Multi-enzyme preparation for industrial cell dissociation as well as use method and application of multi-enzyme preparation

Abstract

The invention discloses a multi-enzyme preparation for industrial cell dissociation as well as a use method and application thereof, and belongs to the technical field of extraction of seed cells for industrial culture of meat. According to the invention, a multi-enzyme preparation composed of a component 1 and a component 2 is adopted; the component 1 comprises neutral protease, hyaluronidase and elastase; and the component 2 comprises matrix metalloproteinase, papain and plasmin. The invention also discloses a preparation method and application of the multienzyme preparation. The reasonable enzyme ratio and the optimized treatment conditions are beneficial to protecting the integrity of cell membranes, maintaining high cell activity and remarkably improving the dissociation efficiency and the cell recovery amount; the complex enzyme preparation is high in adaptability, can be used for various tissue types and dissociation requirements, and is convenient for standardized and large-scale production; in combination with automatic dissociation equipment, the compound enzyme preparation can realize efficient and stable industrial operation, and provides important technical support and wide application prospects for the fields of meat culture industry, tissue engineering, cell therapy and the like.

Description

Technical Field

[0001] The invention relates to the technical field of industrially cultivated meat seed cell extraction, in particular to a multi-enzyme preparation for industrial cell dissociation and a use method and application thereof. Background Art

[0002] Cell dissociation is a key step in cell culture, tissue engineering and cultured meat production, which aims to efficiently and gently separate highly active single cells from tissue samples. At present, the enzymes commonly used for cell dissociation include matrix metalloproteinases (MMPs), trypsin, hyaluronidase and elastase. These enzymes achieve separation between cells or between cells and matrix by degrading specific components of the extracellular matrix (such as collagen, hyaluronic acid, elastin, etc.). However, traditional enzyme preparations have key problems such as high enzymatic hydrolysis requirements, cumbersome operation, high cost, single dissociation effect, low efficiency and extremely low total amount of dissociated cells. At the same time, in the industrial application of technologies such as biomanufacturing, cell agriculture, and cell-cultured meat, there are extremely high requirements for cell activity and total cell amount. Traditional methods are generally limited to laboratory-scale research, and the key problems highlighted in the industrialization process cannot be solved.

[0003] Existing single enzyme or simple enzyme combination methods are suitable for small-scale, laboratory-scale cell extraction, but have significant limitations in high-density, high-volume industrial dissociation scenarios. The dissociation efficiency of single enzyme methods is often low, and prolonged processing time is required in complex tissues, which may lead to decreased cell activity. In addition, due to the large differences in the composition of the extracellular matrix in different tissue types, it is difficult for a single enzyme to achieve a comprehensive effect and has poor adaptability. More importantly, these methods are difficult to meet the needs of efficient, stable and large-scale operations in industrial production, and cannot take into account both cell yield and quality.

[0004] The development of combined complex enzyme preparations provides a new solution for industrial cell dissociation. Through the synergistic action of multiple enzymes, combined complex enzyme preparations can comprehensively degrade the diverse extracellular matrix components in complex tissues, thereby significantly improving the dissociation efficiency. Summary of the invention

[0005] The purpose of the present invention is to provide a multi-enzyme preparation for industrial cell dissociation and its use method and application. In view of the problems of low efficiency, insufficient cell recovery, and difficulty in ensuring cell activity in existing enzymatic hydrolysis technology in industrial cell dissociation, the present invention innovatively proposes a multi-enzyme preparation and use method to meet the needs of large-scale cell culture.

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

[0007] Preferably, 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;

[0008] 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 plasmin is 0.5 mg / ml.

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

[0010] Preferably, the neutral protease is neutral protease II, and its optimum pH is 7.5.

[0011] Preferably, the matrix metalloproteinase solution, the papain solution, and the plasmin solution can be prepared from any one of TESCA buffer, PBS containing calcium and magnesium, Hanks solution containing calcium and magnesium, or a serum-free culture medium.

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

[0013] Preferably, the neutral protease, the hyaluronidase and the elastase are all prepared with PBS without calcium or magnesium, or Hanks solution without calcium or magnesium, or culture medium without serum.

[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 component 1 solution and the component 2 solution:

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

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

[0018] S2: Preparation of target enzymatic hydrolysate: dissecting muscle tissue of 12-day-old chicken embryos; pre-treating the muscle tissue to 0.5-1.0 mm 3 small pieces of meat;

[0019] S3: preliminary digestion and dissociation of tissue: using the solution of component 1, enzymatically dissociating tissue fragments, and then dissociating with a tissue dissociator to obtain preliminary digestion and dissociation solution of tissue;

[0020] S4: secondary digestion and dissociation of tissue: adding component 2 solution to the tissue primary digestion and dissociation solution, performing enzymatic secondary digestion, and dissociating using a tissue dissociator;

[0021] S5: Obtain single cell suspension: Filter through a strainer, collect the supernatant and centrifuge, and resuspend in cell culture medium.

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

[0023] The specific steps of S3 are: using the solution of component 1, enzymatically digesting the tissue fragments at 37° C. for 10 minutes, dissociating them in a tissue dissociator for 30 seconds, and obtaining a preliminary tissue digestion and dissociation solution;

[0024] The specific steps of S4 are as follows: adding the solution of component 2 to the tissue preliminary digestion and dissociation solution, performing secondary enzymatic digestion at 37° C. for 5 minutes, and dissociating in a tissue dissociator for 30 seconds;

[0025] The specific steps of S5 are as follows: filtering with a 100 μm filter, collecting the supernatant and centrifuging at 1000 rpm for 3 min, and resuspending with 20% FBSDMEM cell culture medium.

[0026] An application of a multi-enzyme preparation for industrial cell dissociation or a method for 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 seed cells that meet the required number of muscle cells for industrial-grade chicken embryos required for cultivating the meat industry.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The present invention significantly improves the dissociation efficiency and cell activity.

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

[0030] (3) The enzyme used in the present invention has excellent stability and biocompatibility, ensuring the quality and purity of the cells.

[0031] (4) The present invention realizes automation and process-based dissociation process, which is convenient for industrialized mass production and process-based quality control.

[0032] (5) The present invention not only solves the key problems in the prior art, but also broadens the application prospects of cell dissociation technology in the fields of food science, cell agriculture, etc., and provides reliable technical support for the efficient development of the meat industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a microscopic picture of tissue and cell suspension after trypsin digestion and dissociation in Experimental Example 1 of the present invention.

[0034] Figure 2 This is a microscopic picture of tissue and cell suspension after matrix metalloproteinase digestion and dissociation in Comparative Example 1 of the present invention.

[0035] Figure 3 This is a microscopic picture of the tissue and cell suspension after hyaluronidase digestion and dissociation in Comparative Example 2 of the present invention.

[0036] Figure 4 This is a microscopic picture of the tissue and cell suspension after neutral protease digestion and dissociation in Comparative Example 3 of the present invention.

[0037] Figure 5 This is a microscopic picture of the tissue and cell suspension after elastase digestion and dissociation in Comparative Example 4 of the present invention.

[0038] Figure 6 This is a microscopic picture of tissue and cell suspension after digestion and dissociation with a mixed solution of papain and plasmin in Comparative Example 5 of the present invention.

[0039] Figure 7 This is a microscopic picture of tissue and cell suspension after digestion and dissociation by complex enzyme I in Experimental Example 2 of the present invention.

[0040] Figure 8 This is a microscopic picture of tissue and cell suspension after digestion and dissociation with complex enzyme II in comparative example 6 of the present invention.

[0041] Fig. 9 This is a microscopic picture of tissue and cell suspension after digestion and dissociation by complex enzyme III in comparative example 7 of the present invention.

[0042] Fig.10 This is a microscopic picture of tissue and cell suspension after digestion and dissociation with complex enzyme IV in comparative example 8 of the present invention.

[0043] Fig.11 This is a microscopic picture of tissue and cell suspension after digestion and dissociation by complex enzyme V in comparative example 9 of the present invention.

[0044] Fig.12These are microscopic images of tissue and cell suspension after digestion and dissociation with composite 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] Fig.13 This is the cell counting result after the cell suspension was diluted 100 times after digestion and dissociation with complex enzyme IV (multi-enzyme preparation) in Example 1 of the present invention. DETAILED DESCRIPTION

[0046] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. The experimental methods described in the following embodiments are conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, can be obtained from commercial channels.

[0047] The following raw materials, including neutral protease, hyaluronidase, elastase, matrix metalloproteinase, papain, etc., were purchased from Beijing Solebow.

[0048] From the inside to the outside, muscles are myofibrils, muscle fibers, and muscle bundles, which are wrapped by perimysium, endomysium, and epimysium respectively. The connective tissues of each membrane are continuous with each other, and blood vessels and nerves are distributed to various parts of the muscle along the connective tissue membrane. Muscle tissue is a complex structure, and to obtain muscle cells, each part of the muscle tissue needs to be enzymatically hydrolyzed layer by layer. In order to efficiently obtain muscle cells, trypsin, hyaluronidase, neutral protease, matrix metalloproteinase, elastase, and papain were selected according to the characteristics of muscle tissue and the enzymatic hydrolysis range of digestive enzymes currently on the market to digest chicken embryo muscle tissue fragments and record the enzymatic hydrolysis effect.

[0049] Trypsin is the most widely used digestion agent. It acts on peptide tendons connected to lysine or arginine, removes intercellular mucins and glycoproteins, affects the cytoskeleton, and thus separates cells.

[0050] Experimental Example 1

[0051] The specific steps are as follows:

[0052] S1: Enzyme solution preparation: The pancreatic enzyme solution is a 0.25% commercial enzyme solution without EDTA.

[0053] S2: Tissue block pretreatment: 12-day-old chicken embryos were selected and muscle tissue was obtained by dissection. 5 g of muscle tissue blocks were cut into small meat cubes with a particle size of 0.5-1 mm using a medical scalpel. Preferably, the particle size of the meat cubes was about 0.5 mm.

[0054] S3: Preliminary digestion of tissue: Use 0.125% pancreatic enzyme solution without EDTA to digest tissue fragments at 37°C for 15, 30, 45, and 60 min. Use 5 ml of enzyme solution to digest 5 g of muscle tissue.

[0055] S4: Dissociation: After enzymatic digestion every 15 minutes, the tissue is dissociated once by a tissue dissociator;

[0056] S5: Obtain single cell suspension: After 15min, 30min, 45min, and 60min of enzymatic hydrolysis and dissociation, tissues and cell suspensions with different degrees of dissociation were obtained, the state of tissues and cells was observed, and the digestion effect of trypsin was evaluated.

[0057] Experimental Example 1 0.125% pancreatin without EDTA was used for digestion. Figure 1 It can be seen that no tissue fragments were observed under the microscope after 30 minutes of digestion, and the muscle fibers were further digested and shortened after 45-60 minutes of digestion. After 60 minutes of digestion, the digestion fluid was all fiber fragments. Therefore, 0.125% EDTA-free trypsin digestion has a good effect, and the digestion time should be controlled between 15-30 minutes.

[0058] Comparative Example 1

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

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

[0061] Step S1-2:

[0062] 1. Prepare Tris-HCl buffer (1 L) on ice: dissolve 2.422 g Tris and 0.147 g CaCl2 in 900 mL distilled water, adjust pH to 7.5 and make up to 1 L.

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

[0064] 3. Add 490 μL Tris-HCl buffer to the tube and mix well with a vortex mixer. After dissolution, place at 4°C for stabilization for 30 minutes to restore the enzyme to its natural conformation.

[0065] 4. Filter the solution through a 0.22 μm sterile filter membrane for sterilization, and store at -80°C after aliquoting.

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

[0067] Matrix metalloproteinases hydrolyze proline in the extracellular matrix to disperse cells. Matrix metalloproteinases have a strong digestive effect on the extracellular matrix and only digest the extracellular matrix, with little damage to cells. They are suitable for fibrous tissue and harder tissue.

[0068] according to Figure 2 The results of microscopic observations show that after 15 minutes of digestion, there are no tissue fragments under the microscope and the fibers are relatively long; after 45 minutes of digestion, the fibers are further shortened.

[0069] Comparative Example 2

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

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

[0072] Step S1-2:

[0073] 1. Prepare 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.

[0075] 3. Place the centrifuge tube in an ice bath, put it into an ultrasonic water bath, and perform low-power ultrasonic treatment for 5 minutes, gently shaking every 1 minute to mix.

[0076] 4. After the ultrasonic treatment is completed, slowly aspirate the bottom solution 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 (no more than 24 hours).

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

[0079] Hyaluronidase randomly degrades the β-N-acetylglucosamine-[1-4] glycosidic bonds of hyaluronic acid, thereby reducing the activity of hyaluronic acid and is used to separate connective tissue.

[0080] according to Figure 3The results of comparative example 2 show that the fiber length and tissue state under the microscope did not change significantly after 15-45 minutes of digestion; after 60 minutes of digestion, most muscle bundles were opened, but the muscle fiber length was further shortened. The effect of digestion with 0.4 mg / ml hyaluronidase for more than 60 minutes was better.

[0081] Comparative Example 3

[0082] The difference between Comparative Example 3 and Experimental Example 1 is only the preparation of S1 enzyme solution: neutral protease powder is prepared into a 0.7 mg / ml neutral protease solution using a 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 35 mg of neutral protease powder using an electronic analytical balance to ensure accuracy.

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

[0087] 3. On a magnetic stirrer, stir at medium speed and gradually sprinkle the neutral protease powder into the PBS to avoid directly adding the powder to cause agglomeration.

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

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

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

[0091] 7. Each time you use it, place it at 4°C to melt, then shake it gently to ensure that the enzyme is evenly distributed.

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

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

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

[0095] Comparative Example 4

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

[0097] S1-1 Reagent preparation: elastase, PBS (pH 8.0) without calcium and magnesium ions, and sterile test tubes.

[0098] Step S1-2:

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

[0100] 2. Add 15 mg of elastase powder to a sterile test tube, gradually add 1 ml of buffer, and vortex at high speed for 3 minutes to ensure that there is no lumps.

[0101] 3. Check the solubility. If there are undissolved particles, use a sterile pipette to repeatedly blow and dissolve them.

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

[0103] 5. Use immediately after dissolution to avoid inactivation due to long-term exposure to air.

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

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

[0106] according to Figure 5 The results of comparative example 4 show that: after 15 minutes of digestion with 0.3 mg / ml elastase, the tissue has been basically digested and separated under the microscope, and there is no long fiber segment. After 30 minutes to 60 minutes of digestion, there is no fiber segment under the microscope. The elastase digestion effect is significant. The longer the digestion time, the more cell fragments there are, and the cell damage is significant. The digestion time of 0.3 mg / ml elastase should be controlled within 15 minutes or the elastase concentration should be reduced to avoid damage to the cells.

[0107] Comparative Example 5

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

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

[0110] Step S1-2:

[0111] 1. Dissolve 0.8 g NaCl, 0.144 g Na2HPO4, 0.024 g KH2PO4, and 0.02 g KCl in 100 mL distilled water. Add appropriate amount of distilled water until completely dissolved, then add 0.0184 g calcium chloride dihydrate (CaCl2·2H2O) and 0.01 g 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 plasmin and 25 mg of papain powder into a sterile beaker and add 50 mL of the above buffer.

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

[0114] 4. If the solubility is insufficient, place the beaker in a 37°C incubator for 10 minutes.

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

[0116] 6. Store on ice after filtering through a 0.22 μm filter membrane. The solution must be used on the same day.

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

[0118] Papain is a sulfhydryl (-SH) endopeptidase with protease and esterase activity. It can decompose proteins in acidic, neutral and alkaline environments. Papain is a food additive with the characteristics of being cheap and easy to obtain. Plasmin can decompose muscle fibers, but has a weak effect on fibrin.

[0119] according to Figure 6The results of comparative example 5 show that: after 15 minutes of digestion, all the fibers under the microscope are short segments; as time increases, the fiber segments become shorter but the cell fragments increase. The mixed solution of 0.5 mg / ml papain and 0.5 mg / ml plasmin has a strong digestion ability, and the effect is better when the digestion time is controlled within 15 minutes.

[0120] Based on the final effect of the single enzyme solution in Experimental Example 1 and Comparative Examples 1-5 on tissue dissociation, various types of digestive enzymes were compounded and used according to their main action modes and principles in the tissue dissociation process. Experimental Example 2 and Comparative Examples 6-9 are the experimental steps and final digestion effects of different types of compound enzymes.

[0121] Experimental Example 2

[0122] Complex enzyme I components: pancreatin, neutral protease, hyaluronidase, elastase

[0123] The experimental steps are as follows:

[0124] Preparation of S1 complex enzyme I: The specific concentration of each enzyme is: 0.125% pancreatin, 0.7 mg / ml neutral protease, 0.4 mg / ml hyaluronidase, 0.3 mg / ml elastase.

[0125] S2 tissue block pretreatment: 12-day-old chicken embryos were selected and muscle tissue was obtained by dissection. 5 g of muscle tissue blocks were cut into small meat cubes with a particle size of 0.5 to 1 mm using a medical scalpel. Preferably, the particle size of the meat cubes was about 0.5 mm.

[0126] Initial digestion of S3 tissue: Use complex enzyme I solution to enzymatically digest tissue fragments at 37℃ for 30 minutes.

[0127] S4 dissociation: dissociation once with tissue dissociator;

[0128] S5 Obtain single cell suspension: 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 enzymatically hydrolyzed using 5ml of enzyme solution.

[0130] according to Figure 7 Observation of the cell suspension in Experimental Example 2 showed that the enzymatic hydrolysis effect of compound enzyme I was poor and could not completely digest the tight junctions between tissues. After 30 minutes of digestion, there were still a large number of tissue fragments.

[0131] Comparative Example 6

[0132] The difference between Comparative Example 6 and Experimental Example 2 is only the preparation of S1 complex enzyme II: the specific concentration of each enzyme is: 0.125% pancreatic enzyme, 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 attached Figure 8 Observation of the cell suspension in Comparative Example 6 showed that the enzymatic hydrolysis effect of compound enzyme II was average. After 30 minutes of digestion and dissociation, there were a small amount of myofilaments and partially digested single cells in the suspension.

[0134] Comparative Example 7

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

[0136] S1 complex enzyme III preparation: The specific concentration of each enzyme is: 0.7 mg / ml neutral protease, 0.4 mg / ml hyaluronidase, 0.3 mg / ml elastase.

[0137] Initial digestion of S3 tissue: digest with complex enzyme III at 37°C for 15 min.

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

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

[0140] according to Fig. 9 Observation of the cell suspension of Comparative Example 7 showed that the enzymatic hydrolysis effect of compound enzyme III was too strong. After digestion and dissociation, the suspension contained only completely digested single cells without tissue fragments and myofilaments, which caused severe damage to the cells.

[0141] Comparative Example 8

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

[0143] S1 complex enzyme IV preparation:

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

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

[0146] Initial digestion of S3 tissue: digest for 15 min using component 1;

[0147] S4 tissue secondary digestion: add component 2 for secondary digestion for 15 min;

[0148] according to Fig.10 The observation of the cell suspension of Comparative Example 8 shows that the enzymatic hydrolysis effect of compound enzyme IV is too strong. There are no tissue fragments in the suspension after digestion and dissociation, and there are a large number of completely digested single cells and a small amount of myofilaments, which causes little damage to cells. The results show that compound enzyme IV has the enzymatic hydrolysis ability to efficiently digest and separate cells.

[0149] Comparative Example 9

[0150] The difference between Comparative Example 9 and Experimental Example 2 is that the S1 compound enzyme V is prepared: the specific concentration of each enzyme is: 0.7 mg / ml neutral protease, 0.4 mg / ml hyaluronidase, 0.3 mg / ml elastase

[0151] according to Fig.11 The observation of the cell suspension of Comparative Example 9 shows that the enzymatic hydrolysis effect of compound enzyme V is poor, and there are a large number of tissue fragments, a small amount of myofilaments and a small number of cells in the suspension after digestion and dissociation. The results show 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 the present invention):

[0155] Component 1: Dissolve neutral protease, hyaluronidase, and elastase in a PBS solution without calcium and magnesium. The specific concentration of each enzyme is: 0.7 mg / ml neutral protease, 0.4 mg / ml hyaluronidase, and 0.3 mg / ml elastase.

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

[0157] Reagent and material preparation:

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

[0159] Sterile materials: sterile centrifuge tubes; micropipette and sterile tips; electronic balance; magnetic stirrer and stirring rod

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

[0161] 1. Prepare PBS without calcium and magnesium: Take 100 mL of sterile PBS solution without calcium and magnesium and pour it into a sterile beaker or container. Stir gently on a magnetic stirrer to maintain homogeneity.

[0162] 2. Dissolve the neutral protease: Weigh 70 mg of neutral protease powder and carefully transfer it to the PBS solution with a sterile pipette tip; stir at medium speed on a magnetic stirrer for 10 minutes to ensure that it is completely dissolved; use a pipette to aspirate the bottom liquid 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 in which the neutral protease has been dissolved; 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 it with a pipette.

[0164] 4. Dissolve elastase: Weigh 30 mg of elastase and add it to the above mixed solution in batches to avoid agglomeration caused by adding it all at once; continue stirring 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 a sterile centrifuge tube or storage bottle; store on ice and label with usage information. Note: The mixed solution should be used within a short period of time (it is recommended to be stored at 4°C for no more than 24 hours).

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

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

[0168] Reagents: Matrix metalloproteinase: 30 mg; Papain: 50 mg; Plasmin: 50 mg; DMSO: 1 mL (Matrix metalloproteinase additive, final solution content 1%)

[0169] CaCl2·2H2O (calcium chloride): 0.1 g (for preparing a 10 mM stock solution, final concentration is 1 mM)

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

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

[0172] 1. Prepare 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) and dissolve it in a small amount of water, then add PBS or Hanks solution to adjust the volume to 10 mL to obtain a 10 mM CaCl2 stock solution; add 10 mL of the 10 mM CaCl2 stock solution to 100 mL of Hanks solution to ensure that the final CaCl2 concentration is 1 mM; continue stirring to ensure that the CaCl2 is completely dissolved.

[0173] 2. Dissolve matrix metalloproteinase: Weigh the required amount of matrix metalloproteinase (30 mg), add 1 ml DMSO and blow to pre-dissolve, slowly add matrix metalloproteinase to calcium and magnesium Hanks solution, and stir with a magnetic stirrer to ensure complete dissolution. If the enzyme is difficult to dissolve, it can be slightly heated, 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 papain to the solution of dissolved matrix metalloproteinases and continue stirring; stir for about 10-15 minutes to ensure that the solution is completely transparent and free of particles or precipitation; if necessary, filter the solution using a sterile 0.22 μm filter membrane to remove possible precipitation or particles.

[0175] 4. Dissolve plasmin: Weigh the required amount of plasmin (50 mg); add the plasmin to the solution of dissolved papain in portions to avoid adding too much at one time to prevent 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 whether the solution is completely dissolved and ensure that there is no precipitation. If there are particles, filter with a sterile filter membrane; divide the final dissolved enzyme solution into sterile centrifuge tubes or reagent bottles, and clearly mark the solution information; store the solution at 4°C (if used within 24 hours). If long-term storage is required, the solution can be divided and stored at -20°C to avoid repeated freezing and thawing.

[0177] S2 tissue block pretreatment: 12-day-old chicken embryos were selected and muscle tissue was obtained by dissection. 5 g of muscle tissue blocks were cut into small meat cubes with a particle size of 0.5 to 1 mm using a medical scalpel. Preferably, the particle size of the meat cubes was about 0.5 mm.

[0178] S3 preliminary tissue digestion and dissociation: use the complex enzyme component 1 solution, enzymatically dissociate the tissue fragments at 37℃ for 10 minutes, and dissociate in a tissue dissociator for 30 seconds;

[0179] S4 tissue secondary digestion and dissociation: add component 2 solution, perform secondary digestion at 37℃ for 5min, and dissociate in tissue dissociator for 30s;

[0180] S5 Obtain single cell suspension: filter using a 100 μm filter, collect the supernatant and centrifuge at 1000 rpm for 3 min, and resuspend in 20% FBSDMEM cell culture medium.

[0181] Among them, 5g muscle tissue uses 2.5ml component 1 and 2.5ml component 2. The molecular weight of matrix metalloproteinase is 68000-125000, and the optimal pH is 6.3-8.8; the matrix metalloproteinase is matrix metalloproteinase II. The neutral protease is neutral protease II, and the optimal pH is 7.5. Matrix metalloproteinase solution, papain solution, and plasmin solution can all be prepared by any reagent of TESCAbuffer or PBS containing calcium and magnesium or Hanks solution containing calcium and magnesium or serum-free culture medium. Matrix metalloproteinase II uses DMSO reagent as a cosolvent to pre-dissolve the matrix metalloproteinase II powder. Neutral protease, hyaluronidase, and elastase are all prepared by PBS without calcium and magnesium or Hanks solution without calcium and magnesium or serum-free culture medium.

[0182] According to the attached Fig.12 Cell suspension observation diagram and attached Fig.13 The cell counting result after the cell suspension was diluted 100 times showed that after the enzymatic hydrolysis of compound enzyme IV, there were no tissue fragments in the suspension after digestion and dissociation, but there were a large number of completely digested single cells and a small amount of myofilaments. The counting result showed that the number of single cells in the original solution was 1×10 8 / ml or more. The final cell suspension obtained can adhere to the wall in large quantities after one day of culture, indicating that the enzymatic hydrolysis conditions of compound enzyme IV are mild and cause little damage to cells. Compound enzyme IV can efficiently and gently enzymatically digest and separate cells from tissues. The multi-enzyme preparation prepared in Example 1 is used to prepare a single-cell suspension of muscle tissue of chicken embryos; it can also be used to dissociate seed cells to obtain the required number of muscle cells for industrial-grade chicken embryos required for the meat industry. In addition, the scope of application of the multi-enzyme preparation has been expanded to the dissociation of embryonic muscle tissue of other poultry animals (for example: pigeons, ducks, and geese).

[0183] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A multi-enzyme preparation for industrial cell dissociation, characterized in that: The multi-enzyme preparation consists of component 1 and component 2; component 1 comprises neutral protease, hyaluronidase and elastase; and component 2 comprises matrix metalloproteinase, papain and plasmin.

2. The multi-enzyme preparation for industrial cell dissociation according to claim 1, characterized in that: 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 plasmin is 0.5 mg / ml.

3. A multi-enzyme preparation for industrial cell dissociation according to claim 1 or 2, characterized in that: The number average molecular weight of the matrix metal protein is 68000-125000, and the optimum pH is 6.3-8.8; the matrix metal proteinase is matrix metal proteinase II.

4. A multi-enzyme preparation for industrial cell dissociation according to claim 1 or 2, characterized in that: The neutral protease is neutral protease II, and its optimum pH is 7.

5.

5. A multi-enzyme preparation for industrial cell dissociation according to claim 1 or 2, characterized in that: The matrix metalloproteinase solution, the papain solution, and the plasmin solution can be prepared from any one of TESCA buffer, PBS containing calcium and magnesium, Hanks solution containing calcium and magnesium, or a culture medium without serum.

6. The multi-enzyme preparation for industrial cell dissociation according to claim 3, characterized in that: The matrix metalloproteinase II uses DMSO reagent as a co-solvent to pre-dissolve the matrix metalloproteinase II powder.

7. A multi-enzyme preparation for industrial cell dissociation according to claim 1 or 2, characterized in that: The neutral protease, the hyaluronidase and the elastase are all prepared with PBS without calcium or magnesium, or Hanks solution without calcium or magnesium, or culture medium without serum.

8. The method for using the multi-enzyme preparation for industrial cell dissociation according to claim 1, characterized in that: The method for using the multi-enzyme preparation is prepared as follows: S1: Prepare the component 1 solution and the component 2 solution: The solution of component 1 is prepared by dissolving the neutral protease, the hyaluronidase and the elastase in a PBS solution free of calcium and magnesium; The solution of component 2 is prepared by dissolving the matrix metalloproteinase, the papain and the plasmin in Hanks solution containing calcium and magnesium; S2: Preparation of target enzymatic hydrolysate: dissecting muscle tissue of 12-day-old chicken embryos; pre-treating the muscle tissue to 0.5-1.0 mm 3 small pieces of meat; S3: preliminary digestion and dissociation of tissue: using the solution of component 1, enzymatically dissociating tissue fragments, and then dissociating with a tissue dissociator to obtain preliminary digestion and dissociation solution of tissue; S4: secondary digestion and dissociation of tissue: adding component 2 solution to the tissue primary digestion and dissociation solution, performing enzymatic secondary digestion, and dissociating using a tissue dissociator; S5: Obtain single cell suspension: Filter through a strainer, collect the supernatant and centrifuge, and resuspend in cell culture medium.

9. The method for using the multi-enzyme preparation for industrial cell dissociation according to claim 8, characterized in that: In the step S2: the mass of the muscle tissue is 5 g; the particle size of the pre-processed diced muscle tissue is 0.5 mm; The specific steps of S3 are: using the solution of component 1, enzymatically digesting the tissue fragments at 37° C. for 10 minutes, dissociating them in a tissue dissociator for 30 seconds, and obtaining a preliminary tissue digestion and dissociation solution; The specific steps of S4 are as follows: adding the solution of component 2 to the tissue preliminary digestion and dissociation solution, performing secondary enzymatic digestion at 37° C. for 5 minutes, and dissociating in a tissue dissociator for 30 seconds; The specific steps of S5 are as follows: filtering with a 100 μm filter, collecting the supernatant and centrifuging at 1000 rpm for 3 min, and resuspending with 20% FBSDMEM cell culture medium.

10. Use of the multi-enzyme preparation for industrial cell dissociation according to claim 1 or the method for using the multi-enzyme preparation for industrial cell dissociation according to claim 8, characterized in that: The multi-enzyme preparation is used in preparing single-cell suspension of muscle tissue of chicken embryos; the multi-enzyme preparation is used in dissociating seed cells that meet the muscle cell quantity requirements of industrial-grade chicken embryos required for cultivating the meat industry.

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