Method for maintaining tissue activity by hydrogel

Abstract

The invention relates to the technical field of surgical biomedical materials, and provides a method for maintaining tissue activity by hydrogel, which comprises the following steps: S1, dissolving a photo-crosslinkable natural polymer material in a solvent, and standing to obtain a first mixed solution; s2, adding a photo-initiation cross-linking agent into the first mixed solution to obtain a photo-curing adhesive precursor solution; s3, pouring the photocuring adhesive precursor solution into a pore plate mold to enable the volume of the photocuring adhesive precursor solution to be matched with the size of the tissue; and S4, completely immersing the tissue into the photocuring glue precursor solution, and carrying out photocuring by adopting in-situ light irradiation to obtain the hydrogel wrapping the tissue. The hydrogel has the advantages that the hydrogel has excellent tissue fixing performance and biocompatibility, and the original activity of a sample is kept for slice preparation under the condition that the tissue sample is not dehydrated and inactivated.

Description

Technical Field

[0001] The present invention relates to the technical field of surgical biomedical materials, and more specifically, to a method for maintaining tissue activity using hydrogel. Background Art

[0002] In the 16th century, the invention of the microscope allowed humans to observe cells and tissue structures, opening up a new world in the field of histology. After nearly 400 years of development, the application scenarios of histological examinations are wide and diverse. It not only plays a core role in clinical diagnosis and treatment, but also provides important support for basic research and public health. Among them, tissue fixation technology is a crucial step in histological examination, which is related to the integrity of tissue structure and the stability of cells. At present, the most widely used tissue fixation technology is to maintain the stability of tissue structure through various chemical fixatives, such as formalin, paraformaldehyde and acetic acid. Although chemical tissue fixation plays an important role in histology, it inevitably requires the use of organic solvents such as alcohol and xylene, which may lead to tissue structure denaturation, loss of tissue antigenicity, and degradation of RNA and DNA.

[0003] Maintaining tissue cell activity is of great significance for the accuracy of histological examination, the effectiveness of clinical diagnosis, and the depth of basic research. For example, in tumor diagnosis, maintaining cell activity can help evaluate the proliferation ability and invasiveness of tumor cells, thereby providing more accurate diagnostic information and prognosis evaluation. In recent years, in order to preserve cell activity and morphological structure, rapid freezing sectioning technology has become increasingly popular. This technology can quickly make sections by rapidly freezing tissue samples at low temperatures while maintaining cell activity and structure. However, due to high technical requirements, fluctuations in sample quality, and long-term preservation issues, it is limited in conventional applications. Therefore, the development of a new generation of tissue fixation technology, aimed at effectively preserving cell activity and morphological structure, is of great significance for promoting the development of the field of biopsy. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for maintaining tissue activity using a hydrogel, which maintains the activity of tissue specimens and is simple and efficient.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is: to provide a method for maintaining tissue activity by hydrogel, comprising the steps of:

[0006] S1. dissolving several photo-crosslinkable natural polymer materials in a solvent and allowing to stand to obtain a first mixed solution;

[0007] S2. Adding a photoinitiator crosslinking agent to the first mixed solution to obtain a photocurable adhesive precursor solution;

[0008] S3. Pour the photocurable adhesive precursor solution into the well plate mold so that its volume is adapted to the size of the tissue;

[0009] S4. Completely immerse the tissue in the photocurable adhesive precursor solution and perform photocuring using in situ light irradiation to obtain a hydrogel that wraps the tissue.

[0010] Furthermore, in step S1, the photo-crosslinkable natural polymer material includes polyethylene glycol diacrylate PEGDA and methacryloyl gelatin GelMA.

[0011] Furthermore, in step S1, dissolving a plurality of photo-crosslinkable natural polymer materials in a solvent is specifically performed by first dissolving PEGDA in a solvent and then adding GelMA.

[0012] PEGDA is liquid and easily soluble. It is dissolved first and then solid GelMA is added. In one or more embodiments of the present invention, GelMA is dissolved at 37°C.

[0013] Furthermore, the content of PEGDA in the precursor solution is 5% to 10%. Preferably, the content of PEGDA is in the range of 7% to 8%. In one or more embodiments of the present invention, living tissue hydrogels with better mechanical strength can be obtained within this preferred range.

[0014] Furthermore, each 1 mL of photocurable adhesive precursor solution contains 50 to 100 μL of PEGDA, 50 to 100 mg of GelMA, and 5 to 10 μL of photoinitiator. Preferably, the volume ratio of PEGDA to photoinitiator is (5 to 20): 1, and 0.5 to 2 mg of photoinitiator is added to each 1 mL of photocurable adhesive precursor solution. In each 1 mL of photocurable adhesive precursor solution, PEGDA accounts for 5wt% to 10wt%, GelMA accounts for 5wt% to 10wt%, and photoinitiator accounts for 0.5wt% to 1wt%. In one or more embodiments of the present invention, each 1 mL of photocurable adhesive precursor solution contains 75 μL of PEGDA, 100 mg of GelMA, and 5 μL of photoinitiator.

[0015] Further, the photoinitiator crosslinking agent includes one or more of benzoylphenylphosphine oxide, ethyl 2,4,6-trimethylbenzoylphenylphosphonate, and 2-hydroxy-2-methyl-1-phenyl-1-propanone. Preferably, the photoinitiator is 2-hydroxy-2-methyl-1-phenyl-1-propanone.

[0016] Furthermore, in step S1, the ambient temperature of the static environment is 4-40°C, and the air humidity is 10%-100%. Preferably, the ambient temperature is 25°C, and the air humidity is 70%.

[0017] Furthermore, in step S1, the solvent is physiological saline or PBS buffer solution, and the dissolving is stirring dissolving, stirring until completely mixed.

[0018] In one or more embodiments of the present invention, the stirring and dissolving time is not less than 3 minutes.

[0019] Furthermore, in step S4, the ultraviolet light irradiation time is 10 to 15 minutes, preferably 10 minutes.

[0020] Furthermore, the area loading of the photocurable adhesive precursor solution is 100-1000 μL cm -2 .

[0021] Within a certain range, with the changes in the composition of the photocurable adhesive precursor solution and the light curing conditions, the tissue encapsulation performance and biocompatibility performance will have significant differences. At the same time, with the increase or decrease in the surface load of the precursor solution, the solution will also seep out of the PEGDA / GelMA tissue gel surface. Taking all factors into consideration, the present invention preferably limits the amount of PEGDA, GelMA, and photoinitiator to prepare the photocurable adhesive precursor solution, and according to 100 μL cm -2 A surface loading of 200 μg / ml was added to the composite solution and photocured for 10-15 min under aerobic conditions to obtain a better tissue hydrogel.

[0022] In one or more embodiments of the present invention, the tissue includes heart, artery, lung, stomach, intestine, liver, kidney and the like.

[0023] Compared with the prior art, the present invention has the following beneficial effects: the hydrogel has excellent tissue wrapping performance and biocompatibility, and maintains the original activity of the sample for slice preparation without dehydrating and inactivating the tissue sample. That is, the method of the present invention is used to wrap and fix the tissue, so that it has excellent slice performance and can maintain good activity, and the method is simple and efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the rheological curve of the PEGDA / GelMA gel-encapsulated tissue in Example 1;

[0025] Figure 2 This is the rheological curve of the PEGDA / GelMA gel-encapsulated tissue in Example 2;

[0026] Figure 3 This is the rheological curve of the PEGDA / GelMA gel-encapsulated tissue of Example 3;

[0027] Figure 4 This is the rheological curve of the PEGDA / GelMA gel-encapsulated tissue of Example 4;

[0028] Figure 5 This is the rheological curve of the PEGDA / GelMA gel-encapsulated tissue of Example 5;

[0029] Figure 6 This is a physical picture of the PEGDA / GelMA gel-encapsulated tissue of Example 1 ( Figure 6 a hydrogel, Figure 6 b Colon tissue hydrogel, Figure 6 c Liver tissue hydrogel, scale bar is 1 cm);

[0030] Figure 7 This is a tissue fluorescence image of the tissue slice of the PEGDA / GelMA gel-wrapped tissue of Example 1 after culturing for 24 hours ( Figure 7 a Colon tissue, Figure 7 b Liver tissue, scale bar is 50 μm);

[0031] Figure 8 This is the tissue fluorescence image of the tissue slice of the PEGDA / GelMA gel-wrapped tissue of Example 2 after culturing for 24 hours ( Figure 8 a Colon tissue, Figure 8 b Liver tissue, scale bar is 50 μm);

[0032] Fig. 9 This is the tissue fluorescence image of the tissue slice of the PEGDA / GelMA gel-wrapped tissue of Example 3 after culturing for 24 hours ( Fig. 9 a Colon tissue, Fig. 9 b Liver tissue, scale bar is 50 μm);

[0033] Fig.10 is a line graph of the swelling rate of Example 1;

[0034] Fig.11 is a line graph of the swelling rate of Example 2;

[0035] Fig.12 is a line graph of the swelling rate of Example 3;

[0036] Fig.13 is a line graph of the swelling rate of Example 4;

[0037] Fig.14 is a line graph of the swelling rate of Example 5;

[0038] Fig.15 This is a bar graph showing the biocompatibility of the PEGDA / GelMA gel-encapsulated tissue of Example 1. DETAILED DESCRIPTION

[0039] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0040] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0041] The present invention is now further described in conjunction with specific examples. The following examples are only for explaining the present invention, but do not constitute a limitation of the present invention. The test samples and test processes used in the following examples include the following (if the specific experimental conditions are not indicated in the examples, they are usually in accordance with conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples, if not otherwise specified, can all be obtained from commercial channels).

[0042] Example 1

[0043] This embodiment provides a method for maintaining tissue activity using a hydrogel, comprising the following steps:

[0044] (1) 75 μL of polyethylene glycol diacrylate PEGDA (average molecular weight ≈700, containing 100-300 ppm BHT stabilizer, MacLean, China) was dissolved in 800 μL of PBS buffer solution to obtain solution A;

[0045] (2) 100 mg of methacryloyl gelatin GelMA (GelMA DS90-20 g, Wenzhou Shuhe Biotechnology Co., Ltd., China) was added to solution A to obtain solution B; solution B was poured into a centrifuge tube and placed in a constant temperature water bath (digital display constant temperature water bath, Lichen Technology Co., Ltd., China) for incubation for 5 min, the water bath temperature was 37°C, the air humidity was 25%, and the GelMA was dissolved after incubation;

[0046] (3) Then, 5 μL of the photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone was added to solution B, and then PBS was added to balance to 1 mL to obtain solution C. Solution C was poured into a 24-well plate (the solution thickness was about 1 cm) and placed in room temperature air (humidity of 70% and temperature of 25°C);

[0047] (4) Dissect the fluorescent mice and obtain their liver and colon tissues. Cut the fresh liver and colon tissues into appropriate sizes and place them in solution C.

[0048] (5) Solution C was placed under an ultraviolet UV lamp (LED UV lamp AC85-260 v, Tiandou Lighting, China) and photocured for 10 min to obtain a PEGDA / GelMA hydrogel that encapsulated the tissue. The hydrogel was then sliced ​​using a pathology slicer or a vibrating microtome to test its slicing effect.

[0049] Example 2

[0050] This embodiment provides a method for maintaining tissue activity using a hydrogel, comprising the following steps:

[0051] (1) 50 μL of polyethylene glycol diacrylate PEGDA (average molecular weight ≈700, containing 100-300 ppm BHT stabilizer, MacLean, China) was dissolved in 800 μL PBS buffer solution to obtain solution A;

[0052] (2) 100 mg of methacryloyl gelatin GelMA (GelMA DS90-20 g, Wenzhou Shuhe Biotechnology Co., Ltd., China) was added to solution A to obtain solution B; solution B was poured into a centrifuge tube and placed in a constant temperature water bath (digital display constant temperature water bath, Lichen Technology Co., Ltd., China) for incubation for 5 min, the water bath temperature was 37°C, the air humidity was 25%, and the GelMA was dissolved after incubation;

[0053] (3) Then, 5 μL of the photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone was added to solution B, and then PBS was added to balance to 1 ml to obtain solution C, which was poured into a 24-well plate (the solution thickness was about 1 cm) and placed in room temperature air (humidity of 70% and temperature of 25°C);

[0054] (4) Cut fresh liver and colon tissue into appropriate sizes and place them in solution C;

[0055] (5) Solution C was placed under an ultraviolet UV lamp (LED UV lamp AC85-260 v, Tiandou Lighting, China) and photocured for 10 min to obtain a PEGDA / GelMA hydrogel that encapsulated the tissue. The hydrogel was then sliced ​​using a pathology slicer or a vibrating microtome to test its slicing effect.

[0056] Example 3

[0057] This embodiment provides a method for maintaining tissue activity using a hydrogel, comprising the following steps:

[0058] The steps include:

[0059] (1) 100 μL of polyethylene glycol diacrylate PEGDA (average molecular weight ≈700, containing 100-300 ppm BHT stabilizer, MacLean, China) was dissolved in 800 μL of PBS buffer solution to obtain solution A;

[0060] (2) 100 mg of methacryloyl gelatin GelMA (GelMA DS90-20 g, Wenzhou Shuhe Biotechnology Co., Ltd., China) was added to solution A to obtain solution B; solution B was poured into a centrifuge tube and placed in a constant temperature water bath (digital display constant temperature water bath, Lichen Technology Co., Ltd., China) for incubation for 5 min, the water bath temperature was 37°C, the air humidity was 25%, and the GelMA was dissolved after incubation;

[0061] (3) Then, 5 μL of the photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone was added to solution B, and then PBS was added to balance to 1 ml to obtain solution C. The mixed solution C was poured into a 24-well plate (the solution thickness was about 1 cm) and placed in room temperature air (humidity of 70% and temperature of 25°C);

[0062] (4) Cut fresh liver and colon tissue into appropriate sizes and place them in solution C;

[0063] (5) Solution C was placed under an ultraviolet UV lamp (LED UV lamp AC85-260 v, Tiandou Lighting, China) and photocured for 10 min to obtain a PEGDA / GelMA hydrogel that encapsulated the tissue. The hydrogel was then sliced ​​using a pathology slicer or a vibrating microtome to test its slicing effect.

[0064] Example 4

[0065] This embodiment provides a method for maintaining tissue activity using a hydrogel, comprising the following steps:

[0066] (1) 75 μL of polyethylene glycol diacrylate PEGDA (average molecular weight ≈700, containing 100-300 ppm BHT stabilizer, MacLean, China) was dissolved in 800 μL PBS buffer solution to obtain solution A;

[0067] (2) 50 mg of methacryloyl gelatin GelMA (GelMA DS90-20 g, Wenzhou Shuhe Biotechnology Co., Ltd., China) was added to solution A to obtain solution B; solution B was poured into a centrifuge tube and placed in a constant temperature water bath (digital display constant temperature water bath, Lichen Technology Co., Ltd., China) for incubation for 5 min. The water bath temperature was 37°C and the air humidity was 25%. After incubation, GelMA was dissolved;

[0068] (3) Then, 5 μL of the photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone was added to solution B, and then PBS was added to balance to 1 ml to obtain solution C. The mixed solution C was poured into a 24-well plate (the solution thickness was about 1 cm) and placed in room temperature air (humidity of 70% and temperature of 25°C);

[0069] (4) Cut fresh liver and colon tissue into appropriate sizes and place them in solution C;

[0070] (5) Solution C was placed under an ultraviolet UV lamp (LED UV lamp AC85-260 v, Tiandou Lighting, China) and photocured for 10 min to obtain a PEGDA / GelMA hydrogel that encapsulated the tissue. The hydrogel was then sliced ​​using a pathology slicer or a vibrating microtome to test its slicing effect.

[0071] Example 5

[0072] This embodiment provides a method for maintaining tissue activity using a hydrogel, comprising the following steps:

[0073] (1) 75 μL of polyethylene glycol diacrylate PEGDA (average molecular weight ≈700, containing 100-300 ppm BHT stabilizer, MacLean, China) was dissolved in 800 μL PBS buffer solution to obtain solution A;

[0074] (2) 100 mg of methacryloyl gelatin GelMA (GelMA DS90-20 g, Wenzhou Shuhe Biotechnology Co., Ltd., China) was added to solution A to obtain solution B; solution B was poured into a centrifuge tube and placed in a constant temperature water bath (digital display constant temperature water bath, Lichen Technology Co., Ltd., China) for incubation for 5 min at a water bath temperature of 37°C and an air humidity of 25% until GelMA was dissolved;

[0075] (3) Then, 10 μL of the photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone was added to solution B, and then PBS was added to balance to 1 ml to obtain solution C. The mixed solution C was poured into a 24-well plate (the solution thickness was about 1 cm) and placed in room temperature air (humidity of 70% and temperature of 25° C.);

[0076] (4) Cut fresh liver and colon tissue into appropriate sizes and place them in solution C;

[0077] (5) Solution C was placed under an ultraviolet UV lamp (LED UV lamp AC85-260 v, Tiandou Lighting, China) and photocured for 10 min to obtain a PEGDA / GelMA hydrogel that encapsulated the tissue. The hydrogel was then sliced ​​using a pathology slicer or a vibrating microtome to test its slicing effect.

[0078] Effect Example 1

[0079] [1] This example uses a rotational rheometer (Kinexus pro+) to measure the rheological parameters of PEGDA / GelMA hydrogel. The specific implementation method is: the gel sample is prepared into a round block (diameter 1-2cm), the frequency range is 1HZ, and the results of Examples 1 to 5 are as follows: Figures 1 to 5 As shown: PEGDA / GelMA tissue gel can exhibit stable gel properties.

[0080] Effect Example 2

[0081] [2] This example uses colon tissue and liver tissue to encapsulate tissue gel. The specific implementation method is: add colon tissue and liver tissue to the hydrogel photocurable precursor solution respectively, and perform UV light curing and cross-linking encapsulation. The results are as follows Figure 6 As shown: PEGDA / GelMA tissue gel can show good colon tissue ( Figure 6 b) and liver tissue ( Figure 6 c) Package performance.

[0082] Effect Example 3

[0083] [3] This example uses an automatic vibrating slicer (Leica VT1200s) to slice the gel of fluorescent mouse liver tissue and colon tissue. The specific implementation method is: cut the gel sample into cubes, the cutting frequency is 100Hz, the slice thickness is 100μm, the slice speed is 2.5mm / s, the sliced ​​tissue is placed in DMEM cell culture medium for culture, and after 24h of culture, it is observed using a multiphoton laser confocal microscope (FVMPE-RS). The results are shown in Figure 7-9 As shown: PEGDA / GelMA tissue gel can show good tissue encapsulation performance and can maintain colon tissue after slicing ( Figure 7 a. Figure 8 a. Fig. 9 a) and liver tissue ( Figure 7 b. Figure 8 b. Fig. 9 b) activity.

[0084] Effect Example 4

[0085] [4] This effect example uses a weighing method to measure the swelling rate of the PEGDA / GelMA tissue gel prepared by the preparation method provided in Examples 1 to 5. The specific test method is: weigh the initial mass of the PEGDA / GelMA tissue gel sample as w0, immerse it in deionized water at 37°C, and shake it at a speed of 120 rpm. After a predetermined time, weigh the weight of the PEGDA / GelMA tissue gel sample as w0. tThe swelling ratio Δw is calculated according to the following equation:

[0086]

[0087] The results of Example 1 to Example 5 are as follows: Figure 10 to Figure 14 As shown: PEGDA hydrogels all have a lower swelling rate and can better maintain the gel morphology. For example, the swelling rate of the PEGDA / GelMA tissue gel provided in Example 1 after being immersed in water for 3 days is 44.38%.

[0088] Effect Example 5

[0089] [5] This effect example uses CCK 8 kit to test the cytotoxicity of PEGDA / GelMA tissue gel prepared by the preparation method provided in Example 1. The results are as follows Fig.15 As shown: Compared with the control group, there was no significant difference in the proliferation of L929 fibroblasts in the PEGDA / GelMA tissue gel group on days 1, 2, and 3, demonstrating that the PEGDA / GelMA tissue gel has good biocompatibility.

[0090] [6] Obviously, the above embodiments of the present invention are examples for clearly illustrating the technical solutions of the present invention, and are not intended to limit the specific implementation methods of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the claims of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A method for maintaining tissue activity using a hydrogel, characterized in that: Includes steps: S1. dissolving several photo-crosslinkable natural polymer materials in a solvent and allowing to stand to obtain a first mixed solution; S2. Adding a photoinitiator crosslinking agent to the first mixed solution to obtain a photocurable adhesive precursor solution; S3. Pour the photocurable adhesive precursor solution into the well plate mold so that its volume is adapted to the size of the tissue; S4. Completely immerse the tissue in the photocurable adhesive precursor solution and perform photocuring using in situ light irradiation to obtain a hydrogel that wraps the tissue.

2. The method for maintaining tissue activity by hydrogel according to claim 1, characterized in that: In step S1, the photo-crosslinkable natural polymer material includes polyethylene glycol diacrylate PEGDA and methacryloyl gelatin GelMA.

3. The method for maintaining tissue activity by hydrogel according to claim 2, characterized in that: In step S1, a plurality of photo-crosslinkable natural polymer materials are dissolved in a solvent. Specifically, PEGDA is first dissolved in a solvent, and then GelMA is added.

4. The method for maintaining tissue activity using hydrogel according to claim 2, characterized in that: The concentration of the PEGDA is 5% to 10%.

5. The method for maintaining tissue activity using hydrogel according to claim 2, characterized in that: Each 1 mL of the photocurable adhesive precursor gas solution contains 50-100 μL of PEGDA, 50-100 mg of GelMA, and 5-10 μL of a photoinitiator.

6. The method for maintaining tissue activity using hydrogel according to claim 1, characterized in that: The photoinitiator crosslinking agent includes one or a combination of benzoylphenylphosphine oxide, ethyl 2,4,6-trimethylbenzoylphenylphosphonate, and 2-hydroxy-2-methyl-1-phenyl-1-propanone.

7. The method for maintaining tissue activity using hydrogel according to claim 1, characterized in that: In step S1, the static environment temperature is 37-40° C., and the air humidity is 10%-100%.

8. The method for maintaining tissue activity using hydrogel according to claim 1, characterized in that: In step S1, the solvent is physiological saline, PBS buffer solution or deionized water, and the dissolving is stirring dissolving until completely mixed.

9. The method for maintaining tissue activity using hydrogel according to claim 1, characterized in that: In step S4, the ultraviolet light irradiation time is 10 to 15 minutes.

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