A cell washing solution and use thereof

Abstract

The application discloses a kind of cell washing liquid and its application, belong to the field of biotechnology, cell washing liquid includes PBS solution, glucose, EDTA and collagen;Further, the method for preparing cytoplasm control product using the cell washing liquid is also disclosed.The washing liquid provides basic buffer system with PBS buffer as base, collagen provides protection for cells, ensures the morphological integrity of cell membrane and intracellular components;Glucose and collagen jointly change the density and viscosity of buffer, increase the ability of washing liquid to remove impurities in cells;Surfactant EDTA increases the dispersibility of cytoplasm control product, ensures the staining effect of final IHC detection;Finally, whether in washing or dispersion, ideal effect is achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a cell cleaning solution and application thereof. BACKGROUND

[0002] In the fields of medicine, biology, life science, etc., the cell suspension extracted from the cell sampling site of tissue, organ, culture solution, etc. usually contains impurities other than cells, which may have adverse effects on research and analysis. Cell quality control product is a cell control product for monitoring the IHC detection process. Some cells are prone to bring impurities during the preparation of the quality control product, which affect the IHC detection staining result. Therefore, the impurities need to be removed by cell cleaning to improve the reliability of the research and analysis result; cell cleaning is an important work. However, the PBS buffer solution commonly used for cell cleaning cannot effectively remove the staining impurities, resulting in poor cleaning effect. SUMMARY

[0003] In order to solve at least one of the above problems, the present application provides a cell cleaning solution and application thereof.

[0004] In order to achieve the above purpose, the present application adopts the following technical means:

[0005] The first aspect of the present application provides a cell cleaning solution, comprising a PBS solution, glucose, EDTA and collagen.

[0006] In some embodiments of the present application, the concentration of the PBS is 8-10 mM; preferably, the concentration of the PBS is 10 mM.

[0007] In some embodiments of the present application, the mass-volume percentage concentration of the glucose is 1%-7%; preferably, the mass-volume percentage concentration of the glucose is 4%.

[0008] In some embodiments of the present application, the mass-volume percentage concentration of the EDTA is 0.01%-0.07%. Preferably, the mass-volume percentage concentration of the EDTA is 0.04%.

[0009] In some embodiments of the present application, the mass-volume percentage concentration of the collagen is 0.005%-0.15%. Preferably, the mass-volume percentage concentration of the collagen is 0.01%.

[0010] The second aspect of the present application provides the application of the cell cleaning solution of the first aspect in the preparation of a cleaning solution for cell immunohistochemical quality control product.

[0011] The third aspect of the present application provides a preparation method of a cell quality control product, comprising the following steps,

[0012] (1) preparing the cell washing solution of the first aspect;

[0013] (2) collecting target cells, resuspending after washing 1-2 times by centrifugation using the cell washing solution;

[0014] (3) preparing the cell semi-solid suspension after fixing, dehydrating, embedding and slicing the washed cells, thereby obtaining the cell quality control product. The preparation of the cell semi-solid suspension refers to the related method in patent CN 202011356471.8.

[0015] In some embodiments of the present application, the centrifugation condition is 800-1000 RPM, preferably, the centrifugation condition is 900 RPM.

[0016] The fourth aspect of the present application provides a cell quality control product prepared by the method of the third aspect.

[0017] The fifth aspect of the present application provides a cell immunohistochemical detection kit, which comprises the cell washing solution of the first aspect.

[0018] Advantages of the present application

[0019] Compared with the prior art, the present application has the following advantages: the present application provides a cell washing solution, which provides a basic buffer system with PBS buffer as the base, collagen provides protection for cells to ensure the integrity of cell membranes and intracellular components; glucose and collagen jointly change the density and viscosity of the buffer, increasing the ability of the washing solution to remove impurities in the cells; the surfactant EDTA increases the dispersibility of the cell quality control product, ensuring the final IHC detection staining effect; finally, whether in terms of washing or dispersing, the ideal effect is achieved, which can be applied to the washing of cell quality control products, and the cell washing solution has the same effect of removing impurities for different cell strains. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The staining effect diagrams of 5 groups of experiments in Example 1 of the present application are shown;

[0021] Figure 2 The staining effect diagrams of 4 groups of experiments in Example 2 of the present application are shown;

[0022] Figure 3 The staining effect diagrams of 3 groups of experiments in Example 3 of the present application are shown;

[0023] Figure 4 The staining effect diagrams of 2 groups of experiments in Example 4 of the present application are shown. DETAILED DESCRIPTION

[0024] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present application, and are not intended to limit the scope of what the inventors regard as their application. One skilled in the art will recognize that the examples set forth herein represent techniques that can be used in carrying out the present application, and that changes can be made in the art without departing from the spirit and scope of the application.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, and the materials described herein will be referred to by the citation of the reference that first introduced such term into the art. As will be apparent to those of ordinary skill in the art, many of the features described herein can be implemented in a variety of different embodiments and of the application and that the terminology used is for the purpose of describing particular embodiments only and is not intended to be limiting of the scope of the present application.

[0026] The technical solutions of the present application are further described in detail below in conjunction with the specific embodiments.

[0027] Example 1

[0028] 1. Resuscitate one strain of MCF7 cells (human breast cancer cells, Guangzhou Sike CC0302), and expand the culture.

[0029] 2. Collect 100M of cells in the logarithmic growth phase, with a cell viability of 97.3%. Divide 20M of the cells into 5 groups: Group 1, Group 2, Group 3, Group 4, and Group 5.

[0030] The composition of the washing solution is shown in Table 1 below:

[0031] Table 1 Composition of the washing solution

[0032]

[0033] Note: The glucose, EDTA, and collagen in the table are mass / volume percentage concentrations.

[0034] The cells in Groups 2 to 5 were resuspended to 40mL using different component washing solution formulations, and centrifuged and washed once at 900RPM.

[0035] 3. Group 1 collect cells after resuspension to 10 mL of 10 mM PBS; prepare 1 50 mL centrifuge tube, add 10 mL of cell separation solution (human peripheral blood lymphocyte separation solution, Tianjin Haoyang LTS10771), then add 10 mL of cell suspension slowly along the side wall of the centrifuge tube on the cell separation solution (the action is slow and does not break the separation solution layer). The separated centrifuge tube is placed in the centrifuge, set to 1000 RPM for 30 minutes, and the centrifuge acceleration is set to 5 and the centrifuge deceleration is set to 0. The cell suspension after separation is taken with a pipette to the cell layer between PBS and separation solution, and 40 mL of 10 mM PBS is added for mixing, and centrifuged at 900 RPM for 1 time.

[0036] 4. Groups 1 to 5 after washing, the cells are fixed with 10% neutral formalin fixing solution for 2 hours, and then the fixing solution is removed.

[0037] 5. After fixation, the cells are dehydrated with 50%, 75%, 95% and anhydrous ethanol for 1 hour respectively, and then immersed in xylene for 30 minutes.

[0038] 6. After transparency, the cells are immersed in paraffin for 3 hours and embedded into blocks.

[0039] 7. The wax blocks are sliced and prepared into cell suspensions according to the patent CN 202011356471.8 A method for preparing a cell semi-solid suspension and application thereof to prepare cell suspensions for immunohistochemical staining experiments; the immunohistochemical staining experiment is performed using the LYNX480 full-automatic immunohistochemical staining machine of Huzhou Bio-Medical Technology Co., Ltd., and the target primary antibody (I1219) and auxiliary reagent (I20032C) used in conjunction are purchased from the commercial kit of Huzhou Bio-Medical Technology Co., Ltd., and the experiment adopts the conventional immunohistochemical experiment process according to the instructions of the kit.

[0040] 8. The experimental results are shown in Figure 1

[0041] The results show that the commercial cell separation solution has no obvious effect on removing impurities in the quality control cell, and the impurity removal is basically not improved; only PBS is used for removal, the cells are aggregated, and large pieces of impurities are adhered; only carbohydrates are added in the 10 mM PBS solution, and the impurity removal is basically not improved; only EDTA is added, the cell dispersion degree is improved, but the impurity removal is basically not improved; only collagen is added, and the cells are not obviously dispersed, and the impurity removal basically has no effect. It can be seen that the addition of EDTA has a significant effect on the dispersion degree of the cells.

[0042] Example 2

[0043] 1. One strain of MCF7 cells (human breast cancer cells, Guangzhou Sailu CC0302) is recovered and expanded. ​

[0044] 2. Collect 80M cells in the logarithmic growth phase with a cell viability of 96.7%; 20M cells were grouped into 4 groups: group 6, group 7, group 8, and group 9.

[0045] 3. Cells from groups 6 to 9 were resuspended in different component washing buffer formulations to 40 mL and washed once by centrifugation at 900 RPM. The composition of the washing buffer is shown in Table 2 below:

[0046] Table 2 Composition of the cleaning solution

[0047]

[0048] Note: The glucose, EDTA, and collagen in the table are mass-volume percentage concentrations.

[0049] 4. After washing, the cells in groups 6 to 9 were fixed with 10% neutral formalin for 2 hours, and then washed to remove the fixative.

[0050] 5. After fixation, the cells were dehydrated for 1 hour each with 50%, 75%, 95%, and anhydrous ethanol, and then immersed in xylene for 30 minutes to clear the cells.

[0051] 6. After transparency, the cells are immersed in paraffin for 3 hours and then embedded into blocks.

[0052] 7. The paraffin blocks were sliced ​​and prepared into cell suspensions according to patent CN 202011356471.8, "A Method for Preparing Semi-Solid Cell Suspension and Its Application". Immunohistochemical staining experiments were performed using a LYNX480 fully automated immunohistochemical staining machine from Tuling (Hangzhou) Biomedical Co., Ltd. The target primary antibody (I1219) and auxiliary reagents (I20032C) were all purchased from commercial kits of Tuling (Hangzhou) Biomedical Co., Ltd. The experiments were conducted according to the conventional immunohistochemical experimental procedure and the instructions of the kit.

[0053] 8. Experimental results are as follows Figure 2 As shown.

[0054] The results showed that adding glucose and EDTA to 10mM PBS solution could improve the washing effect, increase cell dispersion, and reduce impurities; adding glucose and collagen reduced cell aggregation and impurities; adding EDTA and collagen did not improve cell dispersion or impurity removal; adding glucose, EDTA, and collagen simultaneously significantly improved cell dispersion and impurity removal, resulting in a better washing effect.

[0055] Example 3

[0056] 1. Revive a strain of MCF7 cells (human breast cancer cells, Guangzhou Sike CC0302), expand culture.

[0057] 2. Collect 60M of logarithmic growth phase cells, cell viability 96.9%; 20M of cells are divided into 3 groups: group 10, group 11, and group 12.

[0058] 3. The cells in groups 10 to 12 are resuspended to 40mL using different concentrations of component cleaning solution formulations, and centrifuged at 900RPM for cleaning once. The composition of the cleaning solution is shown in Table 3:

[0059] Table 3 Composition of cleaning solution

[0060]

[0061] Note: The glucose, EDTA, and collagen in the table are mass / volume percentage concentrations.

[0062] 4. After cleaning, the cells are fixed with 10% neutral formalin for 2 hours, and then washed to remove the fixing solution.

[0063] 5. After fixation, the cells are dehydrated with 50%, 75%, 95%, and anhydrous ethanol for 1 hour each, and then immersed in xylene for 30 minutes.

[0064] 6. After transparency, the cells are immersed in paraffin for 3 hours and embedded into blocks.

[0065] 7. The wax blocks are sliced and prepared into cell suspensions according to the patent CN 202011356471.8 A method for preparing a cell semi-solid suspension and its application to prepare cell suspensions for immunohistochemical staining experiments; the immunohistochemical staining experiment is performed using the LYNX480 full-automatic immunohistochemical staining machine from Huzhou Bio-Medical Technology Co., Ltd., and the target primary antibody (I1219) and auxiliary reagent (I20032C) used are purchased from the company's commercial kit, and the experiment adopts the conventional immunohistochemical experiment process according to the instructions of the kit.

[0066] 8. The experimental results are shown in Figure 3 .

[0067] The results show that different concentrations of component cleaning solution formulations have good cleaning effect.

[0068] Example 4

[0069] 1. Revive a strain of IMR-32 cells (neuroblastoma cells, Guangzhou Keluojie KC2102), expand culture.

[0070] 2. Collect 40M of cells in logarithmic growth phase, cell viability 95.4%; 20M of cells are divided into two groups: group 13 and group 14. The cells in group 13 are resuspended to 40mL of 10mM PBS after collection and washed once.

[0071] 3. The cells in group 14 are resuspended to 40mL using the same formula of the washing solution as that in group 11 in Example 3, and centrifuged at 900RPM for washing once.

[0072] 4. After washing, the cells are fixed with 10% neutral formalin solution for 2 hours, and then washed to remove the fixing solution.

[0073] 5. After fixation, the cells are dehydrated with 50%, 75%, 95% and anhydrous ethanol for 1 hour respectively, and then immersed in xylene for transparency for 30 minutes.

[0074] 6. After transparency, the cells are immersed in paraffin for 3 hours and embedded into blocks.

[0075] 7. The wax blocks are sliced and prepared into cell suspensions according to the method for preparing a cell semi-solid suspension in patent CN 202011356471.8 A method for preparing a cell semi-solid suspension and application thereof, and then subjected to immunohistochemical staining experiments. The immunohistochemical staining experiments are performed using a LYNX480 full-automatic immunohistochemical staining machine from Huzhou Linge Biological Medicine Co., Ltd., and the target primary antibody (I1178) and auxiliary reagent (I20032C) used in conjunction are purchased from the commercial kit of Huzhou Linge Biological Medicine Co., Ltd. The experiments adopt a conventional immunohistochemical experimental process according to the instructions of the kit.

[0076] 8. The experimental results are shown in Figure 4 .

[0077] The results show that the quality control product washing solution of the formula can achieve the same effect of removing staining impurities on different cell strains, and is obviously superior to the conventional PBS solution washing.

[0078] All the documents mentioned in the present application are cited as references in the present application, just as each document is cited as a reference individually. In addition, it should be understood that various modifications or changes can be made to the present application by those skilled in the art after reading the above teaching of the present application, and these equivalent forms also fall within the scope defined by the present application.

Claims

1. A cell washing solution, characterized by, The cell washing solution is composed of glucose, EDTA and collagen added in a PBS solution, wherein the concentration of the PBS solution is 8-10 mM; the mass-volume percentage concentration of the glucose is 1%-7%; the mass-volume percentage concentration of the EDTA is 0.01%-0.07%; and the mass-volume percentage concentration of the collagen is 0.005%-0.15%.

2. Use of the cell washing solution of claim 1 in the preparation of a washing solution for cell immunohistochemical quality control products.

3. A method of preparing a cytocontrol, characterized by: The method comprises the following steps, (1) preparing the cell washing solution of claim 1; (2) collecting target cells, resuspending the cells with the cell washing solution, and then centrifugally washing the cells 1-2 times; (3) preparing a cell semi-solid suspension by fixing, dehydrating, embedding and slicing the washed cells, thereby obtaining a cell quality control product.

4. The method of claim 3, wherein: The centrifugal condition is 800-1000 RPM.

5. The cell quality control product prepared by the method of any one of claims 3-4.

6. A cellular immunohistochemical detection kit, characterized by: The method comprises the cell washing solution of claim 1.

Images