Cell dilution buffer solution and application thereof

Abstract

According to the cell dilution buffer solution provided by the invention, an anti-adsorption inerting component, namely phosphorylcholine, is added to form an inerting layer similar to a biofilm, so that the adsorption of consumables to cells is delayed and inhibited, and natural high-hydrophilic macromolecular sodium polyglutamate is added to prevent cell agglomeration, so that the cell dilution buffer solution is prepared. The common problems of the existing diluent are solved by cooperating with a multi-component synergistic mechanism of proline and / or hydroxyectoine, polystyrene-based acetal trehalose and the like, the cell dispersity is improved, the cell loss and adsorption are reduced, and the experimental consistency is improved.

Description

Technical Field

[0001] This invention relates to the field of cell biology, and in particular to a cell dilution buffer and its applications. Background Technology

[0002] In cell counting, flow cytometry, imaging analysis, and dilution steps during culture, basal buffers such as PBS and HBSS are commonly used. However, these traditional diluents have the following common drawbacks: 1) Cells are prone to aggregation and clumping. Conventional PBS does not have the ability to stably disperse cells, especially in high-density cell suspensions, where cells are easily aggregated due to electrostatic shielding, van der Waals forces, or shear stress, affecting counting accuracy and imaging quality; 2) Cells easily adsorb onto consumable surfaces. Cells are easily adsorbed onto the surfaces of consumables such as centrifuge tubes, counting plates, and chip channels, leading to reduced recovery rates, increased detection bias, and even differences between different batches of consumables; 3) They lack adequate cell protection capabilities. Digested cells or cells processed over long periods are sensitive to osmotic pressure and oxidative stress, which conventional PBS cannot protect against; 4) Existing solutions have significant limitations. While adding EDTA can reduce aggregation, it can affect some metal ion-sensitive experiments. Adding BSA or FBS can reduce adsorption, but their sources are complex, they are not conducive to standardization, and they are costly.

[0003] Most commercially available buffer solutions currently on the market only address a single problem (either reducing aggregation or reducing adsorption), lacking a comprehensive diluent that combines anti-agglomeration, anti-adsorption, and stability protection.

[0004] Therefore, there is an urgent need for an innovative cell dilution buffer with multiple functions to improve the reliability and reproducibility of cell analysis and preparation processes. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention provides a trypan blue dye composition and its application.

[0006] The first aspect of the present invention provides a cell dilution buffer, comprising:

[0007] Cell stabilizing and protective components with a weight-to-volume ratio (w / v) of 0.2%-0.8%;

[0008] Anti-agglomeration and dispersion component with a mass-to-volume ratio (w / v) of 0.005%-0.02%;

[0009] An anti-adsorption inert component with a mass-to-volume ratio (w / v) of 0.005%-0.02%;

[0010] 10mM-200mM buffer conditioning components;

[0011] The anti-adsorption inertizing component is phosphorylcholine, which is used to form a biofilm-like inert layer to improve cell recovery rate.

[0012] Preferably, the anti-agglomeration and dispersing component is a natural, highly hydrophilic macromolecule. More preferably, the anti-agglomeration and dispersing component is sodium polyglutamate. Preferably, the molecular weight of the sodium polyglutamate is between 100 and 300 kDa.

[0013] Preferably, the cell stabilizing and protective component includes proline and / or hydroxytetrahydropyrimidine.

[0014] Preferably, the buffer conditioning component is one or more of PBS buffer, HEPES buffer, and MOPS buffer.

[0015] Preferably, the cell dilution buffer further includes an auxiliary stabilizing component with a weight-to-volume ratio (w / v) of 0.005%-0.02%, wherein the auxiliary stabilizing component is polystyrene acetal trehalose.

[0016] A second aspect of the present invention provides a cell dilution method, comprising:

[0017] Add the cell dilution buffer as described in the first aspect of the present invention to the cells to obtain a diluted cell solution.

[0018] A third aspect of the present invention provides a cell dilution buffer kit, comprising the cell dilution buffer as described in the first aspect of the present invention and the cell dilution buffer method as described in the second aspect of the present invention.

[0019] The fourth aspect of the present invention provides the application of the cell dilution buffer as provided in the first aspect of the present invention, the cell dilution method as described in the second aspect of the present invention, or the cell dilution buffer kit as described in the third aspect of the present invention in cell dilution.

[0020] Compared with the prior art, the advantages of the present invention are:

[0021] The cell dilution buffer provided by this invention contains an anti-adsorption inertizing component, phosphorycholine, which forms a biomembrane-like inert layer, delaying the adsorption of cells by consumables and thus improving cell recovery rate. The anti-agglomeration and dispersing component, sodium polyglutamate, is a natural, highly hydrophilic macromolecule. Unlike the traditional EDTA chelation pathway for metal ions, sodium polyglutamate reliably prevents cell aggregation through "flexible steric hindrance." The cell stabilizing and protecting components, proline and / or hydroxytetrahydropyrimidine, provide osmotic protection and stress-resistant membrane stability. The auxiliary stabilizing component, polystyrene acetal trehalose, possesses multiple functions including slow-release weak osmotic protection, antioxidant properties, and mild surface regulation. It can synergistically work with the cell stabilizing and protecting components to further enhance the cell dilution buffer's osmotic protection and stress-resistant membrane stability. This invention addresses common problems of existing diluents through a multi-component synergistic mechanism, improving cell dispersibility, reducing cell loss and adsorption, and enhancing experimental consistency. Attached Figure Description

[0022] Figure 1 These are microscopic imaging results of Jurkat cells diluted with different diluents, as provided by this invention. Figure 1 A is a cell microscopic image after dilution in Comparative Example 1, showing a large number of aggregated structures of 2-5 cells with blurred edges and poor dispersion. Figure 1 B is a microscopic image of the cells after dilution in Comparative Example 2, showing a slight reduction in aggregation, but still obvious two-cell and three-cell aggregations are present. Figure 1 C is a cell microscopic image after dilution in Example 2. The cells are highly dispersed with almost no visible aggregates and clear boundaries.

[0023] Figure 2 The bar chart shows the cell aggregation rate of Jurkat cells provided by the present invention after being diluted with different diluents. As can be seen from the figure, the cell aggregation rate of Comparative Example 1 is the highest, followed by Comparative Example 2, while Examples 1-3 show better anti-agglomeration properties than Comparative Examples 1 and 2.

[0024] Figure 3 The bar chart shows the cell adsorption rate of the Urkat cells provided by this invention after being diluted with different diluents. As can be seen from the figure, the cell adsorption rate of Comparative Example 1 is the highest, followed by Comparative Example 3, while Examples 1-3 show better anti-adsorption properties than Comparative Example 1 and Comparative Example 2. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0026] A cell dilution buffer comprising:

[0027] Cell stabilizing and protective components with a weight-to-volume ratio (w / v) of 0.2%-0.8%;

[0028] Anti-agglomeration and dispersion component with a mass-to-volume ratio (w / v) of 0.005%-0.02%;

[0029] An anti-adsorption inert component with a mass-to-volume ratio (w / v) of 0.005%-0.02%;

[0030] 10mM-200mM buffer conditioning components;

[0031] The anti-adsorption inertizing component is phosphorylcholine, which is used to form a biofilm-like inert layer to improve cell recovery rate.

[0032] In some embodiments, the cell stabilizing and protective component includes proline and / or hydroxytetrahydropyrimidine.

[0033] In some embodiments, the anti-agglomeration and dispersing component is a natural, highly hydrophilic macromolecule. In some preferred embodiments, the anti-agglomeration and dispersing component is sodium polyglutamate. In some preferred embodiments, the molecular weight of the sodium polyglutamate is between 100 and 300 kDa.

[0034] In some embodiments, the buffer conditioning component is one or more of PBS buffer, HEPES buffer, and MOPS buffer.

[0035] In some embodiments, the cell dilution buffer further includes an auxiliary stabilizing component, which is polystyrene acetal trehalose.

[0036] Example

[0037]

[0038] Application Example 1: Anti-coagulation Performance Evaluation Experiment

[0039] I. Experimental Objective

[0040] The anti-agglomeration ability of the buffer solution of the present invention against suspended cells was verified, with cell aggregation rate (%) as the main indicator.

[0041] II. Experimental Methods

[0042] 1. Culture Jurkat cells to the logarithmic growth phase to obtain Jurkat cell solution.

[0043] 2. Take Jurkat cell solution, centrifuge, and resuspend in Examples 1, 2, and 3, and Comparative Examples 1 and 2, respectively. Resuspension concentration: 1 × 10⁻⁶ 6 cells / mL.

[0044] 3. Gently mix repeatedly in a 50mL centrifuge tube to simulate the counting / dispensing process.

[0045] 4. Samples were taken at 0 min, 10 min, 30 min, and 60 min, and the following were measured using a cell counter: 1) cell aggregation rate (two-cell, three-cell, and multi-cell clusters); 2) automatic cell count (CV).

[0046] III. Experimental Results

[0047] 1. Aggregation rate

[0048]

[0049] 2. Automatic CV% counting

[0050]

[0051] IV. Results Analysis

[0052] 1. Combination Figure 2 It can be seen that the cell aggregation rate of conventional PBS dilution (see Comparative Example 1) is between 18% and 41%, and the cell aggregation rate becomes more severe over time. After adding EDTA to conventional PBS dilution, the cell aggregation rate is between 12% and 25%, and cell clumping is alleviated to some extent. In contrast, the polyglutamate sodium (PGA) in Examples 1-3 significantly inhibits cell approach and bridging through flexible steric hindrance, maintaining a significantly low aggregation level throughout the entire cycle from 0 to 60 minutes, with a cell aggregation rate between 6% and 13%. This indicates that the cell dilution buffer provided by the present invention has strong anti-agglomeration properties and can effectively prevent cell clumping.

[0053] 2. After dilution with conventional PBS, the cells exhibited high cell clumping, leading to counting and signal discrimination errors, resulting in a high auto-counting coefficient of variation (CV%) of 12.8% (see Comparative Example 1). Adding EDTA to the conventional PBS reduced the CV% to 8.5%, indicating that the addition of EDTA alleviated cell clumping to some extent. Furthermore, the CV% of auto-counting in Examples 1-3 was only between 4.1% and 5.2%, further demonstrating that the cell dilution buffer provided by this invention possesses strong anti-agglomeration properties and can effectively prevent cell clumping.

[0054] 3. The cell dilution buffer provided by this invention has clear advantages compared to existing solutions. EDTA only provides a weak anti-agglomeration effect through metal chelation and cannot solve shear-induced aggregation. PBS has no anti-agglomeration function at all. The sodium polyglutamate in the cell dilution buffer provided by this invention can provide steric hindrance and has a highly hydrophilic layer structure, which is significantly superior to traditional cell dilution solutions.

[0055] 4. Synergistic effect of multiple components to produce stable anti-agglomeration effect. The proline / hydroxytetrahydropyrimidine (osmotic protection) and PGA (anti-agglomeration) in the cell dilution buffer provided by this invention work synergistically to keep the cells in a stable dispersed state during the operation (mixing, counting, dispensing).

[0056] Application Example 2: Anti-adsorption performance evaluation experiment

[0057] I. Experimental Objective

[0058] The effectiveness of the buffer solution of this invention in reducing cell adsorption on the surface of consumables was verified.

[0059] II. Experimental Methods

[0060] 1. Culture HeLa cells to the logarithmic growth phase to obtain HeLa cell solution.

[0061] 2. HeLa cell solutions were centrifuged and resuspended in Examples 1, 2, 3, and Comparative Example 1, respectively. Comparative Example 3 was added as a control (comparative Example 1 was supplemented with bovine serum albumin to a final concentration of 1%). Resuspension concentration: 1 × 10⁻⁶ 6 cells / mL.

[0062] 2. Add 500 μL of cell suspension to a 1.5 mL centrifuge tube and gently invert to mix.

[0063] 3. After standing for 10 minutes, collect the supernatant and determine the number of remaining cells;

[0064] 4. Calculate the adsorption rate:

[0065]

[0066] III. Experimental Results

[0067]

[0068] IV. Results Analysis

[0069] 1. This invention significantly reduces cell adsorption, with superior performance compared to existing technologies. Comparative Example 1 (PBS) showed a high adsorption rate of 22.5%, consistent with the typical phenomenon described in the literature that "high-salt plasma buffers cannot effectively prevent non-specific adsorption between cells and polymer consumables." Under the same conditions, the adsorption rates of Examples 1-3 of this invention were all controlled at 7.8-9.5%, with an adsorption reduction of 57-65%. This indicates that the core components of this invention (such as sodium polyglutamate and phosphorylcholine-based surface inertizers) can effectively form a highly hydrophilic "hydration layer" on the surface of the consumables, significantly reducing van der Waals forces and hydrophobic adsorption between cells and the plastic surface.

[0070] 2. The limited improvement shown in Comparative Example 3 (1% BSA) further validates the advantages of this invention. The adsorption rate of Comparative Example 3, with the addition of 1% BSA, decreased to 14.2%, which is indeed an improvement over PBS, but still approximately 1.5 times higher than the average of the examples in this invention. This indicates that protein-based inerting methods suffer from insufficient stability and consistency (the protein adsorption layer is prone to rearrangement or desorption). The cell dilution buffer provided by this invention achieves a lower adsorption rate without the need for protein, demonstrating that its mechanism does not rely on "protein coverage," but rather on the synergistic effect of a hydration layer, electrostatic repulsion, and steric hindrance to form a stable anti-adsorption interface.

[0071] 3. Example 2 had the lowest adsorption rate (7.8%), consistent with its component combination: Sodium polyglutamate (PGA) forms a strong hydration layer with multiple negatively charged segments; phosphorylcholine (PC) can mimic the zwitterionic structure on the cell membrane surface, exhibiting the strongest resistance to protein and cell adsorption; proline / hydroxytetrahydropyrimidine provides permeation protection and anti-stress membrane stability, maintaining cell structural stability.

[0072] The three elements work synergistically to make the consumable wall surface exhibit an "extremely low adsorption energy state".

[0073] Application Example 3: Verification of Cell State and Biocompatibility

[0074] I. Experimental Objective

[0075] The biocompatibility of the buffer solution of this invention and its effect on cell state were verified. To further improve cell stability in the cell dilution buffer, a co-stabilizing component, polystyrene acetal trehalose, was added to the buffer solution based on Example 2, as follows:

[0076]

[0077] II. Experimental Methods

[0078] 1. CHO-K1 and HEK293T cells were cultured to the logarithmic growth phase, respectively.

[0079] 2. Take the cell solution, centrifuge it, and resuspend it in Example 2, Examples 4-6 and Comparative Example 1, respectively.

[0080] 3. Let stand at room temperature, and take samples every 3 hours.

[0081] 4. Use Trypan Blue to assess survival rate.

[0082] III. Experimental Results

[0083] 1. Effect of different dilutions on cell viability (%)

[0084]

[0085] IV. Results Analysis

[0086] 1. After dilution with Comparative Example 1 (PBS), the viability of CHO-K1 cells was only 80.6% and 73.6% at 3 hours and 6 hours, respectively, while that of HEK293T cells was only 82.9% and 76.5%, exhibiting typical short-term instability. However, after dilution with Example 2, the viability of CHO-K1 cells increased to 89.8% and 85.2% at 3 hours and 6 hours, respectively, while that of HEK293T cells increased to 91.8% and 86.5%, respectively. This indicates that, compared to Comparative Example 1, dilution with Example 2 can improve the short-term stability of cells to some extent.

[0087] 2. In Examples 4-6, the addition of polystyrene-based trehalose further enhanced cell viability, especially after a 6-hour interval. For example, in Example 5, the viability in CHO-K1 reached 92.4%, an increase of approximately 18.8% compared to Comparative Example 1, and in HEK293T, it reached 94.1%, an increase of approximately 22.9% compared to Comparative Example 1. This indicates that such auxiliary stabilizers can effectively enhance the stability of the microenvironment surrounding the cell membrane, reducing membrane damage and viability decline caused by osmotic pressure fluctuations during static incubation.

[0088] 3. Examples 4, 5, and 6 show a clear dose-dependent relationship. Within a certain range, cell viability is positively correlated with the concentration of polystyrene acetal trehalose (see Examples 4 and 5). However, when the concentration of polystyrene acetal trehalose is further increased, the cell viability does not increase but rather decreases slightly, which may be related to the increase in local solution viscosity and decrease in diffusion efficiency caused by excessive macromolecules.

[0089] 4. In summary, the proline, sodium polyglutamate, phosphorylcholine, and polystyrene acetal trehalose components in the cell dilution buffer provided by this invention form a multiple stabilizing mechanism. Through the combined effects of osmotic protection, electrostatic repulsion, hydration layer stabilization, and a weak polymer barrier effect, cells maintain a high viability rate for extended periods under static conditions. The differences between the various examples and the comparative examples are statistically significant (p<0.05), demonstrating that the dilution buffer of this invention possesses excellent biocompatibility and cell state maintenance capabilities.

[0090] Application Example 4: Compatibility Verification for Cell Counters

[0091] I. Experimental Objective

[0092] Verify the compatibility of the buffer solution of this invention with cell counters.

[0093] II. Experimental Methods

[0094] 1. Three different brand counters were used: CountLeader FL 100, LUNA-FX7, and Bio-Rad TC20.

[0095] 2. Culture CHO-K1 cells to the logarithmic growth phase to obtain CHO-K1 cell solution.

[0096] 3. Take CHO-K1 cell solution, centrifuge, and resuspend in Example 5 and Comparative Example 1 respectively.

[0097] 4. Measure the concentration deviation (compared with actual manual counting) and repeatability (CV%) of Example 5 and Comparative Example 1 respectively.

[0098] III. Experimental Results

[0099]

[0100] IV. Results Analysis

[0101] The experimental results showed that the average concentration deviation of Comparative Example 1 was 12.3%, and the repeatability CV% was 9.8%. This may be because PBS is prone to aggregation, local sedimentation, and adsorption loss during cell counting, leading to either over- or under-counting and poor repeatability. In contrast, the concentration deviation of Example 5 was significantly reduced to 4.6%, and the repeatability CV% was reduced to 3.9%, both of which showed statistically significant advantages compared to the Comparative Example (p<0.05).

[0102] The aforementioned improvements are consistent with the mechanism of action of the diluent of this invention. This invention constructs a highly hydrophilic hydration layer using sodium polyglutamate and phosphorycholine, effectively reducing non-specific adhesion between cells and between cells and consumables, thereby ensuring uniform cell dispersion within the counting chamber. Simultaneously, proline and polystyrene acetal trehalose protect and stabilize the cell membrane structure, resulting in more intact cell morphology after settling, reducing interference from debris and dead cells, and further improving counting accuracy and repeatability.

[0103] It is worth noting that the fluorescence background value of Example 5 is 178, which is almost the same as that of Comparative Example 1 (180), indicating that the diluent of the present invention will not affect the fluorescence path of the counter, will not generate additional background signals or suppress dye luminescence, and shows good instrument compatibility.

[0104] In summary, the dilution buffer of this invention can stably achieve lower counting bias and repeatability variation on different brands of cell counters (CountLeader FL100, LUNA-FX7, Bio-Rad TC20), while maintaining full compatibility with the original detection system of the equipment, and is suitable for various mainstream counting platforms.

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A cell dilution buffer, characterized in that, include: Cell stabilizing and protective components with a weight-to-volume ratio (w / v) of 0.2%-0.8%; Anti-agglomeration and dispersion component with a mass-to-volume ratio (w / v) of 0.005%-0.02%; An anti-adsorption inert component with a mass-to-volume ratio (w / v) of 0.005%-0.02%; 10mM-200mM buffer conditioning components; The anti-adsorption inertizing component is phosphorylcholine, which is used to form a biofilm-like inert layer to improve cell recovery rate.

2. The cell dilution buffer as described in claim 1, characterized in that, The anti-coagulation and dispersion component is a natural, highly hydrophilic macromolecule.

3. The cell dilution buffer as described in claim 2, characterized in that, The anti-coagulation and dispersing component is sodium polyglutamate.

4. The cell dilution buffer as described in claim 3, characterized in that, The molecular weight of the polyglutamate sodium is between 100 and 300 kDa.

5. The cell dilution buffer as described in claim 1, characterized in that, The cell stabilizing and protective components include proline and / or hydroxytetrahydropyrimidine.

6. The cell dilution buffer according to claim 1, characterized in that, The buffer conditioning component is one or more of PBS buffer, HEPES buffer, and MOPS buffer.

7. The cell dilution buffer according to claim 1, characterized in that, The cell dilution buffer also includes an auxiliary stabilizing component with a weight-to-volume ratio (w / v) of 0.005%-0.02%, wherein the auxiliary stabilizing component is polystyrene acetal trehalose.

8. A method for diluting cells, characterized in that, include: Adding the cell dilution buffer as described in any one of claims 1 to 7 to the cells yields a diluted cell solution.

9. A cell dilution buffer kit, characterized in that, Includes the cell dilution buffer as described in any one of claims 1 to 7 and the cell dilution method as described in claim 8.

10. The application of the cell dilution buffer as described in claim 1, the cell dilution method as described in claim 8, or the cell dilution buffer kit as described in claim 9 in cell dilution.

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