the required amount of plasmin (50mg); add the plasmin to the solution of dissolved papain in portions, avoiding adding too much at once, which may cause precipitation; continue stirring for 15-20 minutes to ensure that the solution is completely transparent and free of precipitation.

[0176] 5. Inspection and Storage: Check that the solution is completely dissolved and that there is no precipitate. If there are particles, filter using a sterile filter membrane; aliquot the finally dissolved enzyme solution into sterile centrifuge tubes or reagent bottles, clearly labeling the solution information; store the solution at 4°C (if to be used within 24 hours). If long-term storage is required, aliquot the solution and store it at -20°C, avoiding repeated freeze-thaw cycles.

[0177] S2 Tissue Block Pretreatment: Select 12-day-old chicken embryos, dissect to obtain muscle tissue, and cut 5g of muscle tissue block into small meat cubes with a medical scalpel with a particle size of 0.5-1mm. Preferably, the particle size of the meat cubes is about 0.5mm.

[0178] S3 tissue preliminary digestion and dissociation: The tissue fragments were digested using the compound enzyme component 1 solution at 37°C for 10 min, and then dissociated using a tissue dissociator for 30 s.

[0179] S4 tissue secondary digestion and dissociation: Add component 2 solution, enzymatic digestion at 37℃ for 5 min, and dissociation in tissue dissociation apparatus for 30 s;

[0180] S5 Single-cell suspension was obtained by filtering with a 100 μm filter, collecting the supernatant and centrifuging at 1000 rpm for 3 min, and resuspending in 20% FBSDMEM cell culture medium.

[0181] For each 5g muscle tissue sample, 2.5ml of component 1 and 2.5ml of component 2 were used. The matrix metalloproteinase (MMP) had a molecular weight of 68,000–125,000 and an optimal pH of 6.3–8.8; the MMP was matrix metalloproteinase II. The neutral protease was neutral protease II, with an optimal pH of 7.5. The MMP, papain, and plasmin solutions could be prepared using any of the following reagents: TESC Abuffer, calcium-magnesium-containing PBS, calcium-magnesium-containing Hanks solution, or serum-free culture medium. The MMP II powder was pre-dissolved using DMSO as a solubilizer. The neutral protease, hyaluronidase, and elastase were all prepared using calcium-magnesium-free PBS, calcium-magnesium-free Hanks solution, or serum-free culture medium.

[0182] According to the appendix Figure 12 Cell suspension observation diagram and appendix Figure 13 The cell count results after diluting the cell suspension 100-fold show that after digestion and dissociation by complex enzyme IV, there were no tissue fragments in the suspension, but a large number of fully digested single cells and a small number of myofilaments. The count results indicate that the original single cell count was approximately 1 × 10⁻⁶. 8 The cell count was above 100 cells / ml. The resulting cell suspension adhered extensively to the culture vessel after just one day, indicating that the enzymatic digestion conditions of the compound enzyme IV were mild and caused minimal cell damage. The compound enzyme IV can efficiently and gently digest and separate cells from tissues. The multi-enzyme preparation prepared in Example 1 was used in the preparation of single-cell suspensions of chicken embryo muscle tissue; it can also be used to dissociate and obtain the required number of seed cells for the production of industrial-grade chicken embryos for the meat industry. Furthermore, the applicability of the multi-enzyme preparation has been expanded to the dissociation of muscle tissue from other poultry animals (e.g., pigeons, ducks, geese) embryos.

[0183] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for using a multi-enzyme preparation for industrial cell dissociation, characterized by: S1: configuring a component 1 solution and a component 2 solution: The component 1 solution is configured: dissolving neutral protease, hyaluronidase, and elastase in a calcium-magnesium-free PBS solution; The component 2 solution is configured: dissolving matrix metalloproteinase, papain, and fibrinolysin in a calcium-magnesium-containing Hanks solution; In the component 1: the concentration of the neutral protease is 0.7 mg / ml, the concentration of the hyaluronidase is 0.4 mg / ml, and the concentration of the elastase is 0.3 mg / ml; In the component 2: the concentration of the matrix metalloproteinase is 0.3 mg / ml, the concentration of the papain is 0.5 mg / ml, and the concentration of the fibrinolysin is 0.5 mg / ml; S2: Preparation of target enzymatic hydrolysate: dissect muscle tissue of 12-day-old chick embryo; pretreat the muscle tissue to 0.5-1.0 mm 3 small pieces of meat; S3: primary digestion and dissociation of tissues: using the component 1 solution to enzymatically digest tissue clumps, followed by dissociation in a tissue dissociator to obtain a primary digestion and dissociation solution of tissues; S4: secondary digestion and dissociation of tissues: adding the component 2 solution to the primary digestion and dissociation solution of tissues for secondary enzymatic digestion, followed by dissociation in a tissue dissociator; S5: obtaining a single-cell suspension: filtering using a filter screen, collecting the supernatant and centrifuging, and resuspending using cell culture medium.

2. The method of use of a multi-enzyme preparation for industrial cell dissociation according to claim 1, characterized in that: The number-average molecular weight of the matrix metalloproteinase is 68,000-125,000, and the optimum pH is 6.3-8.8; the matrix metalloproteinase is matrix metalloproteinase II.

3. The method of use of a multi-enzyme preparation for industrial cell dissociation according to claim 1, characterized in that: The neutral protease is neutral protease II, and the optimum pH is 7.

5.

4. The method of use of a multi-enzyme preparation for industrial cell dissociation according to claim 2, characterized in that: The matrix metalloproteinase II powder is pre-dissolved using DMSO reagent as a cosolvent. 5.The method for using a multi-enzyme preparation for industrial cell dissociation according to claim 1, characterized by: In the S2 step: the muscle tissue mass is 5 g; and the muscle tissue pretreatment meat cube particle size is 0.5 mm; The S3 specific steps: using the component 1 solution to enzymatically digest tissue clumps at 37℃ for 10 min, followed by dissociation in a tissue dissociator for 30 s to obtain a primary digestion and dissociation solution of tissues; The S4 specific steps: adding the component 2 solution to the primary digestion and dissociation solution of tissues, followed by secondary enzymatic digestion at 37℃ for 5 min and dissociation in a tissue dissociator for 30 s; The S5 specific steps: filtering using a 100 μm filter screen, collecting the supernatant and centrifuging at 1,000 rpm for 3 min, and resuspending using 20% FBS DMEM cell culture medium.

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