Fractionated dissociation digestive solution of mesenchymal stem cell spheres, preparation method and application thereof

Abstract

The application discloses a kind of mesenchymal stem cell ball fractional dissociation digestive juice, its preparation method and application, it is related to cell preparation and three-dimensional cell culture technical field, specifically at to provide C liquid, D liquid and E liquid three kinds of gradient concentration working solution.When dissociating, the mesenchymal stem cell ball to be dissociated is determined its average particle size;According to the average particle size, select C liquid, D liquid or E liquid as digestive juice as described above;The selected digestive juice is mixed with the mesenchymal stem cell ball, and digestion reaction is carried out under oscillation condition, and single cell suspension is obtained.The application establishes the hierarchical matching rule of mesenchymal stem cell ball particle size and specific digestive juice concentration, and optimizes oscillation digestion condition, so as to maintain high activity rate and complete phenotype characteristics while efficiently dissociating, realizes the standardization of process and the controllable of dissociation quality, provides stable and reliable solution for the large-scale preparation and quality control of mesenchymal stem cell ball.

Description

Technical Field

[0001] This invention relates to the field of cell preparation and three-dimensional cell culture technology, specifically to a graded dissociation digestion solution for mesenchymal stem cell spheres, its preparation method, and its application. Background Technology

[0002] MSC cell spheres, as a three-dimensional culture medium, are often used to enhance cell-cell interactions and functional expression.

[0003] In cell passage expansion, downstream detection, formulation preparation or quality control, it is usually necessary to effectively dissociate cell spheres into single-cell suspensions.

[0004] Existing dissociation methods often employ a single pancreatic enzyme or a single collagenase, or use a universal digestion solution for organoids / tissues. These methods are prone to problems such as low dissociation efficiency, high residual aggregation rate, and insufficient adaptability to batches of different bead sizes, making it difficult to form a standardized, evaluable, and release-ready process system.

[0005] Therefore, it is necessary to establish a dedicated digestion solution system for MSC cell spheres that can match the digestion intensity according to the sphere diameter, and to provide clear digestion parameters and quality detection thresholds to meet the needs of large-scale preparation and quality control. Summary of the Invention

[0006] The purpose of this invention is to provide a graded dissociation digestion solution for mesenchymal stem cell spheres, its preparation method, and its application, so as to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: A digestive solution for dissociating mesenchymal stem cell spheres is a Hanks' balanced salt solution containing trypsin, type IV collagenase, and disodium EDTA; wherein the digestive solution is any one of the following three formulations: Solution C contains 1.25% (w / v) trypsin, 0.5% (w / v) type IV collagenase, and 0.08% (w / v) disodium EDTA; Solution D contains 0.875% (w / v) trypsin, 0.35% (w / v) type IV collagenase, and 0.056% (w / v) disodium EDTA; Solution E contains 0.4375% (w / v) trypsin, 0.175% (w / v) type IV collagenase, and 0.028% (w / v) disodium EDTA.

[0008] A method for preparing the aforementioned digestive fluid includes the following steps: S1: Prepare solution A and solution B, wherein solution A is a Hanks balanced salt solution containing 2.5% (w / v) trypsin and 0.16% (w / v) disodium ethylenediaminetetraacetate, and solution B is a Hanks balanced salt solution containing 1% (w / v) type IV collagenase. S2: Mix liquid A and liquid B at a volume ratio of 1:1 to obtain liquid C; S3: Mix the C solution with the Hanks equilibrium salt solution at a volume ratio of 7:3 to obtain the D solution; S4: Mix the D solution with the Hanks equilibrium salt solution at a volume ratio of 1:1 to obtain the E solution.

[0009] A method for dissociating mesenchymal stem cell spheres, characterized by comprising the following steps: Provide mesenchymal stem cell spheres to be dissociated and determine their average particle size; Based on the average particle size, select liquid C, liquid D, or liquid E as described above as the digestion liquid; The selected digestive fluid was mixed with the mesenchymal stem cell spheres and digested under oscillation conditions to obtain a single-cell suspension.

[0010] Preferably, the digestive solution is selected based on the average particle size as follows: When the average particle size is less than 100 μm, liquid E is selected; When the average particle size is between 100 μm and 200 μm, liquid D is selected; When the average particle size is greater than 200 μm and not greater than 300 μm, liquid C is selected.

[0011] Use of the digestive fluid of claim 1 in the preparation of a reagent or kit for dissociating mesenchymal stem cell spheres.

[0012] A kit for dissociating mesenchymal stem cell spheres, comprising at least two of the aforementioned solutions C, D, and E.

[0013] Compared with the prior art, the beneficial effects of the present invention are: This invention establishes a grading matching rule between the particle size of mesenchymal stem cell spheres and the concentration of specific digestion solutions (C / D / E solutions), and optimizes the shaking digestion conditions. This achieves efficient dissociation (single cell rate ≥94%, recovery rate ≥85%) while maintaining high cell viability (≥90%) and complete phenotypic characteristics (CD73 / CD90 / CD105 positivity rate ≥95%, impurity markers ≤2%). It realizes the standardization of the process and the controllability of dissociation quality, providing a stable and reliable solution for the large-scale preparation and quality control of mesenchymal stem cell spheres. Attached Figure Description

[0014] Figure 1 These are morphological images of the three groups of cell spheres before and after digestion in this invention.

[0015] Figure 2 This is a comparison chart of the single-cell rate between Example 2 and Comparative Example 1 in this invention.

[0016] Figure 3 This is a comparison chart of the recovery rates of Example 2 and Comparative Example 1 in this invention.

[0017] Figure 4 This is a comparison chart of cell viability between Example 2 and Comparative Example 1 in this invention. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example

[0019] A method for preparing a digestive solution for dissociating mesenchymal stem cell spheres is provided: Weigh 2.5g of trypsin and 0.16g of disodium ethylenediaminetetraacetate (EDTA), add them to Hanks balanced salt solution to dissolve and bring the volume to 100mL. After mixing, filter through a 0.22μm filter to obtain solution A. Weigh 1.0g of type IV collagenase, dissolve it in Hanks balanced salt solution, and bring the volume to 100mL. After mixing, filter through a 0.22μm filter to obtain solution B.

[0020] Solution C is obtained by mixing solution A and solution B in a 1:1 volume ratio. Solution C contains 1.25% trypsin, 0.5% type IV collagenase, and 0.08% disodium EDTA.

[0021] Solution C and Hanks' solution were mixed at a volume ratio of 7:3 to obtain solution D. Solution D contained 0.875% trypsin, 0.35% type IV collagenase, and 0.056% disodium EDTA.

[0022] Solution D and Hanks' solution were mixed at a volume ratio of 1:1 to obtain solution E. Solution E contained 0.4375% trypsin, 0.175% type IV collagenase, and 0.028% disodium EDTA. Example

[0023] A method for dissociating mesenchymal stem cell spheres is provided: Provide MSC cell spheres and group them by size: less than 100 μm, 100–200 μm, and 200–300 μm.

[0024] Prewash the cell spheres 1–3 times with Hanks balanced salt solution to remove culture medium residue.

[0025] Centrifuge to collect cell spheres to form a precipitate and record the precipitate volume.

[0026] Select the digestion solution according to the particle size and add it at 3–10 times the volume of the precipitate: When the average particle size is less than 100 μm, liquid E is selected; When the average particle size is between 100 μm and 200 μm, liquid D is selected; When the average particle size is greater than 200 μm and not greater than 300 μm, liquid C is selected.

[0027] Digest at 37°C with shaking at 50 rpm for 5 min to obtain a single-cell suspension.

[0028] Digestion was terminated by adding buffer or culture medium for dilution, followed by centrifugation, washing, and resuspending to obtain a single-cell suspension for quality testing.

[0029] The results are as follows Figure 1 The image shows the morphology of three groups of cell spheres before and after digestion.

[0030] To verify the effectiveness of the digestive fluid in this application, a commercially available organoid digestive fluid was used as a comparative example.

[0031] Comparative Example 1 MSC cell spheres of the same origin and particle size group as in Example 2 were provided and digested and dissociated using a commercially available organoid digestion solution according to the recommended conditions in its instructions; the remaining dilution termination, centrifugation, washing and resuspension steps were the same as in Example 2, and the same quality tests were performed.

[0032] Experimental Example 1: Single Cell Rate The single-cell rate is defined as: number of single cells / total number of cells × 100%.

[0033] The single-cell rate in Example 2 was 94.8 ± 2.5% (n = 10); the single-cell rate in Comparative Example 1 was 76.7 ± 3.1% (n = 10).

[0034] The results are as follows Figure 2 The figure shown is a comparison of the single-cell rate between Example 2 and Comparative Example 1.

[0035] Table 1: Summary of single cell rate results (mean ± SD, n).

[0036] Sample number Example 2 Single cell rate Comparative Example 1: Single Cell Rate 1 95.1 78.2 2 93.0 74.5 3 96.8 79.5 4 94.6 77.0 5 95.2 75.8 6 92.9 76.3 7 97.1 78.9 8 94.8 76.1 9 95.4 75.0 10 93.1 78.7 Statistical value 94.8±2.5 76.7±3.1 Experimental Example 2: Recovery Rate Recovery rate is defined as: number of cells obtained after dissociation / theoretical number of cells before dissociation × 100%.

[0037] The recovery rate of Example 2 was 86.2 ± 3.4% (n=10); the recovery rate of Comparative Example 1 was 78.2 ± 4.0% (n=10).

[0038] The results are as follows Figure 3 The figure shown is a comparison chart of the recovery rates of Example 2 and Comparative Example 1.

[0039] Table 2: Summary of recovery results (mean ± SD, n).

[0040] Sample number Example 2 Cell recovery rate Comparative Example 1: Cell Recovery Rate 1 88.5 81.0 2 82.9 75.3 3 89.1 82.6 4 86.0 77.8 5 87.2 76.5 6 83.5 79.2 7 85.8 74.9 8 86.4 80.1 9 84.7 77.0 10 86.3 79.0 Statistical value 86.2±3.4 78.2±4.0

[0041] Experimental Example 3: Cell Viability Viability was detected using an AOPI fluorescence counter. The viability of the example group was 90.5±1.9%, and the viability of the comparative example group was 84.3±2.4%.

[0042] The results are as follows Figure 4 The figure shown is a comparison of cell viability between Example 2 and Comparative Example 1.

[0043] Table 3 Summary of Cell Viability Detection Results (AOPI Fluorescence Counter) Sample number Example 2 Cell viability Comparative Example 1 Cell Viability 1 91.2 85.7 2 88.9 81.6 3 92.0 86.2 4 90.3 84.5 5 91.5 83.8 6 88.5 82.9 7 90.1 85.1 8 90.8 86.0 9 89.7 83.5 10 90.5 84.3 Statistical value 90.5±1.9 84.3±2.4 Experimental Example 4: MSC Phenotypic Retention Flow cytometry was performed on the cells after dissociation in the example group, and the results met the following criteria: the positive rates of CD73 (5'-nucleotidase), CD90 (Thy-1 antigen, thymocyte antigen-1), and CD105 (endothelial glycoprotein) were all ≥95%, and the positive rates of CD11b (integrin Am), CD45 (leukocyte common antigen), and HLA-DR (human leukocyte antigen-DR) were all ≤2%.

[0044] Table 4: Summary of Phenotypic Positive Rates

[0045] Sample number CD73 positivity rate (%) CD90 positivity rate (%) CD105 positivity rate (%) CD11b positivity rate (%) CD45 positivity rate (%) HLA-DR positivity rate (%) 1 97.2 98.5 96.8 0.7 0.5 0.8 2 98.6 99.1 97.5 1.2 0.9 1.0 3 96.5 97.8 98.2 0.4 0.3 0.6 4 99.1 99.5 97.9 1.5 1.1 1.2 5 97.8 98.2 96.7 0.8 0.6 0.9 6 98.3 98.9 98.6 0.6 0.4 0.7 7 96.9 97.5 97.3 1.1 0.8 1.1 8 98.7 99.2 99.0 0.9 0.7 0.8 9 97.4 98.1 97.7 1.3 1.0 1.3 10 98.0 98.7 98.1 0.5 0.5 0.5 Mean ± SD 97.8±0.8 98.5±0.7 97.8±0.7 0.9±0.4 0.7±0.3 0.9±0.2

[0046] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. Furthermore, the contents not described in detail in this specification are all prior art known to those skilled in the art.

Claims

1. A digestive solution for dissociating mesenchymal stem cell spheres, characterized in that, It is a Hanks balanced salt solution containing trypsin, type IV collagenase, and disodium EDTA; wherein the digestive solution is any one of the following three formulations: Solution C contains 1.25% (w / v) trypsin, 0.5% (w / v) type IV collagenase, and 0.08% (w / v) disodium EDTA; Solution D contains 0.875% (w / v) trypsin, 0.35% (w / v) type IV collagenase, and 0.056% (w / v) disodium EDTA; Solution E contains 0.4375% (w / v) trypsin, 0.175% (w / v) type IV collagenase, and 0.028% (w / v) disodium EDTA.

2. A method for preparing the digestive fluid of claim 1, characterized in that, Includes the following steps: S1: Prepare solution A and solution B, wherein solution A is a Hanks balanced salt solution containing 2.5% (w / v) trypsin and 0.16% (w / v) disodium ethylenediaminetetraacetate, and solution B is a Hanks balanced salt solution containing 1% (w / v) type IV collagenase. S2: Mix liquid A and liquid B at a volume ratio of 1:1 to obtain liquid C; S3: Mix the C solution with the Hanks equilibrium salt solution at a volume ratio of 7:3 to obtain the D solution; S4: Mix the D solution with the Hanks equilibrium salt solution at a volume ratio of 1:1 to obtain the E solution.

3. A method for dissociating mesenchymal stem cell spheres, characterized in that, Includes the following steps: Provide mesenchymal stem cell spheres to be dissociated and determine their average particle size; Based on the average particle size, liquid C, liquid D, or liquid E as described in claim 1 is selected as the digestion liquid; The selected digestive fluid was mixed with the mesenchymal stem cell spheres and digested under oscillation conditions to obtain a single-cell suspension.

4. The dissociation method according to claim 3, characterized in that, The digestive solution is selected based on the average particle size as follows: When the average particle size is less than 100 μm, liquid E is selected; When the average particle size is between 100 μm and 200 μm, liquid D is selected; When the average particle size is greater than 200 μm and not greater than 300 μm, liquid C is selected.

5. The dissociation method according to claim 3, characterized in that, The volume of the digestive fluid added is 3-10 times the volume of the mesenchymal stem cell spheres precipitated.

6. The dissociation method according to any one of claims 3-5, characterized in that, The digestion reaction is followed by the addition of a culture medium or buffer solution containing serum to terminate the digestion reaction.

7. The dissociation method according to any one of claims 3-5, characterized in that, It also includes quality testing of the single-cell suspension.

8. The dissociation method according to claim 7, characterized in that, The quality testing includes at least one of single-cell rate, cell viability, and cell recovery rate.

9. The dissociation method according to claim 8, characterized in that, The quality testing must meet the following standards: single cell rate not less than 90%, cell recovery rate not less than 85%, and cell viability not less than 90%.

10. The dissociation method according to claim 7, characterized in that, The quality detection also includes phenotypic detection, and the expression of surface markers of the cells after dissociation meets the following requirements: the positive rates of CD73, CD90, and CD105 are all ≥95%, and the positive rates of CD11b, CD19, CD45, and HLA-DR are all ≤2%.

11. Use of the digestive fluid of claim 1 in the preparation of a reagent or kit for dissociating mesenchymal stem cell spheres.

12. A kit for dissociating mesenchymal stem cell spheres, characterized in that, The kit contains: At least two of the C liquid, D liquid and E liquid as described in claim 1.

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