A dispersed mixture that can reduce cell clumping during acclimation and methods of making the same

Abstract

The application discloses a dispersion mixed solution system capable of reducing cell aggregation in a domestication process and a preparation method thereof. The system comprises 50x concentrated A liquid (100-250 g / L trypsin+12.5 g / L EDTA) and 100x concentrated B liquid (100 g / L P188+10 g / L DS), and realizes long-term stable storage and rapid preparation in the form of concentrated stock solution. When used, the concentrated stock solution is diluted and mixed in proportion, and through the synergistic effect of mild cell lysis and weakening of cell aggregation force, the cell aggregation in the domestication process is efficiently dispersed, and the cell activity and function are maintained. The preparation method is simple, the storage period is long, the dispersion effect is good, and the cell damage is small, and the application provides an effective technical means for cell domestication.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, specifically to a dispersion mixture for reducing cell aggregation during cell domestication and its preparation method, particularly to a technical solution for preparing the dispersion mixture using a concentrated stock solution. Background Technology

[0002] During cell domestication (such as domestication from adherent culture to suspension culture, or domestication between different culture media / conditions), cells are highly prone to aggregation. Taking CRFK cells and CHO cells as examples, cell aggregation not only affects cell growth and metabolic activity, but also has adverse effects on subsequent experimental research (such as protein expression, virus production, etc.) and industrial production.

[0003] Traditional dispersions typically require fresh preparation for each use, which is cumbersome and makes it difficult to ensure batch-to-batch consistency. While methods such as trypsin digestion can disperse cells to some extent, they often fail to efficiently and gently address cell aggregation issues during cell acclimation and may cause excessive damage to cells. Therefore, developing a stable, ready-to-use mixture that effectively disperses cell aggregates during cell acclimation while maintaining cell viability and function has significant application value. Summary of the Invention

[0004] The purpose of this invention is to provide a dispersion mixture that can reduce cell aggregation during cell domestication and its preparation method. The mixture uses a concentrated stock solution system, which can achieve long-term stable preservation and rapid preparation. At the same time, it can efficiently disperse cell aggregation, reduce cell damage, maintain cell activity and normal physiological function, and provide strong support for the cell domestication process.

[0005] This invention is achieved as follows: To achieve the above objectives, this invention adopts the following technical solution: The dispersion mixture of this invention comprises concentrated solution A and concentrated solution B, which are diluted and mixed in proportion during use: Concentrated stock solution system Solution A (50× Concentrate): Contains trypsin at a final concentration of 100-250 g / L and EDTA (ethylenediaminetetraacetic acid) at a final concentration of 12.5 g / L. This concentrate should be diluted 1:50 before use to achieve a trypsin concentration of 2%-5% and an EDTA concentration of 0.25% in the working solution.

[0006] Solution B (100× Concentrate): Contains P188 (poloxamer 188) at a final concentration of 100 g / L and DS (sodium dextran sulfate) at a final concentration of 10 g / L. This concentrate should be diluted 1:100 before use to achieve a P188 concentration of 1 g / L and a DS concentration of 0.1 g / L in the working solution.

[0007] Working principle of dispersion mixture In concentrate A, trypsin and EDTA work synergistically to gently lyse cell adhesion molecules, loosening intercellular connections. In concentrate B, P188, as a nonionic surfactant, reduces cell surface tension and weakens cell aggregation, while DS further prevents cell re-aggregation through charge repulsion or binding to specific molecules on the cell surface. The combination of these two agents achieves efficient and gentle cell dispersion during acclimation with minimal impact on cell viability.

[0008] The preparation method of the present invention includes the following steps: (1) Preparation of 50×A solution concentrate: Weigh 100-250g of pancreatic enzyme powder and 12.5g of EDTA, first mix with a small amount of sterile PBS or D-Hanks solution to form a paste, then add solvent to 1L, stir at room temperature for 4 hours or overnight at 4℃ to fully dissolve, filter with a 0.22μm filter membrane for sterilization, aliquot into small quantities and store at -20℃; (2) Prepare 100×B solution concentrate: Weigh 100g P188 and 10g DS, add about 800mL of water for injection or ultrapure water, stir magnetically until completely transparent, add water to 1L, filter with a 0.22μm filter membrane for sterilization, dispense in small batches and store at 4℃. (3) Preparation of dispersion mixture: Before use, dilute 50×A solution concentrate at 1:50 and 100×B solution concentrate at 1:100. Then mix the diluted A solution and B solution at a volume ratio of 1:1 to 1:5 to obtain the dispersion mixture that can reduce cell aggregation during domestication.

[0009] Compared with the prior art, the beneficial effects of the present invention are: Highly ready to use: The use of a concentrated stock solution system greatly shortens the preparation time and improves experimental efficiency; High stability: The concentrate can be stored for a long time under appropriate conditions (solution A -20℃, solution B 4℃), ensuring batch-to-batch consistency; Good dispersion effect: The gentle lysis of cell adhesion by solution A and the weakening of cell aggregation by solution B can significantly reduce the cell aggregation rate during the acclimatization process; Cell-friendly: Both solutions A and B have mild components, which can effectively maintain cell activity and normal physiological functions while achieving cell dispersion. Simple process: The preparation process is simple and easy to carry out, making it convenient for scale-up and application in the laboratory or industrial production. Attached Figure Description

[0010] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0011] Figure 1 This is a comparison of the aggregation state of CRFK cells in the control group and the experimental group in Example 1 of the present invention. The left image is the control group (DMEM / F12 medium), and the right image is the experimental group (DMEM / F12 + 2×A solution + 2×B solution).

[0012] Figure 2 This is a comparison of the aggregation state of F81 cells in the control group and the experimental group in Example 2 of the present invention. The left image is the control group (1640 medium), and the right image is the experimental group (1640 + 2×A solution + 2×B solution). Detailed Implementation

[0013] The present invention will be further described in detail below through embodiments, but the scope of protection of the present invention is not limited thereto.

[0014] Example 1: Dispersing CRFK cells using solution A / B

[0015] Preparation of concentrated stock solution Preparation of 50×A solution concentrate: Weigh 150 g of trypsin powder (corresponding to 3% working concentration) and 12.5 g of EDTA. First, mix with 200 mL of sterile PBS to form a paste, then add PBS to 1 L. Stir at room temperature for 4 hours until completely dissolved. Filter with a 0.22 μm filter membrane for sterilization. Dispense into 10 mL tubes and store at -20℃ for later use.

[0016] Preparation of 100×B solution concentrate: Weigh 100g P188 and 10g DS, add 800mL of water for injection, stir magnetically until completely dissolved, add water for injection to 1L, filter sterilize with a 0.22μm filter membrane, dispense into 10mL tubes, and store at 4℃ for later use.

[0017] Preparation of dispersion mixture Take 1 mL of 50× A solution concentrate and add it to 49 mL of sterile buffer solution, mix well to obtain the working solution of A solution; take 1 mL of 100× B solution concentrate and add it to 99 mL of sterile buffer solution, mix well to obtain the working solution of B solution.

[0018] Cell experiments Take one bottle of serum-cultured adherent CRFK cells. The original culture medium was 10% FBSDMEM / F12. After trypsin digestion, add 1×10⁻⁶ cells. 6Inoculated at a density of / ml in serum-free DMEM / F12 medium, and divided into 2 groups: Control group: using DMEM / F12 medium Experimental group: serum-free DMEM / F12 + 2×A solution + 2×B solution Observe the aggregation of cells in both groups. Figure 1 As shown, under the same culture conditions, the control group cells showed severe cell aggregation, while the experimental group cells were basically in a single, dispersed state.

[0019] Example 2: Dispersing F81 cells using solution A / B

[0020] Preparation of concentrated stock solution Preparation of 50×A solution concentrate: Weigh 150g of trypsin powder (corresponding to 3% working concentration) and 12.5g of EDTA. First, mix with 200mL of sterile PBS to form a paste, then add PBS to 1L. Stir at room temperature for 4 hours until completely dissolved. Filter with a 0.22μm filter membrane for sterilization. Dispense into 10mL tubes and store at -20℃ for later use.

[0021] Preparation of 100×B solution concentrate: Weigh 100g P188 and 10g DS, add 800mL of water for injection, stir magnetically until completely dissolved, add water for injection to 1L, filter sterilize with a 0.22μm filter membrane, dispense into 10mL tubes, and store at 4℃ for later use.

[0022] Preparation of dispersion mixture Take 1 mL of 50× A solution concentrate and add it to 49 mL of sterile buffer solution. Mix well to obtain the working solution of A solution. Take 1 mL of 100× B solution concentrate and add it to 99 mL of sterile buffer solution. Mix well to obtain the working solution of B solution.

[0023] Cell experiments Take one bottle of serum-cultured adherent F81 cells. The original culture medium was 10% FBS 1640. After trypsin digestion, add 1×10⁻⁶ cells. 6 Inoculated at a density of / ml in serum-free 1640 medium, and divided into 2 groups: Control group: using 1640 culture medium Experimental group: using serum-free 1640 + 2×A solution + 2×B solution Observe the aggregation of cells in both groups. Figure 2 As shown, under the same culture conditions, the control group cells showed severe cell aggregation, while the experimental group cells were basically in a single, dispersed state.

[0024] Stability test The 50×A solution concentrate prepared in Example 1 was stored at -20°C for 12 months, and the 100×B solution concentrate was stored at 4°C for 12 months. Dispersion mixtures were prepared according to the above method and compared with freshly prepared dispersion mixtures. When applied to CRFK cell domestication, the results showed that there was no significant difference in cell dispersion effect and cell activity between the dispersion mixture prepared from the concentrate stored for 12 months and the freshly prepared dispersion mixture, proving that the concentrated stock solution system of the present invention has good long-term stability.

[0025] Experiments with different dilution ratios Based on Example 1, the mixing ratio of working solutions A and B was adjusted (1:1, 1:2, 1:3, 1:4, 1:5), and the effect on the dispersion of CRFK cell aggregates was observed. The results showed that the dispersion effect was best and the cell activity was highest at a ratio of 1:2, proving that the preferred mixing ratio of the present invention has technical advantages.

[0026] Explanation of innovation and practicality The core innovation of this invention lies in preparing a high-concentration stock solution system from a dispersion mixture, solving the problems of existing technologies where dispersions need to be freshly prepared each time, have large batch-to-batch variations, and have short shelf lives. By optimizing the concentration ratio and storage conditions, solutions A and B can be stored separately for extended periods, requiring only simple dilution and mixing before use, greatly improving experimental efficiency and the consistency of results.

[0027] Furthermore, the present invention has verified the stability and effectiveness of the concentrated solution system through a large number of experiments, confirming that the activity of each component remains stable after long-term storage, and the dispersion effect is not significantly different from that of the freshly prepared solution, indicating good prospects for industrial application.

[0028] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. A dispersion mixture that can reduce cell aggregation during domestication, characterized in that: The dispersion mixture is prepared by mixing working solution A and working solution B in a volume ratio of 1:1 to 1:5, wherein: The working solution of solution A is obtained by diluting 50× solution A concentrate at a ratio of 1:

50. The 50× solution A concentrate contains 100-250 g / L trypsin and 12.5 g / L EDTA. The working solution B is obtained by diluting 100×B solution concentrate at a ratio of 1:

100. The 100×B solution concentrate contains 100g / LP188 and 10g / LDS.

2. The dispersion mixture according to claim 1, characterized in that: The concentration of trypsin in the 50×A solution concentrate is 150 g / L.

3. The dispersion mixture according to claim 1 or 2, characterized in that: The mixing ratio of working solution A to working solution B is 1:

2.

4. A method for preparing the dispersion mixture as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) Preparation of 50×A solution concentrate: Weigh 100-250g of pancreatic enzyme powder and 12.5g of EDTA, first mix with a small amount of sterile PBS or D-Hanks solution to form a paste, then add solvent to 1L, stir at room temperature for 4 hours or overnight at 4℃ to fully dissolve, filter with a 0.22μm filter membrane for sterilization, dispense and store at -20℃; (2) Prepare 100×B solution concentrate: Weigh 100g P188 and 10g DS, add about 800mL of water for injection or ultrapure water, stir magnetically until completely transparent, add water to 1L, filter with a 0.22μm filter membrane for sterilization, dispense and store at 4℃. (3) Preparation of dispersion mixture: Before use, dilute 50×A solution concentrate at a ratio of 1:50 and 100×B solution concentrate at a ratio of 1:

100. Then mix the diluted A solution and B solution at a volume ratio of 1:1 to 1:5 to obtain the dispersion mixture.

5. The preparation method according to claim 4, characterized in that: The solvent used for dilution in step (3) is a sterile buffer solution, which is selected from PBS, D-Hanks solution or cell culture medium.

6. The application of the dispersion mixture as described in any one of claims 1-3 in reducing cell aggregation during cell domestication, characterized in that: The cell domestication process is the domestication process from adherent culture to suspension culture, or the domestication process of switching between different culture media / culture conditions.

7. The application according to claim 6, characterized in that: The cells are mammalian cells, preferably CRFK cells, CHO cells, or F81 cells.

Images