Preparation method and application of cell preservation solution and liquid cell quality control product

Abstract

The present invention relates to a preparation method and application of a cell preservation solution and a liquid cell quality control product, wherein the cell preservation solution comprises chloride, phosphate, sodium ascorbate, alcohol, ethylenediaminetetraacetic acid and water. The cell preservation solution of the present invention can better fix cells, protect cell antigens, and prolong the shelf life of cells for slice making. Among them, alcohol can fix cells and maintain cell morphology; sodium ascorbate, as an antioxidant, can improve the accuracy and stability of staining; ethylenediaminetetraacetic acid can reduce the influence of metal ions on cell nucleic acids. The liquid cell quality control product prepared by the cell preservation solution of the present invention can be directly dripped for slice making, the method is simple and efficient, and the slice will not be quickly air-dried during making, can be conveniently stored and transported, and has a good staining effect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and medicine, and specifically relates to a preparation method and application of a cell preservation solution and a liquid cell quality control product. Background Art

[0002] In situ section staining techniques such as immunohistochemistry and in situ hybridization are widely used, but in actual work, negative and positive control photos must be set up. The positive control photos are generally slices made of human diseased tissue. During the experiment, the operating procedures and reagents that are completely consistent with those of the slices to be tested are used to monitor the reliability of the experiment itself.

[0003] At present, there are three major defects in using slices made from human diseased tissue as positive controls: first, there are individual differences in such human diseased tissue and the source is unstable; second, because the tissue wax blocks are derived from the patient's human tissue, their ownership belongs to the patient. Strictly speaking, there are ethical and legal ownership risks in the pathology department using the patient's wax blocks to make positive control photos, and it is even more impossible to use the patient's wax blocks for commercial development; third, because each slice to be tested requires a positive control for monitoring, the experimental workload and film reading workload of the pathology department are greatly increased.

[0004] At present, the cell wax block method is widely used in the preparation of immunohistochemical quality control products, and the commonly used methods are cell precipitation method and agarose clot paraffin embedding method. However, these two methods firstly require a large amount of cells, and secondly, before the sample is sliced, it is necessary to go through a series of long and tedious sample pre-treatment processes, such as ethanol dehydration, xylene transparency, wax immersion, embedding, slicing, dewaxing, etc., and the subsequent embedding and slicing processes have relatively high technical requirements for equipment and operators, and the concentration of cells during embedding is difficult to control, which easily leads to unsatisfactory image observation results.

[0005] CN112611614A provides a method for preparing a semi-solid cell suspension and its application, which can reduce the experimental amount by half and ensure a complete control of the experiment. However, this method is still too complicated and still requires the cells to be embedded in paraffin first.

[0006] Therefore, the present invention provides a preparation method and application of a cell preservation fluid and a liquid cell quality control product, which simplifies the operation process while ensuring the effect. Summary of the invention

[0007] In view of the deficiencies of the prior art, the object of the present invention is to provide a preparation method and application of a cell preservation fluid and a liquid cell quality control product.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a cell preservation solution, which comprises chloride, phosphate, sodium ascorbate, alcohol, ethylenediaminetetraacetic acid and water.

[0010] The cell preservation solution of the present invention can better fix cells, protect cell antigens, and prolong the shelf life of cells for slice preparation. Among them, alcohol can fix cells and maintain cell morphology; sodium ascorbate as an antioxidant can improve the accuracy and stability of staining; ethylenediaminetetraacetic acid can reduce the influence of metal ions on cell nucleic acids. The liquid cell quality control product prepared by the cell preservation solution of the present invention can be directly dripped for slice preparation, the method is simple and efficient, and the slice preparation will not be quickly air-dried, can be conveniently stored and transported, and has a good staining effect.

[0011] Preferably, the chloride salt comprises sodium chloride and potassium chloride.

[0012] Preferably, the phosphate includes any one of sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate or dipotassium hydrogen phosphate, or a combination of at least two thereof.

[0013] Preferably, the alcohol includes glycerol, ethanol, 1,3-propylene glycol and polyethylene glycol.

[0014] The present invention creatively finds that glycerol, ethanol and 1,3-propylene glycol have a certain synergistic effect in fixing cells and maintaining cell morphology.

[0015] Preferably, the molecular weight of the polyethylene glycol is 2000-6000; for example, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, etc. Other specific points within the above numerical range can be selected and will not be described one by one here.

[0016] Preferably, the cell preservation solution comprises, by mass percentage, 0.1%-1% sodium chloride, 0.01%-0.1% potassium chloride, 0.05%-0.5% phosphate, 0.01%-0.1% sodium ascorbate, 3%-10% glycerol, 3%-10% 1,3-propylene glycol, 10%-20% ethanol, 0.01%-0.1% ethylenediaminetetraacetic acid, 0.05%-0.5% polyethylene glycol and water;

[0017] The mass percentage of the sodium chloride can be selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, etc.; the mass percentage of the potassium chloride can be selected from 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, etc.; the mass percentage of the phosphate The mass percentage of sodium ascorbate can be 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, etc. The mass percentage of glycerol can be 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, etc.; the mass percentage of sodium ascorbate can be 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, etc. 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, etc.; the mass percentage of the 1,3-propylene glycol can be selected from 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, etc.; the mass percentage of the ethanol can be selected from 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%; the mass percentage of the ethylenediaminetetraacetic acid can be selected from 0.01%, 0. 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, etc.; the mass percentage of the polyethylene glycol can be selected as 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, etc., and other specific point values ​​within the above numerical range can be selected, which will not be repeated here one by one.

[0018] When sodium chloride, potassium chloride, phosphate, sodium ascorbate, glycerol, 1,3-propylene glycol, ethanol, polyethylene glycol and ethylenediaminetetraacetic acid are present in the cell preservation solution at the above-mentioned specific concentrations, the cells can be better fixed, the cell antigens can be protected and the shelf life of the cells for slice preparation can be extended.

[0019] Preferably, the cell preservation solution further comprises glacial acetic acid.

[0020] The present invention creatively finds that sodium ascorbate and glacial acetic acid can positively influence each other and exert a better effect in anti-oxidation.

[0021] Preferably, the mass percentage of the glacial acetic acid is 1%-5%; for example, 1%, 2%, 3%, 4%, 5%, etc. Other specific point values ​​within the above numerical range can be selected and will not be described here one by one.

[0022] When the mass percentage of glacial acetic acid is the above concentration, the antioxidant effect of the cell preservation solution is the best.

[0023] Preferably, the cell preservation solution is obtained by physically mixing various preparation raw materials.

[0024] In a second aspect, the present invention provides a liquid cell quality control product, which is prepared by a preparation method comprising the following steps: collecting cells, and mixing the cells with the cell preservation solution described in the first aspect.

[0025] The preparation method of the liquid cell quality control product of the present invention does not include cumbersome steps such as paraffin embedding and sectioning. It only needs to collect cells by centrifugation and then resuspend the cells in a cell preservation solution. The method is simple and efficient. When the experiment requires, it can be directly dripped and prepared, saving time. The liquid cell quality control product can be directly stored and transported, and storage and transportation are convenient. Moreover, after the liquid cell quality control product is dripped onto the glass slide, it will not quickly air dry, resulting in further destruction of the epitopes on the cells, and has a good staining effect.

[0026] Preferably, the density of cells in the cell preservation solution is 10 5 -10 6 / ml; for example 1×10 5 / ml, 2×10 5 / ml, 3×10 5 / ml, 4×10 5 / ml, 5×10 5 / ml, 6×10 5 / ml, 7×10 5 / ml, 8×10 5 / ml, 9×10 5 / ml, 1×10 6 Other specific point values ​​within the above numerical range can be selected and will not be described in detail here.

[0027] In a third aspect, the present invention provides a use of the liquid cell quality control product according to the second aspect in preparing a reference product suitable for in situ detection or analysis methods of tissue cells.

[0028] Preferably, the tissue cell in situ detection or analysis method comprises immunohistochemistry, in situ hybridization or immunocytochemical staining.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The cell preservation solution of the present invention can better fix cells, protect cell antigens, and prolong the storage period of cells for slide preparation. Among them, alcohol can fix cells and maintain cell morphology; sodium ascorbate can improve the accuracy and stability of staining as an antioxidant; ethylenediaminetetraacetic acid can reduce the influence of metal ions on cell nucleic acids. The liquid cell quality control product prepared by the cell preservation solution of the present invention can be conveniently stored and transported, and can be drop-sliced ​​when needed. It will not dry quickly during the preparation, and the staining effect is good.

[0031] The preparation method of the liquid cell quality control product of the present invention does not include cumbersome steps such as paraffin embedding and sectioning. It only needs to collect cells by centrifugation and then resuspend the cells in a cell preservation solution. The method is simple and efficient. When the experiment requires, it can be directly dripped and prepared, saving time. The liquid cell quality control product can be directly stored and transported, and storage and transportation are convenient. Moreover, after the liquid cell quality control product is dripped onto the glass slide, it will not quickly air dry, resulting in further destruction of the epitopes on the cells, and has a good staining effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is a microscopic examination result diagram of Application Example 1 of the present invention;

[0033] Figure 2 is a microscopic examination result diagram of Application Example 2 of the present invention;

[0034] Figure 3 1 is a microscopic examination result diagram of Application Example 3 of the present invention;

[0035] Figure 4 is a microscopic examination result diagram of Application Example 4 of the present invention;

[0036] Figure 5 is a microscopic examination result diagram of Application Example 5 of the present invention;

[0037] Figure 6 is a microscopic examination result diagram of Application Example 6 of the present invention;

[0038] Figure 7 is a microscopic examination result diagram of Application Example 7 of the present invention;

[0039] Figure 8 is a microscopic examination result diagram of comparative application example 1 of the present invention;

[0040] Fig. 9 It is a microscopic examination result diagram of comparative application example 2 of the present invention. DETAILED DESCRIPTION

[0041] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0042] Example 1

[0043] The present embodiment provides a cell preservation solution, which includes 0.55% sodium chloride, 0.05% potassium chloride, 0.2% sodium dihydrogen phosphate, 0.05% sodium ascorbate, 6% glycerol, 6% 1,3-propylene glycol, 15% ethanol, 0.05% ethylenediaminetetraacetic acid, 0.3% polyethylene glycol 4000 and water to 100%.

[0044] Preparation method: Physically mix sodium chloride, potassium chloride, sodium dihydrogen phosphate, sodium ascorbate, glycerol, 1,3-propylene glycol, ethanol, ethylenediaminetetraacetic acid, polyethylene glycol 4000 and water.

[0045] Example 2

[0046] The present embodiment provides a cell preservation solution, which includes 0.18% sodium chloride, 0.01% potassium chloride, 0.5% sodium dihydrogen phosphate, 0.1% sodium ascorbate, 4% glycerol, 3% 1,3-propylene glycol, 20% ethanol, 0.01% ethylenediaminetetraacetic acid, 0.4% polyethylene glycol 2000 and water to 100%.

[0047] The preparation method is as in Example 1.

[0048] Example 3

[0049] The present embodiment provides a cell preservation solution, which includes 0.8% sodium chloride, 0.1% potassium chloride, 0.14% sodium dihydrogen phosphate, 0.01% sodium ascorbate, 9% glycerol, 8% 1,3-propylene glycol, 10% ethanol, 0.1% ethylenediaminetetraacetic acid, 0.05% polyethylene glycol 6000 and water to 100%.

[0050] The preparation method is as in Example 1.

[0051] Example 4

[0052] The present embodiment provides a cell preservation solution, which includes 0.55% sodium chloride, 0.05% potassium chloride, 0.2% sodium dihydrogen phosphate, 0.05% sodium ascorbate, 6% glycerol, 6% 1,3-propylene glycol, 15% ethanol, 0.05% ethylenediaminetetraacetic acid, 0.3% polyethylene glycol 4000, 3% glacial acetic acid and water to 100%.

[0053] The preparation method is as in Example 1.

[0054] Example 5

[0055] This embodiment provides a cell preservation solution, which differs from Embodiment 1 only in that it does not contain glycerol, and the mass percentage of glycerol is proportionally distributed to the mass percentage of 1,3-propylene glycol and ethanol, and other components and contents remain unchanged.

[0056] The preparation method is as in Example 1.

[0057] Example 6

[0058] This embodiment provides a cell preservation solution, which differs from Embodiment 1 only in that 1,3-propylene glycol is not contained, the mass percentage of 1,3-propylene glycol is proportionally distributed to the mass percentage of glycerol and ethanol, and other components and contents remain unchanged.

[0059] The preparation method is as in Example 1.

[0060] Example 7

[0061] This embodiment provides a cell preservation solution, which differs from Embodiment 1 only in that it does not contain ethanol, and the mass percentage of ethanol is proportionally distributed to the mass percentage of glycerol and 1,3-propylene glycol, and other components and contents remain unchanged.

[0062] The preparation method is as in Example 1.

[0063] Comparative Example 1

[0064] This comparative example provides a cell preservation solution, which is purchased from BD Company in the United States and has the trade name of cell preservation solution.

[0065] Application Example 1

[0066] This application example provides a liquid cell quality control product, and the preparation of the liquid cell quality control product includes the following steps:

[0067] (1) Take the adherent Hela cells with positive expression of P16 protein, digest them, centrifuge and take 10 6 cells;

[0068] (2) Resuspend the Hela cells in 1 ml of the cell preservation solution described in Example 1 to prepare a cell suspension, and then transfer the cells to 10 ml of the cell preservation solution described in Example 1.

[0069] Application Example 2-7

[0070] This application example provides six types of liquid cell quality control products, which differ from application example 1 only in that the cell preservation solution described in example 1 is replaced by the cell preservation solution described in examples 2-7 in equal amounts, and other preparation methods remain unchanged.

[0071] Comparative application example 1

[0072] This comparative application example provides a liquid cell quality control product, which differs from Application Example 1 only in that the cell preservation solution described in Example 1 is replaced by the cell preservation solution described in Comparative Example 1 in equal amounts, and other preparation methods remain unchanged.

[0073] Comparative Application Example 2

[0074] This comparative application example provides a cervical cancer tissue wax block with positive P16 protein expression, and the tissue wax block is stored in the tissue wax block sample library of Guangzhou Anping Pharmaceutical Technology Co., Ltd.

[0075] Test Example 1

[0076] 15 μl of the Hela cell suspension described in Application Examples 1-7 and Comparative Application Example 1 was dripped onto a glass slide.

[0077] The tissue wax block described in Comparative Application Example 2 was sliced ​​to a thickness of 3 μm. After scooping and spreading the slices, the slices were baked in an oven at 60° C. for 15 minutes.

[0078] 1. Dewaxing and hydration:

[0079] Before dewaxing, the samples were placed horizontally in a 60°C constant temperature oven and baked for 2 h.

[0080] (1) After soaking in xylene for 10 min, rinse from dewaxing solution I to dewaxing solution III in turn, and soak in dewaxing solution III for 10 min;

[0081] (2) Soak in anhydrous ethanol I, II and 95% ethanol III for 5 min each;

[0082] (3) Rinse with distilled water.

[0083] 2. Perform high temperature and high pressure heat antigen repair:

[0084] Take 1000ml of 1mmol / L EDTA (pH8.0) in a pressure cooker, place the dewaxed and hydrated sample slides on a high-temperature plastic slice rack, and heat them in a pressure cooker until boiling. Start with the jet, and stop heating after 2 minutes. After standing for 5 minutes, move it horizontally to the sink, pour water on the lid to force cooling, until the automatic lock core falls to its original position, remove the pressure limiting valve, open the lid, take out the slides, and rinse the slides with tap water.

[0085] 3. Perform peroxidase blocking:

[0086] (1) Remove excess liquid, immerse in blocking agent (3% hydrogen peroxide) solution, incubate in a wet box at room temperature for 10 minutes, and then rinse with tap water.

[0087] (2) Use an immunohistochemistry pen to draw a hydrophobic circle 2 mm from the edge of the cells and tissues, and wash three times in tap water.

[0088] 4. Add primary antibody:

[0089] (1) Remove excess liquid, add corresponding primary antibody working solution, and incubate in a wet box at room temperature for 30 min;

[0090] (2) Rinse with PBS.

[0091] 5. Add secondary antibody:

[0092] Excess liquid was removed, and one-step secondary antibody (LBP-VBright I universal secondary antibody polymer) was added dropwise, incubated in a wet box at room temperature for 20 minutes, and then rinsed with PBS.

[0093] 6. Add DAB to develop color:

[0094] (1) DAB working solution: DAB chromogen:DAB buffer = 1:20;

[0095] (2) Remove excess liquid from the slide;

[0096] (3) Add DAB working solution to the sample to completely cover it. After incubation for 5 min, stop the color development with distilled water.

[0097] 7. Counterstain with hematoxylin for 10 seconds and blue with bluing solution for 10 seconds.

[0098] 8. Soak in 95% ethanol, anhydrous ethanol I and II for 1 min each for dehydration.

[0099] 9. Air-dry, seal and examine under a microscope.

[0100] The microscopic examination results of application examples 1-7 are as follows Figure 1-7 , compared with the microscopic examination results of application example 1-2, Figure 8-9. It can be concluded that: by comparing the results of Application Example 2, it can be seen that cervical cancer tissue expresses P16, and the immunohistochemical staining effect is obvious; the results of Application Examples 1-3 show that Hela cells express P16, and the immunohistochemical staining effect is obvious. It can be concluded that the method for preparing liquid cell quality control products using the cell preservation solution of the present invention is simple, and the steps of paraffin embedding and sectioning are omitted, and liquid cell quality control products can be prepared quickly, efficiently and stably. The staining effect of Application Example 4 is more obvious than that of Application Example 1, indicating that sodium ascorbate and glacial acetic acid can have a positive influence on each other in improving the immunohistochemical staining effect. The staining effects of Application Examples 5-7 are inferior to those of Application Example 1, indicating that propylene glycol, ethanol, and 1,3-propylene glycol have a synergistic effect in improving the immunohistochemical staining effect. Compared with Application Example 2, there is no immunohistochemical staining, which indicates that the conventional cell preservation solution on the market has no function of protecting cell antigen epitopes, and the air drying in the immunohistochemical step causes the epitopes on the cells to be further destroyed, resulting in poor staining effect.

[0101] Test Example 2

[0102] The liquid cell quality control products described in Application Examples 1-7 and Comparative Application Example 1 were stored at 55°C for 15 days. On the 0th day, 3rd day, 6th day, 9th day, 12th day, and 15th day, the cell suspension under the above storage conditions was subjected to P16 antibody immunohistochemical staining. The experimental steps were referred to Test Example 1. The scoring criteria for immunohistochemical staining are shown in Table 1, and the P16 antibody immunohistochemical staining effect is shown in Table 2.

[0103] Table 1

[0104]

[0105] Table 2

[0106] 0d 3d 6d 9d 12d 15d Application Example 1 +++ +++ ++ ++ ++ ++ Application Example 2 +++ ++ ++ ++ ++ ++ Application Example 3 +++ ++ ++ ++ ++ ++ Application Example 4 +++ +++ +++ +++ +++ +++ Application Example 5 ++ ++ ++ ++ ++ ++ Application Example 6 ++ ++ ++ ++ ++ ++ Application Example 7 ++ ++ ++ ++ ++ ++ Comparative application example 1 + + + + + +

[0107] The experimental results show that the liquid cell quality control product described in Application Example 4 is stored at 55°C and maintains the cell immunohistochemical staining +++ effect for a longer time than Application Example 1, indicating that sodium ascorbate and glacial acetic acid can have a positive influence on each other in improving the preservation performance of the cell preservation solution. The liquid cell quality control products described in Application Examples 5-7 are stored at 55°C, and the immunohistochemical staining effect is inferior to that of Application Example 1, indicating that propylene glycol, ethanol, and 1,3-propylene glycol have a synergistic effect in improving the immunohistochemical staining effect. According to the Arrhenius formula, storing cells at 55°C for 14 days is equivalent to storing cells at room temperature for 1 year, so the cell preservation solution described in the present invention can be stored at room temperature for 10-12 months, and the cells can still ensure that the cells have a good immunohistochemical staining effect.

[0108] The applicant declares that the present invention illustrates the preparation method and application of the cell preservation fluid and liquid cell quality control product of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0109] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0110] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A cell preservation solution, characterized in that: The cell preservation solution includes, by mass percentage, 0.1%-1% sodium chloride, 0.01%-0.1% potassium chloride, 0.05%-0.5% phosphate, 0.01%-0.1% sodium ascorbate, 3%-10% glycerol, 3%-10% 1,3-propylene glycol, 10%-20% ethanol, 0.01%-0.1% ethylenediaminetetraacetic acid, 0.05%-0.5% polyethylene glycol, 1%-5% glacial acetic acid and water.

2. The cell preservation solution according to claim 1, characterized in that The molecular weight of the polyethylene glycol is 2000-6000.

3. The cell preservation solution according to claim 1, characterized in that The cell preservation solution is obtained by physically mixing various preparation raw materials.

4. A liquid cell quality control product, characterized in that: The liquid cell quality control product is prepared by a preparation method comprising the following steps: collecting cells, and mixing the cells with the cell preservation solution according to any one of claims 1 to 3.

5. The liquid cell quality control product according to claim 4, characterized in that: The density of cells in the cell preservation solution is 10 5 -10 6 Pieces / ml.

6. Use of the liquid cell quality control product according to claim 4 or 5 in the preparation of a reference product suitable for in situ detection or analysis of tissue cells.

7. The use according to claim 6, characterized in that: The tissue cell in situ detection or analysis method includes immunohistochemistry, in situ hybridization or immunocytochemical staining.

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