Cell preservation method and application
By using a gel-encapsulated cell method, and employing components such as carbomer, cells can be rapidly fixed and preserved, solving the problem of time-consuming processes in existing technologies. This method achieves rapid and long-term cell preservation and highly efficient immunohistochemical staining.
Patent Information
- Application Number
- CN202511905372.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-17
- Publication Date
- 2026-03-17
AI Technical Summary
Existing cell preservation methods require multiple steps and are time-consuming, especially the protein denaturation and dehydration processes, which make the operation cumbersome and time-consuming, making it difficult to achieve rapid and long-term cell preservation.
The method of encapsulating cells in a gel, which contains carbomer, protease inhibitors, cell permeability agents, preservatives and buffering components, simplifies the operation process and reduces dehydration steps by rapidly fixing cells and preserving them directly or embedding them in paraffin.
It achieves rapid cell fixation, reduces dehydration and clearing steps, allows for long-term preservation of cell proteins without complete denaturation, saves time, improves cell preservation efficiency, and produces good immunohistochemical staining results.
Smart Images
Figure CN121667208A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of cell immunohistochemistry technology, specifically relating to a cell preservation method and its application. Background Technology
[0002] Existing methods often use protein cross-linking agents such as formaldehyde, paraformaldehyde, glutaraldehyde, methanol, and ethanol to denature proteins, followed by dehydration and embedding in paraffin blocks for long-term preservation of cell antigen epitope proteins.
[0003] However, the protein denaturation process includes cross-linking fixation, dehydration, and embedding steps, which can take anywhere from 8 to 48 hours depending on the fixation time. Furthermore, before use, cells require dewaxing, hydration, and repair, each taking 2 to 3 hours. Therefore, the entire process is lengthy.
[0004] Therefore, there is an urgent need to develop a new cell preservation method that can protect cell proteins from incomplete denaturation, preserve cells for a long time, and save time. Summary of the Invention
[0005] To address at least one of the above problems, the present invention provides a cell preservation method and its application.
[0006] To achieve the above objectives, the present invention employs the following technical means: A first aspect of the present invention provides a cell preservation method, comprising the following steps: S1. Obtain the target cells and fix them; S2. The fixed target cells are encapsulated in a gel; the gel contains carbomer, protease inhibitors, cell permeability enhancers, preservatives, and buffering components; S3. The target cells encapsulated in gel can be directly preserved or further gelled and embedded in paraffin to obtain cell blocks for preservation.
[0007] In some embodiments of the present invention, the fixation method is as follows: immersion in 4% paraformaldehyde, followed by washing with PBS solution. The immersion time is generally 5-20 minutes, more preferably 6-12 minutes, and even more preferably 10 minutes.
[0008] In some embodiments of the present invention, the carbomer is prepared into a carbomer stock solution with a mass-volume concentration of 0.1% using anhydrous ethanol; and the mass-volume fraction of carbomer in the gel is 0.0167%.
[0009] In some embodiments of the present invention, the volume fraction of the protease inhibitor in the gel is 0.00417%-0.0417%; In some embodiments of the present invention, the cell permeability agent in the gel is DMSO, and the volume fraction of DMSO is 0.00417%-0.0167%.
[0010] In some embodiments of the present invention, the preservative in the gel is one of sodium azide, benzalkonium chloride and Proclin series preservatives; the preservative mass-volume fraction is 0.00833%-0.0833%.
[0011] In some embodiments of the present invention, the buffer component in the gel is one of PBS buffer or Tris-HCl buffer.
[0012] In some embodiments of the present invention, in the step of encapsulating the target cells with a gel, a cell preservation solution A is prepared by dispersing protease inhibitors, cell permeability agents, preservatives, and buffering components, and a cell preservation solution B is prepared by dispersing carbomer; the target cells are encapsulated by mixing cell preservation solution A and cell preservation solution B in steps, with the volume ratio of cell preservation solution A to cell preservation solution B being 5:1.
[0013] In some embodiments of the present invention, the cell preservation solution A contains 0.005%-0.05% protease inhibitor by volume.
[0014] In some embodiments of the present invention, the cell permeabilizer in the cell preservation solution A is DMSO, with a DMSO volume fraction of 0.005%-0.02%.
[0015] In some embodiments of the present invention, the preservative in the cell preservation solution A is one of sodium azide, benzalkonium chloride and Proclin series preservatives; the preservative mass-volume fraction is 0.01%-0.1%.
[0016] In some embodiments of the present invention, the buffer component in the cell preservation solution A is one of PBS buffer and Tris-HCl buffer.
[0017] A second aspect of this invention provides a cell quality control product, which is prepared using the cell preservation method described in the first aspect. The cell quality control product can be used directly with embedded cells, or it can be used after further embedding paraffin blocks into sections, or it can be further prepared into a cell suspension for use.
[0018] The third aspect of the present invention provides the application of cells preserved using the method described in the first aspect in immunohistochemistry, wherein the cell wax block is sectioned to obtain paraffin sections, the paraffin sections are dewaxed, the gel is dissolved in anhydrous ethanol to obtain cells, and the cells are sampled for immunohistochemical staining.
[0019] In some embodiments of the present invention, the cells preserved by the method described in the first aspect are used in immunohistochemistry. The gel containing the cells is taken, anhydrous ethanol is added and mixed thoroughly, the gel is removed by centrifugation, and the cells are obtained after resuspending and washing with PBS by centrifugation. The cells are then directly spotted for immunohistochemical staining.
[0020] Beneficial effects of the present invention Compared with existing technologies, the present invention has the following beneficial effects: The present invention provides a cell preservation method and application, in which target cells are encapsulated in a gel, the gel containing carbomer, protease inhibitors, cell permeability agents, preservatives, and buffering components. The cell permeability agents and protease inhibitors rapidly inhibit the activity of endogenous proteases in cells or bind to protease binding sites, thus inhibiting cell autolysis; the antibacterial agent prevents microbial proliferation; and by forming a gel with carbomer, the gel encapsulation method blocks the contact between cells and the external environment, thereby preventing oxidation and protecting cellular antigenic epitope proteins.
[0021] After treatment using the cell preservation method described in this application, cells can be rapidly fixed. Cell clusters can be quickly shaped and cell density and spacing can be adjusted by regulating the gel coating concentration, facilitating uniform cell dispersion and morphological changes. The gel can be easily removed for direct use. Cells fixed using this method can be stored long-term at 4°C without dehydration, clearing, or embedding. Before use, the gel concentration can be adjusted to liquefaction, and the fixing gel can be removed by centrifugation. Alternatively, cells can be embedded in paraffin using standard procedures and sectioned in paraffin before use. This allows for long-term preservation of cell proteins without complete denaturation. Furthermore, the antigen retrieval process is unnecessary during use, saving time and improving efficiency. Attached Figure Description
[0022] Figure 1 The immunohistochemical staining results of groups I and II in Example 1 of this invention are shown; Figure 2 The immunohistochemical staining results of groups III, IV, and V in Example 2 of this invention are shown. Figure 3 The immunohistochemical staining results of groups VI, VII, VIII, and IX in Example 3 of this invention are shown. Figure 4 The immunohistochemical staining results of groups X and XI in Example 4 of this invention are shown; Figure 5 The results of immunohistochemical staining of cells in groups XII and XIII in Example 5 of the present invention are shown. Detailed Implementation
[0023] The following examples are used to illustrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the examples represent techniques discovered by the inventors that can be used to implement the invention, and therefore can be considered preferred embodiments for implementing the invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of the invention.
[0024] 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 invention pertains, and all materials disclosed herein and cited therein are incorporated herein by reference. Many equivalent techniques of specific embodiments of the invention described herein will be recognized or can be understood by ordinary experimentation by those skilled in the art. These equivalents will be included in the claims.
[0025] A cell preservation method includes the following steps: S1. Obtain the target cells and fix them; S2. The fixed target cells are encapsulated in a gel; the gel contains carbomer, protease inhibitors, cell permeability enhancers, preservatives, and buffering components; S3. The target cells encapsulated in the gel can be directly preserved, or the bubble-free gel can be embedded in paraffin to obtain cell blocks for preservation.
[0026] The fixation method was as follows: the sample was soaked in 4% paraformaldehyde and then washed with PBS solution.
[0027] Carbomer was prepared as a 0.1% (w / v) carbomer stock solution using anhydrous ethanol; the carbomer content in the gel was 0.0167% (w / v); the protease inhibitor content was 0.00417%-0.0417% (w / v); the cell permeabilizer was DMSO, with a DMSO content of 0.00417%-0.0167% (w / v); the preservative was one of sodium azide, benzalkonium chloride, and Proclin series preservatives, with a preservative content of 0.00833%-0.0833% (w / v); and the buffer was one of PBS buffer or Tris-HCl buffer.
[0028] There are various methods for preparing gels, as long as the target cells and components are uniformly dissolved and distributed in the gel. To ensure uniform gel mixing and shorten the mixing time, subsequent embodiments employed the method of preparing cell preservation solution A and cell preservation solution B separately, as follows: cell preservation solution A was prepared by dissolving protease inhibitors, cell permeability enhancers, preservatives, and buffers, and cell preservation solution B was prepared by dissolving carbomer; cell preservation solution A and cell preservation solution B were mixed stepwise to form a gel that encapsulated the target cells, with a volume ratio of cell preservation solution A to cell preservation solution B of 5:1.
[0029] The technical solution of this application will be further described in detail below with reference to specific embodiments.
[0030] Example 1 One Jurkat cell line (human acute T-lymphoblastic leukemia cell) (Guangzhou Saiku Biotechnology, CC1902) was revived and cultured on a large scale.
[0031] Cells were collected, counted, and viability recorded. Cell viability was >95%. 200M cells were washed twice with 10mM PBS. The 200M cells were then divided into two groups: Group I and Group II.
[0032] (1) Group I cells were fixed with 4% formaldehyde for 2 hours, then dehydrated with 75% ethanol for 2 hours, 95% ethanol for 1 hour, and anhydrous ethanol for 1 hour. After clearing with xylene for 1 hour, they were embedded in paraffin to form blocks. They were stored at 4-8℃, usually in a refrigerator or cold storage. All the paraffin blocks / gel clumps below were stored under the same conditions.
[0033] After one month of storage, the paraffin block was sectioned and adhered to a glass slide for dewaxing for 30 minutes, hydration for 30 minutes, boiling and heat repair under alkaline conditions for 20 minutes, and then allowed to return to room temperature before immunohistochemical staining.
[0034] (2) Group II cells were soaked in 10 mL of 4% paraformaldehyde for 10 minutes, and then washed twice with 10 mM PBS before use.
[0035] (3) Prepare cell preservation solution A. Take 8 mL of 10 mM PBS, add 2 mg of sodium azide, 1 μL of DMSO and 1 μL of protease inhibitor, mix thoroughly and dissolve, then add 10 mM PBS to make up to 10 mL. Prepare cell preservation solution A. The formula of cell preservation solution A is shown in Table 1 below.
[0036] Table 1. Formulation of Cell Preservation Solution A
[0037] Protease inhibitor (100X) (Thermo Scientific, 78429) (4) Prepare cell preservation solution B. Weigh 1 mg of carbomer 980 and add 1 mL of anhydrous ethanol and shake to form a uniform suspension. Prepare cell preservation solution B. The formula of cell preservation solution B is shown in Table 2 below.
[0038] Table 2. Formulation of Cell Preservation Solution B
[0039] (5) Gel preparation: After washing the group II cells, resuspend them evenly in 1 mL of cell preservation solution A, add 0.5 mL of cell preservation solution B, and quickly pipette to mix. Pour 0.5 mL of the mixture into a suitable mold, add another 0.5 mL of cell preservation solution A to the mold, mix well to form a gel, and then shake on a horizontal shaker at 120 RPM for 30 minutes to remove air bubbles. Then embed the three gel clumps together in paraffin wax. After one month of preservation, the paraffin block is sectioned and adhered to a glass slide. After dewaxing for 30 minutes, the gel is dissolved in anhydrous ethanol for 2 minutes and then ready for immunohistochemical staining.
[0040] It should be noted that steps (3)-(5) above can also be prepared by adding conventional solutes (protease inhibitors, cell permeabilizers, preservatives, carbomer) to the solvent (buffered saline, PBS or others) in sequence and stirring evenly. In actual operation, this method of preparing gel requires stirring overnight and finally adding cells and stirring evenly, which is time-consuming and laborious. Therefore, the above steps are preferred.
[0041] Immunohistochemical staining was performed using the LYNX480 fully automated immunohistochemical staining machine from Tuling (Hangzhou) Biomedical Co., Ltd. The primary antibody (I1199) and auxiliary reagents (I20032C) were all purchased from commercial kits of Tuling (Hangzhou) Biomedical Co., Ltd. The experiments followed standard immunohistochemical procedures and were conducted according to the kit instructions.
[0042] The immunohistochemical staining results of groups I and II are as follows: Figure 1 As shown.
[0043] The results showed that the staining results of group II cells were comparable to those of group I cells using conventional preservation methods.
[0044] Example 2 One Jurkat cell line (human acute T-lymphoblastic leukemia cell) (Guangzhou Saiku Biotechnology, CC1902) was revived and cultured on a large scale.
[0045] Cells were collected, counted, and viability recorded. Cell viability was >95%. 300M cells were washed twice with 10mM PBS. The 300M cells were then divided into three groups: Group III, Group IV, and Group V.
[0046] The cells in the above three groups were soaked in 10 mL of 4% paraformaldehyde for 10 minutes, and then washed twice with 10 mM PBS before use.
[0047] Prepare cell preservation solutions A1 to A3 containing different components according to Table 3 below to treat the above three groups of cells.
[0048] Table 3. Formulations of cell preservation solutions A1 to A3
[0049] Referring to the gel preparation process in Example 1, after cleaning the three groups of cells to be used, gels were prepared using the corresponding cell preservation solutions A (A1-A3) and B, and then embedded in paraffin into blocks. After preservation for 1 month, the paraffin blocks were sectioned and adhered to glass slides. After dewaxing for 30 minutes, the gels were dissolved by soaking in anhydrous ethanol for 2 minutes, and then immunohistochemical staining could be performed.
[0050] Immunohistochemical staining was performed using the LYNX480 fully automated immunohistochemical staining machine from Tuling (Hangzhou) Biomedical Co., Ltd. The primary antibody (I1199) and auxiliary reagents (I20032C) were all purchased from commercial kits of Tuling (Hangzhou) Biomedical Co., Ltd. The experiments followed standard immunohistochemical procedures and were conducted according to the kit instructions.
[0051] The immunohistochemical staining results of groups III, IV, and V are as follows: Figure 2 As shown.
[0052] The results showed that group III cells were weakly positive, group IV cells were negative, and group V cells showed loss of cell morphology.
[0053] Example 3 One Jurkat cell line (human acute T-lymphoblastic leukemia cell) (Guangzhou Saiku Biotechnology, CC1902) was revived and cultured on a large scale.
[0054] Cells were collected, counted, and viability recorded. Cell viability was >95%. 400M cells were washed twice with 10mM PBS. The 400M cells were then divided into four groups: Group VI, Group VII, Group VIII, and Group IX.
[0055] The four groups of cells were soaked in 10 mL of 4% paraformaldehyde for 12 minutes, 10 minutes, 8 minutes and 6 minutes respectively, and then washed twice with 10 mM PBS before use.
[0056] Based on this, cell preservation solutions A4-A7 containing different components were prepared according to Table 4 below to treat the above four groups of cells.
[0057] Table 4. Formulations of Cell Preservation Solutions A4-A7
[0058] Referring to the gel preparation process in Example 1, after cleaning the four groups of cells to be used, gels were prepared using the corresponding cell preservation solutions A (A4-A7) and B, and then embedded in paraffin into blocks. After preservation for one month, the paraffin blocks were sectioned and adhered to glass slides. After dewaxing for 30 minutes, the gels were dissolved by soaking in anhydrous ethanol for 2 minutes, and then immunohistochemical staining could be performed.
[0059] Immunohistochemical staining was performed using the LYNX480 fully automated immunohistochemical staining machine from Tuling (Hangzhou) Biomedical Co., Ltd. The primary antibody (I1199) and auxiliary reagents (I20032C) were all purchased from commercial kits of Tuling (Hangzhou) Biomedical Co., Ltd. The experiments followed standard immunohistochemical procedures and were conducted according to the kit instructions.
[0060] The immunohistochemical staining results of groups VI, VII, VIII, and IX are as follows: Figure 3 As shown.
[0061] The results showed that the staining results of cells in groups VI to IX were all normal.
[0062] Example 4 One Jurkat cell line (human acute T-lymphoblastic leukemia cell) (Guangzhou Saiku Biotechnology, CC1902) was revived and cultured on a large scale.
[0063] Cells were collected, counted, and recorded as viable. Cell viability was >95%. 200M cells were washed twice with 10mM PBS.
[0064] Cells were infiltrated with 10 mL of 4% paraformaldehyde for 10 minutes, then washed twice with 10 mM PBS. The 200 M cells were divided into two groups: group X and group XI.
[0065] Take 8 mL of 10 mM PBS, add 2 mg of sodium azide, 1 μL of DMSO and 1 μL of protease inhibitor, mix thoroughly to dissolve, and then add 10 mM PBS to bring the volume to 10 mL to prepare cell preservation solution A.
[0066] Weigh 1 mg of carbomer 980 and add 1 mL of anhydrous ethanol, shake to form a homogeneous suspension, and prepare cell preservation solution B.
[0067] Group X cells were prepared into gels according to Example 1 and embedded in paraffin into blocks. After one month of storage, the paraffin blocks were sectioned and adhered to glass slides. After dewaxing for 30 minutes, the gels were dissolved by soaking in anhydrous ethanol for 2 minutes, and then immunohistochemical staining could be performed.
[0068] Cells in group XI were resuspended in 2 mL of cell preservation solution A.
[0069] Weigh 0.5g of agarose, add 10mL of pure water and boil. Cool to about 60℃. Add 1mL of the above agarose solution to the cell suspension, mix quickly before the agarose solidifies, and shake to remove air bubbles. Embed the above agarose block in paraffin wax to form a block.
[0070] After one month of storage, the paraffin blocks were sectioned and adhered to glass slides. After dewaxing for 30 minutes, they were baked at 70°C to melt and remove the agarose. After washing, they were subjected to immunohistochemical staining.
[0071] Immunohistochemical staining was performed using the LYNX480 fully automated immunohistochemical staining machine from Tuling (Hangzhou) Biomedical Co., Ltd. The primary antibody (I1199) and auxiliary reagents (I20032C) were all purchased from commercial kits of Tuling (Hangzhou) Biomedical Co., Ltd. The experiments followed standard immunohistochemical procedures and were conducted according to the kit instructions.
[0072] The immunohistochemical staining results of groups X and XI are as follows: Figure 4 As shown, group X showed normal staining, while group XI showed negative results.
[0073] Example 5 One HeLa (human cervical cancer cell line) (Guangzhou Saiku Biotechnology, CC1101) was revived and cultured on a large scale.
[0074] Cells were collected, counted, and viability recorded. Cell viability was >95%. 200M cells were washed twice with 10mM PBS. The 200M cells were then divided into two groups: Group XII and Group XIII.
[0075] Group XII cells were treated with 4% formaldehyde fixative for 2 hours, then dehydrated with 75% ethanol for 2 hours, 95% ethanol for 1 hour, anhydrous ethanol for 1 hour, and xylene for 1 hour before being embedded in paraffin to form blocks.
[0076] After one month of storage, the paraffin blocks were sectioned and adhered to glass slides. They were dewaxed for 30 minutes, hydrated for 30 minutes, boiled for 20 minutes under alkaline conditions for heat repair, and then brought to room temperature for immunohistochemical staining.
[0077] Group XIII cells were soaked in 10 mL of 4% paraformaldehyde for 10 minutes, then washed twice with 10 mM PBS before use.
[0078] Take 8 mL of 10 mM PBS, add 2 mg of sodium azide, 1 μL of DMSO and 1 μL of protease inhibitor, mix thoroughly to dissolve, and then add 10 mM PBS to bring the volume to 10 mL to prepare cell preservation solution A.
[0079] Weigh 1 mg of carbomer 980 and add 1 mL of anhydrous ethanol, shake to form a homogeneous suspension, and prepare cell preservation solution B.
[0080] After washing the XIII group cells, resuspend them evenly with 1 mL of cell preservation solution A, add 0.5 mL of cell preservation solution B, and quickly pipette to mix. Pour 0.5 mL into a suitable mold, add another 0.5 mL of cell preservation solution A to the mold, mix well to form a gel, and then shake on a horizontal shaker at 120 RPM for 30 minutes to remove air bubbles and form three gel clumps, which can be stored directly.
[0081] After one month of storage, before the immunohistochemical experiment, place three gel clumps in a container, add 45 mL of anhydrous ethanol, mix thoroughly to dissolve the gel, centrifuge at 1500 RPM for 5 minutes to remove the dissolved gel, resuspend the cells in 10 mM PBS, wash once by centrifugation at 1500 RPM, and then resuspend the cells in 1 mL of 10 mM PBS. Take 2 μL of the cell suspension and spot it directly onto a glass slide, allow it to air dry, and then perform immunohistochemical staining.
[0082] Immunohistochemical staining was performed using the LYNX480 fully automated immunohistochemical staining machine from Tuling (Hangzhou) Biomedical Co., Ltd. The primary antibody (I1189) and auxiliary reagents (I20032C) were all purchased from commercial kits of Tuling (Hangzhou) Biomedical Co., Ltd. The experiments followed standard immunohistochemical procedures and were conducted according to the kit instructions.
[0083] Immunohistochemical staining results of groups XII and XIII are as follows: Figure 5 As shown.
[0084] The results showed that the cell staining results in group XIII were better than those in group XII, which used conventional fixation methods.
[0085] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by this application.
Claims
1. A method of cell preservation, characterized by, The method comprises the following steps: S1, obtaining target cells and fixing the same; S2, uniformly dispersing the fixed target cells in a gel to form a gel package, wherein the gel comprises carbomer, protease inhibitor, cell permeation agent, preservative and buffer component; S3, directly storing the target cells after the gel package or further embedding the same in paraffin to obtain a cell paraffin block.
2. The method of claim 1, wherein the cell is a stem cell. The fixing method is: infiltrating with 4% paraformaldehyde and cleaning with PBS solution after fixing.
3. The cell preservation method according to claim 1, characterized in that, In the gel, the mass-volume fraction of carbomer is 0.0167%.
4. The method of claim 1, wherein the cell is a stem cell. In the gel, the volume fraction of protease inhibitor is 0.00417%-0.0417%.
5. The method of claim 1, wherein the cell suspension is cooled to a temperature of about - 80°C to about - 196°C. In the gel, the cell permeation agent is DMSO, and the volume fraction of DMSO is 0.00417%-0.0167%.
6. The method of claim 1, wherein the cell suspension is cooled to a temperature of about - 80°C to about - 196°C. In the gel, the preservative is one of sodium azide, benzalkonium chloride and Proclin series preservatives, and the mass-volume fraction of the preservative is 0.00833%-0.0833%.
7. The method of claim 1, wherein the cell suspension is cooled to a temperature of about - 80°C to about - 196°C. In the gel, the buffer component is one of PBS buffer and Tris-HCl buffer.
8. A cell quality control product, characterized by: The method is prepared by any one of claims 1-7.
9. Use of cells preserved according to the method of any one of claims 1 to 7 in immunohistochemistry, characterized in that, Obtaining paraffin sections by slicing the cell paraffin block, deparaffinizing the paraffin sections, dissolving the gel with anhydrous ethanol to obtain cells, and taking the cells for immunohistochemical staining.
10. Use of cells preserved according to the method of any one of claims 1 to 7 in immunohistochemistry, characterized in that, Taking the gel with packaged cells, adding anhydrous ethanol and mixing well, removing the gel by centrifugation, resuspending and cleaning by centrifugation with PBS to obtain cells, and taking the cells for direct immunohistochemical staining.