Cell fixatives, cell fixation methods and their applications

Abstract

This invention relates to the field of cell biology, and particularly to cell fixation solutions, cell fixation methods, and their applications. This invention utilizes low-concentration ethanol (<50%) to treat cells, achieving effective cell fixation. This not only fixes the cell morphology and structure but also increases cell membrane permeability without damaging intracellular nucleic acids. Furthermore, when combined with a cell preservation solution, it ensures stable long-term storage of cells at -20°C. It also does not affect the extraction of intracellular nucleic acids and significantly reduces the impact of one-step nucleic acid detection on the results.

Description

Technical Field

[0001] This invention relates to the field of cell biology, and in particular to cell fixation solutions, cell fixation methods, and their applications. Background Technology

[0002] Nucleic acid testing is a method that uses molecular biology and other techniques to qualitatively and quantitatively analyze the structure, content, and subcellular localization of nucleic acids (DNA or RNA), thereby aiding in clinical disease diagnosis. Commonly used detection techniques include polymerase chain reaction (PCR), isothermal amplification, Western blotting, gene sequencing, and gene chips.

[0003] In clinical testing, drug metabolism gene testing, HPV DNA testing, and tumor gene testing often require whole blood and cell samples, which are often difficult to store stably for long periods. Fixing cell samples allows nucleic acids to remain stable, greatly simplifying sample storage and reducing false negatives in retests. Cell fixation is a technique that uses physical or chemical methods to fix free microorganisms (cells) or enzymes in a defined spatial region, maintaining their activity and allowing for repeated use. From a histological perspective, the purpose of cell fixation is to preserve the inherent morphology and structure of cells as much as possible, maintain cell membrane integrity, prevent autolysis of endogenous lysosomal enzymes on the cell's own tissues and cells, inhibit bacterial and fungal growth, prevent blurred sections due to autolysis after cell death, and prevent excessive cell shrinkage or expansion that results in loss of original morphology. From an immunohistochemical perspective, fixation can cross-link or coagulate intracellular proteins, thereby preserving the cell's antigenicity.

[0004] Current cell fixation techniques are based on protecting antigens, cell membranes, and cell morphology, with few techniques specifically designed to protect intracellular nucleic acids. Commonly used cell fixatives include aldehydes (formaldehyde, glutaraldehyde, glyoxal, and paraformaldehyde), alcohols (methanol), and ketones (acetone). Aldehydes are the most commonly used fixatives, primarily for stabilizing the fine structural details of cells and tissues before optical or electron microscopy examination; however, aldehydes can damage cellular nucleic acids. There is an urgent need for a cell fixation method and cell preservation solution that can ensure the structural stability of cells and preserve intracellular nucleic acids.

[0005] Existing cell fixation techniques only fix proteins within cells, neglecting nucleic acids. Furthermore, after cell fixation, nucleic acids cannot be stably preserved for a long period; the cells become very hard, making it difficult to extract nucleic acids compared to unfixed cells, thus affecting the extraction efficiency. Summary of the Invention

[0006] In view of this, the present invention aims to fix cells and ensure the long-term stable storage of cells and intracellular nucleic acids, as well as to improve the extraction efficiency of nucleic acids from fixed cells. The present invention provides cell fixation solutions, cell fixation methods, and their applications. Experiments have shown that treating cells with low-concentration ethanol (<50%) can achieve effective cell fixation, not only fixing the cell morphology and structure but also increasing cell membrane permeability without damaging intracellular nucleic acids. Furthermore, when combined with a cell preservation solution, it can ensure long-term stable storage of cells at -20℃ without affecting the extraction of intracellular nucleic acids, and also significantly reduces the impact of one-step nucleic acid detection on the detection results.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides the application of ethanol in cell fixation; the volume fraction of the ethanol includes 20% to 50%.

[0009] In some specific embodiments of the present invention, the cells include one or more of Siha cells, HeLa cells, or leukocytes from whole blood.

[0010] The present invention also provides a cell fixation solution, which, by volume fraction, comprises 20-50% ethanol and 50-80% diluent; the diluent comprises cell culture medium and / or buffer solution.

[0011] In some specific embodiments of the present invention, the volume fraction of ethanol includes 27% to 45%.

[0012] In some specific embodiments of the present invention, the cell culture medium includes one or more of MEM, DMEM, BME, IMDM or DMEM / F12.

[0013] In some specific embodiments of the present invention, the cell culture medium further includes fetal bovine serum and / or antibiotics.

[0014] In some specific embodiments of the present invention, the antibiotic includes one or more of penicillin, streptomycin, or gentamicin sulfate.

[0015] In some specific embodiments of the present invention, the volume fraction of the calf serum includes 44.4%; the concentration of the antibiotic includes 100 μg / mL.

[0016] In some specific embodiments of the present invention, the buffer solution includes one or more of PBS buffer, Tris buffer, or physiological saline.

[0017] In some specific embodiments of the present invention, the cell culture medium is selected from at least one of the following: basal culture medium, incomplete or complete culture medium prepared by adding at least one of serum and antibiotics to basal culture medium, and serum-free culture medium prepared by adding at least one of adhesion-promoting substances, growth factors, hormones, binding proteins, transport proteins, and trace elements to basal culture medium.

[0018] The present invention also provides a combined reagent, which, by volume fraction, comprises 10% of the cell fixative and 90% of the cell preservation solution; the cell preservation solution comprises 20% to 50% of a cryoprotectant and 50% to 80% of the diluent in the cell fixative; the cryoprotectant comprises one or more of DMSO, ethylene glycol and glycerol.

[0019] In some specific embodiments of the present invention, the volume ratio of DMSO, ethylene glycol and glycerol in the cryoprotectant is 2:1:1.

[0020] The present invention also provides a cell fixation method, comprising: taking a cell suspension of cells to be fixed, mixing it with the cell fixation solution or the cell fixation solution in the combined reagent, fixing it, and mixing it with the cell preservation solution in the combined reagent to obtain fixed cells.

[0021] In some specific embodiments of the present invention, the temperature of the fixed cells includes 20℃±5℃; the time for fixing the cells includes 0.5~2h; preferably, the time for fixing the cells includes 1h.

[0022] In some specific embodiments of the present invention, the cell density of the cell suspension includes (1~10)×10⁻¹⁰. 7 Cells / mL; preferably, the cell density of the cell suspension includes (1~2) × 10⁻⁶ cells / mL. 7 per mL.

[0023] This invention also provides the application of any of the following in the preservation or extraction of samples containing nucleic acids:

[0024] (I) the cell fixative; and / or

[0025] (II) The aforementioned combined reagents.

[0026] This invention provides the following beneficial effects:

[0027] This invention utilizes low-concentration ethanol (<50%) to treat cells, achieving effective cell fixation. This not only fixes the cell morphology and structure but also increases cell membrane permeability without damaging intracellular nucleic acids. Furthermore, when combined with a cell preservation solution, it ensures long-term stable storage of cells at -20°C. Moreover, it does not affect the extraction of intracellular nucleic acids and greatly reduces the impact of one-step nucleic acid detection on the detection results. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0029] Figure 1 The cell membrane and cell morphology of fresh Siha cells before fixation are shown (400×).

[0030] Figure 2 The cell morphology of the fixed Siha cells is shown (400×).

[0031] Figure 3 The cell morphology (400×) of Siha cells after fixation and preservation for 6 months is shown. Detailed Implementation

[0032] This invention discloses cell fixation solutions, cell fixation methods, and their applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0033] The purpose of this invention is:

[0034] It fixes cells without damaging intracellular nucleic acids and ensures the stability of nucleic acids in cells and the body;

[0035] This ensures that nucleic acids within cells can be easily extracted without affecting nucleic acid detection.

[0036] The present invention provides the application of ethanol in cell fixation to solve at least one of the above-mentioned technical problems.

[0037] Ethanol is a commonly used dehydrating agent in cell fixation, with a common concentration of 95%. It is mainly used for tissue dehydration, but it also causes cell dehydration and shrinkage. Therefore, it is often used in combination with sodium acetate to ensure the normal morphology and structure of cells.

[0038] During the preparation of human papillomavirus cell quality control samples, this invention unexpectedly discovered that treating cells with a low concentration of ethanol (<50%) can effectively fix the cells. This not only fixes the cell morphology and structure but also increases cell membrane permeability without damaging the intracellular nucleic acids. Furthermore, when combined with a cell preservation solution, it can ensure long-term stable storage of cells at -20°C. Moreover, it does not affect the extraction of intracellular nucleic acids and greatly reduces the impact of one-step nucleic acid detection on the detection results.

[0039] In some implementations, the final concentration of ethanol, by volume fraction, is 20-50% when applying cell fixation.

[0040] The present invention also provides a cell preservation solution to solve at least one of the above-mentioned technical problems.

[0041] According to one aspect of the present invention, a cell fixation reagent is provided, comprising, by volume fraction, 20-50% ethanol and 50-80% ethanol dilution.

[0042] The ethanol diluent can be cell culture medium or buffer solution.

[0043] The buffer solution can be selected from, but is not limited to, PBS buffer, Tris buffer, or physiological saline.

[0044] In some embodiments, the cell culture medium may be selected from at least one of the following: basal culture medium, incomplete or complete culture medium prepared by adding at least one of the following additives to the basal culture medium: serum, antibiotics, etc.; or serum-free culture medium prepared by adding at least one of the following additives to the basal culture medium: adhesion promoters, growth factors, hormones, binding proteins, transport proteins, trace elements, etc.; wherein the basal culture medium may be selected from, but is not limited to, one or more mixtures of MEM, DMEM, BME, IMDM, and DMEM / F12. Using cell culture medium as an ethanol dilution to prepare cell fixation solution helps reduce the influence of the external environment on cells and maintain cell morphology.

[0045] In this invention, the additives used for preparing incomplete culture media, complete culture media, and serum-free culture media can be any one or more additives known to those skilled in the art that can be used to prepare incomplete culture media, complete culture media, and serum-free culture media, and are not limited to the few listed above.

[0046] According to another aspect of the present invention, a cell preservation solution is provided, comprising a cell culture medium and a cryoprotectant, wherein the components in the cell culture medium can play a protective role for the cell membrane; at the same time, the combination of buffer and cryoprotectant was attempted, and the buffer played a role in maintaining the cell pH and osmotic pressure, but the optimal protective effect was not achieved.

[0047] The combined action of cell preservation solution and cryoprotectant not only protects fixed cells at low temperatures, but also reduces ice crystal formation, protects the stability of intracellular nucleic acids, prolongs cell viability, and extends the stability of cell structure at low temperatures.

[0048] In some embodiments, when used to preserve fixed cells, the cell culture medium is 50-80% by volume; the cryoprotectant is applied at a final concentration of 20-50% by volume.

[0049] This invention relates to a method for fixing cells and a cell preservation solution;

[0050] This invention is obtained through the following steps:

[0051] (1) After preparing the fixative with anhydrous ethanol using buffer solution, the fixative is pre-cooled under low temperature conditions, where the low temperature conditions can be -20℃ to 4℃.

[0052] (2) Add a pre-cooled fixative to the cell suspension to obtain a cell-containing mixture; wherein the volume fraction of ethanol in the cell-containing mixture is 20-50%;

[0053] (3) Then the mixture containing cells is placed at 20℃±5℃ for 1~2h, during which time the cells are kept in a well mixed state to prevent cell precipitation;

[0054] Mixing the pre-cooled ethanol solution with the cell suspension reduces cell aggregation and helps maintain cell condition.

[0055] After cell fixation, the cells can be directly added to a cell preservation solution containing cell culture medium and cryoprotectant and stored at -20°C for a long period of time.

[0056] Unless otherwise specified, the cell fixation solution, cell fixation method and raw materials and reagents used in the application of the present invention can all be purchased from the market.

[0057] The present invention will be further illustrated below with reference to the embodiments:

[0058] Example 1: Cell fixation

[0059] Cell fixation methods: 20%~50% ethanol cell preservation solution: cell culture medium + cryoprotectant

[0060] Siha cell fixation includes the following steps:

[0061] 1. Reagent preparation

[0062] (1) Preparation of complete culture medium: Fetal bovine serum (Nanjing Aofan, catalog number: 20200304), penicillin (Beyotime, catalog number: ST488-1), streptomycin (Beyotime, catalog number: ST488-2) and gentamicin sulfate (Beyotime, catalog number: ST2398-1) were added to DMEM medium to prepare complete culture medium. In the complete culture medium, the volume percentage of fetal bovine serum was 44.4%, and the final concentrations of penicillin, streptomycin and gentamicin sulfate were all 100 μg / mL.

[0063] (2) Preparation of cryoprotectant: DMSO, ethylene glycol and glycerol are mixed to prepare cryoprotectant, wherein the volume percentage of DMSO in the cryoprotectant is 50%; the final concentration of ethylene glycol and glycerol is 25% each;

[0064] (3) Preparation of cell preservation solution: Take 5.4 mL of the complete culture medium prepared in step (1) and 3.6 mL of the cryoprotectant prepared in step (2) and add it to a 50 mL centrifuge tube and mix well to obtain 10 mL of cell preservation solution. Place the cell preservation solution in a refrigerator at 4°C for pre-cooling.

[0065] (4) Preparation of cell fixation solution: Take 300 μL of anhydrous ethanol and add it to 700 μL of PBS solution (0.01 mol / L, pH 7.4) to obtain a 30% ethanol solution (cell fixation solution).

[0066] 2. Fixation of Siha cells

[0067] (5) After digesting the cultured Siha cells (human cervical squamous cell carcinoma cells) with digestive solution, resuspend them in complete culture medium, count the cells, and adjust the cell density to (1~2)×10⁻⁶. 7 Cells / mL, then add 900 μL of cell fixation solution (30% ethanol solution) to every 100 μL of cell suspension, mix well, and fix the mixture containing cells and ethanol at 20℃±5℃ for 1 h, keeping the cells in a mixed state during the process.

[0068] (6) Add 1 mL of the mixture containing cells and ethanol, which has been placed at 20℃±5℃ for 1 h, to 9 mL of cell preservation solution that has been pre-cooled at 4℃. After mixing, a mixture of ethanol containing Siha cells with a final concentration of about 2.7% is obtained; thus, fixed Siha cells are obtained.

[0069] (7) After immobilization, Siha cells were aliquoted and stored at -20°C;

[0070] Cell membrane and cell morphology of fresh Siha cells before fixation, as shown in the figure. Figure 1 As shown, the cell morphology of fixed Siha cells and Siha cells preserved for 6 months after fixation are respectively as follows. Figure 2 , Figure 3 As shown. By Figures 1-3 It can be seen that after Siha cells are fixed by the fixation method of the present invention, the cell membrane structure is intact and the morphology is full, and there is no significant change in the structure and morphology of the cell membrane after 6 months.

[0071] Example 2

[0072] The preparation steps are the same as in Example 1. The difference is that the complete culture medium in the cell preservation solution is replaced with PBS buffer, specifically: 60% PBS buffer and 40% cryopreservation solution.

[0073] Example 3

[0074] The preparation steps are the same as in Example 1. The difference is that the cell fixative is a 27% ethanol solution.

[0075] Example 4

[0076] The preparation steps are the same as in Example 1. The difference is that the cell fixation solution is a 27% ethanol solution, and the cell preservation solution is a mixture of 60% PBS buffer and 40% cryopreservation solution.

[0077] Example 5

[0078] The preparation steps are the same as in Example 1. The difference is that the cell fixative is a 50% ethanol solution.

[0079] Example 6

[0080] The preparation steps are the same as in Example 1. The difference is that the cell fixation solution is a 50% ethanol solution, and the cell preservation solution is a mixture of 60% PBS buffer and 40% cryopreservation solution.

[0081] Example 7

[0082] The preparation steps are the same as in Example 1. The difference is that the cell preservation solution is 80% complete culture medium (20% fetal bovine serum and 60% DMEM) and 20% cryopreservation solution (10% DMSO, 5% ethylene glycol and 5% glycerol).

[0083] Example 8

[0084] The preparation steps are the same as in Example 1. The difference is that the cell preservation solution is 80% complete culture medium (50% fetal bovine serum and 30% DMEM) and 20% cryopreservation solution (5% DMSO, 5% ethylene glycol and 10% glycerol).

[0085] Example 9

[0086] The preparation steps are the same as in Example 1. The difference is that the cell preservation solution is 50% complete culture medium (20% fetal bovine serum and 30% DMEM) and 50% cryopreservation solution (40% DMSO, 5% ethylene glycol and 5% glycerol).

[0087] Example 10

[0088] The preparation steps are the same as in Example 1. The difference is that the cell preservation solution is 50% complete culture medium (20% fetal bovine serum and 30% DMEM) and 50% cryopreservation solution (5% DMSO, 40% ethylene glycol and 5% glycerol).

[0089] Example 11

[0090] The preparation steps are the same as in Example 1. The difference is that the cell preservation solution is 50% complete culture medium (20% fetal bovine serum and 30% DMEM) and 50% cryopreservation solution (5% DMSO, 5% ethylene glycol and 40% glycerol).

[0091] Comparative Example 1

[0092] Siha cells were fixed using 0.075% or 0.2% formaldehyde, with the remaining steps described in Example 2.

[0093] Comparative Example 2

[0094] Siha cells were fixed with 0.1% or 0.5% glutaraldehyde; the remaining preparation steps are the same as those in Comparative Example 1.

[0095] Comparative Example 3

[0096] The fixation method is the same as in Example 1, except that the final concentration of ethanol in the cell fixation solution is 10%, and the cell preservation solution is 60% PBS buffer and 40% cryopreservation solution.

[0097] Comparative Example 4

[0098] The fixation method is the same as in Example 1, except that the final concentration of ethanol in the cell fixation solution is 70%, and the cell preservation solution is 60% PBS buffer and 40% cryopreservation solution.

[0099] Example of effect

[0100] Experimental subjects: Freshly cultured Siha cells, fixed tumor cells from Examples 1-11 and Comparative Examples 1-4, stored at -20°C and then at 2-8°C, while fresh cells were cultured under standard conditions. Cells stored for 1-7 days, 30 days, and 60 days after fixation, as well as conventionally cultured cells, were selected for experiments.

[0101] Experimental methods:

[0102] (1) Boiling method: Extraction of nucleic acid: Take 200 μL of the mixture containing fixed cells, centrifuge at 130,000 rpm for 1 min, discard the supernatant, add 0.5 mL of physiological saline to resuspend the cells, centrifuge again at 130,000 rpm for 1 min, discard the supernatant; add 50 μL of cell lysis buffer (high-risk human papillomavirus detection kit, purchased from Guangdong Kaipu Biotechnology Co., Ltd.), shake well to resuspend the cells, boil for 10 min; centrifuge at 130,000 rpm for 10 min, and keep the supernatant for later use;

[0103] DNA amplification: Mix 17.5 μL of PCR mixture with 0.5 μL of DNA polymerase (high-risk human papillomavirus detection kit, purchased from Guangdong Kaipu Biotechnology Co., Ltd.); take 2 μL of the nucleic acid extracted in the previous step as template; add the template to the mixture; the reaction conditions are 95℃, 10 min; 45 cycles (95℃, 15 s; 60℃, 60 s, single fluorescence collection; 38℃, 5 s).

[0104] (2) One-step method: Nucleic acid extraction: Take 200 μL of the mixture containing fixed cells, centrifuge at 12000 rpm for 5 min, discard the supernatant and add 50~100 μL of sample release agent to the precipitate, mix with the precipitate by shaking or pipetting, and wait at least 10 min for later use.

[0105] DNA amplification: Mix 36 μL of PCR reaction solution with 4 μL of DNase mixture; add 10 μL of sample; reaction conditions are 50℃, 2 min; 94℃, 5 min; 45 cycles (94℃, 15 s; 57℃, 30 s, single fluorescence sampling; 25℃, 10 s).

[0106] The sample release agent (model S1013) and PCR reaction solution DNase (human papillomavirus (16,18) nucleic acid detection kit) were purchased from Hunan Shengxiang Biotechnology Co., Ltd.

[0107] Experimental results:

[0108] Nucleic acid extraction and detection: The detection results are shown in Table 2; As can be seen from Table 2, with the increase of storage time, the cells preserved by the cell fixation method and cell preservation solution provided by this invention have a better preservation effect on nucleic acids.

[0109] Nucleic acid one-step detection: The detection results are shown in Table 3; As can be seen from Table 3, compared with the comparative example, the cells preserved by the cell fixation method and cell preservation solution provided by this invention are closer to fresh unfixed cells in the one-step amplification.

[0110] Table 1. Composition of cell fixation solution and cell preservation solution in each project

[0111]

[0112]

[0113] Table 2 Nucleic Acid Extraction and Detection

[0114]

[0115] Table 3 One-step nucleic acid detection

[0116]

[0117] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of ethanol in cell fixation; wherein the volume fraction of ethanol is 20% to 50%.

2. A cell fixative, characterized in that, The cell fixative comprises 20-50% ethanol and 50-80% diluent by volume fraction; the diluent comprises cell culture medium and / or buffer.

3. The cell fixative as described in claim 2, characterized in that, The cell culture medium includes one or more of MEM, DMEM, BME, IMDM, or DMEM / F12.

4. The cell fixative as described in claim 2 or 3, characterized in that, The cell culture medium also includes calf serum and / or antibiotics.

5. The cell fixative according to any one of claims 2 to 4, characterized in that, The buffer solution includes one or more of PBS buffer, Tris buffer, or physiological saline.

6. A combination reagent, characterized in that, By volume fraction, the combined reagent comprises 10% of the cell fixative and 90% of the cell preservation solution as described in any one of claims 2 to 5; the cell preservation solution comprises 20% to 50% of a cryoprotectant and 50% to 80% of the diluent in the cell fixative as described in any one of claims 2 to 5; the cryoprotectant comprises one or more of DMSO, ethylene glycol and glycerol.

7. A cell fixation method, characterized in that, include: Take the cell suspension of the cells to be fixed, mix it with the cell fixation solution as described in any one of claims 2 to 5 or the cell fixation solution in the combined reagent as described in claim 6, fix it, and mix it with the cell preservation solution as described in the combined reagent as described in claim 6 to obtain fixed cells.

8. The cell fixation method as described in claim 7, characterized in that, The temperature for fixing the cells is 20℃±5℃; the fixation time is 0.5~2h; preferably, the fixation time is 1h.

9. The cell fixation method as described in claim 7 or 8, characterized in that, The cell density of the cell suspension includes (1~10)×10⁻¹⁰ cells. 7 Cells / mL; preferably, the cell density of the cell suspension includes (1~2) × 10⁻⁶ cells / mL. 7 per mL.

10. Any of the following applications in the preservation of samples containing nucleic acids or in nucleic acid extraction: (I) The cell fixative as described in any one of claims 2 to 5; and / or (II) The combined reagent as described in claim 6.

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