A low-damage cell solidification and permeation integrated method

Abstract

The application discloses a low-damage cell solidification and permeation integrated method, and relates to the technical field of cell biology and analytical detection. The method comprises the following steps: preparing a single-cell suspension of cells to be detected; constructing a solidification-permeation gradient system, wherein the system comprises at least three levels of treatment solutions with increasing alcohol concentration, and each level of the treatment solution contains a cross-linking agent and an osmotic pressure regulator; sequentially passing the cell suspension through the solidification-permeation gradient system for gradient treatment, gradually improving the cell membrane permeability with alcohol solvents, and synchronously entering the cell interior with the cross-linking agent to in-situ low-density cross-linking anchoring of the cell skeleton and nuclear protein, so as to realize the kinetics synchronization of cell solidification and permeation; and after the gradient treatment is completed, the cells are balanced and solidified at low temperature for preservation. The method solves the problems of cell morphology damage, DNA loss, high fragment rate and easy clumping in the cell fixation and permeation process in the prior art, and further improves the cell sample preparation quality and the accuracy of detection results.

Description

Technical Field

[0001] This invention relates to the field of cell biology and analytical detection technology, specifically to a low-damage integrated method for cell solidification and permeation. Background Technology

[0002] Modern cell detection techniques such as flow cytometry, immunofluorescence imaging, and single-cell omics analysis all require cell fixation and permeabilization: fixation stabilizes cell structure and intracellular components, preventing degradation or loss during subsequent operations; permeabilization increases cell membrane permeability, allowing large molecular reagents such as dyes and antibodies to enter the cell and bind to targets. The quality of sample preparation directly determines the accuracy and reliability of the final detection results.

[0003] Current mainstream methods mainly include ethanol fixation and aldehyde fixation. While ethanol fixation offers good permeation, the instantaneous impact of high-concentration ethanol causes rapid cell dehydration and shrinkage, disrupting the cytoskeleton and easily leading to DNA loss from the cell nucleus. This results in broadened G0 / G1 peaks or the appearance of numerous fragments in cell cycle assays. Aldehyde fixation, while better preserving cell morphology, can cause excessive cross-linking that blocks antigenic epitopes. Furthermore, aldehyde fixation alone cannot effectively permeate the cell membrane, usually requiring subsequent use of detergents, which increases the procedural steps and the risk of cell damage.

[0004] While some existing technologies attempt to combine fixation and permeabilization, they often overlook the osmotic pressure balance and oxidative stress damage of cells during the processing, resulting in cells that are prone to clumping and have a high fragmentation rate, which seriously affects the data quality of flow cytometry.

[0005] Therefore, developing an integrated method that can achieve both efficient curing and permeation while minimizing cell damage is a pressing technical problem that needs to be solved. Summary of the Invention

[0006] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a low-damage integrated method for cell fixation and permeation, which solves problems such as cell morphology damage, DNA loss, high fragmentation rate, and easy agglomeration in the cell fixation and permeation process of existing technologies, and further improves the quality of cell sample preparation and the accuracy of detection results.

[0007] (II) Technical Solution To achieve the above objectives, the present invention provides the following technical solution: a low-damage integrated method for cell solidification and permeation, comprising the following steps: Step S1: Prepare a single-cell suspension of the cells to be tested; Step S2: Construct a curing-permeation gradient system, wherein the system contains at least three stages of treatment solutions with increasing alcohol concentrations, and each stage of treatment solution contains a crosslinking agent and an osmotic pressure regulator; Step S3: The cell suspension is sequentially passed through the solidification-permeability gradient system for gradient treatment. As the alcohol solvent gradually increases the cell membrane permeability, the cross-linking agent enters the cell interior simultaneously to perform in-situ low-density cross-linking and anchoring of the cytoskeleton and nucleoproteins, thereby achieving kinetic synchronization of cell solidification and permeability. Step S4: After the gradient treatment is completed, the cells are subjected to low-temperature equilibrium solidification and preservation.

[0008] Furthermore, the curing-permeation gradient system comprises three stages of treatment liquid, with alcohol volume fractions of 20%–35%, 45%–55%, and 65%–75% respectively; the crosslinking agent volume fraction in each stage of treatment liquid is 0.01%–0.2%.

[0009] Furthermore, the crosslinking agent is selected from one or more of glyoxal, paraformaldehyde, glutaraldehyde, genipin, and water-soluble carbodiimide.

[0010] Furthermore, in step S3, the residence time of cells in each gradient treatment solution is 1 to 5 minutes, and the treatment temperature is controlled at 0℃ to 8℃. As the alcohol concentration increases step by step, the cell membrane permeability gradually increases, and the cross-linking agent simultaneously penetrates into the cell to cross-link the chromatin, thus matching the progress of the solidification and permeation processes.

[0011] Furthermore, the treatment solution also includes a buffer system, wherein the osmotic pressure regulator is selected from one or more of trehalose, sucrose, and mannitol, with a concentration of 50 mM to 200 mM; the buffer system contains 10 to 30 mM HEPES, 100 to 200 mM NaCl, 0.5 to 5 mM MgCl2, and 0.5 to 5 mM N-acetylcysteine, and the pH of the treatment solution is 7.0 to 7.6.

[0012] Furthermore, after step S3 and before step S4, an aldehyde quenching step is included: treating the cells with a quenching solution containing a reducing agent or an amino compound for 1 to 3 minutes to neutralize residual aldehyde groups and eliminate autofluorescence background; the alcohol concentration of the quenching solution is consistent with the alcohol concentration of the last-stage gradient treatment solution in step S3.

[0013] Furthermore, the reducing agent in the quenching solution is sodium borohydride, with a final mass-volume fraction of 0.05% to 0.1%; the amino compound is glycine, with a final concentration of 50 to 100 mM.

[0014] Furthermore, the treatment solution also contains 0.02% to 0.05% saponins as synergistic permeabilizing agents, which work together with alcohols to achieve mild and controllable permeabilization of the cell membrane.

[0015] Furthermore, after low-temperature equilibrium solidification and preservation, anti-agglomeration treatment is also included: ethylenediaminetetraacetic acid (EDTA) at a final concentration of 1–5 mM and bovine serum albumin at a final concentration of 0.2%–1% are added to the cell suspension, mixed well, and then filtered through a 40 μm pore size filter membrane.

[0016] Furthermore, this method has been applied to cell sample preparation in flow cytometry cell cycle analysis, cell immunofluorescence staining, and single-cell sequencing sample pretreatment.

[0017] (III) Beneficial Effects This invention provides a low-damage, integrated method for cell solidification and permeation. It offers the following advantages: (1) The cross-linking and permeation are promoted in a coordinated manner through the solidification-permeation gradient system. The cross-linking agent gradually penetrates into the cell as the membrane permeability increases. The anchoring timing is precisely matched with the degree of permeation, which effectively avoids the instantaneous impact of high concentration alcohol and structural damage caused by excessive cross-linking, reduces the proportion of cell debris, and preserves the original morphology of the cell and the integrity of nucleic acids and proteins to the greatest extent.

[0018] (2) Each treatment solution is equipped with an osmotic pressure regulator, which, together with the gradient alcohol concentration, maintains a stable transition of the system's osmotic pressure and avoids cell shrinkage or rupture due to sudden changes in osmotic pressure; the matching isotonic quenching scheme further eliminates secondary damage in the quenching step and maintains cell structure stability throughout the process.

[0019] (3) The method is compatible with a variety of cross-linking agents such as glyoxal and paraformaldehyde, and is suitable for various sample types such as adherent cells, suspension cells and primary cells. It can be widely used in multiple technical scenarios such as flow cytometry, immunofluorescence, and single-cell sequencing. Attached Figure Description

[0020] Figure 1 This is a flowchart illustrating the operation of the method of the present invention; Figure 2 This is a bar chart comparing the proportion of cell debris and aggregation rate between the embodiments and comparative examples of the present invention; Figure 3 This is a bar chart comparing the G0 / G1 peak CV values ​​and Sub-G1 ratios of embodiments and comparative examples of the present invention. Detailed Implementation

[0021] 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.

[0022] Example 1: Integrated treatment of HeLa cells using a three-level gradient solidification-permeation process In this embodiment, the human cervical cancer cell line HeLa (adherent cells) was used as the test sample, and a three-level solidification-permeation gradient system was used for integrated processing. The specific steps are as follows: Preparation of single-cell suspension: Take HeLa cells in logarithmic growth phase, discard the culture supernatant, and wash once with PBS; add 0.25% trypsin-EDTA digestion solution and digest at 37℃ for 2-3 minutes; add complete culture medium containing 10% fetal bovine serum to stop digestion, gently pipette to prepare single-cell suspension, filter through a 300-mesh nylon mesh to remove clumps; centrifuge at 4℃ and 400g for 5 minutes, discard the supernatant, and gently tap to disperse the cell pellet.

[0023] Preparation of the solidification-permeability gradient system: Three-stage treatment solutions were prepared in advance, all in isotonic buffer solution (20 mM HEPES, 140 mM NaCl, 1 mM MgCl2, 100 mM trehalose, 1 mM N-acetylcysteine, pH 7.4). The components of each stage are as follows: First stage: 25% (volume fraction) ethanol + 0.1% (volume fraction) glyoxal; Second stage: 50% (volume fraction) ethanol + 0.1% (volume fraction) glyoxal; Grade 3: 70% (volume fraction) ethanol + 0.1% (volume fraction) glyoxal; All treatment solutions were pre-cooled to 4°C.

[0024] Gradient integrated treatment: Maintaining a low-speed vortex, slowly add the first-stage treatment solution to the cell pellet to resuspend the cells, and react at 4°C for 2 minutes; then add the second-stage treatment solution dropwise, adjusting the ethanol concentration to 50%, and react at 4°C for 2 minutes; continue adding the third-stage treatment solution dropwise, adjusting the ethanol concentration to 70%, and react at 4°C for 10 minutes. Throughout the process, the cells gradually permeate as the ethanol concentration increases, and glyoxal simultaneously penetrates into the cells to complete cross-linking.

[0025] Isotonic aldehyde quenching: Prepare a quenching solution with the same concentration as the tertiary alcohol (70% ethanol + 0.1% sodium borohydride + 50mM glycine, pre-cooled), add an equal volume of quenching solution to the cell suspension, mix gently, and react at 4°C for 2 minutes to neutralize residual aldehyde groups.

[0026] Low-temperature equilibration preservation: Place the treated cell suspension in a -20°C freezer for equilibration and solidification preservation for 12 hours.

[0027] Subsequent pretreatment: Remove the cell suspension, centrifuge at 400g for 5 minutes at 4℃, and discard the supernatant; wash once with PBS, resuspend in PBS containing 2mMEDTA + 0.5%BSA, filter through a 40μm filter membrane, and perform PI staining and flow cytometry cell cycle detection according to the standard procedure.

[0028] Example 2: Comparison of the effects of different types of crosslinking agents This embodiment examines the application effects of different types of crosslinking agents in gradient systems and verifies the universality of the method.

[0029] The difference between this embodiment and Example 1 is that the crosslinking agent in the three-stage treatment solution is replaced with 0.1% paraformaldehyde, 0.05% glutaraldehyde, 0.08% genipin, and 0.1% water-soluble carbodiimide, respectively. The remaining steps and parameters are the same as in Example 1.

[0030] The results showed that the above crosslinking agents could achieve simultaneous curing and permeation in the gradient system, with cell debris ratios all below 5% and G0 / G1 peak CV values ​​all below 3.5%, which were significantly better than the traditional stepwise method. Among them, the glyoxal group had the lowest cell damage and the best detection accuracy.

[0031] Example 3: Optimization Verification with Different Gradient Parameters This embodiment examines the impact of the number of gradients and the dwell time at each level on the processing effect.

[0032] Two control groups were set up: Group A used a two-stage gradient (35% ethanol, 70% ethanol, each stage containing 0.1% glyoxal), with residence times of 3 minutes and 12 minutes for each stage, respectively; Group B used a four-stage gradient (20%, 40%, 55%, 70% ethanol, each stage containing 0.1% glyoxal), with residence times of 1.5 minutes, 1.5 minutes, 2 minutes, and 8 minutes for each stage, respectively. The remaining procedures were the same as in Example 1.

[0033] The results showed that the proportion of cell debris in the two-level gradient group was slightly higher than that in the three-level gradient group, while there was no significant difference in effect between the four-level gradient group and the three-level gradient group. Considering both ease of operation and treatment effect, the three-level gradient was the optimal solution. Good simultaneous solidification-permeation effects could be achieved at each stage within the range of 1–5 minutes, with 2–3 minutes balancing efficiency and quality.

[0034] Example 4: Implementation plan for saponin-containing synergistic permeation In this embodiment, saponins were added to the gradient treatment solution as a synergistic permeabilizing agent to verify the mild permeabilizing effect.

[0035] The difference between this embodiment and Example 1 is that 0.03% (mass-volume fraction) of saponin was added to all three treatment solutions, while the remaining steps and parameters were the same as in Example 1.

[0036] The results showed that the synergistic permeation of saponins and alcohols could achieve the same permeation effect at a lower alcohol concentration, further reducing the damage to cells caused by alcohol dehydration. The cell aggregation rate was reduced by about 15% compared with Example 1, making it suitable for primary cell samples that are highly sensitive to alcohols.

[0037] Example 5: Validation of Primary Mouse Spleen Cells This embodiment uses primary mouse spleen cells as the test sample to verify the applicability of the method to primary cells.

[0038] Spleens from C57BL / 6 mice were harvested, ground, filtered, and used to prepare a single-cell suspension. Red blood cells were lysed and collected by centrifugation. The cells were then processed using the three-stage gradient solidification-permeation system described in Example 1, with the glyoxal concentration adjusted to 0.08% at each stage and the residence time at each stage extended to 3 minutes. The remaining procedures were the same as in Example 1.

[0039] The results showed that after treatment with this method, the proportion of cell fragments in primary spleen cells was 4.2%, and the CV value of the G0 / G1 peak was 3.4%, which was far superior to the traditional 70% ethanol method (fragmentation 15.8%, CV value 9.2%), proving that this method has excellent protective effect on sensitive primary cells.

[0040] Comparative Example 1: Traditional 70% Cold Ethanol Direct Fixation Method HeLa cells from the same batch were fixed using the standard laboratory method of 70% cold ethanol: the cell pellet was collected by centrifugation, and pre-cooled 70% ethanol was added dropwise under low-speed vortexing, and the cells were fixed at -20°C for 12 hours; the subsequent washing, staining, and detection steps were completely consistent with those in Example 1.

[0041] Comparative Example 2: Two-step method of glyoxal prefixation + progressive alcohol permeation HeLa cells from the same batch were processed using a two-step method: pre-fixation followed by permeabilization. Pre-fixation: Add isotonic buffer containing 0.1% glyoxal to the cell pellet and react at 4°C for 5 minutes; Gradual permeation: Pre-cooled ethanol was added in steps to bring the system concentration to 25%, 50%, and 70% sequentially, with each step taking 2 minutes, 2 minutes, and 10 minutes respectively. The subsequent quenching, preservation, and testing steps are completely consistent with those in Example 1.

[0042] Effect verification example The samples from Example 1, Comparative Example 1, and Comparative Example 2 were tested in parallel, and the key indicators were compared. The results are as follows: (1) Comparison of cell debris and aggregation rate The fragmentation ratio was determined by FSC-SSC scatter plot using flow cytometry, and the aggregation rate was determined by FSC-A / FSC-W scatter plot. The results are shown in Table 1.

[0043] Table 1 Comparison of cell debris ratio and aggregation rate in each group

[0044] As shown in Table 1, the proportion of cell debris in Example 1 of the present invention is reduced by about 77.2% compared with the traditional ethanol method and by about 50.0% compared with the two-step pre-fixation method; the cell aggregation rate is reduced by about 79.8% compared with the traditional ethanol method and by about 60.0% compared with the two-step pre-fixation method, demonstrating the significant advantages of the simultaneous curing-permeation process in reducing cell damage.

[0045] (2) Comparison of CV values ​​of G0 / G1 peak with Sub-G1 ratio The DNA content histogram was fitted using ModFitLT software, and the CV values ​​of the G0 / G1 peaks and the proportion of Sub-G1 fragments were recorded. The results are shown in Table 2.

[0046] Table 2 Comparison of G0 / G1 peak CV values ​​and Sub-G1 ratio for each group

[0047] As shown in Table 2, the G0 / G1 peak CV value of the method of the present invention is significantly lower than that of the two control methods, indicating better DNA staining uniformity; the Sub-G1 ratio is much lower than that of the control group, indicating better cell DNA integrity and extremely low degradation and damage.

[0048] (3) Comparison of the accuracy of cell cycle detection HeLa cells enriched in the S phase were obtained using the thymine blocking method. The detection deviations of each group were compared with the theoretical reference value. The results are shown in Table 3.

[0049] Table 3 Comparison of Cell Cycle Distribution Detection Results

[0050] As shown in Table 3, the detection results of the method of the present invention are in high agreement with the theoretical values, and the deviation of each phase is within 1%. The accuracy is significantly better than that of the traditional method and the two-step pre-fixation method, and it can more realistically reflect the cell cycle distribution state.

[0051] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A low-damage integrated method for cell solidification and permeation, characterized in that, Includes the following steps: Step S1: Prepare a single-cell suspension of the cells to be tested; Step S2: Construct a curing-permeation gradient system, wherein the system contains at least three stages of treatment solutions with increasing alcohol concentrations, and each stage of treatment solution contains a crosslinking agent and an osmotic pressure regulator; Step S3: The cell suspension is sequentially passed through the solidification-permeability gradient system for gradient treatment. As the alcohol solvent gradually increases the cell membrane permeability, the cross-linking agent enters the cell interior simultaneously to perform in-situ low-density cross-linking and anchoring of the cytoskeleton and nucleoproteins, thereby achieving kinetic synchronization of cell solidification and permeability. Step S4: After the gradient treatment is completed, the cells are subjected to low-temperature equilibrium solidification and preservation.

2. The low-damage cell solidification and permeation integrated method according to claim 1, characterized in that: The curing-permeation gradient system comprises three stages of treatment liquid, with alcohol volume fractions of 20%–35%, 45%–55%, and 65%–75% respectively; the crosslinking agent volume fraction in each stage of treatment liquid is 0.01%–0.2%.

3. The low-damage cell solidification and permeation integrated method according to claim 1, characterized in that: The crosslinking agent is selected from one or more of glyoxal, paraformaldehyde, glutaraldehyde, genipin, and water-soluble carbodiimide.

4. The low-damage cell solidification and permeation integrated method according to claim 1, characterized in that: In step S3, the residence time of cells in each gradient treatment solution is 1 to 5 minutes, and the treatment temperature is controlled between 0°C and 8°C. As the alcohol concentration increases step by step, the cell membrane permeability gradually increases, and the cross-linking agent simultaneously penetrates into the cell to cross-link the chromatin, thus matching the progress of the solidification and permeation processes.

5. The low-damage integrated method for cell solidification and permeation according to claim 1, characterized in that: The treatment solution also includes a buffer system, wherein the osmotic pressure regulator is selected from one or more of trehalose, sucrose, and mannitol, with a concentration of 50 mM to 200 mM; the buffer system contains 10 to 30 mM HEPES, 100 to 200 mM NaCl, 0.5 to 5 mM MgCl2, and 0.5 to 5 mM N-acetylcysteine, and the pH of the treatment solution is 7.0 to 7.

6.

6. The low-damage integrated cell solidification and permeation method according to claim 1, characterized in that: After step S3 and before step S4, an aldehyde quenching step is also included: the cells are treated with a quenching solution containing a reducing agent or an amino compound for 1 to 3 minutes to neutralize residual aldehydes and eliminate autofluorescence background; the alcohol concentration of the quenching solution is consistent with the alcohol concentration of the last-stage gradient treatment solution in step S3.

7. The low-damage cell solidification and permeation integrated method according to claim 1, characterized in that: The reducing agent in the quenching solution is sodium borohydride, with a final mass-volume fraction of 0.05% to 0.1%; the amino compound is glycine, with a final concentration of 50 to 100 mM.

8. The low-damage integrated method for cell solidification and permeation according to claim 1, characterized in that: The treatment solution also contains 0.02% to 0.05% saponins as synergistic permeabilizing agents, which work together with alcohols to achieve mild and controllable permeabilization of the cell membrane.

9. The low-damage integrated method for cell solidification and permeation according to claim 1, characterized in that: After low-temperature equilibrium solidification and preservation, anti-agglomeration treatment is also included: ethylenediaminetetraacetic acid (EDTA) at a final concentration of 1–5 mM and bovine serum albumin at a final concentration of 0.2%–1% are added to the cell suspension, mixed well, and then filtered through a 40 μm pore size filter membrane.

10. The low-damage integrated cell solidification and permeation method according to claim 1, characterized in that: This method is applied to cell sample preparation in flow cytometry cell cycle analysis, cell immunofluorescence staining, and single-cell sequencing sample pretreatment.

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