Neutrophil matrix tablet and preparation method thereof

Abstract

The invention relates to a neutrophile granulocyte matrix sheet and a preparation method thereof. The neutrophile granulocyte matrix sheet comprises a modified cell matrix sheet and neutrophile granulocytes flatly laid on the modified cell matrix sheet, wherein the modified cell matrix sheet is obtained by reacting chitosan, modified sodium alginate and a silicon dioxide glass slide grafted with acrylic acid. The modified cell matrix sheet is prepared through reaction of the chitosan, the modified sodium alginate and the silicon dioxide glass slide grafted with the acrylic acid, a more stable and suitable culture environment is provided for the neutrophile granulocyte, and the survival rate and the functional stability of the neutrophile granulocyte are effectively improved. Compared with a traditional matrix material, the modified matrix can reduce cell apoptosis in long-term culture, significantly prolong the function retention time of neutrophils, optimize the adhesiveness and metabolic activity of neutrophils, and ensure the good state of cells in an in-vitro environment.

Description

Technical Field

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

[0002] With the continuous development of regenerative medicine and biomaterial technology, the application of cell matrix materials in tissue repair, drug screening and biological research has received widespread attention. Among them, neutrophils are an important part of the human immune system, and their functional stability is of great significance for studying immune response mechanisms, developing immunotherapy technologies and promoting tissue regeneration. However, neutrophils are easily affected by external conditions in an in vitro environment, and their survival rate, activity and functional stability are difficult to guarantee, which limits their application potential in the biomedical field.

[0003] In the prior art, cell matrix materials are usually based on natural or modified polysaccharides (such as chitosan, sodium alginate, etc.) as the main components. These matrix materials have good biocompatibility and low toxicity, but there is still a problem of insufficient physical and chemical stability of the matrix materials when used for neutrophil loading and preservation. Natural polysaccharide matrix materials have limited performance in terms of swelling, mechanical strength and degradation rate, and are prone to deformation or degradation during storage and operation, resulting in the stability of the cell microenvironment being difficult to maintain. In addition, the surface properties (such as hydrophilicity, surface energy, etc.) of ordinary matrix materials are difficult to meet the requirements of neutrophil attachment and survival, the cell attachment rate is low, and long-term cultivation is prone to loss of activity. Although the existing modification methods can improve cell attachment to a certain extent, they are still insufficient for maintaining the metabolic activity and functional performance of neutrophils.

[0004] The function of neutrophils depends on a specific microenvironment. Existing matrix materials are prone to affect the release or stability of certain cells when loaded with cells, thereby affecting the normal function of cells. Especially in culture systems containing complex cells, the compatibility problem between the matrix and cells is particularly prominent.

[0005] Therefore, it is necessary to develop a new technical solution to solve the deficiencies in the prior art. Summary of the invention

[0006] Based on this, the present invention prepares a modified cell matrix sheet by reacting chitosan, modified sodium alginate and a silica glass slide grafted with acrylic acid, which can significantly improve the physicochemical stability of the matrix, so that it can maintain structural integrity during operation and storage, and reduce the interference of the matrix to the neutrophil microenvironment. The adhesion ability of neutrophils is significantly improved by surface modification, and its survival microenvironment is optimized, effectively maintaining the activity and functional stability of the cells. In addition, the modified matrix sheet enhances the compatibility of the matrix with cells, so that it can better support the normal metabolism and immune function of neutrophils in complex culture systems.

[0007] One object of the present invention is to provide a neutrophil matrix sheet, the neutrophil matrix sheet comprising: a modified cell matrix sheet and neutrophils laid flat on the modified cell matrix sheet;

[0008] Wherein, the modified cell matrix sheet is obtained by the reaction of chitosan, modified sodium alginate and a silicon dioxide glass slide grafted with acrylic acid.

[0009] 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.

[0010] Furthermore, the red blood cell sedimentation buffer is selected from Dextran solution.

[0011] Furthermore, the red blood cell lysing solution is selected from ACK lysing solution.

[0012] Furthermore, the concentration of the Dextran solution is 5-12%.

[0013] Furthermore, the volume ratio of the whole blood sample to the Dextran solution is 4-5:1.

[0014] Furthermore, the viscosity of the chitosan is less than 200 mPa.s.

[0015] The present invention also provides a method for preparing the neutrophil matrix sheet, comprising the following steps:

[0016] A1. Soaking the silica slide in a sodium hydroxide solution, ultrasonically treating, and purifying to obtain a hydroxylated silica slide;

[0017] A2. The hydroxylated silica glass slide and acrylic acid are blended and heated to react to obtain a silica glass slide grafted with acrylic acid;

[0018] A3. Add allyl alcohol glycidyl ether to the sodium alginate solution and heat the reaction to obtain modified sodium alginate;

[0019] A4. Chitosan and modified sodium alginate are added to a solvent, and then a crosslinker and a photoinitiator are added, and the mixture is coated on a silica glass slide grafted with acrylic acid, and then irradiated with ultraviolet light to obtain a modified cell matrix sheet;

[0020] A5. Spread the neutrophils on the modified cell matrix sheet to obtain a neutrophil matrix sheet.

[0021] Furthermore, in step A2, the heating temperature is 50-70°C.

[0022] Furthermore, in step A3, the heating temperature is 70-75°C.

[0023] The present invention has the following beneficial effects:

[0024] The present invention provides a modified cell matrix sheet prepared by reacting chitosan, modified sodium alginate and a silica glass slide grafted with acrylic acid, which provides a more stable and suitable culture environment for neutrophils, and effectively improves the survival rate and functional stability of neutrophils. Compared with traditional matrix materials, the modified matrix can reduce cell apoptosis in long-term culture, significantly prolong the functional retention time of neutrophils, and optimize its adhesion and metabolic activity at the same time, ensuring the good state of cells in an in vitro environment. The modified matrix sheet makes it easier for neutrophils to adhere and maintain activity through the improvement of surface properties. The optimized matrix surface properties regulate the cell microenvironment, thereby promoting the survival and function of cells, and effectively supporting the immune response ability of neutrophils in vitro. The design of the material fully considers the compatibility with cells, and the modified matrix sheet can avoid the adverse interactions between traditional matrix materials and cells, and ensure the integrity of neutrophil function. The enhanced compatibility enables the matrix sheet to better support neutrophils in complex culture systems while maintaining their biological functions. DETAILED DESCRIPTION

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] Photoinitiator, 2-hydroxy-2-methylpropiophenone.

[0030] Cross-linking agent, N,N'-methylenebisacrylamide, MBA.

[0031] Example 1

[0032] A neutrophil matrix sheet, comprising: a modified cell matrix sheet and neutrophils spread on the modified cell matrix sheet;

[0033] 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.

[0034] The preparation method of the neutrophil matrix sheet comprises the following steps:

[0035] 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;

[0036] 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;

[0037] A3. 10 g of sodium alginate was added to 300 ml of deionized water, and the pH was adjusted to 8.5 using a 50 wt % sodium hydroxide solution. Then 8 g of allyl alcohol glycidyl ether was added, and the reaction was heated to 75 ° C for 8 h. After cooling, the pH of the solution was adjusted to 4, acetone was added, and the solution was allowed to stand for 12 h. After precipitation, it was filtered and dried to obtain modified sodium alginate;

[0038] A4. Add 5 g of chitosan to 100 ml of 1 wt% acetic acid solution, stir evenly, add 20 g of modified sodium alginate, then add 0.05 g of photoinitiator and 0.05 g of cross-linking agent, stir evenly, apply on a silica glass slide grafted with acrylic acid, irradiate under ultraviolet light for 10 min, wash, and obtain a modified cell matrix sheet;

[0039] A5. Spread the neutrophils on the modified cell matrix sheet to obtain a neutrophil matrix sheet.

[0040] Example 2

[0041] A neutrophil matrix sheet, comprising: a modified cell matrix sheet and neutrophils spread on the modified cell matrix sheet;

[0042] 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 12:1), 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.

[0043] The preparation method of the neutrophil matrix sheet comprises the following steps:

[0044] 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;

[0045] 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;

[0046] A3. 10 g of sodium alginate was added to 300 ml of deionized water, and the pH was adjusted to 8.5 using a 50 wt % sodium hydroxide solution. Then 8 g of allyl alcohol glycidyl ether was added, and the reaction was heated to 75 ° C for 8 h. After cooling, the pH of the solution was adjusted to 4, acetone was added, and the solution was allowed to stand for 12 h. After precipitation, it was filtered and dried to obtain modified sodium alginate;

[0047] A4. Add 5 g of chitosan to 100 ml of 1 wt% acetic acid solution, stir evenly, add 20 g of modified sodium alginate, then add 0.05 g of photoinitiator and 0.05 g of cross-linking agent, stir evenly, apply on a silica glass slide grafted with acrylic acid, irradiate under ultraviolet light for 10 min, wash, and obtain a modified cell matrix sheet;

[0048] A5. Spread the neutrophils on the modified cell matrix sheet to obtain a neutrophil matrix sheet.

[0049] Example 3

[0050] A neutrophil matrix sheet, comprising: a modified cell matrix sheet and neutrophils spread on the modified cell matrix sheet;

[0051] The neutrophil extraction method comprises the following steps: adding ml of whole blood sample into a centrifuge tube, adding 5wt% Dextran solution (the volume ratio of the whole blood sample to the Dextran solution is 6:1), leaving it to stand at room temperature for 30 minutes, transferring the supernatant into a centrifuge tube containing 10ml of PBS buffer, centrifuging at 1000rpm for 5 minutes, discarding the supernatant, adding 3ml of PBS buffer for resuspending, then adding ACK lysis buffer to lyse red blood cells (the volume ratio of the ACK lysis buffer to the resuspending liquid is 15:1), mixing for 5 minutes, centrifuging at 1000rpm for 5 minutes, pouring out the supernatant and resuspending it with 1ml of PBS, blowing and collecting cells again, centrifuging at 1000rpm, and obtaining neutrophils after resuspending.

[0052] The preparation method of the neutrophil matrix sheet comprises the following steps:

[0053] 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;

[0054] 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;

[0055] A3. 10 g of sodium alginate was added to 300 ml of deionized water, and the pH was adjusted to 8.5 using 45 wt % sodium hydroxide solution. Then 8 g of allyl alcohol glycidyl ether was added, and the reaction was heated to 70 ° C for 10 h. After cooling, the pH of the solution was adjusted to 4, acetone was added and allowed to stand for 12 h. After precipitation, the solution was filtered and dried to obtain modified sodium alginate;

[0056] A4. Add 5 g of chitosan to 100 ml of 1 wt% acetic acid solution, stir evenly, add 20 g of modified sodium alginate, then add 0.05 g of photoinitiator and 0.05 g of cross-linking agent, stir evenly, apply on a silica glass slide grafted with acrylic acid, irradiate under ultraviolet light for 10 min, wash, and obtain a modified cell matrix sheet;

[0057] A5. Spread the neutrophils on the modified cell matrix sheet to obtain a neutrophil matrix sheet.

[0058] Comparative Example 1

[0059] A neutrophil matrix sheet. The difference between this comparative example and Example 1 is that step A3 is omitted, and in step A4, the modified sodium alginate is removed and replaced with chitosan of equal quality, and the other components and preparation methods are the same.

[0060] Test Case

[0061] The neutrophil uniformity of the neutrophil matrix sheets prepared in Example 1 and Comparative Example 1 was tested

[0062] 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.

[0063] Table 1 Performance test results of neutrophil matrix sheets of Example 1 and Comparative Example 1

[0064] project Example 1 Comparative Example 1 Uniformity % 86 78

[0065] It can be concluded from Table 1 that the neutrophil 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.

[0066] 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.

[0067] 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 neutrophil matrix sheet, characterized in that: The neutrophil 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 the reaction of chitosan, modified sodium alginate and a silicon dioxide glass slide grafted with acrylic acid.

2. The neutrophil 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.

3. The neutrophil matrix sheet according to claim 2, characterized in that: The erythrocyte sedimentation buffer is selected from Dextran solution.

4. The neutrophil matrix sheet according to claim 2, characterized in that: The red blood cell lysing solution is selected from ACK lysing solution.

5. The neutrophil matrix sheet according to claim 2, wherein the concentration of the Dextran solution is 5-12%.

6. According to the neutrophil matrix sheet of claim 2, the volume ratio of the whole blood sample to the Dextran solution is 4-5:

1.

7. The neutrophil matrix sheet according to claim 1, characterized in that: The viscosity of the chitosan is <200 mPa.s.

8. The method for preparing the neutrophil matrix sheet according to any one of claims 1 to 7, 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 silica glass slide grafted with acrylic acid; A3. Add allyl alcohol glycidyl ether to the sodium alginate solution and heat the reaction to obtain modified sodium alginate; A4. Chitosan and modified sodium alginate are added to a solvent, and then a crosslinker and a photoinitiator are added, and the mixture is coated on a silica glass slide grafted with acrylic acid, and then irradiated with ultraviolet light to obtain a modified cell matrix sheet; A5. Spread the neutrophils on the modified cell matrix sheet to obtain a neutrophil matrix sheet.

9. The method for preparing the neutrophil matrix sheet according to claim 8, characterized in that: In step A2, the heating temperature is 50-70°C.

10. The method for preparing the neutrophil matrix sheet according to claim 8, characterized in that: In step A3, the heating temperature is 70-75°C.