CN115655834A - Transparent reagent for transparentizing artificial micro-organs and application thereof - Google Patents
Transparent reagent for transparentizing artificial micro-organs and application thereof Download PDFInfo
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- CN115655834A CN115655834A CN202211121775.5A CN202211121775A CN115655834A CN 115655834 A CN115655834 A CN 115655834A CN 202211121775 A CN202211121775 A CN 202211121775A CN 115655834 A CN115655834 A CN 115655834A
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Abstract
The invention belongs to the technical field of biomedical optical imaging, and particularly relates to a transparent reagent for transparentizing an artificial micro-organ and application thereof. The transparent reagent for transparentizing the artificial micro-organ comprises m-xylylenediamine with hydration and degreasing functions, and also comprises alcohol or sugar alcohol substances capable of maintaining the shape of a sample, wherein the solvent is water. The fixed artificial micro-organ is placed into the transparent reagent of the invention to be fully soaked for transparentization, and finally, the highly transparent artificial micro-organ with relatively uniform refractive index can be obtained. The artificial micro-organ processed by the method becomes highly transparent, the size of the sample cannot be changed, and the method can be used for three-dimensional integral imaging of the artificial micro-organ so as to obtain high-resolution three-dimensional structure information of the artificial micro-organ.
Description
Technical Field
The invention belongs to the technical field of biomedical optical imaging, and particularly relates to a transparent reagent for transparentizing an artificial micro-organ and application thereof.
Background
Accurate description of the model features of the artificial micro-organ requires high-resolution acquisition of the three-dimensional overall structure of the artificial micro-organ, which is the premise and basis for developing related studies such as drug screening. Modern optical imaging techniques provide an important means for obtaining three-dimensional structures of tissues, but the high scattering properties of the tissues themselves limit the penetration of light into the tissues, which in turn affects the imaging depth.
The recently developed tissue light transparent technology provides a solution to this problem by reducing the scattering and absorption of light by tissue by physical or chemical means, increasing the depth of penetration of light, and thus increasing the imaging depth. However, most of the existing tissue light-transparent methods are directed at real tissues or organs of rodents, and artificial micro-organs formed by gathering a large number of cells are fragile, so that samples are easily damaged or lost in the operation process, and the maintenance of tissue forms of the samples is the basis of structural observation. Most of the existing light transparency methods applied to artificial micro-organs can cause serious deformation of samples, have long processing time and complicated operation, and the development of a rapid deformation-free tissue transparency method is urgently needed to accurately obtain internal structure information.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a transparent reagent for transparently processing an artificial micro-organ and application thereof, so as to solve the technical problems that most of light transparent reagents and methods applied to the artificial micro-organ in the prior art can seriously deform a sample, have long processing time, are complicated to operate and the like.
In order to achieve the above objects, the present invention provides a transparent reagent for transparently treating an artificial micro-organ, comprising m-xylylenediamine having hydration and degreasing effects, and further comprising an alcohol and/or sugar alcohol substance capable of maintaining the form of a sample, wherein the solvent is water;
wherein the concentration of the m-xylylenediamine is 20-40% by volume, and the mass concentration of the alcohols and/or sugar alcohols capable of maintaining the sample morphology is 20-40% by volume.
Preferably, the alcohol capable of maintaining the sample morphology is glycerol; the sugar alcohol substance capable of maintaining the form of the sample is sorbitol.
According to another aspect of the invention, the application of the transparent reagent in the rapid deformation-free transparentizing treatment of the artificial micro-organ is provided.
Preferably, the application comprises the following steps:
and soaking the fixed artificial micro-organ in the transparent reagent to match the refractive indexes of the components in the micro-tissue, thereby finally obtaining the highly transparent artificial micro-organ.
Preferably, the artificial micro-organ is a three-dimensional cell culture tissue.
Preferably, the three-dimensional cell culture tissue is a tumor micro-tissue, a cardiac spheroid, a brain-like or a liver-like organ.
Preferably, the thickness of said artificial micro-organ is between 100 and 3000 microns; the soaking time is 10min-200min.
Preferably, the fixed artificial micro-organ is a paraformaldehyde fixed artificial micro-organ.
According to another aspect of the invention, the application of the transparent artificial micro-organ obtained by the application treatment is provided, and the transparent artificial micro-organ is used for confocal imaging, two-photon imaging or light sheet fluorescence microscopic imaging.
Generally, compared with the prior art, the technical scheme conceived by the invention has the following beneficial effects:
(1) The transparent reagent for transparentizing the artificial micro-organ, provided by the invention, comprises m-xylylenediamine with hydration and degreasing effects, and the m-xylylenediamine can loosen a tissue protein structure; the reagent also comprises alcohol or sugar alcohol substances capable of maintaining the sample form, and the solvent in the transparent reagent is water. M-xylylenediamine in the reagent has high refractive index, low viscosity and small molecular weight, can quickly permeate into tissues to ensure that the tissues are highly transparent, and can further improve the transparent effect of the samples by matching with alcohols or sugar alcohols such as sorbitol and/or glycerol which can maintain the shapes of the samples, keep the sizes of the samples unchanged and protect the fluorescence of the samples, so that the quick and deformation-free (shrinkage-free or expansion-free) transparency of the artificial micro-organ samples becomes possible, and the imaging depth of the samples is greatly improved.
(2) The method for performing transparent treatment on the artificial micro-organ by adopting the transparent reagent adopts single-step soaking, the fixed artificial micro-organ is fully soaked in the transparent reagent, and the sample can reach high transparency after being incubated for a short time, and the transparent treatment method is simple and efficient.
(3) The fixed artificial micro-organ is fully soaked in the transparent reagent to be transparent, so that the highly transparent and deformation-free tissue with relatively uniform refractive index is obtained. The highly transparent and deformation-free tissue obtained by the transparent processing method can be used in confocal, two-photon and light sheet fluorescence microscopic imaging, can greatly improve the fluorescence imaging depth, and realizes high-resolution three-dimensional integral structure imaging of the artificial micro-organ.
In summary, the transparentizing agent and the transparentizing method of the present invention have the following advantages: (i) less deformation; (ii) the transparent time is short, and the transparent speed is high; (iii) The high transparency, the high resolution three-dimensional integral structure imaging of the multi-scale artificial micro-organ can be realized by using an optical microscope; (iiii) simple and convenient to handle.
Drawings
FIGS. 1a and 1b are visual images taken before and after light clearing of 300 μm HCT116 three-dimensional cell spheres of example 1.
FIGS. 2a and 2b are graphs comparing fluorescence signals of three-dimensional cytospheres taken in example 2 before and after light-clearing treatment, and FIG. 2a is a maximum value projection image before light-clearing treatment; FIG. 2b is a maximum projection image after light transparency processing; FIG. 2c is a fluorescence imaging depth map of the three-dimensional cell pellet before and after the light-clearing treatment.
Fig. 3a and 3b show visual images taken before and after the light-transparentization of the myocardial ball of about 1mm in example 3.
FIG. 4 shows fluorescence images of different depths obtained by transillumination imaging of the myocardium ball after example 4 is transparent.
FIGS. 5a and 5b show fluorescence images and size variation statistics of three-dimensional cytospheres before and after the clearing of the prior art clearing reagent and the clearing reagent of the present invention in comparative example 1, respectively.
FIG. 6 shows the fluorescence of the three-dimensional cell pellet of comparative example 2 before and after treatment with the prior art clearing reagent.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Patent document CN109632420A discloses a method for rapid tissue transparency treatment based on water-soluble reagents, which includes multiple steps, first performing swelling treatment on the tissue by using aqueous solution of m-xylylenediamine and sorbitol as tissue swelling treatment agents with low concentration, then performing shrinkage treatment on the tissue by using m-xylylenediamine and sorbitol as tissue shrinkage agents with medium concentration and phosphate buffer, and finally performing refractive index matching on the tissue by using aqueous solution of m-xylylenediamine and sorbitol as refractive index matching agents with high concentration to obtain highly transparent tissue. The tissues targeted by this document include mouse 1-2mm thick brain sections, mouse whole brain, mouse heart, liver, kidney, lung, spleen, stomach, intestine, or mouse embryo, which are the actual tissues or organs of rodents mentioned in the background of the invention. The transparent reagent of the document is directly adopted to carry out transparentization treatment on the artificial micro-organ formed by aggregation or differentiation of a large number of cells, and experiments show that the artificial micro-organ sample is seriously deformed. The inventors of the present invention carried out the transparentization treatment of the artificial micro-organ only with the transparent reagent of a medium concentration in the intermediate step of the patent document, but found that the artificial micro-organ sample was significantly contracted regardless of whether the concentrations of m-xylylenediamine and sorbitol were adjusted to be high or low. Through a large number of experiments, the invention provides a transparent reagent which is suitable for artificial micro-organs and is rapid and free from sample deformation, and the transparent reagent comprises m-xylylenediamine with hydration and degreasing effects, and also comprises alcohols and/or sugar alcohols capable of maintaining the sample form, wherein the solvent is water, namely water such as deionized water is used as a constant volume reagent. Wherein the concentration of m-xylylenediamine is 20% to 40% by volume (also referred to herein as 20v/v% to 40v/v%, which means 20 to 40 ml of m-xylylenediamine per 100 ml of the clearing reagent), preferably 30% to 40% by volume; the mass volume concentration of the alcohol and/or sugar alcohol substance capable of maintaining the sample morphology is 20-40% (also expressed as 20 w/v-40 w/v%, and expressed as 20-40 g per 100 ml of the transparent reagent). Experiments show that the transparent reagent provided by the invention is used for performing transparentization treatment on the artificial micro-organ, so that the artificial micro-organ can reach high transparency in a short time, and a sample cannot be deformed. For analytical reasons, it may be that the multi-step processing method of expansion followed by contraction followed by refractive index matching in the above-mentioned documents is not applicable to the artificial micro-organ of the present invention; the phosphate in the tissue shrinking agent in the above document is replaced by deionized water, and the appropriate concentration range is adjusted to highly transparent the artificial micro-organ and keep the sample from deforming, which may be caused by reducing the osmotic pressure of the solvent, and avoid the problem of tissue shrinkage deformation caused by extracting water from the tissue due to high ion concentration in the transparent agent.
In some embodiments, the alcohol or sugar alcohol capable of maintaining the morphology of the sample is sorbitol and/or glycerol.
The transparent method for carrying out the artificial micro-organ by adopting the transparent reagent comprises the following steps: and fully soaking the fixed artificial micro-organ in the transparent reagent to match the refractive indexes of the components in the tissue, and finally obtaining the highly transparent artificial micro-organ. The term "tissue-internal components" as used herein refers to a broad range of components, such as cells in a microtissue; or between cells and interstitial fluid; or between the cell membrane, cytosol, organelle, nucleus of a single cell, and may also refer to water, protein, lipid, etc.
The artificial micro-organ to which the transparent reagent is applied can be an artificial micro-organ with various scales, including but not limited to a plurality of three-dimensional cell culture tissues such as tumor micro-tissues, myocardial balls, brain-like or liver-like organs and the like.
In some embodiments, the thickness of the artificial micro-organ of the invention is between 100 microns and 3000 microns; the soaking time is 10min-200min. The treatment time of the tissue clearing reagent can be determined according to the tissue size, for example, in some embodiments, a thin sample (e.g., 200 microns to 300 microns) is treated for 10min to 15min, and a thicker sample (e.g., 800 microns to 1200 microns) is treated for 1h to 3h.
In some embodiments, the fixed artificial tissue of the invention is a paraformaldehyde fixed artificial micro-organ.
The tissue processed by the rapid non-deformation tissue transparentization method of the artificial micro-organ is used for confocal, two-photon or light sheet fluorescence microscopic imaging, so that the fluorescence imaging depth can be greatly improved, and the high-resolution three-dimensional integral structure imaging of the artificial micro-organ can be realized.
After the artificial micro-organ is processed by the processing method provided by the embodiment of the invention, the tissue can become highly transparent in a short time, and the penetration depth of light in the artificial micro-organ model is greatly improved, so that the imaging depth is improved, and an important tool is provided for acquiring the three-dimensional integral structure information of the artificial micro-organ. The method has the advantages of high speed, simple and convenient operation, good compatibility of the transparent effect and the fluorescence, and no change of the size of the processed sample.
In order to make the aforementioned and other objects, features and advantages of the present invention comprehensible, several embodiments accompanied with the present invention are described below to illustrate the method for rapid non-deformation transparentization for artificial micro-organs and the application thereof.
The artificial micro-organ used in the embodiment of the invention, such as HCT116 three-dimensional cell balls, is cultured and manufactured by the method of the prior art, and only needs to add cell suspension (50-100 ten thousand per milliliter) into a low-attachment multi-hole plate, centrifuge and then soak the cell suspension in culture solution for culture; the cardiomyocytes were obtained from commercial sources (e.g., the Suzhou institute of medical instrumentation, university of southeast).
Example 1
The artificial micro-organ is HCT116 three-dimensional cell balls fixed by paraformaldehyde and with the thickness of about 300 microns, and the artificial micro-organ is fully soaked in a transparent reagent to be incubated for 15min, and transparent front and back visual images are shot by combining a USAF 1951 resolution plate. The transparent reagent comprises the following components: the concentration of m-xylylenediamine was 40v/v%, the concentration of sorbitol was 30w/v%, and the solvent was double distilled water.
FIGS. 1a and 1b show visual images taken before and after light clearing of 300 micron thick HCT116 three-dimensional cell spheres. Wherein FIG. 1a is the condition before transparency treatment, the USAF 1951 resolution plate line pair under the tissue is completely covered due to the turbid nature of the tissue; FIG. 1b shows the situation after the light transparency process, where the tissue has become highly transparent and the USAF 1951 resolution plate line below the tissue is clearly visible.
Example 2
In this example, the artificial micro-organ is a 300 μm thick HCT116 three-dimensional cell pellet fixed by paraformaldehyde and carrying endogenous green fluorescent protein, and is fully immersed in a transparent reagent to incubate for 15min, followed by confocal fluorescence imaging. The transparent reagent comprises the following components: the concentration of m-xylylenediamine was 40v/v%, the concentration of sorbitol was 30w/v%, and the solvent was double distilled water.
FIGS. 2a, 2b and 2c show fluorescence images of three-dimensional cell spheres taken before and after the cell spheres are subjected to light-clearing treatment according to example 2 of the present invention. Wherein, FIGS. 2a and 2b are fluorescence signal comparison graphs before and after light clearing treatment of the three-dimensional cell ball, FIG. 2a is a maximum value projection image before light clearing treatment, and FIG. 2b is a maximum value projection image after light clearing treatment; FIG. 2c is a depth map of fluorescence imaging of the three-dimensional cell pellet taken before and after the light-clearing treatment. The result shows that the tissue is subjected to transparent treatment without influencing the fluorescent signal of green fluorescent protein expressed in cells, and the fluorescent imaging depth of the transparent three-dimensional cell spheres under a confocal microscope is greatly increased.
Example 3
The artificial micro-organ of this example is a myocardial ball of about 1mm thickness fixed by paraformaldehyde, the tissue was embedded using agarose at a concentration of 0.6% (wt/vol) and incubated in a transparent reagent for 3h, and a transparent front and back visual image was taken in combination with a USAF 1951 resolution plate. The transparent reagent comprises the following components: the concentration of m-xylylenediamine was 40v/v%, the concentration of sorbitol was 20w/v%, and the solvent was double distilled water.
Fig. 3a and 3b show visual images taken before and after the myocardial ball is subjected to light transparency processing. Wherein FIG. 3a is a condition before transparency treatment, the USAF 1951 resolution plate line pair under the tissue is completely covered due to the turbid nature of the tissue; FIG. 3b shows the case after light transparency treatment, when the tissue becomes highly transparent and the USAF 1951 resolution plate line below the tissue is clearly visible.
Example 4
The artificial micro-organ is a 1mm thick myocardial ball dyed by PI, and is fully soaked in a transparent reagent for incubation for 3 hours and then subjected to light sheet illumination imaging. The transparent reagent comprises the following components: the concentration of m-xylylenediamine was 40v/v%, the concentration of sorbitol was 20w/v%, and the solvent was double distilled water.
FIG. 4 shows a fluorescence image obtained by the transillumination imaging of the myocardium ball after the transparency of the present invention. It can be seen that the fluorescence signal of the deep position of the myocardial sphere can also be detected through the light transparency processing, which shows that the method can realize the acquisition of the three-dimensional overall structure information of the myocardial sphere.
Example 5
The artificial micro-organ is HCT116 three-dimensional cell balls fixed by paraformaldehyde and with the thickness of about 300 microns, and the artificial micro-organ is fully soaked in a transparent reagent to be incubated for 15min, and transparent front and back visual images are shot by combining a USAF 1951 resolution plate. The transparent reagent comprises the following components: the concentration of m-xylylenediamine was 40w/v%, the concentration of sorbitol was 20w/v%, the concentration of glycerin was 10w/v%, and the solvent was double distilled water.
Experiments showed that the 300 micron thick HCT116 three-dimensional cell spheres had been light-transparent and the tissue became highly transparent, and the USAF 1951 resolution plate line below the tissue was clearly visible.
Comparative example 1
The artificial micro-organ model of the comparative example is a 300-micron-thick HCT116 three-dimensional cell ball carrying endogenous green fluorescent protein, which is respectively treated by transparent reagents (named MACS-R0, MACS-R1 and MACS-R2) of CN109632420A and transparent reagents (named MACS-W) of the invention, and the specific steps are as follows:
clearing method and clearing reagent using CN 109632420A: the incubation was carried out for 3h, 1h and 1h in turn in MACS-R0 (20 v/v% m-xylylenediamine, 15w/v% sorbitol in water), MACS-R1 (40 v/v% m-xylylenediamine, 30w/v% sorbitol in PBS) and MACS-R2 (40 v/v% m-xylylenediamine, 50w/v% sorbitol in water) transparent reagents. The transparent reagent MACS-W is used for soaking a sample subjected to transparent treatment, and the sample is incubated in the transparent reagent for 15min, wherein the transparent reagent comprises 40v/v% of m-xylylenediamine, 30W/v% of sorbitol and water as a solvent. Fluorescence imaging was then performed using a fluorescence zoom stereomicroscope.
FIG. 5a shows fluorescence images taken before and after the light transparency treatment of the three-dimensional cell channels CN109632420A, the transparent reagent and the invention. Wherein the sample is seriously deformed after the transparency treatment of the transparent method and the transparent reagent of CN109632420A, generates swelling in the MACS-R0 reagent, and generates swelling in the MACS-R1,
Shrinkage in MACS-R2 reagent occurred, whereas the sample size did not change after the clearing treatment of the present invention; FIG. 5b is a statistical chart of the size change of three-dimensional cell spheres before and after being transparent by MACS (MACS-R0-3 h, MACS-R1-1h, MACS-R2-1 h) and the inventive transparent reagent (MACS-W-15 min), 3 groups of three-dimensional cell spheres are selected, and the area before and after being transparent is measured and counted, so that the sample is found to have no deformation after being transparent by the invention. In the figure, no clean indicates an initial sample image.
Comparative example 2
The other conditions were the same as example 1, except that the HCT116 three-dimensional cell spheres of example 1 having a thickness of about 300 μ M were subjected to the vitrification treatment by replacing the aqueous solvent in the vitrification reagent with a phosphate buffer solution having a concentration of 0.01M, and the resulting HCT116 three-dimensional cell spheres were found to have severe shrinkage with a shrinkage of about 77%.
FIG. 6 shows fluorescence of three-dimensional cell spheres before and after the above-mentioned treatment with a clearing reagent. It was found that when phosphate buffer was used as a solvent, the three-dimensional cell spheres contracted significantly.
The artificial micro-organ transparent processing method adopts single-step soaking, the fixed artificial micro-organ model is fully soaked in the transparent reagent, and m-xylylenediamine in the reagent has high refractive index, low viscosity and small molecular weight, can quickly permeate into tissues and proteolipid to generate hydration reaction, so that the tissues are transparent; sorbitol in the reagent has the functions of keeping the shape of a sample and protecting fluorescence; the glycerol in the reagent has high biocompatibility, and can further keep the size of the sample unchanged by matching with other reagents, and the transparent effect of the sample is improved. The transparent reagent selected in the processing method of the invention is obtained by introducing m-xylylenediamine with high refractive index and strong hydration, sorbitol and glycerol into the tissue, which can keep the sample shape and protect fluorescent protein in the tissue, and mixing the materials in proportion, thereby matching the refractive index of each component in the tissue, obtaining the highly transparent tissue and further improving the imaging depth.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (9)
1. A transparent reagent for transparentizing an artificial micro-organ, which is characterized by comprising m-xylylenediamine with hydration and degreasing effects and also comprising alcohols and/or sugar alcohols capable of maintaining the form of a sample, wherein the solvent is water;
wherein the concentration of the m-xylylenediamine in percentage by volume is 20-40%, and the mass concentration of the alcohols and/or sugar alcohols capable of maintaining the sample morphology is 20-40%.
2. The transparent reagent of claim 1, wherein the alcohol capable of maintaining the form of the sample is glycerol; the sugar alcohol capable of maintaining the form of the sample is sorbitol.
3. Use of the clearing agent according to claim 1 or 2 for rapid deformation-free clearing treatment of artificial micro-organs.
4. Use according to claim 3, characterized in that it comprises the following steps:
immersing the fixed artificial micro-organ in the transparent reagent according to claim 1 or 2 to match the refractive index of each component inside the micro-tissue, thereby obtaining a highly transparent artificial micro-organ.
5. The use of claim 4, wherein said artificial micro-organ is a three-dimensional cell culture tissue.
6. The use of claim 5, wherein the three-dimensional cell culture tissue is a tumor micro-tissue, a cardiomyocyte, a brain-like or a liver-like organ.
7. The use of claim 4, wherein said artificial micro-organ has a thickness of 100 to 3000 microns; the soaking time is 10min-200min.
8. The use of claim 4, wherein said fixed artificial micro-organ is a paraformaldehyde-fixed artificial micro-organ.
9. Use of a transparentized artificial micro-organ obtained by the application process according to any one of claims 3 to 8, in confocal imaging, two-photon imaging or light sheet fluorescence microscopy.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115216447A (en) * | 2022-07-06 | 2022-10-21 | 华中科技大学 | Online optical transparent reagent and method for organ chip |
| CN115216447B (en) * | 2022-07-06 | 2024-05-10 | 华中科技大学 | On-line light transparent reagent and transparent method for organ chip |
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