Cell matrix sheet and preparation method thereof

Abstract

The invention relates to a cell matrix sheet and a preparation method thereof. The cell matrix sheet comprises a modified cell matrix sheet and neutrophils flatly laid on the modified cell matrix sheet, wherein the modified cell matrix sheet is obtained by reacting modified cellulose with a modified silicon dioxide glass slide. Acrylic anhydride is introduced into modified cellulose molecules, so that double bonds are introduced into cellulose, free radical polymerization reaction can be carried out on the cellulose molecules and the modified silicon dioxide glass slide cut off acrylic acid under the catalysis of BPO, stable chemical combination is formed, and the adhesive force of antigen protein on the surface of a matrix sheet is remarkably improved. In addition, the surface of cellulose is rich in functional groups such as hydroxyl groups and carboxyl groups, and the hydroxyl groups can generate hydrogen-bond interaction or electrostatic interaction with polar groups such as amino groups on the surface of the antigen protein, so that the adhesive force of the antigen protein is remarkably enhanced.

Description

Technical Field

[0001] The present invention relates to the technical field of cell detection, and in particular to a cell matrix sheet and a preparation method thereof. Background Art

[0002] In biomedical research, cell matrix sheets are an important biomaterial widely used in the fields of cell culture, cell adhesion, tissue engineering, and immunology. The surface properties of cell matrix sheets play a key role in cell growth, differentiation, and interaction with the external environment. Therefore, the development and optimization of modified cell matrix materials, especially their effects on the attachment and stability of cells, proteins, and other biomolecules, has become a hot topic in current research.

[0003] In the prior art, silicon dioxide slides are usually used as the basic material of the cell matrix, and their surfaces are modified by chemical or physical methods to improve the performance of the cell matrix sheet. For example, researchers have tried to promote the interaction between cells and the matrix by changing the hydrophilicity of the slide surface, increasing chemical groups and other means. However, although these methods have improved cell adhesion to a certain extent, they have limited effects in improving the adhesion and stability of antigen proteins.

[0004] Cellulose is a natural polymer material with good biocompatibility and adjustability. In recent years, cellulose-based materials have been widely used in the biomedical field, especially in cell culture and biosensors.

[0005] However, in the prior art, the binding of cellulose to the surface of the glass slide mostly relies on relatively simple physical adsorption or chemical cross-linking methods. These methods may have the problem of insufficient stability of the attached antigen protein in practical applications and be easily affected by environmental changes (such as temperature, humidity, etc.).

[0006] Therefore, it is necessary to develop a new technical solution to solve the deficiencies in the prior art. Summary of the invention

[0007] Based on this, the cell matrix sheet of the present invention exhibits excellent effects in improving the stability of antigen proteins, can maintain the attachment of antigen proteins for a longer period of time, and reduce the impact of environmental changes on the stability of antigen proteins, thereby improving the reliability and consistency of experimental results.

[0008] One object of the present invention is to provide a cell matrix sheet, the cell matrix sheet comprising: a modified cell matrix sheet and neutrophils plated on the modified cell matrix sheet;

[0009] Wherein, the modified cell matrix sheet is obtained by reacting modified cellulose with a hydroxylated silica glass slide.

[0010] Furthermore, the molecular weight of the cellulose is 5W-250W.

[0011] The present invention also provides a method for preparing the cell matrix sheet, comprising the following steps:

[0012] A1. Soaking the silica slide in a sodium hydroxide solution, ultrasonically treating, and purifying to obtain a hydroxylated silica slide;

[0013] A2. The hydroxylated silica glass slide and acrylic acid are blended and heated to react to obtain a modified silica glass slide;

[0014] A3. Add methacrylic anhydride and cellulose to a solvent and react at room temperature to obtain modified cellulose;

[0015] A4. adding the modified silica glass slide and the modified cellulose to a solvent, heating and reacting under the action of a catalyst to obtain a modified cell matrix sheet;

[0016] A5. Spread the neutrophils on the modified cell matrix sheet to obtain a cell matrix sheet.

[0017] Furthermore, the method for extracting neutrophils comprises the following steps: adding red blood cell sedimentation buffer to a whole blood sample to obtain a supernatant, adding a red blood cell lysing solution, lysing the red blood cells in the supernatant to obtain neutrophils.

[0018] Furthermore, the red blood cell sedimentation buffer is selected from Dextran solution.

[0019] Furthermore, the red blood cell lysing solution is selected from ACK lysing solution.

[0020] Furthermore, the concentration of the Dextran solution is 5-10%.

[0021] Furthermore, the volume ratio of the whole blood sample to the Dextran solution is 4-5:1.

[0022] Furthermore, in step A3, the mass ratio of methacrylic anhydride to cellulose is 0.8-1:50-52.

[0023] Furthermore, in step A4, the heating temperature is 90-100°C.

[0024] Specifically, cellulose, as a natural polymer material, has excellent biocompatibility. Introducing modified cellulose into the surface of the matrix sheet can not only enhance the adhesion of antigen proteins, but also improve the adhesion performance of cells on the matrix sheet. The modified cell matrix sheet can provide a more suitable microenvironment, promote cell attachment and proliferation, and is suitable for the culture of various cell types, especially in immunological research and cell-matrix interaction experiments. It has good application prospects.

[0025] Furthermore, because cellulose can form a stable multi-point interaction network, it can help the antigen protein maintain stability on the surface of the slide for a long time, reducing the impact of environmental factors on the structure and function of the antigen protein. This stability is of great significance for experiments such as antigen detection and immunoassay.

[0026] The present invention has the following beneficial effects:

[0027] The modified cellulose molecules introduced by the invention introduce double bonds into cellulose by introducing acrylic anhydride, and can undergo free radical polymerization reaction with the modified silica glass slide cut off by acrylic acid under the catalysis of BPO, forming a stable chemical bond, which significantly improves the adhesion of antigen protein on the surface of the matrix sheet. In addition, the cellulose surface is rich in functional groups such as hydroxyl and carboxyl, and these hydroxyl groups can hydrogen bond or electrostatic interaction with polar groups such as amino groups on the surface of antigen protein, significantly enhancing the adhesion of antigen protein. This hydrophilicity and biocompatibility of cellulose can provide a more stable adsorption environment, so that the antigen protein forms a more firmly bond on the surface of the glass slide. DETAILED DESCRIPTION

[0028] In order to more clearly illustrate the technical solution of the present invention, the following examples are listed. Unless otherwise stated, the raw materials, reactions and post-treatment methods shown in the examples are common raw materials on the market and technical methods well known to those skilled in the art.

[0029] The words "preferred", "preferably", "more preferably", etc. in the present invention refer to embodiments of the present invention that can provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same circumstances or other circumstances. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not applicable, nor is it intended to exclude other embodiments from the scope of the present invention.

[0030] It should be understood that, except in any operating examples, or where otherwise indicated, all numbers expressing, for example, the amounts of ingredients used in the specification and claims should be understood to be modified in all instances by the term "about". Therefore, unless indicated to the contrary, the numerical parameters set forth in the following specification and the appended claims are approximate values ​​that vary depending on the desired properties to be obtained by the present invention.

[0031] Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related conventional techniques in the art. These techniques have been fully described in existing literature.

[0032] Cellulose was purchased from Adamas Reagent Co., Ltd., Shanghai, China.

[0033] Example 1

[0034] A cell matrix sheet, comprising: a modified cell matrix sheet and neutrophils spread on the modified cell matrix sheet;

[0035] The method for extracting neutrophils comprises the following steps: adding ml of whole blood sample to a centrifuge tube, adding 5wt% Dextran solution (the volume ratio of whole blood sample to Dextran solution is 5:1), leaving it at room temperature for 30 minutes, transferring the supernatant to a centrifuge tube containing 10ml PBS buffer, centrifuging at 1000rpm for 5 minutes, discarding the supernatant, adding 3ml PBS buffer to resuspend, then adding ACK lysis solution to lyse red blood cells (the volume ratio of ACK lysis solution to lyse red blood cells to resuspending liquid is 15:1), mixing for 5 minutes, and centrifuging at 1000rpm for 5 minutes. After pouring out the supernatant, resuspending with 1ml PBS, blowing and collecting cells again, centrifuging at 1000rpm, and obtaining neutrophils after resuspending.

[0036] The preparation method of the cell matrix sheet comprises the following steps:

[0037] A1. Soak the silica slide in a sufficient amount of 5 mol / L sodium hydroxide solution, ultrasonically treat for 3 h, wash and dry to obtain a hydroxylated silica slide;

[0038] A2. The hydroxylated silica glass slide was immersed in a sufficient amount of methacrylic acid, ultrasonically heated to 80°C for 4h, filtered and dried to obtain a silica glass slide grafted with acrylic acid;

[0039] A3. Add 0.8 g of methacrylic anhydride to 250 ml of ethanol, add 50 g of cellulose, react at room temperature for 4 h, adjust the pH to neutral, filter, wash, and obtain modified cellulose;

[0040] A4. Add the modified silica slide and 50 g of modified cellulose to 250 ml of toluene, add 0.1 g of BPO under the protection of nitrogen, heat to 90 ° C for 5 h, filter and dry to obtain a modified cell matrix sheet;

[0041] A5. Spread the neutrophils on the modified cell matrix sheet to obtain a cell matrix sheet.

[0042] Example 2

[0043] A cell matrix sheet, comprising: a modified cell matrix sheet and neutrophils spread on the modified cell matrix sheet;

[0044] The method for extracting neutrophils comprises the following steps: adding ml of whole blood sample to a centrifuge tube, adding 5wt% Dextran solution (the volume ratio of whole blood sample to Dextran solution is 5:1), leaving it at room temperature for 30 minutes, transferring the supernatant to a centrifuge tube containing 10ml PBS buffer, centrifuging at 1000rpm for 5 minutes, discarding the supernatant, adding 3ml PBS buffer to resuspend, then adding ACK lysis solution to lyse red blood cells (the volume ratio of ACK lysis solution to lyse red blood cells to resuspending liquid is 15:1), mixing for 5 minutes, and centrifuging at 1000rpm for 5 minutes. After pouring out the supernatant, resuspending with 1ml PBS, blowing and collecting cells again, centrifuging at 1000rpm, and obtaining neutrophils after resuspending.

[0045] The preparation method of the cell matrix sheet comprises the following steps:

[0046] A1. Soak the silica slide in a sufficient amount of 6 mol / L sodium hydroxide solution, ultrasonically treat for 3 h, wash and dry to obtain a hydroxylated silica slide;

[0047] A2. The hydroxylated silica glass slide was immersed in a sufficient amount of methacrylic acid, ultrasonically heated to 80°C for 4h, filtered and dried to obtain a silica glass slide grafted with acrylic acid;

[0048] A3. Add 0.8 g of methacrylic anhydride to 250 ml of ethanol, add 50 g of cellulose, react at room temperature for 4 h, adjust the pH to neutral, filter, wash, and obtain modified cellulose;

[0049] A4. Add the modified silica slide and 40 g of modified cellulose to 250 ml of toluene, add 0.1 g of BPO under the protection of nitrogen, heat to 90 ° C for 4 h, filter and dry to obtain a modified cell matrix sheet;

[0050] A5. Spread the neutrophils on the modified cell matrix sheet to obtain a cell matrix sheet.

[0051] Example 3

[0052] A cell matrix sheet, comprising: a modified cell matrix sheet and neutrophils spread on the modified cell matrix sheet;

[0053] The method for extracting neutrophils comprises the following steps: adding ml of whole blood sample to a centrifuge tube, adding 5wt% Dextran solution (the volume ratio of whole blood sample to Dextran solution is 4:1), leaving it at room temperature for 30 minutes, transferring the supernatant to a centrifuge tube containing 10ml PBS buffer, centrifuging at 1000rpm for 5 minutes, discarding the supernatant, adding 3ml PBS buffer to resuspend, then adding ACK lysis solution to lyse red blood cells (the volume ratio of ACK lysis solution to lyse red blood cells to resuspending liquid is 15:1), mixing for 5 minutes, and centrifuging at 1000rpm for 5 minutes. After pouring out the supernatant, resuspending with 1ml PBS, blowing and collecting cells again, centrifuging at 1000rpm, and obtaining neutrophils after resuspending.

[0054] The preparation method of the cell matrix sheet comprises the following steps:

[0055] A1. Soak the silica slide in a sufficient amount of 5 mol / L sodium hydroxide solution, ultrasonically treat for 3 h, wash and dry to obtain a hydroxylated silica slide;

[0056] A2. The hydroxylated silica glass slide was immersed in a sufficient amount of methacrylic acid, ultrasonically heated to 75 ° C for 5 h, filtered and dried to obtain a silica glass slide grafted with acrylic acid;

[0057] A3. Add 0.8 g of methacrylic anhydride to 250 ml of ethanol, add 50 g of cellulose, react at room temperature for 4 h, adjust the pH to neutral, filter, wash, and obtain modified cellulose;

[0058] A4. Add the modified silica slide and 50 g of modified cellulose to 250 ml of toluene, add 0.1 g of BPO under the protection of nitrogen, heat to 95 ° C for 4 h, filter and dry to obtain a modified cell matrix sheet;

[0059] A5. Spread the neutrophils on the modified cell matrix sheet to obtain a cell matrix sheet.

[0060] Comparative Example 1

[0061] A cell matrix sheet. The difference between this comparative example and Example 1 is that step A3 is omitted, and in step A4, modified cellulose is removed. Other components and preparation methods are the same.

[0062] Test Case

[0063] The neutrophil uniformity of the cell matrix sheets prepared in Example 1 and Comparative Example 1 was tested

[0064] Test method: A qualitative comparison was made based on the light microscopic images at the same dilution and the same microscope magnification, and the uniformity was estimated based on the degree of close aggregation between individual cells. The test results are shown in Table 1.

[0065] Table 1 Cell matrix sheet performance test results of Example 1 and Comparative Example 1

[0066] project Example 1 Comparative Example 1 Uniformity % 88 75

[0067] It can be concluded from Table 1 that the cell matrix sheet of Example 1 of the present invention can better support the growth and functional performance of neutrophils during the culture process, thereby improving the stability and uniformity of neutrophil functions.

[0068] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0069] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A cell matrix sheet, characterized in that: The cell matrix sheet comprises: a modified cell matrix sheet and neutrophils spread on the modified cell matrix sheet; Wherein, the modified cell matrix sheet is obtained by reacting modified cellulose with a modified silica glass slide.

2. The cell matrix sheet according to claim 1, characterized in that: The molecular weight of the cellulose is 5W-250W.

3. The method for preparing the cell matrix sheet according to any one of claims 1 to 2, characterized in that: The steps include: A1. Soaking the silica slide in a sodium hydroxide solution, ultrasonically treating, and purifying to obtain a hydroxylated silica slide; A2. The hydroxylated silica glass slide and acrylic acid are blended and heated to react to obtain a modified silica glass slide; A3. Add methacrylic anhydride and cellulose to a solvent and react at room temperature to obtain modified cellulose; A4. adding the modified silica glass slide and the modified cellulose to a solvent, heating and reacting under the action of a catalyst to obtain a modified cell matrix sheet; A5. Spread the neutrophils on the modified cell matrix sheet to obtain a cell matrix sheet.

4. The method for preparing the cell matrix sheet according to claim 1, characterized in that: The method for extracting neutrophils comprises the following steps: adding red blood cell sedimentation buffer to a whole blood sample to obtain a supernatant, adding a red blood cell lysing solution, lysing the red blood cells in the supernatant to obtain neutrophils.

5. The method for preparing the cell matrix sheet according to claim 4, characterized in that: The erythrocyte sedimentation buffer is selected from Dextran solution.

6. The method for preparing the cell matrix sheet according to claim 4, characterized in that: The red blood cell lysing solution is selected from ACK lysing solution.

7. The method for preparing a cell matrix sheet according to claim 4, wherein the concentration of the Dextran solution is 5-10%.

8. According to the method for preparing a cell matrix sheet according to claim 4, the volume ratio of the whole blood sample to the Dextran solution is 4-5:

1.

9. The method for preparing the cell matrix sheet according to claim 3, characterized in that: In step A3, the mass ratio of methacrylic anhydride to cellulose is 0.8-1:50-52.

10. The method for preparing the cell matrix sheet according to claim 3, characterized in that: In step A4, the heating temperature is 90-100°C.