Preparation method and application of collagenase based on Clostridium histolyticum

Abstract

This invention relates to a method for preparing collagenase based on Clostridium histolyticum and its application. The preparation method includes the following steps: inoculating Clostridium histolyticum into a fermentation medium for anaerobic fermentation to obtain a fermentation product; wherein the anaerobic fermentation pH is 6.8–7.2; microcarriers and gelatin are added to the fermentation medium; and the fermentation product is separated and extracted to obtain a collagenase composition. Thus, by using gelatin + microcarriers as dual collagen inducers, replacing the traditional single gelatin induction method, a collagenase enzyme spectrum highly adapted to the Huakan three-dimensional collagen microcarrier can be targeted and induced, significantly improving the microcarrier lysis specificity and efficiency, and significantly increasing collagenase activity and expression levels.

Description

Technical Field

[0001] This invention relates to the field of collagenase fermentation production technology, and in particular to a method for preparing collagenase based on Clostridium histolyticum and its application. Background Technology

[0002] Three-dimensional microcarrier culture is a core technology for the large-scale industrial expansion of stem cells and immune cells. The 3DRecomTrix® / TableTrix® microcarrier series uses collagen as the main framework and has advantages such as biomimetic elasticity, good biocompatibility, uniform pore structure, and suitability for high-density cell attachment and growth. It has been widely used in the large-scale culture of various adherent cells such as MSCs, fibroblasts, Vero, human diploid cells, and 293T.

[0003] Currently, cell harvesting after microcarrier culture has the following main defects: (1) Trypsin digestion: non-specific lysis of cell-cell and cell-matrix connections, which can easily damage cell membrane proteins and receptors, reducing cell viability and function; (2) Physical blowing: incomplete cell shedding and microcarrier breakage residue can easily cause mechanical damage to cells; (3) Common collagenase: single component, low lysis efficiency, violent reaction, which can easily lead to cell apoptosis.

[0004] Collagenase derived from Clostridium histolyticum is a complex enzyme system that can specifically degrade the triple helix structure of natural / recombinant collagen without acting on cell membrane proteins. It is an ideal tool for achieving gentle, specific, and non-destructive lysis of collagen microcarriers.

[0005] However, the existing preparation processes yield collagenases with low activity and expression levels, limiting their application. For example, Chinese patent CN120624417A discloses a fermentation and purification method for Clostridium histolyticum collagenase. This method determines 37℃ as the optimal fermentation temperature, 1% nitrogen source concentration, and 24-hour fermentation time, and innovatively introduces 2.8 mg / 100 mL Fe... 2+ and 10.0 mmol / L Ca 2+ As a key additive, Clostridium histolytica collagenase with high expression levels and high enzyme activity was obtained, increasing collagenase activity from 0.790 U / L to 0.903 U / L while maintaining a high protein concentration of 40-45 mg / mL. This allowed for the rapid digestion of mouse liver and gallbladder tissue, enabling the extraction of bile duct cells for organoid culture. However, the collagenase activity and expression levels remain low, indicating significant room for improvement. Summary of the Invention

[0006] Therefore, it is necessary to provide a method for preparing collagenase based on Clostridium histolytica that improves collagenase activity and expression levels, and its application.

[0007] A method for preparing collagenase based on Clostridium histolyticum includes the following steps: Clostridium histolyticum was inoculated into a fermentation medium for anaerobic fermentation to obtain the fermentation product; wherein the anaerobic fermentation pH was 6.8–7.2; and the fermentation medium contained microcarriers and gelatin. The fermentation product was separated and extracted to obtain a collagenase composition.

[0008] In one embodiment, the particle size of the microcarrier is in the range of 50-500 μm; In one embodiment, the raw materials for preparing the microcarrier are artificially synthesized biomaterials or natural biomaterials. The artificially synthesized biomaterials are selected from at least one of polyethylene glycol, polyethylene glycol derivatives, polyethylene glycol diacrylate, polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxy acid, polylactic-alkyd copolymer, polydimethylsiloxane, polyanhydride, polyester, polyamide, polylysine, polyacetal, polycyanoacrylate, polyurethane, polypyrrole, polymethacrylate, polyethylene, polycarbonate, and polyethylene oxide. The natural biomaterials are selected from at least one of collagen, denatured collagen, collagen protein, proteoglycans, glycoproteins, gelatin, gelatin derivatives, chitin, alginate, alginate derivatives, agar, agarose, dextran, starch, whey protein, pectin, fibrinogen, matrix gum, hyaluronic acid, laminin, fibronectin, and fibronectin. In one embodiment, the concentration of the microcarrier in the fermentation medium is 2-6 g / L; In one embodiment, the microcarrier is a 3D TableTrix® microcarrier; In one embodiment, the collagenase composition includes type I collagenase, type II collagenase, neutral protease, and serine protease, with the abundance ratio of type I collagenase, type II collagenase, neutral protease, and serine protease being 56.20:4.70:12.40:25.20.

[0009] In one embodiment, the concentration of gelatin in the fermentation medium is 10–18 g / L; In one embodiment, the fermentation medium further includes L-cysteine ​​hydrochloride, the concentration of which is 0.7–0.9 g / L. In one embodiment, the fermentation medium further includes vitamin B1 at a concentration of 0.015–0.025 g / L; In one embodiment, the fermentation medium further includes soybean peptone, yeast extract, maltose, beef extract, gelatin, CaCl2, and MgSO4·7H2O.

[0010] In one embodiment, the collagenase composition includes type I collagenase, type II collagenase, neutral protease, and serine protease, with the abundance ratio of type I collagenase, type II collagenase, neutral protease, and serine protease being 56.20:4.70:12.40:25.20.

[0011] In one embodiment, the fermentation medium comprises the following components at the following mass concentrations: soybean peptone 13–17 g / L, yeast extract 7–9 g / L, maltose 5–8 g / L, beef extract 4–6 g / L, gelatin 10–18 g / L, 3DTableTrix® microcarriers 2–6 g / L, potassium dihydrogen phosphate 2.5–3.5 g / L, dipotassium hydrogen phosphate 0.8–1.2 g / L, magnesium sulfate heptahydrate 0.7–0.9 g / L, calcium chloride 0.15–0.25 g / L, L-cysteine ​​hydrochloride 0.7–0.9 g / L, and vitamin B1 0.015–0.025 g / L; the pH of the fermentation medium is controlled at 7.0 ± 0.1. In one embodiment, the fermentation medium comprises the following components at the following mass concentrations: 15 g / L soybean peptone, 8 g / L yeast extract, 6 g / L maltose, 5 g / L beef extract, 15 g / L gelatin, 4 g / L 3D TableTrix® microcarriers, 3 g / L potassium dihydrogen phosphate, 1 g / L dipotassium hydrogen phosphate, 0.8 g / L magnesium sulfate heptahydrate, 0.2 g / L calcium chloride, 0.8 g / L L-cysteine ​​hydrochloride, and 0.02 g / L vitamin B1.

[0012] In one embodiment, during fermentation, a replenishing culture medium is added, comprising soybean peptone, yeast extract, gelatin, microcarriers, maltose, and L-cysteine ​​hydrochloride. The replenishment begins 12 hours after fermentation, with the replenished volume accounting for 0.01% to 8% of the initial fermentation medium volume; for example, replenishment is done in multiple batches, with each replenishment volume accounting for 0.01% to 3% of the initial fermentation medium volume; or, replenishment is continuous. Alternatively, batch replenishment can be initiated after 12 hours of fermentation, with 3% added at 12 hours, 3% at 20 hours, and 2% at 28 hours.

[0013] In one embodiment, the replenishing culture medium comprises the following components at the following mass concentrations: 20 g / L soybean peptone, 15 g / L yeast extract, 80 g / L gelatin, 20 g / L 3D TableTrix® microcarriers, 30 g / L maltose, and 1.0 g / L L-cysteine ​​hydrochloride. In one embodiment, the replenishment method is either continuous replenishment or batch replenishment. Preferably, batch replenishment is started after 12 hours of fermentation, with 3% added at the 12th hour, 3% added at the 20th hour, and 2% added at the 28th hour. In one embodiment, the fermentation time was controlled at 30±2h, the fermentation temperature was 37±0.5℃, and the dissolved oxygen concentration during anaerobic fermentation was less than 6% of the air saturation. In one embodiment, during anaerobic fermentation, 10% ammonia water or 5% phosphoric acid solution is added to stabilize the fermentation pH value within the range of 6.8 to 7.2. In one embodiment, the stirring speed is controlled at 60-90 rpm during anaerobic fermentation.

[0014] In one embodiment, the preparation method further includes culturing Clostridium histolyticum using an enrichment medium before anaerobic fermentation of the Clostridium histolyticum inoculated with it. In one embodiment, the enrichment medium comprises the following components at the following mass concentrations: soybean peptone 8–12 g / L, yeast extract 4–6 g / L, glucose 4–6 g / L, gelatin 0.3–0.7 g / L, potassium dihydrogen phosphate 1.5–2.5 g / L, dipotassium hydrogen phosphate 0.8–1.2 g / L, magnesium sulfate heptahydrate 0.4–0.6 g / L, sodium chloride 1–3 g / L, and L-cysteine ​​hydrochloride 0.4–0.6 g / L; the pH value is 7.0 ± 0.1. In one embodiment, the enrichment medium comprises the following components at the following mass concentrations: 10 g / L soybean peptone, 5 g / L yeast extract, 5 g / L glucose, 0.5 g / L gelatin, 2 g / L potassium dihydrogen phosphate, 1 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 2 g / L sodium chloride, and 0.5 g / L L-cysteine ​​hydrochloride. In one embodiment, the proliferation culture time is 8-24 hours.

[0015] In one embodiment, the step of separating and extracting the collagenase composition from the fermentation product includes: sequentially filtering and centrifuging the fermentation product to collect the supernatant; adding ammonium sulfate to the supernatant to adjust the ammonium sulfate saturation of the system to 60-80%, allowing it to stand at a low temperature of 4°C for salt precipitation, and collecting the precipitated product; and sequentially desalting, concentrating, sterilizing, filtering, and freeze-drying the precipitated product to prepare the collagenase composition.

[0016] In one embodiment, the desalting process is performed by dialysis. The resulting precipitate is reconstituted in 20 mM Tris-HCl buffer solution with a pH of 7.2. The reconstituted solution is dialyzed using a dialysis bag with a molecular weight cutoff of 10 kDa to remove residual salts and obtain a desalted collagenase solution. In one embodiment, the concentration is achieved through an ultrafiltration concentration process, whereby the concentration is achieved via an ultrafiltration system. In one embodiment, sterile filtration is performed by aseptic filtration of the concentrated collagenase solution using a 0.22 μm polyethersulfone (PES) filter membrane; In one embodiment, freeze drying is performed by quick freezing with liquid nitrogen followed by freeze drying at -40 to -50°C to obtain a freeze-dried powder of the collagenase composition.

[0017] A collagenase composition is prepared using the method for preparing collagenase based on Clostridium histolyticum as described in any of the above embodiments.

[0018] Application of the collagenase composition in microcarrier cell culture as described in any of the above embodiments.

[0019] In one embodiment, applying the collagenase composition to cell harvesting from microcarrier cell culture includes the following steps: Preparation of collagenase stock solution: Dissolve the prepared lyophilized collagenase composition in Hanks balanced salt solution, DMEM medium or 3D FloTrix® three-dimensional stem cell basal medium to prepare a 10X collagenase stock solution with a concentration of 10 mg / mL. After sterilization by filtration through a 0.22 μm filter membrane, store at 4℃ for later use. Microcarrier lysis process: Cells cultured on the three-dimensional microcarrier for 3 days or more are allowed to settle statically, some of the culture medium supernatant is removed, and 10X collagenase stock solution is added to dilute to 1X working concentration; lysis is carried out by stirring at 40 rpm and 37°C. Timing starts when the system temperature rises above 36°C and lysis is continued for 30-40 minutes until the three-dimensional microcarrier is completely lysed. Cell harvesting: After complete lysis of the microcarriers, the cell suspension in the system is pumped out and washed and harvested using either manual centrifugation or an automated cell harvesting device. The manual centrifugation process involves transferring the harvested cell suspension to a centrifuge container and centrifuging at 1500 rpm for 5 minutes. After centrifugation, the supernatant is discarded, and the cells are washed with PBS solution or physiological saline containing 1% human albumin, repeating this washing process 2-3 times to obtain a single-cell suspension. The automated cell harvesting process uses a 3D FloTrix® vivaPREP PLUS or 3D FloTrix® vivaPREPULTRA automated cell harvesting device, and the cell washing and harvesting operations are performed according to the corresponding instruction manual.

[0020] The above-described method for preparing collagenase based on Clostridium histolyticum involves simultaneously introducing microcarriers and gelatin into the fermentation medium and maintaining the anaerobic fermentation pH stably between 6.8 and 7.2. The microcarriers, acting as the core specific enzyme-inducing substance, also provide sustained release of organic carbon and nitrogen nutrition, increasing the effective cell density in the fermentation system. This increase in cell volume directly boosts the total collagenase yield. Gelatin serves as a specific inducing substrate, stimulating efficient collagenase gene expression. Using gelatin + microcarriers as dual collagen inducers in the Clostridium histolyticum fermentation medium, instead of the traditional single gelatin induction method, can target and induce collagenase enzymes highly compatible with the Huakan three-dimensional collagen microcarriers, significantly improving the microcarrier cleavage specificity and efficiency, and significantly increasing both collagenase activity and expression levels. The neutral to mild pH range ensures normal cell proliferation while reducing the degradation and inactivation of extracellular collagenase. These three factors synergistically enhance the overall collagenase expression level, significantly increasing the enzyme activity level and yield per unit fermentation broth of the final collagenase composition.

[0021] This invention utilizes a combination of gelatin and 3D TableTrix® microcarriers for dual collagen induction and fed-batch anaerobic fermentation, along with salting-out precipitation, dialysis desalination, ultrafiltration concentration, sterile filtration, and freeze-drying processes to produce a highly active collagenase composition containing collagenase I, collagenase II, and clostridium protease. The fermentation process of this invention is stable and controllable, with collagenase activity reaching 2000–3000 CDU / mL and uniform enzyme component ratios. This composition can specifically and gently degrade the collagen backbone of the three-dimensional collagen microcarriers without damaging cell membrane proteins and cell surface markers. It can be applied to the three-dimensional culture and harvesting of adherent cells such as mesenchymal stem cells, immune cells, and human diploid cells, achieving a cell recovery rate ≥95%, a viability rate ≥90%, and microcarrier residue <0.1%. It can be seamlessly integrated with three-dimensional cell culture and automated cell harvesting devices. This invention solves the technical problems of cell damage caused by traditional pancreatic enzyme digestion, incomplete harvesting by physical blowing, and low cleavage efficiency of ordinary collagenase. The process can be scaled up industrially and is low in cost, and has good application prospects and industrial practical value in cell therapy, biopharmaceuticals and other fields.

[0022] The collagenase composition of this invention is prepared by standardized anaerobic fermentation of Clostridium histolyticum, which is stable, scalable, and low-cost. For the first time, gelatin + 3D TableTrix® microcarriers are used as dual collagen inducers, which can target and induce the production of collagenase composition highly adapted to Huakan 3D collagen microcarriers. This significantly improves the lysis specificity of microcarriers, gently lyses 3D collagen microcarriers, and achieves high recovery rate, high viability, and non-destructive harvesting of adherent cells. It fully meets the needs of large-scale and standardized production of cell therapy products and can be widely used in biopharmaceutical companies, cell preparation centers, and research institutions, with good industrial applicability and market application prospects. Attached Figure Description

[0023] Figure 1 The OD of the Clostridium histolytica fermentation process in specific embodiment 1 of the present invention 600 Growth curve and collagenase activity change curve; Figure 2 This is an SDS-PAGE purity analysis chromatogram of the collagenase composition extracted from Clostridium histolyticum fermentation products in Specific Example 1 of the present invention; Figure 3 This is a mass spectrometry analysis result of the collagenase composition extracted from the Clostridium histolyticum fermentation product in Specific Embodiment 1 of the present invention; Figure 4 This is a microscopic morphological image of the three-dimensional microcarrier before lysis in a specific embodiment 1 of the present invention; Figure 5 This is a morphological diagram of the cell release after complete lysis of the three-dimensional microcarrier by the collagenase composition in a specific embodiment 1 of the present invention. Figure 6This is a graph showing the cell proliferation and viability test results after cell harvesting in a specific embodiment 1 of the present invention. Detailed Implementation

[0024] To facilitate understanding of the present invention and to make the above-mentioned objects, features, and advantages of the present invention more apparent, a detailed description of specific embodiments of the present invention is provided below in conjunction with the accompanying drawings. Numerous specific details are set forth in the following description to provide a thorough understanding of the present invention, and preferred embodiments are shown in the accompanying drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of the present invention. The present invention can be implemented in many other ways than those described herein, and similar modifications can be made by those skilled in the art without departing from the spirit of the invention; therefore, the present invention is not limited to the specific embodiments disclosed below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0025] In one embodiment, this application provides a method for preparing collagenase based on Clostridium histolyticum, comprising the following steps: Clostridium histolyticum was inoculated into a fermentation medium for anaerobic fermentation to obtain the fermentation product; wherein the anaerobic fermentation pH was 6.8–7.2; and the fermentation medium contained microcarriers and gelatin. The fermentation product was separated and extracted to obtain a collagenase composition.

[0026] The above-described method for preparing collagenase based on Clostridium histolyticum involves simultaneously introducing microcarriers and gelatin into the fermentation medium and maintaining the anaerobic fermentation pH stably between 6.8 and 7.2. The microcarriers, acting as the core specific enzyme-inducing substance, also provide sustained release of organic carbon and nitrogen nutrition, increasing the effective cell density in the fermentation system. This increase in cell volume directly boosts the total collagenase yield. Gelatin serves as a specific inducing substrate, stimulating efficient collagenase gene expression. Using gelatin + microcarriers as dual collagen inducers in the Clostridium histolyticum fermentation medium, instead of the traditional single gelatin induction method, can target and induce collagenase enzymes highly compatible with the Huakan three-dimensional collagen microcarriers, significantly improving the microcarrier cleavage specificity and efficiency, and significantly increasing both collagenase activity and expression levels. The neutral to mild pH range ensures normal cell proliferation while reducing the degradation and inactivation of extracellular collagenase. These three factors synergistically enhance the overall collagenase expression level, significantly increasing the enzyme activity level and yield per unit fermentation broth of the final collagenase composition.

[0027] For example, *Clostridium histolyticum* was tested using the standard strain ATCC19401. However, *Clostridium histolyticum* is not limited to this strain.

[0028] For example, the particle size of the microcarrier is in the range of 50-500 μm; thus, as a core specific enzyme-inducing substance, it also has the dual function of slow-release organic carbon and nitrogen nutrition, which helps to improve the overall expression level and enzyme activity of collagenase.

[0029] For example, the raw materials for preparing the microcarrier are artificially synthesized biomaterials or natural biomaterials. The artificially synthesized biomaterials are selected from at least one of the following: polyethylene glycol, polyethylene glycol derivatives, polyethylene glycol diacrylate, polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxy acid, polylactic-alkyd copolymer, polydimethylsiloxane, polyanhydride, polyester, polyamide, polylysine, polyacetal, polycyanoacrylate, polyurethane, polypyrrole, polymethacrylate, polyethylene, polycarbonate, and polyethylene oxide. The natural biomaterials are selected from at least one of the following: collagen, denatured collagen, collagen protein, proteoglycans, glycoproteins, gelatin, gelatin derivatives, chitin, alginate, alginate derivatives, agar, agarose, dextran, starch, whey protein, pectin, fibrinogen, matrix gum, hyaluronic acid, laminin, fibronectin, and fibronectin. For example, the microcarrier is a 3D TableTrix® microcarrier. Thus, by selecting 3D TableTrix® gelatin microcarriers as the microcarrier, with a carrier matrix of pharmaceutical-grade gelatin collagen material, collagen-inducing signals can be continuously released, further improving the overall expression level and enzyme activity of collagenase. For example, the microcarrier is a 3D TableTrix® microcarrier from Huakan Biotechnology. For example, the microcarrier has a porosity >90% and a particle size controlled between 50 and 500 μm, forming a continuous three-dimensional porous structure. As a core specific enzyme-inducing substance, it also has the dual function of slow-release organic carbon and nitrogen nutrition, which helps to improve the overall expression level and enzyme activity of collagenase.

[0030] This invention pioneers a dual collagen-induced fermentation system composed of gelatin and 3D TableTrix® microcarriers, which differs from the traditional single-induction mode that only adds gelatin. The microcarriers serve as the core specific enzyme-inducing substance, while also providing sustained release of organic carbon and nitrogen nutrition. Simultaneously, the gelatin matrix of the microcarriers, together with the gelatin in the culture medium, provides dual stimulation for the continuous and efficient transcription and translation of collagenase-encoding genes. Combined with a neutral to mild fermentation pH range of 6.8–7.2, this system ensures the normal proliferation of Clostridium histolytica under obligate anaerobic conditions and reduces the degradation and inactivation of extracellular collagenase by miscellaneous proteases during fermentation. These multiple conditions synergistically significantly enhance the overall expression level of collagenase, with collagenase activity in the fermentation broth reaching 2000–3000 U / mL. At the same time, it induces the generation of a composite collagenase profile that is highly matched with the collagen-based three-dimensional microcarriers, resulting in significantly improved specificity and lysis efficiency during subsequent lysis of the microcarriers.

[0031] In one embodiment, the concentration of the microcarrier in the fermentation medium is 2-6 g / L. Thus, controlling the amount of microcarrier added to 2-6 g / L can balance the total amount of inducible substrate and the mass transfer efficiency of the fermentation system. If the amount added is too low, the induction signal will be insufficient and the cell enrichment effect will be poor. If the amount added is too high, the viscosity of the medium will increase, dissolved oxygen conduction will be blocked, and cell metabolism will be inhibited. This concentration range can achieve the dual optimal balance between cell enrichment and collagen induction.

[0032] In one embodiment, the concentration of gelatin in the fermentation medium is 10–18 g / L. Thus, limiting the gelatin concentration in the fermentation medium to 10–18 g / L ensures that sufficient exogenous collagen substrate can continuously activate the Clostridium histolytica collagenase synthesis pathway and eliminate the inhibition of enzyme-producing genes by carbon metabolism repression. When the concentration is below 10 g / L, the induction signal is weak and the collagenase expression level decreases significantly. When the concentration is above 18 g / L, the viscosity of the medium will increase significantly, resulting in uneven mass transfer and insufficient oxygen supply to the cells. This range can balance strong induction effect with the fluidity of the fermentation broth, maximizing the total collagenase yield.

[0033] In one embodiment, the fermentation medium further includes L-cysteine ​​hydrochloride, the concentration of which is 0.7–0.9 g / L. Thus, L-cysteine ​​hydrochloride contains reducing sulfhydryl groups, which can continuously lower the redox potential of the fermentation system, thus aiding in the growth and metabolism of Clostridium histolyticum. Simultaneously, it can provide sulfhydryl synthesis raw materials for the active site of collagenase protein, maintaining the spatial conformational stability of collagenase and reducing enzyme activity loss.

[0034] In one embodiment, the fermentation medium further includes vitamin B1 at a concentration of 0.015–0.025 g / L; thus, vitamin B1, as a key coenzyme for sugar metabolism and amino acid synthesis in Clostridium histolyticum, can enhance the basal metabolic level of the bacteria by adding 0.015–0.025 g / L, thereby increasing the bacterial proliferation rate and biomass, and indirectly increasing the total secretion of collagenase.

[0035] In one embodiment, the fermentation medium further includes soybean peptone, yeast extract, maltose, beef extract, gelatin, CaCl2, and MgSO4·7H2O. Thus, by using a complex organic nitrogen source of peptone, yeast extract, and beef extract combined with a slow-release carbon source of maltose, supplemented with phosphate, magnesium, and calcium metal ions to maintain cell osmotic pressure and meet the enzyme catalytic cofactor requirements, and combined with gelatin, 3DTableTrix® microcarriers as dual inducers, the reducing agent L-cysteine ​​hydrochloride, and coenzyme vitamin B1, and maintaining a stable pH of 7.0±0.1 suitable for initial cell growth, the entire set of nutrient components synergistically achieves high-density cell enrichment. This continuous collagen-inducing culture system significantly improves the unit yield of collagenase compared to a simple basal medium, and the enzyme component ratio is uniform and stable.

[0036] In one embodiment, the fermentation medium comprises the following components at the following mass concentrations: soybean peptone 13–17 g / L, yeast extract 7–9 g / L, maltose 5–8 g / L, beef extract 4–6 g / L, gelatin 10–18 g / L, 3DTableTrix® microcarriers 2–6 g / L, potassium dihydrogen phosphate 2.5–3.5 g / L, dipotassium hydrogen phosphate 0.8–1.2 g / L, magnesium sulfate heptahydrate 0.7–0.9 g / L, calcium chloride 0.15–0.25 g / L, L-cysteine ​​hydrochloride 0.7–0.9 g / L, and vitamin B1. 0.015~0.025g / L; the pH of the fermentation medium is controlled at 7.0±0.1; for example, the fermentation medium includes the following components at the following mass concentrations: soybean peptone 15g / L, yeast extract 8g / L, maltose 6g / L, beef extract 5g / L, gelatin 15g / L, 3DTableTrix® microcarrier 4g / L, potassium dihydrogen phosphate 3g / L, dipotassium hydrogen phosphate 1g / L, magnesium sulfate heptahydrate 0.8g / L, calcium chloride 0.2g / L, L-cysteine ​​hydrochloride 0.8g / L, and vitamin B1 0.02g / L. Thus, when the components are selected at the optimal intermediate concentration ratio, the nutrient supply, inducing substrate content, anaerobic protectant, and trace elements reach an optimal balance, and the cell OD... 600 The peak value can reach 3.29, and the collagenase activity at the fermentation endpoint can reach up to 2880 CDU / mL. It is the optimal combination of enzyme production performance in the entire component range, suitable for industrial-scale fermentation production, with small batch-to-batch enzyme activity fluctuations and high product consistency.

[0037] In one embodiment, during fermentation, a replenishing medium is added, comprising soybean peptone, yeast extract, gelatin, microcarriers, maltose, and L-cysteine ​​hydrochloride. The replenishment begins 12 hours after fermentation, with the replenished volume accounting for 0.01% to 3% of the initial fermentation medium volume. For example, the replenishing medium comprises the following components at the following mass concentrations: soybean peptone 20 g / L, yeast extract 15 g / L, gelatin 80 g / L, 3D TableTrix® microcarriers 20 g / L, maltose 30 g / L, and L-cysteine ​​hydrochloride 1.0 g / L. Thus, during the logarithmic growth phase of fermentation, the replenishing medium containing gelatin and 3D TableTrix® microcarriers is continuously added, continuously supplementing collagen-inducing substrates and slow-release carbon and nitrogen nutrients, extending the stable enzyme production cycle of the cells, and alleviating nutrient deficiency and cell autolysis problems in the later stages of fermentation.

[0038] In one embodiment, the replenishment method is either continuous replenishment or batch replenishment. Preferably, batch replenishment is initiated after 12 hours of fermentation, with 3% replenished at 12 hours, 3% at 20 hours, and 2% at 28 hours. Thus, the three-stage gradient replenishment matches the growth cycle of Clostridium histolyticum: at 12 hours, the cells enter the logarithmic enzyme production phase, and the first replenishment enhances induction; at 20 hours, the cell biomass reaches its peak, and a second replenishment maintains enzyme production metabolism; at 28 hours, the cells enter the stable late stage, and a small amount of replenishment delays autolysis. The three-stage replenishment strategy can extend the stable enzyme production cycle by more than 8 hours, significantly increasing the total collagenase yield compared to a single replenishment.

[0039] In one embodiment, the fermentation time is controlled at 30±2h, the fermentation temperature is 37±0.5℃, and the dissolved oxygen concentration during anaerobic fermentation is below 6% air saturation. Thus, 37±0.5℃ is the optimal temperature for the growth of Clostridium histolyticum and the synthesis of collagenase. Temperature deviations from this range will significantly reduce cell proliferation and enzyme activity. The total fermentation time of 30±2h can balance the maximum cell biomass and the highest collagenase activity. Insufficient fermentation time will result in insufficient enzyme production, while fermentation time exceeding 32h will lead to extensive autolysis of the cells and degradation of collagenase. The dissolved oxygen concentration being below 6% air saturation throughout the process can prevent the toxic damage of oxygen to obligate anaerobic bacteria, maintain a high viability rate throughout the process, and ensure continuous enzyme production.

[0040] In one embodiment, the anaerobic environment is controlled as follows: the entire fermentation process is carried out under strict anaerobic conditions, with high-purity nitrogen (purity ≥99.999%) continuously introduced into the fermenter to maintain the dissolved oxygen (DO) concentration below 6% (relative to air), thus ensuring the obligate anaerobic growth characteristics of Clostridium histolyticum.

[0041] In one embodiment, during anaerobic fermentation, a 10% ammonia solution or a 5% phosphoric acid solution is added to stably control the fermentation pH within the range of 6.8 to 7.2. This prevents the pH from continuously rising due to microbial metabolites during fermentation. Adding 10% ammonia or 5% phosphoric acid allows for real-time dynamic pH control, maintaining the pH within this range. This range ensures normal microbial growth while minimizing the degradation of collagenase proteins by the alkaline environment, reducing enzyme loss during fermentation, and stabilizing the enzyme activity of the final product. By automatically adding 10% ammonia or 5% phosphoric acid solution, the fermentation pH is stably controlled within the range of 6.8 to 7.2, avoiding the impact of pH fluctuations on microbial growth and enzyme synthesis.

[0042] In one embodiment, the stirring speed is controlled at 60–90 rpm during anaerobic fermentation. This low-shear stirring speed range ensures uniform mixing of the culture medium, microcarriers, and bacterial cells, guaranteeing sufficient contact between nutrients and inducing substrates with the bacterial cells. Simultaneously, it significantly reduces mechanical shear damage caused by high-speed stirring, preventing microcarrier breakage and bacterial cell rupture and autolysis, thus balancing mass transfer efficiency and bacterial survival rate. Using a low-shear stirring system with a stirring speed controlled at 60–90 rpm ensures uniform mixing of the culture medium while minimizing mechanical damage to the bacterial cells.

[0043] In one embodiment, before anaerobic fermentation of Clostridium histolyticum inoculation, the preparation method further includes propagating Clostridium histolyticum using an enrichment medium. Thus, seed culture in an enrichment medium is added before fermentation, employing a two-stage differentiated culture medium strategy. The enrichment medium is supplemented with glucose, a readily available carbon source, which can rapidly increase the proliferation rate of the strain and obtain a high-activity, high-density seed liquid in a short time, achieving the goal of first strengthening the bacteria and then producing enzymes. The fermentation medium removes glucose, completely eliminating the carbon decomposition and metabolic inhibition caused by glucose, and maximally relieving the inhibition of collagenase synthesis. Compared with direct fermentation without seeds, the increase in collagenase activity at the fermentation endpoint is more significant.

[0044] In one embodiment, the enrichment culture medium comprises the following components at the following mass concentrations: soybean peptone 8-12 g / L, yeast extract 4-6 g / L, glucose 4-6 g / L, gelatin 0.3-0.7 g / L, potassium dihydrogen phosphate 1.5-2.5 g / L, dipotassium hydrogen phosphate 0.8-1.2 g / L, magnesium sulfate heptahydrate 0.4-0.6 g / L, sodium chloride 1-3 g / L, and L-cysteine ​​hydrochloride 0.4-0.6 g / L; the pH value is 7.0 ± 0.1; Example For example, in one embodiment, the enrichment medium comprises the following components at the following mass concentrations: soybean peptone 10 g / L, yeast extract 5 g / L, glucose 5 g / L, gelatin 0.5 g / L, potassium dihydrogen phosphate 2 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium sulfate heptahydrate 0.5 g / L, sodium chloride 2 g / L, and L-cysteine ​​hydrochloride 0.5 g / L. Thus, the enrichment medium uses low-concentration gelatin for basic induction, glucose as a rapid carbon source for rapid propagation of the strain, and a low dose of L-cysteine ​​hydrochloride to maintain an anaerobic environment for the seeds. This low-salt, low-inducing-substrate design is suitable for the rapid proliferation needs of the seeds, preventing premature over-induction and resulting in premature strain decline. The secondary seed OD... 600 It can stably reach above 0.7, indicating vigorous bacterial activity.

[0045] In one embodiment, the proliferation culture time is 8-24 hours. Thus, when the components are taken at the optimal intermediate concentration, the seed proliferation rate is the fastest, reaching the qualified inoculation OD value in 8-12 hours, resulting in high strain survival rate, a short adaptation period after transfer to the fermenter, and rapid entry into the enzyme production stage, thus shortening the overall fermentation cycle. Preferably, the proliferation culture time is 10 hours.

[0046] In one embodiment, the step of separating and extracting the collagenase composition from the fermentation product includes: sequentially filtering and centrifuging the fermentation product to collect the supernatant; adding ammonium sulfate to the supernatant to adjust the ammonium sulfate saturation of the system to 60-80%, allowing it to stand at 4°C for salting-out precipitation, and collecting the precipitate; and sequentially desalting, concentrating, sterilizing, filtering, and freeze-drying the precipitate to prepare the collagenase composition. Thus, 60-80% ammonium sulfate saturation salting-out efficiently precipitates the collagenase complex components, removing most of the impurities, proteins, and bacterial fragments; 10kDa dialysis bag desalting completely eliminates the inhibition of collagenase activity by ammonium sulfate ions; ultrafiltration concentration rapidly increases the enzyme concentration and reduces freeze-drying time; a 0.22μm PES filter membrane achieves sterility, meeting the sterility requirements of cell therapy preparations; liquid nitrogen quick-freezing combined with freeze-drying at -40 to -50°C completely preserves the spatial conformation of the collagenase; the freeze-dried powder exhibits slow enzyme activity decay during long-term storage at room temperature and 4°C, significantly improving the stability of the preparation.

[0047] In one embodiment, the desalting process is performed using dialysis. The resulting precipitate is reconstituted in a 20 mM Tris-HCl buffer solution with a pH of 7.2. The reconstituted solution is then dialyzed using a dialysis bag with a molecular weight cutoff of 10 kDa to remove residual salts and obtain a desalted collagenase solution. In this way, the pH 7.2 Tris-HCl buffer solution matches the stable range of collagenase, and the dialysis process does not cause a decrease in enzyme activity. The 10 kDa molecular weight cutoff can completely retain collagenase protein, allowing only small molecule ammonium sulfate to pass through, resulting in thorough desalting without enzyme protein loss and no significant loss of collagenase activity after desalting.

[0048] In one embodiment, the concentration is achieved through ultrafiltration concentration, which is performed using an ultrafiltration system. In this way, the ultrafiltration system can quickly remove water and small molecule metabolic impurities, and increase the collagenase concentration without introducing exogenous precipitants. Compared with vacuum concentration and heating, it does not cause enzyme protein denaturation, and the collagenase activity retention rate after concentration is ≥95%.

[0049] In one embodiment, sterile filtration is performed by using a 0.22 μm polyethersulfone (PES) filter membrane to sterile filter the concentrated collagenase solution. In this way, the polyethersulfone filter membrane has very low protein adsorption, and the loss of collagenase during the filtration process is minimal. It can completely retain bacterial cells and microbial impurities to obtain sterile collagenase stock solution, which meets the sterile use standards for cell drugs and clinical-grade cell harvesting.

[0050] In one embodiment, freeze-drying involves rapid freezing with liquid nitrogen followed by freeze-drying at -40 to -50°C to obtain a freeze-dried powder of the collagenase composition. This rapid freezing with liquid nitrogen avoids large ice crystals inside the enzyme solution from damaging the tertiary structure of the collagenase protein, while the -40 to -50°C low-temperature vacuum freeze-drying process maintains the collagenase activity to the maximum extent. The freeze-dried powder is convenient to transport and store, whereas the liquid enzyme solution can only be stored at 4°C for a short period, showing a significant difference in stability.

[0051] In one embodiment, the collagenase composition includes type I collagenase, type II collagenase, neutral protease, and serine protease, with an abundance ratio of 56.20:4.70:12.40:25.20. Subsequent mass spectrometry analysis of the lyophilized components revealed that the collagenase fragment contained 56.20% type I collagenase, 4.70% type II collagenase, 12.40% neutral protease, 25.20% serine protease, and 1.50% other miscellaneous enzyme components. Therefore, the abundance ratio of type I collagenase, type II collagenase, neutral protease, and serine protease was 56.20:4.70:12.40:25.20.

[0052] The collagenase composition prepared by the complete fermentation and extraction process of this invention contains a stable ratio of collagenase I, collagenase II and clostridium protease complex enzyme system. The enzyme activity is stable at 2000-3000 CDU / mL, the enzyme components are uniform in proportion, and it can specifically degrade the triple helix structure of collagen without damaging cell membrane proteins or cell surface markers. Unlike single collagenases and trypsin, the cell harvest viability is ≥90%, and the cells retain their original phenotype and proliferative function intact.

[0053] A collagenase composition is prepared using the method for preparing collagenase based on Clostridium histolyticum as described in any of the above embodiments.

[0054] Application of the collagenase composition in microcarrier cell culture as described in any of the above embodiments.

[0055] In one embodiment, applying the collagenase composition to cell harvesting from microcarrier cell culture includes the following steps: Preparation of collagenase stock solution: Dissolve the prepared lyophilized collagenase composition in Hanks balanced salt solution, DMEM medium or 3D FloTrix® three-dimensional stem cell basal medium to prepare a 10X collagenase stock solution with a concentration of 10 mg / mL. After sterilization by filtration through a 0.22 μm filter membrane, store at 4℃ for later use. Microcarrier lysis process: Cells cultured on the three-dimensional microcarrier for 3 days or more are allowed to settle statically, some of the culture medium supernatant is removed, and 10X collagenase stock solution is added to dilute to 1X working concentration; lysis is carried out by stirring at 40 rpm and 37°C. Timing starts when the system temperature rises above 36°C and lysis is continued for 30-40 minutes until the three-dimensional microcarrier is completely lysed. Cell harvesting: After complete lysis of the microcarriers, the cell suspension in the system is pumped out and washed and harvested using either manual centrifugation or an automated cell harvesting device. The manual centrifugation process involves transferring the harvested cell suspension to a centrifuge container and centrifuging at 1500 rpm for 5 minutes. After centrifugation, the supernatant is discarded, and the cells are washed with PBS solution or physiological saline containing 1% human albumin, repeating this washing process 2-3 times to obtain a single-cell suspension. The automated cell harvesting process uses a 3D FloTrix® vivaPREP PLUS or 3D FloTrix® vivaPREPULTRA automated cell harvesting device, and the cell washing and harvesting operations are performed according to the corresponding instruction manual.

[0056] This collagenase composition is specifically designed for use with 3D TableTrix® series collagen microcarriers. At a working concentration of 1×, it can completely lyse the microcarrier backbone in 30–40 minutes at 37°C, with microcarrier residue <0.1%. It is compatible with both manual centrifugation and automated harvesting processes. The lysate residue can be completely removed by washing 2–3 times, with a cell recovery rate of ≥95%. It does not require vigorous physical pipetting, thus avoiding mechanical damage. It can be seamlessly integrated with 3D FloTrix® automated cell harvesting equipment to achieve large-scale, automated, and non-destructive cell harvesting. This solves the industry pain points of traditional trypsin damaging cells, low lysis efficiency of ordinary collagenases, and incomplete harvesting by physical pipetting.

[0057] The above-described method for preparing collagenase based on Clostridium histolyticum and its application involves simultaneously introducing microcarriers and gelatin into the fermentation medium and maintaining the anaerobic fermentation pH stably between 6.8 and 7.2. The microcarriers, acting as the core specific enzyme-inducing substance, also provide sustained release of organic carbon and nitrogen nutrition, increasing the effective cell density in the fermentation system. This increase in cell volume directly boosts the total collagenase yield. Gelatin serves as a specific inducing substrate, stimulating efficient collagenase gene expression. Using gelatin + microcarriers as dual collagen inducers in the Clostridium histolyticum fermentation medium, instead of the traditional single gelatin induction method, can target and induce collagenase enzyme profiles highly compatible with the Huakan three-dimensional collagen microcarriers, significantly improving the microcarrier cleavage specificity and efficiency, and significantly increasing both collagenase activity and expression levels. The neutral to mild pH range ensures normal cell proliferation while reducing the degradation and inactivation of extracellular collagenase. These three factors synergistically enhance the overall collagenase expression level, significantly increasing the enzyme activity level and yield per unit fermentation broth of the final collagenase composition.

[0058] This application discloses a collagenase composition produced by Clostridium histolyticum fermentation and its application in the lysis of three-dimensional collagen microcarriers and cell-free harvesting. Using Clostridium histolyticum ATCC 19401 as the production strain, a two-stage differentiated culture medium system is employed: an enrichment medium containing glucose and a fermentation medium without glucose. Through dual collagen-inducing with gelatin and 3D TableTrix® microcarriers, and strictly anaerobic fermentation with batch feeding, combined with salting-out precipitation, dialysis desalination, ultrafiltration concentration, sterile filtration, and freeze-drying processes, a highly active collagenase composition containing collagenase I, collagenase II, and clostridium protease is obtained. The fermentation process of this invention is stable and controllable, with collagenase activity reaching 2000-3000 CDU / mL and uniform enzyme component ratios. This composition can specifically and gently degrade the collagen backbone of three-dimensional collagen microcarriers without damaging cell membrane proteins and cell surface markers. It can be applied to the three-dimensional culture and harvesting of adherent cells such as mesenchymal stem cells, immune cells, and human diploid cells, achieving a cell recovery rate ≥95%, a viability rate ≥90%, and microcarrier residue <0.1%. It can be seamlessly integrated with three-dimensional cell culture and automated cell harvesting devices. This invention solves the technical problems of traditional trypsin digestion damaging cells, incomplete harvesting by physical pipetting, and low lysis efficiency of ordinary collagenases. The process can be industrially scaled up, is low-cost, and has good application prospects and industrial practical value in cell therapy, biopharmaceuticals, and other fields.

[0059] In one embodiment, the preparation method of collagenase based on Clostridium histolyticum of the present invention uses the standard strain ATCC19401 as the Clostridium histolyticum strain.

[0060] For example, the culture medium of this application uses three types of culture media: enrichment medium, fermentation medium, and fed-batch medium; each culture medium includes the following components at the following mass concentrations: Enrichment medium: Soybean peptone 8–12 g / L, preferably 10 g / L; yeast extract 4–6 g / L, preferably 5 g / L; glucose 4–6 g / L, preferably 5 g / L; gelatin 0.3–0.7 g / L, preferably 0.5 g / L; KH₂PO₄ 1.5–2.5 g / L, preferably 2 g / L; K₂HPO₄ 0.8–1.2 g / L, preferably 1 g / L; MgSO₄•7H₂O 0.4–0.6 g / L, preferably 0.5 g / L; NaCl 1–3 g / L, preferably 2 g / L; L-cysteine ​​hydrochloride 0.4–0.6 g / L, preferably 0.5 g / L; pH 7.0±0.1, prepared as an anaerobic medium; Fermentation medium: Soybean peptone 13–17 g / L, preferably 15 g / L; yeast extract 7–9 g / L, preferably 8 g / L; maltose 5–8 g / L, preferably 6 g / L; beef extract 4–6 g / L, preferably 5 g / L; gelatin 10–18 g / L, preferably 15 g / L; 3DTableTrix® microcarrier 2–6 g / L, preferably 4 g / L; KH₂PO₄ 2.5–3.5 g / L, preferably 3 g / L; KH₂PO₄ 0.8–1.2 g / L, preferably 1 g / L; MgSO₄•7H₂O 0.7–0.9 g / L, preferably 0.8 g / L; CaCl₂ 0.15–0.25 g / L, preferably 0.2 g / L; L-cysteine ​​hydrochloride 0.7–0.9 g / L. g / L, preferably 0.8 g / L; vitamin B1 0.015~0.025 g / L, preferably 0.02 g / L, pH 7.0±0.1, to prepare an anaerobic culture medium; Feeding medium: 20 g / L soybean peptone, 15 g / L yeast extract, 80 g / L gelatin, 20 g / L 3D TableTrix® microcarriers, 30 g / L maltose, 1.0 g / L L-cysteine ​​hydrochloride.

[0061] For example, the fermentation process of this application is as follows: a) Anaerobic environment control: The entire fermentation process is carried out under strict anaerobic conditions. High-purity nitrogen (purity ≥99.999%) is continuously introduced into the fermenter to maintain the dissolved oxygen (DO) concentration below 6% (relative to air) to ensure the obligate anaerobic growth characteristics of Clostridium histolyticum. b) Temperature control: The fermentation temperature is precisely controlled at 37±0.5℃, using a jacketed temperature control system to ensure temperature uniformity; c) pH control: The fermentation pH is stably controlled within the range of 6.8 to 7.2 by automatically adding 10% ammonia water or 5% phosphoric acid solution, so as to avoid the impact of pH fluctuations on cell growth and enzyme synthesis. d) Stirring control: A low-shear stirring system is used, and the stirring speed is controlled at 60-90 rpm to ensure uniform mixing of the culture medium while reducing mechanical damage to the bacteria; e) Feeding strategy: Start feeding in batches after 12 hours of fermentation, adding 3% at 12 hours, 3% at 20 hours, and 2% at 28 hours; (calculated based on the volume of fermentation medium). f) Fermentation cycle: The total fermentation time is controlled at 30±2 hours, and the cell concentration (OD) is monitored in real time. 600 ) and collagenase activity to determine the optimal harvest time; g) Dissolved oxygen monitoring: Equipped with an online dissolved oxygen electrode to monitor the dissolved oxygen content in the fermentation broth in real time, ensuring that it is always maintained in an anaerobic state.

[0062] For example, the extraction process of the collagenase composition after fermentation according to this application is as follows: a) Salting out: Adjust the supernatant after fermentation filtration and centrifugation to a 60-80% saturated ammonium sulfate solution, let it stand at 4℃ for 2 hours to allow salting out, and collect the precipitate; b) Dialysis desalination: Dissolve the salt-out precipitate in 20 mM Tris-HCl buffer (pH 7.2) and dialyze using a dialysis bag with a molecular weight cutoff of 10 kDa to remove residual salts; c) Concentration: The purified collagenase solution is concentrated using an ultrafiltration system; d) Sterile filtration: Aseptic filtration is performed using a 0.22μm polyethersulfone (PES) filter membrane; e) Freeze-dried formulation: The aseptically filtered collagenase solution is rapidly frozen with liquid nitrogen and then freeze-dried at -40 to -50°C to prepare a stable powder.

[0063] For example, the process of applying the collagenase composition of this application to the lysis and cell harvesting of three-dimensional microcarriers is as follows: a) Preparation of lysis buffer: Dissolve the lyophilized collagenase composition in Hanks balanced salt solution, DMEM or 3DFloTrix® three-dimensional stem cell basal culture medium to prepare a stock solution of 10 mg / mL (10X), filter it through a 0.22µm filter membrane for sterilization and store it at 4℃ for later use. b) Allow cells cultured on the three-dimensional microcarrier to settle to day 3 or above (hereinafter referred to as "micro-tissues") to settle, discard part of the culture supernatant, add the 10X stock solution prepared above to the concentration of 1X, turn on the stirrer at 40 rpm (consistent with the culture), heat at 37°C, and lyse for 30-40 min (start timing when the temperature rises above 36°C). c) After all microcarriers have lysed (30-40 min), pump out the cell suspension and wash and harvest the cells using manual centrifugation, or the 3DFloTrix® vivaPREPPLUS or 3D FloTrix® vivaPREP ULTRA automated cell harvesting device. d) Manual centrifugation and washing method: Transfer all the harvested cell suspension to a centrifuge tube or centrifuge cup of appropriate volume, and centrifuge at 1500 rpm for 5 min. After centrifugation, discard the supernatant, and wash the cells with PBS or 1% human albumin + physiological saline 2-3 times. After washing, resuspend the cells for subsequent experiments. e) Automated cell harvesting method: Refer to the instruction manual of the corresponding equipment.

[0064] This application has the following significant advantages: (1) Strict anaerobic fermentation process is stable: collagenase activity can reach 2000-3000U / mL, and the component ratio is uniform; (2) Specific targeted lysis: only degrades the three-dimensional microcarrier collagen skeleton without damaging the cell membrane; (3) Excellent cell harvesting effect: recovery rate ≥95%, viability ≥90%, maintaining the complete phenotype and function of cells; (4) Simple operation and suitable for large-scale production: with an automated cell harvesting device, cells can be cleaned and harvested.

[0065] This application pioneers a composite induced fermentation system. For the first time, this invention uses gelatin + 3D TableTrix® microcarriers as dual collagen inducers in Clostridium histolyticum fermentation medium, replacing the traditional single gelatin induction method. It can target and induce the production of collagenase enzyme profiles that are highly compatible with Huakan 3D collagen microcarriers, significantly improving the lysis specificity and efficiency of microcarriers.

[0066] This application employs a two-stage culture medium differential precision design. By using a two-stage differentiation strategy of enrichment medium containing glucose and fermentation medium without glucose, the enrichment medium ensures rapid cell proliferation with glucose, while the fermentation medium removes glucose to completely eliminate carbon decomposition metabolism inhibition, thereby maximizing the activation of collagenase synthesis gene expression and achieving a highly efficient fermentation mode of "first strengthening the bacteria, then producing enzymes".

[0067] In the anaerobic fermentation process of this application, a targeted and customized feeding strategy is adopted: an anaerobic-specific feeding medium containing gelatin + 3DTableTrix® microcarriers is designed and added in batches during the logarithmic growth phase of fermentation to continuously provide targeted inducing substrates and slow-release carbon and nitrogen sources, prolong the cell stability period, reduce late autolysis, and further improve collagenase yield and component stability, thus meeting the needs of large-scale industrial anaerobic fermentation.

[0068] This application prepares a collagenase composition with precise and controllable components: through fermentation regulation process, a composition of collagenase I, collagenase II and clostridium protease is obtained, which eliminates the defects of single collagenase components, can mildly and specifically degrade the triple helix structure of collagen, without damaging cell membrane proteins and cell surface markers, and achieves cell harvesting without damage. It can be further customized for the Huakan three-dimensional microcarrier lysis scenario.

[0069] This application presents a fully scalable cell harvesting process: adapted to Huakan 3D RecomTrix® / TableTrix® microcarriers, complete lysis can be achieved at 1× working concentration, 37℃, 40rpm, and 20–40min, with microcarrier residue <0.1%. Cells do not require excessive pipetting, and viability and phenotype are fully preserved. Simultaneously, the fermented collagenase composition is seamlessly integrated with Huakan's 3D microcarrier culture and 3D FloTrix® automated cell harvesting device, establishing an integrated process of "fermentation enzyme production - microcarrier lysis - automated harvesting." This process is simple to operate, scalable, and overcomes the industry bottlenecks of traditional trypsin digestion and physical pipetting, which result in significant cell damage, incomplete harvesting, and difficulty in scaling up.

[0070] The present application will be described below with reference to specific embodiments.

[0071] Specific Example 1

[0072] I. Production of collagenase by anaerobic fermentation of Clostridium histolyticum 1) Strain activation and selection: Inoculate Clostridium histolyticum ATCC19401 onto Columbia blood agar plates containing 5% defibrinated sheep blood and incubate anaerobically at 37°C for 24-48 hours until typical single colonies with a diameter of 0.5-2 mm, grayish-white color, and irregular edges grow. Select plump single colonies and inoculate them onto gelatin screening plates. Incubate anaerobically at 37°C for 24-48 hours and select high-yielding strains with large gelatin hydrolysis zones and clear transparent zones as the strains for this fermentation production.

[0073] 2) Seed culture: The selected high-yielding single colonies were inoculated into enrichment medium and anaerobically cultured at 37°C for 14 hours until the OD reached the target level. 600 =0.6~0.8, to obtain primary seed culture, then transfer the primary seed culture to fresh enrichment medium at an inoculation rate of 3% (v / v), and anaerobic incubate at 37℃ for 10 h, OD 600 =0.7, resulting in a vigorous secondary seed solution; 3) Fermentation in a fermenter: Fermentation medium was used, with an inoculum of 8% (v / v). Fermentation parameters were set as follows: temperature 37±0.5℃, pH 6.8~7.2, stirring speed 60 rpm, and anaerobic environment maintained by N2 throughout the process. 3% of the culture medium was added at 12 hours, 3% at 20 hours, and 2% at 28 hours, for a total feed amount of 8%. Fermentation continued for 36 hours, and cell OD was monitored. 600 The peak value reached 3.29, and the collagenase activity reached 2880 CDU / mL, at which point fermentation ended.

[0074] During fermentation, enzyme activity and OD were measured every four hours. 600 The test results are as follows Figure 1It is the OD of the Clostridium histolytica fermentation process. 600 The curve showing changes in collagenase activity was obtained from... Figure 1 It can be seen that bacterial growth follows the following process: Fermentation 0–12 h: Lag phase + Early logarithmic growth phase (before fed-batch initiation); After inoculation, the cells briefly adapt to the anaerobic fermentation environment, OD 600 The growth was slow and gradual; thanks to the pre-enrichment in the enrichment medium and the removal of glucose from the fermentation medium to relieve carbon metabolism inhibition, the cells gradually entered a rapid proliferation phase 12 hours later, and the biomass steadily increased. Regarding the collagenase activity curve, almost no collagenase was secreted in the early stages of fermentation, and enzyme activity remained at a very low baseline level. This is because at this stage, the strain preferentially utilizes carbon and nitrogen sources such as maltose and peptone in the medium to complete cell proliferation, and the collagenase synthesis genes were not significantly activated. The dual inducing substrate of gelatin + 3DTableTrix® microcarriers had not yet fully exerted its inducing effect.

[0075] Fermentation 12–28 h: Late logarithmic growth phase (critical stage of fed-batch feeding); this stage involves three feedings (3% at 12 h, 3% at 20 h, and 2% at 28 h), and is the core region for cell proliferation and massive collagenase synthesis: cell OD 600 The curves show that with continuous supplementation of gelatin, microcarrier-induced substrates, and slow-release carbon and nitrogen sources, the bacteria enter the exponential logarithmic growth phase, and the OD... 600 The growth rate shows a steep upward trend, with rapid accumulation of cell density. Supplementation continuously delays nutrient depletion, significantly extending the window for rapid cell growth. The collagenase activity curve shows that with increased cell biomass and continuous supply of dual collagen-inducing substrates, the collagenase synthesis pathway is continuously activated, and enzyme activity rises rapidly and synchronously. The curve correlates with OD... 600 The highly synchronized growth trends demonstrate that collagenase is a growth-associated secondary metabolite of Clostridium histolyticum. Each addition of feed resulted in a slight increase in the rate of enzyme activity rise, verifying that the targeted fed-batch medium can enhance enzyme production efficiency. Between 28 and 32 hours of fermentation, especially at 30 hours, cell density and enzyme activity both peaked, indicating that collecting the fermentation products at this time yielded high levels of collagenase activator. After 32 hours, both cell density and enzyme activity began to decline.

[0076] II. Preparation of Collagenase Composition through Post-treatment of Fermentation Broth 1) Centrifugation to remove bacteria: Centrifuge the fermentation broth at 4℃ and 8000 rpm for 20 min to remove bacteria and collect the supernatant; 2) Salting out purification: Slowly add ammonium sulfate to the supernatant until 60% saturation, let stand at 4℃ for 2 hours, centrifuge at 10000 rpm for 20 minutes, and collect the collagenase precipitate; 3) Dialysis desalting: The precipitate was redissolved in 20 mM Tris-HCl buffer (pH 7.2), placed in a 10 kDa molecular weight cutoff dialysis bag, and dialyzed at 4°C for 24 h. The dialysis solution was changed every 6 h to remove residual ammonium sulfate. 4) Concentration: The purified collagenase solution is concentrated by ultrafiltration through a 100kd hollow fiber column; 5) Sterile filtration: Aseptic filtration is performed using a 0.22μm polyethersulfone (PES) filter membrane; 6) Freeze-drying: The sterile collagenase solution was dispensed into liquid nitrogen and quick-frozen for 0.5 h, then placed in a freeze dryer and freeze-dried at -45°C and 10 Pa vacuum for 48 h to produce a brown, loose collagenase composition freeze-dried powder, which was then sealed and stored at 4°C for later use.

[0077] To further verify the composition and purity of the collagenase composition, purity analysis and mass spectrometry analysis were performed using SDS-PAGE. The SDS-PAGE experimental results are as follows: Figure 2 The image shows the SDS-PAGE purity analysis chromatogram of the lyophilized collagenase composition. The mass spectrometry analysis results are as follows: Figure 3 . Figure 2 In the image, the left side shows the protein molecular weight marker (10–180 kDa), and the right side shows the bands generated by the collagenase composition. After multi-stage purification through salting out, dialysis, and ultrafiltration, the characteristic bands of the final product are concentrated around 44 kDa. This represents a highly active catalytic domain fragment formed by the controlled cleavage of Clostridium histolyticum collagenases I and II by endogenous proteases. This demonstrates that the fermentation induction and purification process of this invention can stably obtain a homogeneous composite enzyme preparation with a collagenase catalytic fragment of approximately 44 kDa as its core. During fermentation, the Clostridium protease (neutral protease) in the system gently cleaves the collagen-binding region of the full-length 110 kDa collagenase protein, retaining the 44 kDa catalytic domain with complete collagen degradation activity. After purification, the full-length high-molecular-weight collagenase is significantly removed, thus the main band of the final product is concentrated around 44 kDa. Figure 3 To further analyze the composition of the lyophilized collagenase composition powder using mass spectrometry, and to determine the proportion of various proteins, the following analysis was conducted: Figure 3 Mass spectrometry analysis revealed that the approximately 44 kDa collagenase fragment contained 56.20% type I collagenase, 4.70% type II collagenase, 12.40% neutral protease, 25.20% serine protease, and 1.50% other miscellaneous enzyme components; the neutral protease was a Clostridium neutral thiol protease. This confirms that the collagenase composition extracted in this application has a clear core composition, maintains a high catalytic domain, and thus ensures high collagenase activity.

[0078] III. Three-dimensional microcarrier lysis and non-destructive harvesting of MSCs 1) Cell Culture: Human umbilical cord MSCs (mesenchymal stem cells) were seeded onto Huakan 3D TableTrix® microcarriers and cultured in serum-free 3D FloTrix® mesenchymal stem cell culture medium at 37°C in a 5% CO2 incubator. The stirring speed was set to 40 rpm for 5 min and 0 rpm for 25 min for 24 h. After the cells were fully attached to the microcarriers, the stirring speed was set to a constant 40 rpm and cultured for 72-96 h. The cells grew at high density and formed micro-tissues. During the culture period, samples were taken at 24 h and 96 h to observe the cell growth status.

[0079] 2) Preparation of lysis buffer: Dissolve 50 g of the prepared collagenase composition lyophilized powder in 5 mL of Hanks balanced salt solution, DMEM or 3D FloTrix® three-dimensional stem cell basal culture medium, filter with a 0.22µm filter membrane for sterilization and store at 4℃ for later use.

[0080] 3) Microtissue lysis: At the end of culture, discard part of the culture supernatant and transfer the remaining microtissue suspension to a suitable volume centrifuge tube or centrifuge cup. Add the prepared 10X stock solution to a 1X concentration, place in a 37°C water bath, and heat for 15 minutes. Remove and gently mix in a clean bench, observing the lysis process. Lyse for 30-40 minutes until the microcarriers are completely degraded, then centrifuge at 1500 rpm for 5 minutes. After centrifugation, discard the supernatant and wash the cells 2-3 times with PBS or 1% human albumin + physiological saline. After washing, resuspend the cells for further passage.

[0081] 4) Continuous culture: Cells are cultured continuously for 4 generations in this manner to record cell proliferation, viability, and lysis.

[0082] 5) After the culture is completed, cell phenotype, microcarrier residue, and lysis buffer residue tests are performed.

[0083] Please see Figures 4 to 6 ,in Figure 4 This is a microscopic morphological image of the 3D microcarrier before lysis. The image shows the microscopic imaging results of the 3D TableTrix® collagen microcarrier loaded with cells before lysis, simultaneously displaying AM live cell fluorescence, PI dead cell fluorescence, and bright field. Under bright field, the microcarrier skeleton can be observed to be intact, with a large number of cells attached and aggregated to form dense micro-tissue. The AM green fluorescence signal is strong, while the PI red dead cell signal is weak, proving that the cells are in good growth condition and have high viability on the microcarrier, making it a qualified sample for lysis and harvesting experiments.

[0084] Figure 5The images show the morphology of cells released after complete lysis of the three-dimensional microcarriers by the collagenase composition of this invention; and the microscopic images of cells after complete lysis by the collagenase of this invention. No intact collagen microcarrier skeleton remains in the field of view, indicating that the collagen skeleton of the microcarriers has been specifically degraded, and the cells have been completely dissociated into single-cell suspensions with intact cell morphology. This visually demonstrates that the collagenase can gently disintegrate the microcarriers and release the cells intact without significant mechanical or enzymatic damage.

[0085] Figure 6 The first image shows the results of cell proliferation and viability testing after non-destructive harvesting; the second image shows the proliferation capacity and cell viability testing data of cells harvested by this collagenase after continuous passage culture. The results show that the cell viability is stably maintained above 90% after multiple passages of culture, and the cell proliferation rate does not decrease significantly. This proves that the collagenase lysis process does not damage cell viability and proliferation potential, and can achieve non-destructive cell harvesting, which is suitable for the continuous passage expansion of stem cells.

[0086] The harvested cells were subjected to flow cytometry phenotyping, and the results are shown in Table 1: Table 1 Results of non-destructive cell phenotyping

[0087] This table shows the flow rate of surface markers in mesenchymal stem cells after collagenase lysis. The expression levels of positive markers CD105, CD73, and CD90 all exceeded 98%, while the expression levels of negative markers CD19, CD34, CD14, CD45, and HLA were close to 0. This demonstrates that collagenase lysis does not damage characteristic proteins on the cell surface, and the harvested cells maintain the complete phenotype of standard mesenchymal stem cells.

[0088] Further washing and residual detection of microcarriers after cell harvesting were performed. The results of microcarrier residual detection are shown in Table 2: Table 2 Detection results of microcarrier residues

[0089] As can be seen from the above, for each × 10 washes, the effect is different. 7 The residual amount of collagen microcarriers per cell was high before washing, but after 2-3 washes, the residual amount dropped to an extremely low level, meeting the impurity control requirements for cell preparations. This verifies that the matching washing process can effectively remove degraded collagen carrier fragments. Negative values ​​represent "below the detection limit / not detected".

[0090] Please refer to Table 3 below for the results of residual lysis buffer detection: Table 3 Results of residual lysis buffer detection

[0091] As shown above, comparing the residual amount of collagenase lysis buffer in cells after different washing cycles, significant exogenous enzyme residue was observed only after 0 washes, while the residue was almost completely removed after 2 washes. This indicates that simple centrifugation and washing can significantly remove enzyme residue and reduce the interference of exogenous proteins on subsequent cell applications. Negative values ​​represent "below the detection limit / not detected".

[0092] As can be seen from Specific Example 1, the collagenase composition of this invention is prepared by Clostridium histolyticum ATCC 19401 through two-stage differentiated anaerobic fermentation, fed-batch feeding, and purification by salting out-dialysis-ultrafiltration-lyophilization. The fermentation cycle is 36 h, and the endpoint cell OD is [not specified]. 600 The product has a collagenase activity of 2880 CDU / mL and a collagenase activity of 3.29. The product is mainly composed of a highly active collagenase catalytic fragment of about 44 kDa, combined with a controllable proportion of clostridium protease. When used in the 3D TableTrix® microcarrier culture of human umbilical cord MSCs for lysis and harvesting, the microcarriers can be completely degraded by lysis at 37℃ for 30–40 min. The cell recovery rate is ≥95% and the viability is ≥90%. The surface markers of mesenchymal stem cells in the harvested cells are expressed normally. After 2–3 centrifugation and washing, the residues of microcarriers and lysin can be reduced to near the detection limit. The entire fermentation, purification and cell lysis harvesting process parameters are stable and controllable, and can be adapted to the non-destructive preparation of cells on a large scale.

[0093] The collagenase composition of this invention is prepared by standardized anaerobic fermentation of Clostridium histolyticum, which is stable, scalable, and low-cost. For the first time, gelatin + 3D TableTrix® microcarriers are used as dual collagen inducers, which can target and induce the production of collagenase composition highly adapted to Huakan 3D collagen microcarriers. This significantly improves the lysis specificity of microcarriers, gently lyses 3D collagen microcarriers, and achieves high recovery rate, high viability, and non-destructive harvesting of adherent cells. It fully meets the needs of large-scale and standardized production of cell therapy products and can be widely used in biopharmaceutical companies, cell preparation centers, and research institutions, with good industrial applicability and market application prospects.

[0094] The technical features of the embodiments described above can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as the combination of these technical features does not contradict each other, it should be considered within the scope of this specification. It should be noted that the terms "in one embodiment," "for example," and "again," etc., in this application are intended to illustrate the application and not to limit it. The embodiments described above only illustrate several implementations of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be pointed out that for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this patent should be determined by the appended claims.

Claims

1. A method for preparing collagenase based on Clostridium histolyticum, characterized in that, Includes the following steps: Clostridium histolyticum was inoculated into a fermentation medium for anaerobic fermentation to obtain the fermentation product; wherein the anaerobic fermentation pH was 6.8–7.2; and the fermentation medium contained microcarriers and gelatin. The fermentation product was separated and extracted to obtain a collagenase composition.

2. The preparation method according to claim 1, characterized in that, The particle size of the microcarrier is in the range of 50-500 μm; And / or, the raw materials for preparing the microcarriers are artificially synthesized biomaterials or natural biomaterials, wherein the artificially synthesized biomaterials are selected from at least one of polyethylene glycol, polyethylene glycol derivatives, polyethylene glycol diacrylate, polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxy acid, polylactic acid-alkyd copolymer, polydimethylsiloxane, polyanhydride, polyester, polyamide, polylysine, polyacetal, polycyanoacrylate, polyurethane, polypyrrole, polymethacrylate, polyethylene, polycarbonate, and polyethylene oxide; the natural biomaterials are selected from at least one of collagen, denatured collagen, collagen protein, proteoglycan, glycoprotein, gelatin, gelatin derivatives, chitin, alginate, alginate derivatives, agar, agarose, dextran, starch, whey protein, pectin, fibrinogen, matrix gum, hyaluronic acid, laminin, fibronectin, and fibronectin. And / or, the concentration of the microcarrier in the fermentation medium is 2-6 g / L; And / or, the microcarrier is a 3D TableTrix® microcarrier; And / or, the concentration of the gelatin in the fermentation medium is 10–18 g / L; And / or, the fermentation medium further includes L-cysteine ​​hydrochloride, wherein the concentration of L-cysteine ​​hydrochloride in the fermentation medium is 0.7–0.9 g / L; And / or, the fermentation medium further includes vitamin B1 at a concentration of 0.015–0.025 g / L; And / or, the fermentation medium further includes soybean peptone, yeast extract, maltose, beef extract, gelatin, CaCl2 and MgSO4·7H2O; And / or, the collagenase composition includes type I collagenase, type II collagenase, neutral protease, and serine protease, preferably, the abundance ratio of type I collagenase, type II collagenase, neutral protease, and serine protease is 56.20:4.70:12.40:25.

20.

3. The preparation method according to claim 2, characterized in that, The fermentation medium comprises the following components at the following mass concentrations: soybean peptone 13–17 g / L, yeast extract 7–9 g / L, maltose 5–8 g / L, beef extract 4–6 g / L, gelatin 10–18 g / L, microcarrier 2–6 g / L, potassium dihydrogen phosphate 2.5–3.5 g / L, dipotassium hydrogen phosphate 0.8–1.2 g / L, magnesium sulfate heptahydrate 0.7–0.9 g / L, calcium chloride 0.15–0.25 g / L, L-cysteine ​​hydrochloride 0.7–0.9 g / L, and vitamin B1 0.015–0.025 g / L; the pH of the fermentation medium is controlled at 7.0 ± 0.

1. Preferably, the fermentation medium comprises the following components at the following mass concentrations: soybean peptone 15 g / L, yeast extract 8 g / L, maltose 6 g / L, beef extract 5 g / L, gelatin 15 g / L, microcarrier 4 g / L, potassium dihydrogen phosphate 3 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium sulfate heptahydrate 0.8 g / L, calcium chloride 0.2 g / L, L-cysteine ​​hydrochloride 0.8 g / L, and vitamin B1 0.02 g / L.

4. The preparation method according to claim 1, characterized in that, During fermentation, a replenishing medium is added, which includes soybean peptone, yeast extract, gelatin, microcarriers, maltose, and L-cysteine ​​hydrochloride. The replenishment begins 12 hours after fermentation, and the volume of the replenishing medium is 0.01% to 8% of the initial fermentation medium volume. Preferably, the replenishing culture medium comprises the following components at the following mass concentrations: 20 g / L soybean peptone, 15 g / L yeast extract, 80 g / L gelatin, 20 g / L microcarrier, 30 g / L maltose, and 1.0 g / L L-cysteine ​​hydrochloride. Preferably, the replenishment method is continuous replenishment or batch replenishment. Preferably, batch replenishment is started after 12 hours of fermentation, with 3% added at the 12th hour, 3% added at the 20th hour, and 2% added at the 28th hour. Preferably, the fermentation time is controlled at 30±2h, the fermentation temperature is 37±0.5℃, and the dissolved oxygen concentration in anaerobic fermentation is less than 6% of the air saturation. Preferably, during anaerobic fermentation, 10% ammonia water or 5% phosphoric acid solution is added to stabilize the fermentation pH value within the range of 6.8 to 7.2; Preferably, during anaerobic fermentation, the stirring speed is controlled at 60–90 rpm.

5. The preparation method according to claim 4, characterized in that, Before anaerobic fermentation of Clostridium histolyticum inoculation, the preparation method further includes propagating Clostridium histolyticum using an enrichment medium; Preferably, the enrichment culture medium comprises the following components at the following mass concentrations: soybean peptone 8-12 g / L, yeast extract 4-6 g / L, glucose 4-6 g / L, gelatin 0.3-0.7 g / L, potassium dihydrogen phosphate 1.5-2.5 g / L, dipotassium hydrogen phosphate 0.8-1.2 g / L, magnesium sulfate heptahydrate 0.4-0.6 g / L, sodium chloride 1-3 g / L, and L-cysteine ​​hydrochloride 0.4-0.6 g / L; the pH value is 7.0 ± 0.

1. Preferably, the enrichment culture medium comprises the following components at the following mass concentrations: soybean peptone 10 g / L, yeast extract 5 g / L, glucose 5 g / L, gelatin 0.5 g / L, potassium dihydrogen phosphate 2 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium sulfate heptahydrate 0.5 g / L, sodium chloride 2 g / L, and L-cysteine ​​hydrochloride 0.5 g / L; Preferably, the proliferation culture time is 8-24 hours.

6. The preparation method according to claim 1, characterized in that, The step of separating and extracting collagenase composition from the fermentation product includes: sequentially filtering and centrifuging the fermentation product to collect the supernatant; adding ammonium sulfate to the supernatant to adjust the ammonium sulfate saturation of the system to 60-80%, allowing it to stand at low temperature for salt precipitation, and collecting the precipitate; and sequentially desalting, concentrating, sterilizing and filtering the precipitate, and freeze-drying the precipitate to prepare the collagenase composition.

7. The preparation method according to claim 6, characterized in that, In the desalting process, dialysis desalting was performed. The obtained precipitate was reconstituted in 20 mM Tris-HCl buffer with a pH of 7.

2. The reconstituted solution was dialyzed using a dialysis bag with a molecular weight cutoff of 10 kDa to remove residual salts and obtain a desalted collagenase solution. And / or, concentrate to ultrafiltration concentration treatment, concentrate by ultrafiltration system; And / or, sterile filtration is performed by aseptic filtration of the concentrated collagenase solution using a 0.22μm polyethersulfone filter membrane; And / or, freeze drying is the process of quick freezing in liquid nitrogen followed by freeze drying at -40 to -50°C to obtain a freeze-dried powder of the collagenase composition.

8. A collagenase composition, characterized in that, It was prepared using the method for preparing collagenase based on Clostridium histolyticum as described in any one of claims 1 to 7.

9. The application of the collagenase composition as described in claim 8 in microcarrier cell culture.

10. The application according to claim 9, characterized in that, The application of collagenase composition in cell harvesting from microcarrier cell culture includes the following steps: Preparation of collagenase stock solution: Dissolve the prepared lyophilized collagenase composition in Hanks balanced salt solution, DMEM medium or 3D FloTrix® three-dimensional stem cell basal medium to prepare a 10X collagenase stock solution with a concentration of 10 mg / mL. After sterilization by filtration through a 0.22 μm filter membrane, store at 4℃ for later use. Microcarrier lysis process: Cells cultured on the three-dimensional microcarrier for 3 days or more are allowed to settle statically, some of the culture medium supernatant is removed, and 10X collagenase stock solution is added to dilute to 1X working concentration; lysis is carried out by stirring at 40 rpm and 37°C. Timing starts when the system temperature rises above 36°C and lysis is continued for 30-40 minutes until the three-dimensional microcarrier is completely lysed. Cell harvesting: After complete lysis of the microcarriers, the cell suspension in the system is pumped out and washed and harvested using either manual centrifugation or an automated cell harvesting device. The manual centrifugation process involves transferring the harvested cell suspension to a centrifuge container and centrifuging at 1500 rpm for 5 minutes. After centrifugation, the supernatant is discarded, and the cells are washed with PBS solution or physiological saline containing 1% human albumin, repeating this washing process 2-3 times to obtain a single-cell suspension. The automated cell harvesting process uses a 3D FloTrix® vivaPREP PLUS or 3D FloTrix® vivaPREP ULTRA automated cell harvesting device, and the cell washing and harvesting operations are performed according to the corresponding instruction manual.

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