Tissue transparentizing reagent and use method thereof

Abstract

The invention belongs to the technical field of medical materials, and discloses a tissue transparentizing reagent and a use method thereof. The tissue transparentizing reagent comprises sulfur diethylene glycol, hydroxyethyl methylacrylate, a hardening agent and an initiator. The tissue transparentizing reagent provided by the invention has excellent optical clearing capability and good tissue clearing effect, realizes dual effects of tissue transparentizing and embedding, and is beneficial to fixation and imaging of biological tissues. The tissue transparentizing reagent provided by the invention has simple components, and takes thiodiglycol as a solvent and hydroxyethyl methylacrylate as a polymer monomer. Used reagents are non-toxic, do not harm health and do not have negative effects on the environment; and the preparation process is controllable, and the preparation and use methods are simple, green, safe and pollution-free. The formed tissue transparentizing gel is stable in storage and beneficial to research of biological tissues.

Description

Technical Field

[0001] The present invention relates to the technical field of medical materials, and specifically to a tissue clearing agent and a method for using the same. Background Art

[0002] Tissue clearing technology can achieve complete transparency of tissues or organs, making samples visually transparent. When combined with optical microscopy and fluorescent labeling technology, it can achieve three-dimensional reconstruction of tissue structure.

[0003] At present, hydrogel embedding transparent technology has attracted widespread attention for its ability to shape soft tissues. This technology is divided into active and passive types, but the formation of hydrogel and transparent operation are performed independently, and the ingredients are complex and the operation steps are relatively cumbersome. Therefore, it is of great significance to develop a transparent reagent with simple ingredients, easy operation, and the ability to quickly shape biological tissues. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a tissue clearing gel and a method for using the same. The tissue clearing reagent provided by the present invention has simple ingredients and is easy to use. It can simultaneously achieve the clearing and gelation of biological tissues, greatly shorten the experimental steps, simplify the operation process, and achieve a rapid shaping effect on biological tissues.

[0005] The present invention provides a tissue clearing agent.

[0006] Specifically, a tissue clearing agent includes thiodiglycol and hydroxyethyl methacrylate, a hardener and an initiator.

[0007] It should be understood that the tissue clearing reagent generally has its components packaged separately before use and mixed when used.

[0008] Preferably, the tissue clearing agent comprises, by mass percentage, 45% to 58% thiodiglycol, 41% to 54% hydroxyethyl methacrylate, 0.05% to 0.5% hardener and 0.1% to 0.8% initiator; further preferably, the tissue clearing agent comprises, by mass percentage, 46% to 55% thiodiglycol, 44% to 53% hydroxyethyl methacrylate, 0.1% to 0.5% hardener and 0.1% to 0.7% initiator.

[0009] Preferably, the initiator is an azo thermal initiator; further preferably, the initiator is selected from at least one of azobisisobutyronitrile (AIBN), azobisisoheptanenitrile (ABVN), azobisisobutyramidine hydrochloride (AIBA), and azobisisobutylimidazoline hydrochloride (AIBI).

[0010] Preferably, the hardener includes at least one of acrylic acid, methacrylic acid, divinylbenzene, N-hydroxymethyl acrylamide, diacetone acrylamide, polyethylene glycol diacrylate (PEGDA), and 1,4-butylene glycol diacrylate.

[0011] The invention also provides a method for using the tissue clearing reagent.

[0012] Specifically, a method for using a tissue clearing reagent comprises the following steps: Thiodiglycol and hydroxyethyl methacrylate, a hardener and an initiator are mixed to obtain a tissue clearing reagent (prepolymer solution); the fixed biological tissue is placed in the tissue clearing reagent, incubated, and a hydrogel-embedded clearing tissue is obtained.

[0013] Preferably, the incubation temperature is 25 to 50° C., and the incubation time is 6 to 48 hours.

[0014] During the heating and incubation process of the tissue clearing agent, the polymer monomer forms a stable three-dimensional network structure under the action of free radicals, forming a tissue clearing gel, which embeds the biological tissue. The tissue embedded in the hydrogel is shaped and transparent. When the hardener is ethylene glycol dimethacrylate, the resulting hydrogel structure is as follows:

[0015] Where 1≤x≤10, 1≤y≤3, 1≤m≤2, and n is 1 or 2.

[0016] Preferably, the tissue clearing agent forms a hydrogel during the incubation process, and the refractive index of the hydrogel is 1.505-1.550, the stretching ratio is 1.00-1.15, and the hardness is 95N-105N.

[0017] The tissue clearing reagent provided by the present invention uses thiodiglycol as a solvent and hydroxyethyl methacrylate as a polymer monomer, and by selecting a corresponding hardener, it can simultaneously achieve the transparency and gelation of biological tissues, greatly shorten the experimental steps, simplify the operation process, and have a rapid shaping effect on biological tissues. Among them, thiodiglycol is effective in tissue clearance and refractive index matching, can be applied to various applications and imaging techniques, is compatible with immunostaining, and can retain fluorescence without causing deformation at the subcellular level. Using thiodiglycol as a solvent, in the presence of a hardener, the hydroxyethyl methacrylate monomer is free radical polymerized, and the refractive index of the sample is homogenized by removing, replacing and modifying some components in the tissue, thereby making the biological tissue transparent. Further, a strong and abundant non-covalent cross-link is formed between the polymer chains, thereby forming a hard, tough and uniform network. The tissue is embedded in the hydrogel while being transparent, thereby forming a gel with strong mechanical properties.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The tissue clearing reagent provided by the present invention has excellent optical clearing ability and good tissue clearing effect, achieving the dual effects of tissue clearing and embedding, which is beneficial to the fixation and imaging of biological tissues.

[0019] (2) The tissue clearing reagent provided by the present invention has simple ingredients, with thiodiglycol as solvent and hydroxyethyl methacrylate as polymer monomer. The reagent used is non-toxic, does not harm health, and does not have a negative impact on the environment; and its preparation process is controllable, the preparation and use methods are simple, and it is green, safe and pollution-free. The tissue clearing gel formed is stable in storage, safe and reliable, and is conducive to the study of biological tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the SEM image of the tissue clearing gel of Example 1; Figure 2 This is an infrared characterization image of the prepolymer solution and gel of the tissue clearing gel in Example 1; Figure 3 This is a characterization diagram of the mechanical properties of the tissue clearing gel in Example 1; Figure 4 These are images of the tissue clearing gel of Example 1 when it was just prepared and 14 days after preparation; Figure 5 A general experimental procedure diagram of the optical clearing method of tissue clearing gel in Example 1; Figure 6 These are comparison diagrams of tissues before and after transparentization treatment using different transparentization methods in Example 1. DETAILED DESCRIPTION

[0021] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

[0022] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0023] Experiment 1: Study the effects of different solutions and polymer monomer types on the properties of tissue clearing gels.

[0024] The preparation of the gel is as follows: take the solvent and polymer monomer in Table 1, add 0.8% of the weight of the monomer as the initiator azobisisoheptanenitrile, add 0.2% of the weight of the monomer as the hardener methacrylic acid, mix well, and heat at 50°C for 6 hours. The transparency effect, refractive index, hardness and toughness of the gel are shown in Table 1. The transparency effect of the gel is observed by naked eyes; the transmission at a certain angle (including the minimum deflection angle of light) is measured using a goniometer-spectrometer; the hardness of the gel is measured using a texture analyzer; and the tensile properties of the gel are measured using a universal testing machine.

[0025] Table 1

[0026] As can be seen from Table 1, when using tissue clearing reagents to prepare tissue clearing gels, thiodiglycol and hydroxyethyl methacrylate cannot be replaced by other reagents. After thiodiglycol is replaced by water, methanol or ethylene glycol, the refractive index of the solution formed by the two is low and the matching degree of biological tissue is low, and the formed gel cannot be transparent to biological tissue. After hydroxyethyl methacrylate is replaced by other polymerizing monomers such as acrylamide and acrylate, the toughness and hardness of the formed gel cannot achieve tight embedding of tissues, causing tissue shrinkage or swelling. This is only because the polymer formed by hydroxyethyl methacrylate has a higher hardness and is suitable for tissue plasticity when combined with thiodiglycol; the polymer formed by acrylamide has a higher transparency, but has a higher toughness and is easy to stretch, which is not conducive to tissue plasticity; the polymer formed by acrylate has a certain degree of transparency, but does not match tissue transparency.

[0027] Experiment 2: Study the effects of TED, HEMA, hardener and initiator content on the properties of tissue clearing gel. The transparency effect, refractive index, hardness and toughness of the gel are shown in Table 2. The gel was prepared as follows: TED and HEMA in Table 1 were added with initiator azobisisoheptanenitrile and hardener methacrylic acid, mixed well, and heated at 50°C for 6h.

[0028] Table 2 Effects of different material mass ratios on the transparency, refractive index, hardness and toughness of the gel

[0029] As shown in Table 2, when the polymer monomer content is lower, the gel is softer, tougher, and has higher tensile strength, which is not conducive to tissue shaping. When the thiodiglycol content is lower, the solution content is relatively low, the refractive index is reduced, and the low transparency is not conducive to tissue transparency. When the initiator content is higher, the gel hardness is greater.

[0030] Example 1

[0031] A tissue clearing agent, composed of the following components by mass fraction: 46.8% hydroxyethyl methacrylate, 52.7% thiodiglycol; 0.4% initiator (azobisisoheptylnitrile); and 0.1% hardener (ethylene glycol dimethacrylate).

[0032] The method of preparing tissue clearing gel using the tissue clearing reagent comprises the following steps: Hydroxyethyl methacrylate, thiodiglycol, initiator and hardener were mixed evenly to obtain a tissue clearing agent (prepolymer solution); then heated at 50°C for 6 hours to obtain a tissue clearing gel. The prepared tissue clearing agent had high transparency, a refractive index of 1.5491, a hardness of 99.68N, and a stretching ratio of 1.05. Figure 1 This is a microscopic morphology characterization of tissue clearing gel. Figure 1 It can be seen that the gel structure is uniform and has many pores. Figure 2 This is the infrared characterization of the prepolymer solution and gel of the tissue clearing gel. In the FTIR spectrum of the prepolymer solution, the −1 The peak at 1422 cm-1 is attributed to the stretching of C=C. −1 and 650cm −1 The peak at 2921 cm is attributed to the C-H bending of the alkene group. In the FTIR spectrum of tissue clearing gel, the C-H stretching peak of the alkane group is 2921 cm −1 , the C−H bending peak is 1483 cm −1 , the C−C stretching peak is 1174 cm −1 , confirming that the prepolymer solution was copolymerized into copolymer. Figure 3 For example 2, mechanical properties of tissue clearing gel are characterized; Figure 3 It can be seen that HEMA polymer presents constant G′ (elastic modulus) and G″ (loss modulus) values ​​and the relationship of G′>G″ within the test temperature range. This shows that HEMA polymer has high elastic properties and stability. Figure 4 The images of tissue clearing gel just after preparation and 14 days after preparation. Figure 4 It also shows that the prepared tissue clearing gel has excellent stability.

[0033] The method for using the tissue clearing reagent comprises the following steps: Thiodiglycol and hydroxyethyl methacrylate, a hardener and an initiator are mixed to obtain a tissue clearing reagent (prepolymer solution); the fixed biological tissue is placed in the tissue clearing reagent, incubated, and a hydrogel-embedded clearing tissue is obtained.

[0034] The tissue clearing prepolymer solution (i.e., the mixed tissue clearing reagent) was used to treat the heart, liver, spleen, lung, and kidney of mice. The incubation temperature was 37°C, and the incubation time was: 16 hours for the heart of mice, 16 hours for the spleen of mice, 18 hours for the kidney of mice, 16 hours for the lung of mice, and 24 hours for the liver of mice. Figure 5 General experimental procedure for the tissue optical clearing method using tissue clearing gel. Figure 6 The above-mentioned tissues are compared before and after being treated with a transparent reagent. From left to right, they are the heart, liver, spleen, lung, and kidney of a mouse. The upper row of images are images before the tissue transparent reagent treatment. Due to the turbidity of the tissue, the grid below the tissue is covered by the tissue and cannot be seen; the lower row of images are images of the same tissue after being treated with a transparent reagent. At this time, the tissue becomes highly transparent, and the grid below the tissue is clearly visible (the background grid is 2mm*2mm). From the comparison of the pictures taken before and after transparentization by a stereoscope, it can be obtained that the tissue transparentizing reagent provided by the present invention can achieve a better transparent effect by simple immersion on the basis of defatting, and the tissue morphology remains unchanged, and can also achieve the transparentization of lipid-rich brain tissue.

[0035] Example 2

[0036] A tissue clearing agent, composed of the following components by mass fraction: 47.8% hydroxyethyl methacrylate, 51.8% thiodiglycol; 0.25% initiator (azobisisobutylimidazoline hydrochloride); and 0.15% hardener (ethylene glycol dimethacrylate).

[0037] The method of preparing tissue clearing gel using the tissue clearing reagent comprises the following steps: The above components were mixed evenly and heated at 45° C. for 4 hours to obtain a tissue clearing gel. The prepared tissue clearing agent had high transparency, a refractive index of 1.5490, a hardness of 103.38N, and a stretching ratio of 1.09.

[0038] The method for using the tissue clearing reagent comprises the following steps: Thiodiglycol and hydroxyethyl methacrylate, a hardener and an initiator are mixed to obtain a tissue clearing reagent (prepolymer solution); the fixed biological tissue is placed in the tissue clearing reagent, incubated, and a hydrogel-embedded clearing tissue is obtained.

[0039] Example 3

[0040] A tissue clearing agent, which is composed of the following components by mass fraction: 48.78% of hydroxyethyl methacrylate, 50.77% of thiodiglycol; 0.2% of initiator (azobisisoheptanenitrile); and 0.25% of hardener (1,4-butylene glycol diacrylate).

[0041] The method of preparing tissue clearing gel using the tissue clearing reagent comprises the following steps: The above components were mixed evenly and heated at 55° C. for 6 hours to obtain a tissue clearing gel. The prepared tissue clearing agent had high transparency, a refractive index of 1.5489, a hardness of 104.38N, and a stretching ratio of 1.10.

[0042] The method for using the tissue clearing reagent comprises the following steps: Thiodiglycol and hydroxyethyl methacrylate, a hardener and an initiator are mixed to obtain a tissue clearing reagent (prepolymer solution); the fixed biological tissue is placed in the tissue clearing reagent, incubated, and a hydrogel-embedded clearing tissue is obtained.

[0043] Example 4

[0044] A tissue clearing agent, which is composed of the following components by mass fraction: 49.7% hydroxyethyl methacrylate, 49.7% thiodiglycol; 0.5% initiator (azobisisoheptanenitrile); and 0.1% hardener (1,4-butylene glycol diacrylate).

[0045] The method of preparing tissue clearing gel using the tissue clearing reagent comprises the following steps: The above components were mixed evenly and heated at 70° C. for 3 hours to obtain a tissue clearing gel. The prepared tissue clearing agent had high transparency, a refractive index of 1.5486, a hardness of 104.45 N, and a stretching ratio of 1.13.

[0046] The method for using the tissue clearing reagent comprises the following steps: Thiodiglycol and hydroxyethyl methacrylate, a hardener and an initiator are mixed to obtain a tissue clearing reagent (prepolymer solution); the fixed biological tissue is placed in the tissue clearing reagent, incubated, and a hydrogel-embedded clearing tissue is obtained.

[0047] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A tissue clearing reagent, characterized in that: It includes thiodiglycol, hydroxyethyl methacrylate, a hardener and an initiator.

2. The tissue clearing reagent according to claim 1, characterized in that Calculated by mass percentage, it includes: 45% to 58% thiodiglycol, 41% to 54% hydroxyethyl methacrylate, 0.05% to 0.5% hardener and 0.1% to 0.8% initiator.

3. The tissue clearing reagent according to claim 2, characterized in that: Calculated by mass percentage, it includes: 46% to 55% thiodiglycol, 44% to 53% hydroxyethyl methacrylate, 0.1% to 0.5% hardener and 0.1% to 0.7% initiator.

4. The tissue clearing agent according to any one of claims 1 to 3, characterized in that The initiator is an azo thermal initiator.

5. The tissue clearing agent according to any one of claims 1 to 3, characterized in that The hardener includes at least one of acrylic acid, methacrylic acid, divinylbenzene, N-hydroxymethyl acrylamide, diacetone acrylamide, and polyethylene glycol diacrylate.

6. The method for using the tissue clearing agent according to any one of claims 1 to 5, characterized in that: The following steps are involved: Thiodiglycol and hydroxyethyl methacrylate, a hardener and an initiator are mixed to obtain a tissue clearing reagent; fixed biological tissue is placed in the tissue clearing reagent and incubated to obtain a hydrogel-embedded clearing tissue.

7. The method of use according to claim 6, characterized in that: The incubation temperature is 25 to 50° C., and the incubation time is 6 to 48 hours.

8. The method of use according to claim 6, characterized in that: When the hardener is ethylene glycol dimethacrylate, the resulting hydrogel structure is as follows: ； Where 1≤x≤10, 1≤y≤3, 1≤m≤2, and n is 1 or 2.

9. The method of use according to any one of claims 6 to 8, characterized in that: The tissue clearing agent forms a hydrogel during the incubation process, and the refractive index of the hydrogel is 1.505-1.550.

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