Recombinant trypsin cell digestion solution for maintaining high cell viability

Abstract

The present invention relates to the technical field of cell culture. Specifically disclosed is a recombinant trypsin cell digestion solution for maintaining high cell viability. The recombinant trypsin cell digestion solution consists of the following concentrations of components: 5000-8000 mg / L sodium chloride, 500-1500 mg / L glucose, 500-1500 mg / L disodium hydrogen phosphate, 100-400 mg / L sodium bicarbonate, 1000-2000 mg / L potassium dihydrogen phosphate, 1000-2000 mg / L potassium chloride, 0.5-2 mM of EDTA, 100-500 mg / L poloxamer 188 PRO, 100-500 mg / L HEPES, and 100-500 mg / L recombinant trypsin, with the balance being pure water. The provided recombinant trypsin cell digestion solution greatly improves cell viability after digestion, and solves the problem of reduced cell viability after conventional trypsin digestion.

Description

A recombinant trypsin cell digestion solution for maintaining high cell viability Technical Field

[0001] The present application relates to the technical field of cell culture, and more specifically, to a recombinant pancreatic enzyme cell digestion solution for maintaining high cell viability. Background Art

[0002] Trypsin is a serine proteolytic enzyme extracted from the pancreas of cattle, sheep, and pigs. It acts as a digestive enzyme in vertebrates and is widely used in the biological and medical fields. Trypsin exists in the pancreas of animals in the form of inactive zymogen. 2+ In the presence of , it is activated by enterokinase or active trypsin itself, breaking the peptide bond between the lysine and isoleucine residues at the N-terminus of the peptide chain, losing a hexapeptide, and undergoing certain changes in the molecular conformation before transforming into active trypsin.

[0003] Since animal-derived trypsin may introduce mad cow virus (natural bovine pancreatic enzyme) or foot-and-mouth disease virus (natural porcine pancreatic enzyme), which will lead to serious consequences, the use of microbial fermentation to obtain trypsin has gradually become popular due to its high efficiency and specificity.

[0004] In the cell culture process, animal-derived trypsin has a strong digestion ability for cells, which easily leads to cell death and reduces cell viability. CN107090427A discloses a genetically recombinant trypsin cell digestion solution, wherein the components of the genetically recombinant trypsin cell digestion solution are: dipotassium hydrogen phosphate 50-70mg / L, potassium chloride 350-450mg / L, sodium bicarbonate 300-400mg / L, sodium chloride 7000-9000mg / L, disodium hydrogen phosphate heptahydrate 80-100mg / L, glucose 1000-2000mg / L, EDTA-4Na 200-500mg / L, genetically recombinant trypsin 1-60mg / L, and the balance is water. This solution greatly improves the uncontrollable factors of introducing animal-derived trypsin during cell digestion by introducing genetically recombinant trypsin instead of animal-derived trypsin, but the cell viability after digestion in this solution can only be maintained at more than 90%, which fails to meet the high viability requirements of cells.

[0005] Summary of the Invention

[0006] In order to solve the technical problem that trypsin digestion during adherent culture of mesenchymal stem cells causes damage to cells, thereby affecting the quantity and quality of mesenchymal stem cells, the present application provides a recombinant trypsin cell digestion solution that maintains high cell viability, which is mainly used for adherent digestion of mesenchymal stem cells.

[0007] This application uses a high-concentration buffer system to effectively maintain pH stability during cell digestion. At the same time, the addition of poloxamer 188 can effectively reduce the mechanical damage to cells caused by repeated pipetting and other operations during cell digestion. This component also has the function of maintaining cell membrane stability and repairing cell membranes. Therefore, the recombinant pancreatic enzyme cell digestion solution of this application greatly improves the viability of cells after digestion, solving the problem of reduced viability after traditional pancreatic enzyme digestion.

[0008] In a first aspect, the present application provides a recombinant pancreatic enzyme cell digestion solution for maintaining high cell viability, using the following technical solution:

[0009] A recombinant pancreatic enzyme cell digestion solution for maintaining high cell viability is provided. The recombinant pancreatic enzyme cell digestion solution is composed of components with the following concentrations: 5000-8000 mg / L of sodium chloride, 500-1500 mg / L of glucose, 500-1500 mg / L of disodium hydrogen phosphate, 100-400 mg / L of sodium bicarbonate, 1000-2000 mg / L of potassium dihydrogen phosphate, 1000-2000 mg / L of potassium chloride, 0.5-2 mM of EDTA, 100-500 mg / L of poloxamer 188PRO, 100-500 mg / L of HEPES, 100-500 mg / L of recombinant pancreatic enzyme, and the remaining components are pure water.

[0010] HEPES is a zwitterionic buffer containing amino and carboxyl groups. It has a stable molecular structure, a neutral pH, and good thermal stability at room temperature. Due to these properties, HEPES is widely used in biochemistry and cell biology experiments that require precise control of pH.

[0011] Poloxamer 188PRO is a non-ionic polymer surfactant, mainly used as an emulsifier, solubilizer, gene drug carrier and other pharmaceutical excipients. 188PRO is one of the poloxamer excipients with good emulsification and safety, and is currently the only artificial emulsifier used for intravenous injection.

[0012] Poloxamer 188PRO can increase the permeability of cell membranes, protect cell membranes, enhance cell membrane stability, reduce cell fragility, and can directly embed into the lipid layer of the cell membrane to protect cells. It has been widely used in the research of cell membrane repair therapy and is called the "band-aid" of the cell membrane.

[0013] The recombinant trypsin cell digestion solution of the present application, which maintains high cell viability, plays a good protective role in the transformation of cells from an adherent state to a suspended state. Even if the cells are digested for more than 15 minutes in the recombinant trypsin cell digestion solution of the present application, the cell viability can still be maintained at more than 97%, and the state of continued cell passage is completely unaffected. It is suitable for large-scale cell production and solves the technical problem of decreased cell viability, quantity, and quality due to repeated blowing, excessive digestion, etc. during cell digestion.

[0014] In a specific embodiment, the recombinant pancreatic enzyme cell digestion solution is composed of components with the following concentrations: sodium chloride 5000 mg / L, glucose 1500 mg / L, disodium hydrogen phosphate 1500 mg / L, sodium bicarbonate 400 mg / L, potassium dihydrogen phosphate 2000 mg / L, potassium chloride 2000 mg / L, EDTA 2mM, poloxamer 188PRO 500 mg / L, HEPES 400 mg / L, recombinant pancreatic enzyme 500 mg / L, and the remaining components are pure water.

[0015] In a specific embodiment, the recombinant pancreatic enzyme cell digestion solution is composed of components with the following concentrations: sodium chloride 6500 mg / L, glucose 1000 mg / L, disodium hydrogen phosphate 1000 mg / L, sodium bicarbonate 300 mg / L, potassium dihydrogen phosphate 1500 mg / L, potassium chloride 1500 mg / L, EDTA 1 mM, poloxamer 188PRO 300 mg / L, HEPES 300 mg / L, recombinant pancreatic enzyme 350 mg / L, and the remaining components are pure water.

[0016] In a specific embodiment, the recombinant pancreatic enzyme cell digestion solution is composed of components with the following concentrations: sodium chloride 8000 mg / L, glucose 500 mg / L, disodium hydrogen phosphate 500 mg / L, sodium bicarbonate 100 mg / L, potassium dihydrogen phosphate 1000 mg / L, potassium chloride 1000 mg / L, EDTA 0.5 mM, poloxamer 188PRO 100 mg / L, HEPES 100 mg / L, recombinant pancreatic enzyme 100 mg / L, and the remaining components are pure water.

[0017] In a second aspect, the present application provides a method for preparing the above-mentioned recombinant pancreatic enzyme cell digestion solution that maintains high cell viability: the following technical solution is used:

[0018] The preparation method of the recombinant pancreatic enzyme cell digestion solution for maintaining high cell viability in the present application is: dissolve each component separately and mix the solution, add pure water to the concentration of each component, adjust the pH value to 6.9-7.4, and filter to obtain the recombinant pancreatic enzyme cell digestion solution.

[0019] In a third aspect, the present application provides the use of the above-mentioned recombinant pancreatic enzyme cell digestion solution in digesting mesenchymal stem cells.

[0020] In summary, this application has the following beneficial effects:

[0021] 1. This application uses a high-concentration buffer system to maintain pH stability during cell digestion. At the same time, the addition of poloxamer 188 can effectively reduce the mechanical damage to cells caused by repeated pipetting and other operations during cell digestion. This component also maintains cell membrane stability and repairs cell membranes. Therefore, the recombinant pancreatic enzyme cell digestion solution of this application greatly improves the viability of cells after digestion, solving the problem of reduced viability after traditional pancreatic enzyme digestion.

[0022] 2. The recombinant trypsin cell digestion solution of the present application, which maintains high cell viability, plays a good protective role in the transformation of cells from an adherent state to a suspended state. Even if the cells are digested for more than 30 minutes in the recombinant trypsin cell digestion solution of the present application, the cell viability can still be maintained at more than 97%, and the state of continued cell passage is completely unaffected. It is suitable for large-scale cell production and solves the technical problem of decreased cell viability, quantity, and quality due to repeated blowing and excessive digestion during cell digestion. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG1 shows the counting results of mesenchymal stem cells after the first treatment with the recombinant trypsin cell digestion solution of Example 1.

[0024] FIG2 shows the counting results of mesenchymal stem cells after the first treatment with the recombinant trypsin cell digestion solution of Example 2.

[0025] FIG3 shows the counting results of mesenchymal stem cells after the first treatment with the recombinant trypsin cell digestion solution of Example 3.

[0026] FIG4 shows the counting results of mesenchymal stem cells after the first treatment with the recombinant trypsin cell digestion solution of the control group.

[0027] FIG5 is the counting result of mesenchymal stem cells after the first treatment with the recombinant trypsin cell digestion solution of Comparative Example 1.

[0028] FIG6 shows the counting results of mesenchymal stem cells after the first treatment with the recombinant trypsin cell digestion solution of Comparative Example 2.

[0029] FIG7 shows the counting results of mesenchymal stem cells after the second treatment with the recombinant trypsin cell digestion solution of Example 1.

[0030] FIG8 shows the counting results of mesenchymal stem cells after the second treatment with the recombinant trypsin cell digestion solution of Example 2.

[0031] FIG9 shows the counting results of mesenchymal stem cells after the second treatment with the recombinant trypsin cell digestion solution of Example 3.

[0032] FIG10 shows the counting results of mesenchymal stem cells after the second treatment with the recombinant trypsin cell digestion solution of the control group.

[0033] FIG11 shows the counting results of mesenchymal stem cells after the second treatment with the recombinant trypsin cell digestion solution of Comparative Example 1.

[0034] FIG12 shows the counting results of mesenchymal stem cells after the second treatment with the recombinant trypsin cell digestion solution of Comparative Example 2. DETAILED DESCRIPTION

[0035] Before describing the embodiments of the present application in detail, it should be understood that the terminology used herein is only for the purpose of describing particular embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the term belongs.

[0036] The present application provides a recombinant pancreatic enzyme cell digestion solution for maintaining high cell viability. Specifically, it is composed of the following components at the following concentrations: sodium chloride 5000-8000 mg / L, glucose 500-1500 mg / L, disodium hydrogen phosphate 500-1500 mg / L, sodium bicarbonate 100-400 mg / L, potassium dihydrogen phosphate 1000-2000 mg / L, potassium chloride 1000-2000 mg / L, EDTA 0.5-2 mM, poloxamer 188 PRO 100-500 mg / L, HEPES 100-500 mg / L, recombinant pancreatic enzyme 100-500 mg / L, and the remaining components are pure water.

[0037] To make the purpose, technical solutions, and advantages of this application more clear, the technical solutions in the embodiments of this application are clearly and completely described below with reference to the accompanying drawings. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without making any creative efforts shall fall within the scope of protection of this application.

[0038] The present application is further described in detail below with reference to the examples, drawings and test results.

[0039] The components and reagents involved in the following examples are all conventional commercially available products. Among them, sodium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, potassium chloride, and EDTA were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; glucose, sodium bicarbonate, poloxamer 188PRO, and HEPES were purchased from SIGMA; and recombinant pancreatic enzymes were purchased from Wuhan Hanhai New Enzyme Biotechnology Co., Ltd.

[0040] The stem cells involved in the following examples are mesenchymal umbilical cord stem cells. The culture supernatant in the culture flask is aspirated, and the adherent cells are washed 1-2 times with PBS without calcium and magnesium ions. An appropriate amount of recombinant trypsin cell digestion solution to maintain high cell viability is added to the rinsed culture flask, covering the bottom of the culture flask. The flask is placed at room temperature or 37°C for digestion. The cell digestion is observed under a microscope. When the cells shrink and become round, the digestion can be terminated with 2 times the volume of the digestion solution supernatant or fresh culture medium. The digestion time is related to factors such as cell confluence and the degree of adhesion. Digestion can be completed within 2-10 minutes. The cell suspension is collected into a centrifuge tube and centrifuged at 1200 rpm for 5 minutes. The supernatant is discarded and the cell pellet is resuspended in fresh culture medium. After sampling and counting, the cells are passaged or frozen.

[0041] Example

[0042] Example 1

[0043] This embodiment provides a recombinant trypsin cell digestion solution that maintains high cell viability.

[0044] The recombinant pancreatic enzyme cell digestion solution is composed of the following components in the following concentrations: sodium chloride 5000 mg / L, glucose 1500 mg / L, sodium hydrogen phosphate 1500 mg / L, sodium bicarbonate 400 mg / L, potassium dihydrogen phosphate 2000 mg / L, potassium chloride 2000 mg / L, EDTA 2 mM, poloxamer 188PRO 500 mg / L, HEPES 400 mg / L, recombinant pancreatic enzyme 500 mg / L, and the remaining components are pure water.

[0045] The preparation method of the recombinant pancreatic enzyme cell digestion solution is as follows: taking all components, dissolving them separately according to their own characteristics and then mixing the solutions, adding pure water to the concentration of each component, adjusting the pH value to 6.9-7.4, and filtering with a 0.22μm filter element to obtain the recombinant pancreatic enzyme cell digestion solution.

[0046] Example 2

[0047] This embodiment provides a recombinant trypsin cell digestion solution that maintains high cell viability.

[0048] The recombinant pancreatic enzyme cell digestion solution is composed of the following components in the following concentrations: sodium chloride 6500 mg / L, glucose 1000 mg / L, disodium hydrogen phosphate 1000 mg / L, sodium bicarbonate 300 mg / L, potassium dihydrogen phosphate 1500 mg / L, potassium chloride 1500 mg / L, EDTA 1 mM, poloxamer 188PRO 300 mg / L, HEPES 300 mg / L, recombinant pancreatic enzyme 350 mg / L, and the remaining components are pure water.

[0049] The preparation method of the recombinant pancreatic enzyme cell digestion solution is as follows: taking all components, dissolving them separately according to their own characteristics and then mixing the solutions, adding pure water to the concentration of each component, adjusting the pH value to 6.9-7.4, and filtering with a 0.22μm filter element to obtain the recombinant pancreatic enzyme cell digestion solution.

[0050] Example 3

[0051] This embodiment provides a recombinant trypsin cell digestion solution that maintains high cell viability.

[0052] The recombinant pancreatic enzyme cell digestion solution is composed of the following components in the following concentrations: 8000 mg / L sodium chloride, 500 mg / L glucose, 500 mg / L disodium hydrogen phosphate, 100 mg / L sodium bicarbonate, 1000 mg / L potassium dihydrogen phosphate, 1000 mg / L potassium chloride, 0.5 mM EDTA, 100 mg / L poloxamer 188 PRO, 100 mg / L HEPES, 100 mg / L recombinant pancreatic enzyme, and the remaining components are pure water.

[0053] The preparation method of the recombinant pancreatic enzyme cell digestion solution is as follows: taking all components, dissolving them separately according to their own characteristics and then mixing the solutions, adding pure water to the concentration of each component, adjusting the pH value to 6.9-7.4, and filtering with a 0.22μm filter element to obtain the recombinant pancreatic enzyme cell digestion solution.

[0054] Comparative Example

[0055] Comparative Example 1

[0056] This comparative example provides a recombinant pancreatic enzyme cell digestion solution. This comparative example differs from Example 2 in that the recombinant pancreatic enzyme cell digestion solution does not contain poloxamer 188PRO. All other aspects are consistent with Example 2.

[0057] Comparative Example 2

[0058] This comparative example provides a recombinant pancreatic enzyme cell digestion solution. This comparative example differs from Example 2 in that the recombinant pancreatic enzyme cell digestion solution does not contain HEPES. All other components are consistent with Example 2.

[0059] Test results

[0060] (I) Cells were digested using the recombinant pancreatic enzyme digestion solutions provided in the examples and comparative examples, respectively. Trypsin (conventional porcine pancreatic enzyme) was also used as a control. After digestion, cell viability (%) and cell proliferation were measured, as shown in Table 1. Cells were also measured using a cell counter, as shown in Figures 1-6.

[0061] Table 1 Detection results of cells digested with recombinant trypsin cell digestion solution

[0062] As can be seen from Table 1 and Figures 1-4, among the recombinant pancreatic enzyme cell digestion solutions of the present application, the cell viability after digestion with the recombinant pancreatic enzyme cell digestion solutions of Example 1 and Example 2 is higher than that of the other experimental groups, but the cell expansion multiple after digestion with the recombinant pancreatic enzyme cell digestion solution of Example 1 is low, and the recombinant pancreatic enzyme cell digestion solution of Example 3 is over-digested, and the cell viability is reduced.

[0063] Further referring to Figures 5-6, the test results of Example 2 and Comparative Examples 1-2 show that the cell viability after digestion with the recombinant trypsin cell digestion solution of Comparative Examples 1-2 is lower than that of Example 2, and the cell expansion multiple after digestion with the recombinant trypsin cell digestion solution of Comparative Examples 1-2 is relatively low. Therefore, the recombinant trypsin cell digestion solution of the present application, which includes both poloxamer 188PRO and HEPES, can effectively improve cell expansion multiple and cell viability.

[0064] (2) The digested cells were further passaged and digested using recombinant trypsin cell digestion solution. After digestion, the cell viability (%) and cell proliferation rate of the passaged cells were measured, as shown in Table 2. The results were also measured using a cell counter, as shown in Figures 7-12.

[0065] Table 2 Detection results of cells after passage

[0066] As shown in Table 2 and Figures 7-10, after digestion with different digestion solutions, both cell viability and cell expansion were in the optimal state after digestion with the recombinant trypsin cell digestion solution of Example 2. This shows that different formulations of recombinant trypsin cell digestion solutions that maintain high cell viability have a significant impact on cell viability and cell expansion.

[0067] Further referring to Figures 11-12, the results of Example 2 and Comparative Examples 1-2 show that the cell viability and cell expansion multiples after digestion with the recombinant trypsin cell digestion solution of Comparative Examples 1-2 are significantly lower than those of Example 2. Therefore, the recombinant trypsin cell digestion solution of the present application, which includes both poloxamer 188PRO and HEPES, can effectively improve the cell viability and cell expansion multiples of cells after passage.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A recombinant pancreatic enzyme cell digestion solution for maintaining high cell viability, characterized in that: The recombinant pancreatic enzyme cell digestion solution is composed of the following components in the following concentrations: sodium chloride 5000-8000 mg / L, glucose 500-1500 mg / L, disodium hydrogen phosphate 500-1500 mg / L, sodium bicarbonate 100-400 mg / L, potassium dihydrogen phosphate 1000-2000 mg / L, potassium chloride 1000-2000 mg / L, EDTA 0.5-2 mM, poloxamer 188 PRO 100-500 mg / L, HEPES 100-500 mg / L, recombinant pancreatic enzyme 100-500 mg / L, and the remaining components are pure water.

2. The recombinant pancreatic enzyme cell digestion solution according to claim 1, characterized in that The recombinant pancreatic enzyme cell digestion solution is composed of the following components in the following concentrations: sodium chloride 5000 mg / L, glucose 1500 mg / L, disodium hydrogen phosphate 1500 mg / L, sodium bicarbonate 400 mg / L, potassium dihydrogen phosphate 2000 mg / L, potassium chloride 2000 mg / L, EDTA 2 mM, poloxamer 188 PRO 500 mg / L, HEPES 400 mg / L, recombinant pancreatic enzyme 500 mg / L, and the remaining components are pure water.

3. The recombinant pancreatic enzyme cell digestion solution according to claim 1, characterized in that The recombinant pancreatic enzyme cell digestion solution is composed of the following components in the following concentrations: sodium chloride 6500 mg / L, glucose 1000 mg / L, disodium hydrogen phosphate 1000 mg / L, sodium bicarbonate 300 mg / L, potassium dihydrogen phosphate 1500 mg / L, potassium chloride 1500 mg / L, EDTA 1 mM, poloxamer 188 PRO 300 mg / L, HEPES 300 mg / L, recombinant pancreatic enzyme 350 mg / L, and the remaining components are pure water.

4. The recombinant pancreatic cell digestion solution according to claim 1, characterized in that The recombinant pancreatic enzyme cell digestion solution is composed of the following components in the following concentrations: sodium chloride 8000 mg / L, glucose 500 mg / L, disodium hydrogen phosphate 500 mg / L, sodium bicarbonate 100 mg / L, potassium dihydrogen phosphate 1000 mg / L, potassium chloride 1000 mg / L, EDTA 0.5 mM, poloxamer 188 PRO 100 mg / L, HEPES 100 mg / L, recombinant pancreatic enzyme 100 mg / L, and the remaining components are pure water.

5. A method for preparing the recombinant pancreatic cell digestion solution according to any one of claims 1 to 4, characterized in that: The components were dissolved separately to prepare a mixed solution, purified water was added to adjust the concentration of each component, the pH value was adjusted to 6.9-7.4, and the solution was filtered to obtain the recombinant pancreatic enzyme cell digestion solution.

6. Use of the recombinant pancreatic enzyme cell digestion solution according to any one of claims 1 to 4 in digesting mesenchymal stem cells.

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