A transparent reagent for transparentizing artificial micro-organs and its application

Abstract

The present invention belongs to the field of biomedical optical imaging technology, and more specifically, relates to a transparent reagent for transparentizing artificial micro-organs and its application. The transparent reagent for transparentizing artificial micro-organs provided by the present invention includes m-phenylenediamine with hydration and degreasing effects, and also includes alcohols or sugar alcohols that can maintain the sample morphology, and the solvent is water. The fixed artificial micro-organs are placed in the transparent reagent of the present invention and fully soaked to make them transparent, and finally a highly transparent artificial micro-organ with a relatively uniform refractive index can be obtained. The artificial micro-organs treated by the present invention become highly transparent and do not change the sample size. They can be used for three-dimensional overall imaging of artificial micro-organs, thereby obtaining their high-resolution three-dimensional structural information.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical optical imaging, and more particularly, relates to a transparent reagent for transparentizing artificial micro-organs and application thereof. Background Art

[0002] Accurately characterizing artificial micro-organ models requires high-resolution capture of their overall three-dimensional structure, a prerequisite and foundation for conducting research such as drug screening. Modern optical imaging technology provides an important means for capturing the three-dimensional structure of tissues, but the high scattering properties of tissues themselves limit light penetration, thus affecting imaging depth.

[0003] The tissue optical clearing technology developed in recent years provides a solution to this problem. This technology uses physical or chemical means to reduce the scattering and absorption of light by tissues, increasing the depth of light penetration and thus improving the imaging depth. However, most existing tissue optical clearing methods are aimed at real tissues or organs of rodents. Artificial micro-organs formed by a large number of cell aggregations are relatively fragile. Samples are easily damaged or lost during operation. Maintaining the morphology of sample tissue is the basis for structural observation. Most of the optical clearing methods currently used for artificial micro-organs will cause severe deformation of the samples, and the processing time is long and the operation is complicated. To accurately obtain their internal structural information, it is urgent to develop a rapid and non-deformable tissue clearing method. Summary of the Invention

[0004] In response to the shortcomings of the prior art, the purpose of the present invention is to provide a transparent reagent for transparentizing artificial micro-organs and its application, so as to solve the technical problems that the existing optical transparent reagents and methods used in artificial micro-organs mostly cause serious deformation of the sample, have long processing time, and are complicated.

[0005] To achieve the above-mentioned object, the present invention provides a transparent reagent for transparentizing artificial micro-organs, comprising m-xylylenediamine having hydrating and degreasing effects, and alcohols and / or sugar alcohols capable of maintaining the sample morphology, wherein the solvent is water;

[0006] The volume percentage concentration of the m-xylylenediamine is 20%-40%, and the mass volume concentration of the alcohol and / or sugar alcohol substances capable of maintaining the sample morphology is 20%-40%.

[0007] Preferably, the alcohol substance capable of maintaining the sample form is glycerol; and the sugar alcohol substance capable of maintaining the sample form is sorbitol.

[0008] According to another aspect of the present invention, there is provided a use of the transparent reagent in the rapid invisible transparentization treatment of artificial micro-organs.

[0009] Preferably, the application comprises the following steps:

[0010] The fixed artificial micro-organ is immersed in the transparent reagent to match the refractive index of the components inside the micro-tissue, thereby finally obtaining a highly transparent artificial micro-organ.

[0011] Preferably, the artificial micro-organ is a three-dimensional cell culture tissue.

[0012] Preferably, the three-dimensional cell culture tissue is a tumor microtissue, a cardiosphere, a brain organoid or a liver organoid.

[0013] Preferably, the thickness of the artificial micro-organ is 100 micrometers to 3000 micrometers; and the immersion time is 10 minutes to 200 minutes.

[0014] Preferably, the fixed artificial micro-organ is an artificial micro-organ fixed with paraformaldehyde.

[0015] According to another aspect of the present invention, there is provided an application of the transparent artificial micro-organ obtained by the application process, which is used in confocal imaging, two-photon imaging or light-sheet fluorescence microscopy.

[0016] In general, the above technical solutions conceived by the present invention have the following beneficial effects compared with the prior art:

[0017] (1) The transparent reagent provided by the present invention for transparentizing artificial micro-organs includes m-xylenediamine, which has hydrating and degreasing effects and can loosen the tissue protein structure; and also includes an alcohol or sugar alcohol substance that can maintain the sample morphology. The solvent in the transparent reagent is water. The m-xylenediamine in the reagent has a high refractive index, low viscosity, and small molecular weight, and can quickly penetrate into the tissue to make the tissue highly transparent. When combined with an alcohol or sugar alcohol substance such as sorbitol and / or glycerol that can maintain the sample morphology, keep the sample size unchanged, and protect the sample fluorescence, the sample transparency effect can be further improved, making it possible to quickly and non-deformed (without shrinkage or expansion) transparent artificial micro-organ samples, greatly improving the sample imaging depth.

[0018] (2) The transparent treatment method for artificial micro-organs using the transparent reagent of the present invention adopts a single-step immersion method. The fixed artificial micro-organs are fully immersed in the transparent reagent. After a short incubation time, the sample can achieve high transparency. The transparent treatment method is simple and efficient.

[0019] (3) The fixed artificial micro-organ is fully immersed in the transparent reagent of the present invention to make it transparent, thereby obtaining a highly transparent and non-deformed tissue with a relatively uniform refractive index. The highly transparent and non-deformed tissue obtained by the transparent treatment method of the present invention can be used in confocal, two-photon, and light-sheet fluorescence microscopy, greatly improving the fluorescence imaging depth and achieving high-resolution three-dimensional imaging of the entire structure of the artificial micro-organ.

[0020] In summary, the transparentizing reagent and treatment method of the present invention have the following advantages: (i) small deformation; (ii) short transparent time and fast transparent speed; (iii) high transparency, which can achieve high-resolution three-dimensional overall structural imaging of multi-scale artificial micro-organs using an optical microscope; and (iiii) simple operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1a and Figure 1b These are direct images of the 300-micron HCT116 three-dimensional cell spheroids in Example 1 taken before and after light transparency treatment.

[0022] Figure 2a and Figure 2b This is a comparison of the fluorescence signals of the three-dimensional cell spheroids taken in Example 2 before and after the optical transparency treatment. Figure 2a This is the maximum projection image before light transparency processing; Figure 2b It is the maximum projection image after light transparency processing; Figure 2c Fluorescence imaging depth maps of the three-dimensional cell spheroids before and after optical clearing treatment.

[0023] Figure 3a and Figure 3b The following are direct images of the approximately 1 mm myocardial spheres in Example 3 taken before and after light clearing.

[0024] Figure 4 Fluorescence images at different depths obtained by light sheet illumination imaging of the transparent myocardial sphere in Example 4 are given.

[0025] Figure 5a and Figure 5b The three-dimensional fluorescence images and size change statistics of the cell spheroids before and after being transparentized by the prior art transparent reagent and the transparent reagent of the present invention in Comparative Example 1 are respectively given.

[0026] Figure 6 The fluorescence images of the three-dimensional cell spheroids in comparative example 2 before and after treatment with the prior art transparent reagent are given. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0028] Patent document CN109632420A discloses a method for rapid tissue clearing using a water-soluble reagent. The method involves multiple steps: first, swelling the tissue with a relatively low concentration of an aqueous solution of m-xylylenediamine and sorbitol, the tissue shrinking agents, m-xylylenediamine and sorbitol, and a phosphate buffer solution; and finally, refractive index matching with a relatively high concentration of an aqueous solution of m-xylylenediamine and sorbitol, the tissue undergoing refractive index matching, resulting in highly transparent tissue. The tissues targeted in this document include 1-2 mm thick mouse brain slices, whole mouse brain, mouse heart, liver, kidney, lung, spleen, stomach, intestine, or mouse embryos—these are the authentic rodent tissues or organs mentioned in the background of this invention. Direct use of the clearing reagent in this document to clear the artificial micro-organs formed by the aggregation or differentiation of a large number of cells in this invention revealed severe deformation of the artificial micro-organ samples. The inventors used only the intermediate-concentration clearing reagent described in the patent document to clear the artificial micro-organs of the present invention. However, experiments revealed that regardless of whether the concentrations of m-xylenediamine and sorbitol were increased or decreased, the artificial micro-organ samples shrank significantly. After extensive experimental research, the present inventors proposed a clearing reagent suitable for artificial micro-organs that rapidly and without sample deformation. The reagent comprises m-xylenediamine, which has hydrating and degreasing properties, and alcohols and / or sugar alcohols that maintain sample morphology. The solvent is water, i.e., water, such as deionized water, is used as the constant volume reagent. The volume concentration of the meta-xylylenediamine is 20%-40% (also expressed herein as 20 v / v%-40 v / v%, indicating that 20-40 ml of meta-xylylenediamine is contained per 100 ml of the transparent reagent). Preferably, the volume concentration of the meta-xylylenediamine is 30%-40%. The mass volume concentration of the alcohol and / or sugar alcohol capable of maintaining sample morphology is 20%-40% (also expressed herein as 20 w / v%-40 w / v%, indicating that 20-40 grams of the alcohol and / or sugar alcohol are contained per 100 ml of the transparent reagent). Experiments have found that using the transparent reagent of the present invention to transparentize artificial micro-organs can achieve high transparency in a short period of time without causing sample deformation. Analysis suggests that the multi-step treatment method described in the aforementioned literature, which involves expansion followed by contraction and then refractive index matching, is not suitable for the artificial micro-organs of the present invention. Replacing the phosphate in the tissue contraction agent described in the aforementioned literature with deionized water and adjusting the concentration to an appropriate range enables highly transparent artificial micro-organs while maintaining sample deformation. This may be due to the reduced osmotic pressure of the solvent, which avoids the problem of the high ion concentration in the transparent reagent extracting water from the tissue, leading to tissue contraction and deformation.

[0029] In some embodiments, the alcohol or sugar alcohol substance capable of maintaining the sample morphology is sorbitol and / or glycerol.

[0030] The method for making an artificial micro-organ transparent using the transparent reagent of the present invention comprises the following steps: thoroughly immersing the fixed artificial micro-organ in the transparent reagent to match the refractive indices of the various components within the tissue, thereby ultimately obtaining a highly transparent artificial micro-organ. The "components within the tissue" herein refer broadly to components such as between cells within a microtissue; between cells and interstitial fluid; between the cell membrane, cell fluid, organelles, and nucleus of a single cell; and may also refer to the spaces between water, proteins, lipids, and the like.

[0031] The artificial micro-organs to which the transparent reagent of the present invention is applicable can be artificial micro-organs of various scales, including but not limited to tumor microtissues, myocardial spheres, brain-like or liver-like organoids and other three-dimensional cell culture tissues.

[0032] In some embodiments, the thickness of the artificial micro-organs of the present invention ranges from 100 microns to 3000 microns, and the immersion time ranges from 10 minutes to 200 minutes. The treatment time for the tissue clearing agent can vary depending on the size of the tissue. For example, in some embodiments, thin samples (e.g., 200 microns to 300 microns) can be treated for 10 minutes to 15 minutes, while thicker samples (e.g., 800 microns to 1200 microns) require 1 hour to 3 hours of treatment.

[0033] In some embodiments, the fixed artificial tissue of the present invention is an artificial micro-organ fixed with paraformaldehyde.

[0034] Using tissue treated by the rapid non-transforming tissue clearing method of the artificial micro-organ of the present invention for confocal, two-photon or light-sheet fluorescence microscopy can greatly enhance the depth of fluorescence imaging and achieve high-resolution three-dimensional overall structural imaging of the artificial micro-organ.

[0035] The processing method of the present invention can render artificial micro-organs highly transparent in a short period of time, significantly improving the penetration depth of light within the artificial micro-organ model, thereby increasing imaging depth and providing an important tool for obtaining information about the three-dimensional overall structure of the artificial micro-organ. The method is fast, easy to operate, and the transparency effect is well compatible with fluorescence. Furthermore, the sample size remains unchanged after processing.

[0036] In order to make the above and other objects, features, and advantages of the present invention more clearly understood, several embodiments are given below to illustrate the rapid invisible transparentization method for artificial micro-organs and its applications described in the present invention.

[0037] The artificial micro-organs used in the embodiments of the present invention, such as HCT116 three-dimensional cell spheroids, are cultured and produced in accordance with existing methods. Cell suspensions (500,000 to 1,000,000 per milliliter) are added to a low-attachment multi-well plate, centrifuged, and then immersed in culture medium for culture. Cardiomyocyte spheroids can be purchased commercially (e.g., from the Suzhou Medical Device Research Institute of Southeast University).

[0038] Example 1

[0039] The artificial micro-organs in this example consisted of paraformaldehyde-fixed HCT116 3D cell spheroids approximately 300 microns thick. These spheroids were immersed in a clearing agent and incubated for 15 minutes. Images were taken before and after clearing using a USAF 1951 resolution plate. The clearing agent consisted of 40% m-xylenediamine (v / v), 30% sorbitol (w / v), and double-distilled water as the solvent.

[0040] Figure 1a and Figure 1b The following are direct images of 300 μm thick HCT116 3D cell spheroids before and after light clearing. Figure 1a This is the situation before transparent treatment. Due to the turbidity of the tissue, the USAF 1951 resolution plate line pairs below the tissue are completely obscured; Figure 1b This is the situation after optical clearing treatment. At this time, the tissue becomes highly transparent and the USAF 1951 resolution plate line pairs underneath the tissue are clearly visible.

[0041] Example 2

[0042] The artificial micro-organs in this example consisted of 300-micron-thick three-dimensional HCT116 cell spheroids carrying endogenous green fluorescent protein, fixed with paraformaldehyde. These spheroids were immersed in a clearing reagent and incubated for 15 minutes before confocal fluorescence imaging. The clearing reagent consisted of 40% m-xylylenediamine (v / v), 30% sorbitol (w / v), and double-distilled water.

[0043] Figure 2a 、 Figure 2b and Figure 2c The following are fluorescent images of the three-dimensional cell spheroids taken before and after the light transparency treatment according to Example 2 of the present invention. Figure 2a and Figure 2b This is a comparison of the fluorescence signals of the three-dimensional cell spheroids before and after light transparent treatment. Figure 2a is the maximum projection image before light transparency processing, Figure 2b It is the maximum projection image after light transparency processing; Figure 2cThe fluorescence depth images of the 3D spheroids captured before and after optical clearing were taken. The results show that tissue clearing did not affect the fluorescence signal of the green fluorescent protein expressed in the cells, and that the fluorescence imaging depth of the 3D spheroids under a confocal microscope was significantly increased after clearing.

[0044] Example 3

[0045] The artificial micro-organ in this example consisted of approximately 1mm thick myocardial spheres fixed with paraformaldehyde. The tissue was embedded in 0.6% (wt / vol) agarose and thoroughly immersed in a clearing agent for 3 hours. Images of the clearing agent were taken before and after the clearing process using a USAF 1951 resolution plate. The clearing agent consisted of 40% m-xylenediamine (v / v), 20% sorbitol (w / v), and double-distilled water as the solvent.

[0046] Figure 3a and Figure 3b The following are direct images of myocardial spheres taken before and after light clearing. Figure 3a This is the situation before transparent treatment. Due to the turbidity of the tissue, the USAF 1951 resolution plate line pairs below the tissue are completely obscured; Figure 3b This is the situation after optical clearing treatment. At this time, the tissue becomes highly transparent and the USAF 1951 resolution plate line pairs underneath the tissue are clearly visible.

[0047] Example 4

[0048] The artificial micro-organs in this example were 1mm thick myocardial spheres stained with PI. These were immersed in a clearing reagent and incubated for 3 hours before light-sheet imaging. The clearing reagent consisted of 40% m-xylenediamine (v / v), 20% sorbitol (w / v), and double-distilled water (DDI).

[0049] Figure 4 The fluorescence images of the transparent myocardial spheres obtained by light sheet illumination imaging are shown. It can be seen that after the light transparency treatment, the fluorescence signal in the deep layer of the myocardial sphere can also be detected, indicating that this method can realize the acquisition of the three-dimensional overall structural information of the myocardial spheres.

[0050] Example 5

[0051] The artificial micro-organs in this example consisted of paraformaldehyde-fixed HCT116 three-dimensional cell spheroids approximately 300 microns thick. These spheroids were immersed in a clearing agent and incubated for 15 minutes. Images were taken before and after clearing using a USAF 1951 resolution plate. The clearing agent consisted of 40% m-xylenediamine (v / v), 20% sorbitol (w / v), and 10% glycerol (w / v). The solvent was double-distilled water.

[0052] Experiments show that after optical transparency treatment, the 300-micron-thick HCT116 three-dimensional cell spheroid becomes highly transparent, and the USAF 1951 resolution plate line pairs underneath the tissue are clearly visible.

[0053] Comparative Example 1

[0054] The artificial micro-organ model of this comparative example is a 300-μm-thick three-dimensional HCT116 cell spheroid carrying endogenous green fluorescent protein. It was treated with the transparent reagents of CN109632420A (named MACS-R0, MACS-R1, and MACS-R2) and the transparent reagent of the present invention (named MACS-W), respectively. The specific steps are as follows:

[0055] Using the clearing method and clearing reagents described in CN109632420A: incubation in MACS-R0 (20 v / v% m-xylenediamine, 15 w / v% sorbitol, in water), MACS-R1 (40 v / v% m-xylenediamine, 30 w / v% sorbitol, in PBS), and MACS-R2 (40 v / v% m-xylenediamine, 50 w / v% sorbitol, in water) for 3 hours, 1 hour, and 1 hour, respectively. Samples cleared using the present invention's clearing reagent, MACS-W, were then immersed and incubated in the clearing reagent for 15 minutes. The clearing reagent is composed of 40 v / v% m-xylenediamine, 30 w / v% sorbitol, in water. Fluorescence imaging was then performed using a fluorescence zoom stereomicroscope.

[0056] Figure 5a The fluorescence images of three-dimensional cell spheroids before and after the transparent method and transparent reagent of CN109632420A and the light transparent treatment of the present invention are shown. The sample is severely deformed after the transparent method and transparent reagent of CN109632420A, and swells in MACS-R0 reagent.

[0057] Shrinkage occurs in the MACS-R2 reagent, while the sample size does not change after the transparent treatment of the present invention; Figure 5b This graph shows the size changes of three-dimensional spheroids before and after clearing with MACS (MACS-R0 - 3h, MACS-R1 - 1h, MACS-R2 - 1h) and the clearing reagent of the present invention (MACS-W - 15min). Three groups of three-dimensional spheroids were selected and their areas were measured before and after clearing. Clearing with the present invention revealed no change in the samples. The image "No clearing" indicates the initial sample image.

[0058] Comparative Example 2

[0059] Other conditions were the same as in Example 1, except that the aqueous solvent in the transparent reagent was replaced with 0.01 M phosphate buffer. The HCT116 three-dimensional cell spheroids with a thickness of approximately 300 μm from Example 1 were transparentized. It was found that the resulting HCT116 three-dimensional cell spheroids had severe shrinkage, with a shrinkage rate of approximately 77%.

[0060] Figure 6 Fluorescence images of the three-dimensional cell spheroids before and after treatment with the above-mentioned transparent reagents are shown. It can be seen that when phosphate buffer is used as the solvent, the three-dimensional cell spheroids will undergo obvious shrinkage.

[0061] The present invention's method for transparentizing artificial micro-organs employs a single-step immersion process, fully immersing the fixed artificial micro-organ model in a transparent reagent. The m-xylenediamine in the reagent has a high refractive index, low viscosity, and low molecular weight, allowing it to quickly penetrate tissues and react with proteins and lipids to produce a hydration reaction, thereby rendering the tissue transparent. The sorbitol in the reagent maintains sample morphology and protects fluorescence. The glycerol in the reagent has high biocompatibility and, when combined with other reagents, can further maintain sample size and enhance sample transparency. The present invention rapidly renders tissue transparent without deformation. The transparent reagent selected in the present invention's treatment method is prepared by introducing m-xylenediamine, which has a high refractive index and strong hydration properties, into the tissue. Sorbitol and glycerol maintain sample morphology and protect fluorescent proteins within the tissue. The transparent reagent is obtained by mixing them in proportion, thereby matching the refractive indices of the various components within the tissue, resulting in highly transparent tissue and improving imaging depth.

[0062] It will be easily understood by those skilled in the art that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of a transparent reagent in the rapid invisible transparentization treatment of artificial micro-organs, characterized in that: The transparent reagent is composed of m-xylylenediamine with hydration and degreasing effects, alcohols and sugar alcohols capable of maintaining the sample shape, and solvent water; The alcohol substance capable of maintaining the sample shape is glycerol; the sugar alcohol substance capable of maintaining the sample shape is sorbitol; The volume percentage concentration of the m-xylenediamine is 20%-40%, and the mass volume concentration of the alcohol and sugar alcohol substances capable of maintaining the sample morphology is 20%-40%; When the transparent reagent is used for transparentizing an artificial micro-organ, the method comprises the following steps: The fixed artificial micro-organ is immersed in the transparent reagent to match the refractive index of the components inside the micro-tissue, and finally a highly transparent artificial micro-organ is obtained; the artificial micro-organ is a three-dimensional cell culture tissue.

2. The use according to claim 1, characterized in that The three-dimensional cell culture tissue is tumor microtissue, cardiomyocyte sphere, brain-like organ or liver-like organ.

3. The use according to claim 1, characterized in that The thickness of the artificial micro-organ is 100 microns to 3000 microns; and the soaking time is 10 minutes to 200 minutes.

4. The use according to claim 1, wherein The fixed artificial micro-organ is an artificial micro-organ fixed with paraformaldehyde.

5. Use of the transparent artificial micro-organ obtained by the application treatment according to any one of claims 1 to 4, characterized in that: Used in confocal imaging, two-photon imaging, or light-sheet fluorescence microscopy.

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