A cell digestion solution, and a preparation method and application thereof

Abstract

This application relates to the field of cell culture technology, and more particularly to a cell digestion solution, its preparation method, and its application. A cell digestion solution is provided, the effective components of which include trypsin, ethylenediaminetetraacetic acid tetrasodium salt dihydrate, sodium chloride, L-glutamine, human serum albumin, and phosphate buffer. This cell digestion solution uses L-glutamine and human serum albumin as nutrient additives to ensure that cells maintain a high level of viability even during digestion.

Description

Technical Field

[0001] This application belongs to the field of cell culture technology, and in particular relates to a cell digestion solution, its preparation method and application. Background Technology

[0002] Mesenchymal stem cells (MSCs) are pluripotent stem cells derived from the umbilical cord tissue of newborns. They possess a fibroblast-like morphology and can differentiate into various tissue cells such as adipocytes and osteoblasts in vitro. Simultaneously, MSCs exhibit unique immunomodulatory functions, making them highly promising candidates for cell therapy. Based on their self-renewal capacity, multi-lineage differentiation potential, and immunomodulatory properties, combined with the rapid development of regenerative medicine, MSCs have become a research hotspot of widespread interest in academia and the medical community, demonstrating significant value in clinical translational research.

[0003] In the in vitro isolation and expansion of MSCs, cells typically grow in a monolayer adherent state. For clinical research and applications, they need to be dissociated into single-cell suspensions. Therefore, the choice of digestion solution is a crucial step in the expansion process. Trypsin is currently the most commonly used digestive enzyme. It achieves cell separation by cleaving peptide bonds linked to arginine or lysine, degrading intercellular mucins and glycoproteins. Traditional trypsin has been validated through years of practice, offering advantages such as wide application, stable efficacy, and low cost. However, its digestion process inevitably causes mechanical and stress damage to cells. Improper control of digestion time or concentration can easily lead to over-digestion, significantly affecting cell viability and stemness. While existing technologies include improved solutions such as the digestion solution containing trypsin, epidermal growth factor, vitamin C, and serum albumin disclosed in CN116333975A, and the cell digestion solution containing mild digestive enzymes and various inorganic salt components disclosed in CN109439621A, these solutions do not adequately consider providing key nutritional protective factors simultaneously during digestion to mitigate cell damage.

[0004] Glutamine is a key molecule for maintaining cell growth and survival. It can generate ATP through metabolism to support cell proliferation and help cells maintain normal function under stress. Human serum albumin is rich in amino acids, providing nutritional support while protecting cells from oxidative stress damage through its antioxidant properties, reducing cell damage caused by mechanical force, and improving cell survival rate. However, there are currently no reports of a mild cell digestion solution that simultaneously contains both L-glutamine and human serum albumin and can effectively maintain stem cell activity. Therefore, developing a digestion solution with high digestion efficiency, minimal cell damage, and effective maintenance of MSC activity and stemness remains a pressing technical problem to be solved in this field. Summary of the Invention

[0005] The purpose of this application is to provide a cell digestion solution, its preparation method and application, in order to solve the problem that in the prior art, cell digestion solutions can easily cause stress damage to stem cells and affect cell viability and stemness during digestion.

[0006] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows: In a first aspect, this application provides a cell digestion solution, the effective components of which include trypsin, ethylenediaminetetraacetic acid tetrasodium salt dihydrate, sodium chloride, L-glutamine, human serum albumin, and phosphate buffer.

[0007] In some embodiments, the cell digestion solution includes: Trypsin 800-2500 mg / L; EDTA-14 sodium tetrahydrate dihydrate 1000-1200 mg / L; Sodium chloride 4250-4300 mg / L; L-glutamine 146-150 mg / L; Human serum albumin 5000-20000 mg / L; Add phosphate buffer to a total volume of 1L.

[0008] In some embodiments, the pH of the phosphate buffer solution is 7.2-7.4.

[0009] In some embodiments, the pH of the cell digestion solution is 6.8-7.2.

[0010] In some embodiments, the cell digestion solution is used to maintain the cell viability of stem cells.

[0011] Secondly, this application provides a method for preparing the above-mentioned cell digestion solution, comprising the following steps: Mix the trypsin powder and phosphate buffer to obtain the first mixture; The first mixture was prepared by mixing ethylenediaminetetraacetic acid tetrasodium salt dihydrate, sodium chloride, L-glutamine, and human serum albumin with the first mixture and adjusting the pH to 6.8-7.2 to obtain the second mixture. The second mixture was filtered to obtain a cell digestion solution.

[0012] In some embodiments, the cell digestion solution is stored at 2-8°C, and for long-term storage at -20°C.

[0013] Thirdly, this application provides a method for dissociating stem cells, comprising the following steps: The cell digestion solution described above was added to the adherent stem cells and digested in a constant temperature incubator. After digestion, add complete culture medium to stop digestion and collect the cell suspension; The cell suspension was centrifuged, the precipitate was collected, and then resuspended to obtain a single-cell stem cell suspension.

[0014] In some embodiments, the digestion process takes 2-4 minutes.

[0015] In some embodiments, the centrifugation conditions are 1500-1800 rpm for 5-8 minutes.

[0016] Compared with the prior art, the beneficial effects of this application are as follows: (1) The cell digestion solution provided uses L-glutamine and human serum albumin as nutritional supplements. L-glutamine generates ATP through metabolism to support cell growth and maintain normal cell function. Human serum albumin provides abundant amino acids and plays an antioxidant protective role. The two work together to effectively alleviate the stress response of stem cells caused by trypsin digestion, promote the stabilization of cell state, maintain the original function and stemness of stem cells, and reduce mechanical damage caused by cell blowing.

[0017] (2) The provided cell digestion solution has high digestion efficiency, which can cause adherent mesenchymal stem cells to completely detach in a short time, with a cell viability of up to 97%. While maintaining a high cell viability, it has outstanding cell digestion ability.

[0018] (3) The provided cell digestion solution can significantly increase the average expansion fold of stem cells in continuous passage, stabilize cell passage, and reduce loss.

[0019] (4) The method for preparing the cell digestion solution is simple and does not require advanced equipment. Conventional equipment and consumables can meet the preparation conditions. It is low in cost and suitable for industrial production. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 Cell harvesting and counting results after MSCs were digested for 2 minutes with digestion solutions A, B, and C.

[0022] Figure 2 Cell viability test results after MSCs were digested with digestion solutions A, B, and C for 2 minutes.

[0023] Figure 3Cell harvesting and counting results after MSCs were digested for 2 minutes with digestion solutions A, D, and E.

[0024] Figure 4 Cell viability test results after MSCs were digested with digestion solutions A, D, and E for 2 minutes.

[0025] Figure 5 Cell harvesting and counting results after MSCs were digested with F, G, and H digestion solutions for 2 minutes.

[0026] Figure 6 Cell viability assay results after MSCs were digested with F, G, and H digestion solutions for 2 minutes.

[0027] Figure 7 The results show the average cell expansion fold of digestion solutions A, F, G, and H. Detailed Implementation

[0028] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0029] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0030] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0031] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0032] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms "a" and "the" as used in the embodiments of this application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0033] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass in the embodiments of this application can be a well-known unit of mass in the chemical industry, such as µg, mg, g, or kg.

[0034] The terms "first" and "second" are used for descriptive purposes only, to distinguish objects, such as substances, from one another, and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. For example, without departing from the scope of the embodiments of this application, "first XX" may also be referred to as "second XX," and similarly, "second XX" may also be referred to as "first XX." Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of that feature.

[0035] The first aspect of this application provides a cell digestion solution, the effective components of which include trypsin, ethylenediaminetetraacetic acid tetrasodium salt dihydrate, sodium chloride, L-glutamine, human serum albumin, and phosphate buffer.

[0036] The provided cell digestion solution uses L-glutamine and human serum albumin as nutritional supplements. L-glutamine generates ATP through metabolism to support cell growth and maintain normal cell function, while human serum albumin provides abundant amino acids and plays an antioxidant protective role. The two work together to effectively alleviate the stress response of stem cells caused by trypsin digestion, promote cell stability, maintain the original function and stemness of stem cells, and reduce mechanical damage caused by cell blowing.

[0037] In some embodiments, the cell digestion solution includes: Trypsin 800-2500 mg / L; EDTA-14 sodium tetrahydrate dihydrate 1000-1200 mg / L; Sodium chloride 4250-4300 mg / L; L-glutamine 146-150 mg / L; Human serum albumin 5000-20000 mg / L; Add phosphate buffer to a total volume of 1L.

[0038] The concentration of trypsin is limited to the range of 800-2500 mg / L to ensure sufficient digestive capacity while avoiding excessive hydrolysis of cell membrane proteins and decreased cell viability due to excessive concentration. In some specific embodiments, the concentration of trypsin can be typical but not limiting values ​​such as 800 mg / L, 1000 mg / L, 1200 mg / L, 1400 mg / L, 1600 mg / L, 1800 mg / L, 2000 mg / L, 2200 mg / L, 2400 mg / L, and 2500 mg / L.

[0039] The concentration of ethylenediaminetetraacetic acid tetrasodium dihydrate dihydrate is 1000-1200 mg / L, which is beneficial for fully chelating divalent cations in the culture medium and cell surface microenvironment, promoting cell detachment, while avoiding cytotoxicity from excessively high concentrations. In some specific embodiments, the concentration of ethylenediaminetetraacetic acid tetrasodium dihydrate dihydrate can be typical but not limiting values ​​such as 1000 mg / L, 1100 mg / L, and 1200 mg / L.

[0040] The addition of sodium chloride primarily works synergistically with phosphate buffer to maintain the digestive fluid at a near-physiological osmotic pressure, reducing cell swelling or shrinkage during digestion. In some specific embodiments, the concentration of sodium chloride can be typical but not limiting values ​​such as 4250 mg / L, 4260 mg / L, 4270 mg / L, 4280 mg / L, 4290 mg / L, and 4300 mg / L.

[0041] The concentration of L-glutamine is 146-150 mg / L, which can meet the metabolic needs of stem cells during digestion.

[0042] The concentration of human serum albumin is 5000-20000 mg / L, which can provide sufficient protection to prevent cell damage and apoptosis.

[0043] In some embodiments, the pH of the phosphate buffer is 7.2-7.4. A pH of 7.2-7.4 is close to the cell culture and in vivo physiological environment, which reduces the acid-base shock when digestive fluids come into contact with cells, thus helping to maintain stem cell activity. Simultaneously, it allows trypsin to have higher activity in the weakly alkaline to neutral range.

[0044] In some embodiments, the pH of the cell digestion solution is 6.8-7.2. A pH of 6.8-7.2 is close to that of common cell culture media, which reduces the stress on stem cells caused by sudden pH changes during digestion; it also slows down the autolysis of trypsin under weakly acidic conditions, contributing to improved storage stability and reproducibility of the digestion solution. Simultaneously, human serum albumin and L-glutamine exhibit good stability within this pH range, which helps maintain their protective functions.

[0045] In some embodiments, the cell digestion solution is used to maintain the cell viability of stem cells.

[0046] A second aspect of this application provides a method for preparing the above-mentioned cell digestion solution, comprising the following steps: S01. Mix trypsin powder and phosphate buffer to obtain the first mixture; S02. Mix ethylenediaminetetraacetic acid tetrasodium salt dihydrate, sodium chloride, L-glutamine, and human serum albumin with the first mixture, and adjust the pH to 6.8-7.2 to obtain the second mixture; S03. Filter the second mixture to obtain cell digestion solution.

[0047] In this preparation method, trypsin powder is first dissolved in phosphate buffer to prevent aggregation or decreased activity in high-concentration salts or other components, thus ensuring enzyme activity. Subsequently, EDTA, sodium chloride, L-glutamine, and human serum albumin are added to prevent these components from prolonged exposure to high concentrations of trypsin under extreme conditions, reducing adverse effects on nutritional and protective components. Furthermore, the pH is uniformly adjusted to 6.8-7.2 to ensure the digestive solution maintains activity within the optimal pH range, while also improving batch-to-batch stability. Simultaneously, filtration removes bacteria, fungi, and particulate impurities to obtain a sterile digestive solution suitable for cell culture, reducing the risk of contamination. This preparation method is simple, mild, and suitable for both laboratory and large-scale production.

[0048] In some embodiments, the cell digestion solution is stored at 2-8°C, and for long-term storage at -20°C.

[0049] A third aspect of this application provides a method for dissociating stem cells, comprising the following steps: G01. Add the above cell digestion solution to the adherent stem cells and digest them in a constant temperature incubator; G02. After digestion, add complete culture medium to stop digestion and collect the cell suspension; G03. Centrifuge the cell suspension, collect the precipitate, and then resuspend it to obtain a single-cell stem cell suspension.

[0050] In some embodiments, the digestion process takes 2-4 minutes.

[0051] The short digestion time helps avoid over-digestion that could lead to cell membrane damage, loss of surface proteins, or decreased cell activity.

[0052] In some embodiments, centrifugation is performed at 1500-1800 rpm for 5-8 minutes. The relatively low speed of 1500-1800 rpm results in low shear force, reducing mechanical damage to the stem cell membrane and preserving cell integrity and viability. The 5-8 minute centrifugation time is sufficient to precipitate the stem cells, avoiding excessively low speeds or short times that could lead to decreased cell recovery rates. This also facilitates obtaining a uniform single-cell suspension.

[0053] The following description is based on specific embodiments.

[0054] Example 1 Preparation and digestive effect of digestive fluid that effectively maintains stem cell stemness I. Preparation of cell digestion solution The digestive solution A used in this embodiment to maintain stem cell stemness consists of the following components, with each component having a concentration in mg / L: trypsin 2500 mg / L, ethylenediaminetetraacetic acid tetrasodium dihydrate 1000 mg / L, sodium chloride 4250 mg / L, L-glutamine 146 mg / L, and the remainder being phosphate buffer.

[0055] This embodiment of digestive solution B, which can maintain the stemness of stem cells, consists of the following components, each with a concentration in mg / L: trypsin 2500 mg / L, ethylenediaminetetraacetic acid tetrasodium dihydrate 1000 mg / L, sodium chloride 4250 mg / L, L-glutamine 146 mg / L, D-glucose 500 mg / L, sodium pyruvate 55 mg / L, with the remainder being phosphate buffer.

[0056] The digestive solution C used in this embodiment to maintain stem cell stemness consists of the following components, with each component having a concentration in mg / L: trypsin 2500 mg / L, ethylenediaminetetraacetic acid tetrasodium dihydrate 1000 mg / L, sodium chloride 4250 mg / L, L-glutamine 146 mg / L, adenosine 10 mg / L, cytidine 10 mg / L, guanosine 10 mg / L, uridine 10 mg / L, 2'-deoxyadenosine 10 mg / L, 2'-deoxycytidine hydrochloride 11 mg / L, 2'-deoxyguanosine 10 mg / L, deoxythymidine 10 mg / L, with the remainder being phosphate buffer.

[0057] The method for preparing the digestive solution for maintaining stem cell stemness in this embodiment is as follows: Trypsin powder is added to phosphate buffer and mixed well. The mixture is then divided into three equal portions, and the components of digestive solutions A, B, and C are added to phosphate buffer respectively to obtain a mixture. The pH is adjusted to 6.8-7.2, and the mixture is filtered through a 0.22 μm filter membrane into a sterile container to obtain the cell digestive solution. The prepared digestive solution is stored at 2-8℃, and for long-term storage, it is stored at -20℃.

[0058] II. Digestion Effect Experiment (a) Experimental subjects: human umbilical cord mesenchymal stem cells (MSCs).

[0059] (II) Experimental Methods: MSCs were seeded at a density of 8000 cells / cm² in T25 culture flasks and cultured at 37℃ with 5.0% CO₂. When the cells reached 85%-90% confluence, the culture supernatant was removed, and the cells were washed twice with 2 mL of PBS solution. 1 mL each of the digestion solutions A, B, and C were added to the T25 culture flasks, and the flasks were incubated for 2 minutes. After digestion, an equal volume of complete culture medium was added to terminate the digestion, and the cell suspension was collected. The cell suspension was centrifuged at 1500 rpm for 5 minutes. After centrifugation, the supernatant was discarded, and the cells were resuspended in 1 mL of fresh culture medium. After mixing, 20 μL was used for trypan blue staining and cell counting.

[0060] (III) Experimental Results After the above-mentioned digestion solutions A, B, and C were applied to MSCs, microscopic examination showed that the cells were round, in good condition, with clear cell membrane edges, and no cell clumping was observed.

[0061] The digestion results of A, B, and C are shown in Table 1 and... Figure 1 , Figure 2 MSCs were treated with digestion solution A, and the total cell count was 1.08 × 10⁻⁶. 6 The cell count was 1.06 × 10⁶ cells, with a cell viability of 95%. MSCs were digested with B digestion solution, and trypan blue staining showed a total cell count of 1.06 × 10⁶ cells. 6 The cell count was 93%; MSCs were treated with C20 digestion solution, and the total cell count was 0.82 × 10⁻⁶. 6 The number of cells was 95%, and the cell viability was 95%.

[0062] The results of this embodiment indicate that, in terms of total cell count, digestion solution A is superior to digestion solutions B and C, indicating that digestion solution A has a stronger ability to digest cells; in terms of cell viability, digestion solution A is superior to digestion solution B and consistent with the viability of digestion solution C, indicating that digestion solution A can maintain cell viability. These results demonstrate that digestion solution A can more effectively digest cells and improve digestion efficiency while maintaining cell viability.

[0063] Table 1. Results of MSC dissociation from digestion solutions A, B, and C

[0064] Example 2 Optimize trypsin concentration I. Preparation of digestive solutions with different concentrations of trypsin The digestive solution in this embodiment is the same as that in Example 1A.

[0065] In this embodiment, the digestive solution D used to optimize the trypsin concentration has the same composition as digestive solution A, only the concentration of each component is adjusted. The concentration of each component is in mg / L: trypsin 1500, ethylenediaminetetraacetic acid tetrasodium dihydrate 1000, sodium chloride 4250, L-glutamine 146, and the balance is phosphate buffer.

[0066] In this embodiment, the digestive solution E used to optimize the trypsin concentration has the same composition as digestive solution A, only the concentration of each component is adjusted. The concentration of each component is in mg / L: trypsin 800, ethylenediaminetetraacetic acid tetrasodium salt dihydrate 1000, sodium chloride 4250, L-glutamine 146, and the balance is phosphate buffer.

[0067] The preparation and preservation methods of the digestive fluid in this embodiment are the same as those in Example 1.

[0068] II. Digestion Effect Experiment (a) Experimental subjects: Same as in Example 1.

[0069] (II) Experimental method: Except for the digestive fluid, the other parameters are the same as in Example 1.

[0070] (III) Experimental Results In this embodiment, after digestion solutions A, D, and E were applied to MSCs, microscopic examination showed that the cells became rounded and suspended, with no abnormalities and no debris production.

[0071] The digestive effects of A, D, and E are shown in Table 2. Figure 3 , Figure 4 Digestive solution A was applied to MSCs, and trypan blue staining showed a total cell count of 1.4 × 10⁻⁶ cells. 6 The cell count was 95%; when MSCs were treated with D digestion solution, the total cell count was 1.12 × 10⁶ cells / year. 6 The cell count was 97%; when E digestion solution was applied to MSCs, the total cell count was 0.8 × 10⁶ cells / year. 6 The number of cells was 95%, and the cell viability was 95%.

[0072] The results of this embodiment show that, under the same digestion time, digestion solution A collected the most cells and maintained a cell viability of 95%. Overall, digestion solution A demonstrated superior cell digestion ability while maintaining high cell viability.

[0073] Table 2 Results of MSC dissociation from digestion solutions A, D, and E

[0074] Example 3 Preparation of stem cell digestion solution to enhance cell viability I. Preparation of stem cell digestion solution to enhance cell viability The F digestion solution used in this embodiment to improve cell viability consists of the following components, with each component having a concentration in mg / L: trypsin 2500 mg / L, ethylenediaminetetraacetic acid tetrasodium dihydrate 1000 mg / L, sodium chloride 4250 mg / L, L-glutamine 146 mg / L, human serum albumin 20000 mg / L, and the remainder being phosphate buffer.

[0075] In this embodiment, the components of the G and H digestive solutions, which can improve cell viability, are identical to those of the F digestive solution, except for the concentration of human serum albumin. The human serum albumin concentration of the G digestive solution is 10000 mg / L; the human serum albumin concentration of the H digestive solution is 5000 mg / L.

[0076] II. Digestion Effect Experiment (a) Experimental subjects: Same as in Example 1.

[0077] (II) Experimental method: Except for the digestive fluid, the other parameters are the same as in Example 1.

[0078] (III) Experimental Results MSCs were digested using digestion solutions F, G, and H in this embodiment. Under a microscope, the cells were observed to have detached and become rounded, and were dispersed in the digestion solution, forming a single-cell suspension.

[0079] The digestion effects of F, G, and H are shown in Table 3. Figure 5 , Figure 6 Trypan blue staining and cell counting showed that the total number of MSCs collected after treatment with F digestion solution was 1.08 × 10⁻⁶. 6 The number of MSCs was 97%; after digestion with G digestion solution, the total number of cells was 0.95 × 10⁶. 6 The number of MSCs was [number missing], with a cell viability of 95%; after treatment with H digestion solution, the total cell count was 0.9 × 10⁻⁶. 6 The number of cells was 94%, and the cell viability was 94%.

[0080] The results of Example 3 indicate that the addition of human serum albumin improved cell viability, with the preparation of F digestive solution being particularly effective in enhancing cell viability. The counting results demonstrate that F digestive solution not only improved cell viability but also demonstrated stronger digestive capacity.

[0081] Table 3. Results of MSC dissociation from F, G, and H digestion solutions.

[0082] Example 4 Continuous amplification experiment I. Selection of Digestive Fluids The digestive solutions used in this embodiment are digestive solution A, digestive solution F, digestive solution G, and digestive solution H.

[0083] II. Digestion Effect Experiment (a) Experimental subjects: Same as in Example 1.

[0084] (II) Experimental Methods: Cells from three consecutive passages were digested and passaged. Except for the digestion solution, the other parameters were the same as in Example 1.

[0085] (III) Experimental Results MSCs were digested using digestion solutions A, F, G, and H in this embodiment. Under a microscope, all cells detached, forming a single-cell suspension.

[0086] The digestive effects are shown in Table 4 and... Figure 7 MSCs were continuously digested with digestion solution A, with an average fold increase of 6.79. The results of digestion F are shown in Table 5. Figure 7 MSCs were continuously digested with F digestion solution, with an average fold increase of 8.12. The results of G digestion are shown in Table 6. Figure 7 MSCs were continuously digested with G digestion solution, with an average fold increase of 7.51. The results of H digestion are shown in Table 7. Figure 7 MSCs were continuously digested with H digestion solution, and the average fold increase was 6.99.

[0087] The results of Example 4 indicate that after adding human serum albumin, continuous passage of MSCs with cell digestion solution resulted in higher amplification folds, stable cell passage, and reduced loss.

[0088] Table 4A Results of continuous passaging of digestive fluid

[0089] Table 5 shows the results of continuous passaging of F digestive fluid.

[0090] Table 6 shows the results of continuous passaging of G digestate.

[0091] Table 7 Results of continuous passage of H digestive fluid

[0092] Compared with the prior art, the beneficial effects of the embodiments of this application are as follows: (1) The cell digestion solution provided uses L-glutamine and human serum albumin as nutritional supplements. L-glutamine generates ATP through metabolism to support cell growth and maintain normal cell function. Human serum albumin provides abundant amino acids and plays an antioxidant protective role. The two work together to effectively alleviate the stress response of stem cells caused by trypsin digestion, promote the stabilization of cell state, maintain the original function and stemness of stem cells, and reduce mechanical damage caused by cell blowing.

[0093] (2) The provided cell digestion solution has high digestion efficiency, which can cause adherent mesenchymal stem cells to completely detach in a short time, with a cell viability of up to 97%. While maintaining a high cell viability, it has outstanding cell digestion ability.

[0094] (3) The provided cell digestion solution can significantly increase the average expansion fold of stem cells in continuous passage, stabilize cell passage, and reduce loss.

[0095] (4) The method for preparing the cell digestion solution is simple and does not require advanced equipment. Conventional equipment and consumables can meet the preparation conditions. It is low in cost and suitable for industrial production.

[0096] The above are merely preferred embodiments of this application and are not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A cell digestion fluid, characterized in that, The effective components of the cell digestion solution include trypsin, ethylenediaminetetraacetic acid tetrasodium dihydrate, sodium chloride, L-glutamine, human serum albumin, and phosphate buffer.

2. The cell digestion solution according to claim 1, characterized in that, The cell digestion solution includes: Trypsin 800-2500 mg / L; EDTA-14 sodium tetrahydrate dihydrate 1000-1200 mg / L; Sodium chloride 4250-4300 mg / L; L-glutamine 146-150 mg / L; Human serum albumin 5000-20000 mg / L; Add phosphate buffer to a total volume of 1L.

3. The cell digestion solution according to claim 1 or 2, characterized in that, The pH of the phosphate buffer solution is 7.2-7.

4.

4. The cell digestion solution according to claim 1 or 2, characterized in that, The pH value of the cell digestion solution is 6.8-7.

2.

5. The cell digestion solution according to claim 1 or 2, characterized in that, The cell digestion solution is used to maintain the cell viability of stem cells.

6. A method for preparing a cell digestion solution as described in any one of claims 1-5, characterized in that, Includes the following steps: Mix the trypsin powder and phosphate buffer to obtain the first mixture; The first mixture was mixed with ethylenediaminetetraacetic acid tetrasodium salt dihydrate, sodium chloride, L-glutamine, and human serum albumin, and the pH was adjusted to 6.8-7.2 to obtain the second mixture. The second mixture is filtered to obtain the cell digestion solution.

7. The preparation method according to claim 6, characterized in that, The cell digestion solution is stored at 2-8℃, and for long-term storage, it is stored at -20℃.

8. A method for dissociating stem cells, characterized in that, Includes the following steps: The cell digestion solution according to any one of claims 1-5 is added to adherent stem cells and digested in a constant temperature incubator. After digestion, add complete culture medium to stop digestion and collect the cell suspension; The cell suspension was centrifuged, the precipitate was collected, and then resuspended to obtain a single-cell stem cell suspension.

9. The method for dissociating stem cells according to claim 8, characterized in that, The digestion process takes 2-4 minutes.

10. The method for dissociating stem cells according to claim 8, characterized in that, The centrifugation conditions are 1500-1800 rpm for 5-8 minutes.

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