Method for pretreating biological specimens having a size of up to 1 mm to make them transparent, and method for making biological specimens transparent including said method

Abstract

The clearing pretreatment method of the biological sample of spheroid or organoid with a size of up to 1 mm according to the present invention uses phosphate buffered saline (PBS) as pretreatment solution instead of the conventional sucrose solution, thereby solving the problem that spheroid or organoid floats on the water surface due to density difference, causing damage to the structure of the sample.Therefore, it can make spheroid or organoid transparent while maintaining its original shape, thereby enabling imaging of deep areas.

Description

[Technical field]

[0001] Technical Field The present invention relates to a pretreatment method for making a biological sample having a size of 1 mm or less transparent, and a method for making a biological sample transparent that includes said method. [Background technology]

[0002] Background technology Tissue clearing techniques allow for the investigation of protein distribution and internal structure of biological tissues, and transcend the observation limitations of existing techniques for observing the depth of tissue structures. In addition, tissue clearing techniques allow access to integrated structural and molecular information obtained from various organ systems. As a result, tissue clearing techniques have been developed into various methods in recent years.

[0003] Antigen integrity has been reported for tissues processed by clearing techniques such as the Spatleholz, BABB, Scale S, and iDISCO methods, which are tissue clearing processes using organic solvents, and the Activated CLARITY Technology (ACT) method, which is a polymer flooding method. Methods other than ACT have problems with loss of fluorescence and antigen integrity. In the case of ACT, antigen integrity is over 90%, which is higher than methods such as CLARITY, which require additional binding of fixed proteins to hydrogel polymers. However, there are still issues to be considered, such as the strong tissue fixation process causing loss of antigenicity and the reduction in the number of available antibodies. As a result, improvements in various techniques are required.

[0004] On the other hand, clearing of biological tissues with the use of spheroids or organoids with a size of 1 mm or less has another major problem. To carry out the tissue clearing method, preservation of proteins through pretreatment with 40% sucrose (Patent Document 1, KR 10-2017-0105551) is important. However, small spheroids or organoids with a size of 1 mm or less float on the sucrose solution due to their lower density than sucrose. As a result, spheroids or organoids, which are fragile tissues, are seriously damaged when shaking the sample. In addition, the small size of spheroids or organoids makes it easy for researchers to lose sight of the sample because they cannot be observed by the naked eye.

[0005] In addition, rapid 3D imaging and quantification are required to establish a certification system to evaluate the efficacy and toxicity of new drugs for spheroid and organoid screening kits. However, there are currently difficulties in rapid 3D image construction and quantification of organoids.

[0006] To obtain the three-dimensional image of human-derived cancer tissue organoid, a confocal microscope or the like is generally used, which allows obtaining thickness information at the level of several tens of micrometers.The thickness is limited to some extent by the depth that the light source can penetrate.However, since the structure of organoid has a size of several hundred micrometers or more, this method can only obtain partial information.

[0007] Therefore, to obtain information about the inside of a relatively thick tissue, a series of processes are required, including making serial slices each having a thickness of several tens of micrometers, imaging each slice one by one through a microscope, and then reconstructing the slices together. However, since organoid samples are fragile, the problems that may occur during the series of cutting and pasting processes arise exponentially.

[0008] In addition, novel imaging techniques are required to assess the efficacy and toxicity of new drugs in three dimensions in living cells.

[0009] The present invention relates to the development of an alternative solution that makes it possible to solve the problems that arise when making spheroids or organoids with a size of 1 mm or less transparent, where samples such as spheroids or organoids are severely damaged in the pretreatment process with 40% sucrose due to their fragile tissue, and where due to the small size of the spheroids or organoids, they are easily lost as researchers are unable to observe the samples with the naked eye.

[0010] A solution with low density that can protect proteins from damage in the clearing solution was observed through experiments to prevent tissues from floating up due to density. As a result, the same effect as using 40% sucrose could be observed in the brain of fluorescent genetic tissue mice when fixing the tissue with 4% PFA and incubating the same in 1X PBS for 24 hours. The problem of the tissue floating when applying the above solution to small samples such as spheroids or organoids can be solved, thereby solving the problem of tissue damage. In addition, researchers can confirm the location of the sample, solving the existing problems that have been caused so far.

[0011] In addition, the present invention utilizes the recently developed Leica THUNDER Imager 3D microscope for rapid 3D imaging of human-derived cancer organoids, allowing for faster quantification and construction of 3D images of living cells than existing confocal microscopes.

[0012] In addition, the tissue clearing technique of the present invention allows the investigation of internal structure and protein distribution without damaging human-derived cancer organoids, thereby enabling the observation of the depth of tissue while overcoming the observation limit of existing techniques, and allowing access to the integrated structural and molecular information obtained from various organ systems.Furthermore, the kit for human-derived cancer organoids of the present invention is a kit that applies a method that reduces the damage to cancer organoid structure that may occur during the clearing process.

[0013] Therefore, high-throughput imaging and clearing techniques have been applied to human-derived cancer organoids to establish protocols that allow researchers to easily screen the efficacy and toxicity of new drugs. In addition, kits have been invented that allow researchers to easily perform relatively precise analysis and evaluation simultaneously by applying clearing methods to human-derived cancer organoids.

[0014] On the other hand, Patent Document 2 (KR 10-2018-0013747) provides a method for clearing spheroids using a clearing agent (CHAPS and urea). The clearing composition containing the clearing agent (CHAPS and urea) can easily and rapidly clear spheroids, and can therefore be effectively used for imaging of spheroids. In addition, Patent Document 2 discloses that the clearing composition can be effectively used for identifying the causes of various diseases, treating them, and predicting the efficacy and toxicity of drugs.

[0015] In this case, the present invention discloses that sharp fluorescent images of cleared spheroids can be obtained by applying a clearing agent to the spheroids, and such sharp fluorescent images can be effectively used for identifying the causes of various diseases, treating them, and predicting the efficacy and toxicity of drugs.

[0016] Furthermore, damage (denaturation) to spheroids and organoids can be minimized before inducing clearing by treatment with a clearing agent, thereby enabling clearing to be induced sufficiently well for imaging at depth. Moreover, it is different from the related techniques mentioned above in that a pretreatment with PBS solution is additionally performed to overcome the problems arising from the existing pretreatment solution (sugar solution, sucrose).

[0017] That is, the present invention has the effect of overcoming the problem that spheroid or organoid floats on the surface of pretreatment solution due to density difference, which makes pretreatment process difficult and causes damage to fragile tissue.Therefore, the present invention has been proven and completed to be usefully applied to the clarification of spheroid and organoid. Summary of the Invention

[0018] Disclosure technical challenges In one aspect, an object of the present invention is to provide a method for pre-treating a biological sample having a size of 1 mm or less to make it transparent, the method comprising treating a fixed biological sample having a size of 1 mm or less with a pre-treatment solution comprising phosphate-buffered saline (PBS).

[0019] In another aspect, another object of the present invention is to provide a method for clearing a biological sample having a size of 1 mm or less, the method comprising: Pretreating a fixed biological sample having a size of 1 mm or less with a pretreatment solution containing phosphate buffered saline (PBS); and and clarifying the resulting pretreated biological sample by contacting it with a clarifying agent comprising a compound represented by formula 1, an optical isomer of the compound represented by formula 1, a hydrate of the compound represented by formula 1, or a salt of the compound represented by formula 1.

[0020] [Formula 1] [ka] In formula 1, R 1 and R 2 are each independently a straight chain or branched alkyl group having 1 to 10 carbon atoms, and p, q, and r each independently represent an integer ranging from 0 to 10.

[0021] In a further aspect, a further object of the present invention is to provide a clearing pretreatment composition for a biological sample having a size of 1 mm or less, the composition comprising phosphate buffered saline (PBS).

[0022] In yet a further aspect, it is a still further object of the present invention to provide a clearing kit for a biological sample having a size of 1 mm or less, the kit comprising: a clearing pretreatment composition for a biological sample having a size of 1 mm or less, the composition comprising phosphate buffered saline (PBS); and A clarifying agent comprising a compound represented by formula 1, an optical isomer of a compound represented by formula 1, a hydrate of a compound represented by formula 1, or a salt of a compound represented by formula 1.

[0023] [Formula 1] [ka] In formula 1, R 1 and R 2 are each independently a straight chain or branched alkyl group having 1 to 10 carbon atoms, and p, q, and r each independently represent an integer ranging from 0 to 10.

[0024] In yet a further aspect, it is a still further object of the present invention to provide a method for improving the clearing efficiency of a biological sample having a size of 1 mm or less, the method comprising treating a fixed biological sample having a size of 1 mm or less with a pretreatment solution comprising phosphate buffered saline (PBS).

[0025] In yet a still further aspect, it is a still further object of the present invention to provide a method for reducing damage to a biological sample having a size of 1 mm or less during a process of clearing the biological sample, the method comprising treating a fixed biological sample having a size of 1 mm or less with a pretreatment solution comprising phosphate buffered saline (PBS).

[0026] technical solution In order to achieve the above-mentioned object, one aspect of the present invention provides a method for pre-treating a biological sample having a size of 1 mm or less to make it transparent, the method comprising treating a fixed biological sample having a size of 1 mm or less with a pre-treatment solution containing phosphate-buffered saline (PBS).

[0027] Additionally, another aspect of the present invention provides a method for clearing a biological sample having a size of 1 mm or less, the method comprising: pretreating a fixed biological sample having a size of 1 mm or less with a pretreatment solution comprising phosphate buffered saline (PBS); and contacting the resulting pretreated biological sample with a clearing agent comprising a compound represented by formula 1, an optical isomer of the compound represented by formula 1, a hydrate of the compound represented by formula 1, or a salt of the compound represented by formula 1.

[0028] [Formula 1] [ka] In formula 1, R 1 and R 2 are each independently a straight chain or branched alkyl group having 1 to 10 carbon atoms, and p, q, and r each independently represent an integer ranging from 0 to 10.

[0029] Furthermore, a further aspect of the present invention provides a clearing pretreatment composition for a biological sample having a size of 1 mm or less, the composition comprising phosphate buffered saline (PBS).

[0030] Additionally, yet a further aspect of the present invention provides a clearing kit for a biological sample having a size of 1 mm or less, the kit comprising: a clearing pretreatment composition for a biological sample having a size of 1 mm or less, the composition comprising phosphate buffered saline (PBS); and a clearing agent comprising a compound represented by formula 1, an optical isomer of the compound represented by formula 1, a hydrate of the compound represented by formula 1, or a salt of the compound represented by formula 1.

[0031] [Formula 1] [ka] In formula 1, R 1 and R 2 are each independently a straight chain or branched alkyl group having 1 to 10 carbon atoms, and p, q, and r each independently represent an integer ranging from 0 to 10.

[0032] Furthermore, yet a further aspect of the present invention provides a method for improving the clearing efficiency of a biological sample having a size of 1 mm or less, the method comprising treating a fixed biological sample having a size of 1 mm or less with a pretreatment solution comprising phosphate buffered saline (PBS).

[0033] Additionally, a still further aspect of the present invention provides a method for reducing damage to a biological sample having a size of 1 mm or less during a process of clearing the biological sample, the method comprising treating a fixed biological sample having a size of 1 mm or less with a pretreatment solution comprising phosphate buffered saline (PBS).

[0034] Beneficial Effects The clearing pretreatment method of the biological sample of 1 mm or less size, which is spheroid or organoid, according to the present invention uses phosphate buffered saline (PBS) as pretreatment solution instead of the existing sucrose solution, thus solving the problem that spheroid or organoid floats on the water surface due to density difference, which causes damage to the structure of the sample.Therefore, it has the effect that spheroid or organoid can be made transparent while maintaining its original shape, and thus it is possible to image deep inside. Description of the drawings [Brief description of the drawings]

[0035] [Figure 1] FIG. 1 is an image showing the comparative results of transparency and fluorescence levels for mouse brains according to the type of pretreatment solution. [Diagram 2] FIG. 2 is an image of an organoid sample cultured in a neuron culture medium in the preparation process of human-derived organoids in Experimental Example 2, 2-1.

[0036] [Diagram 3] FIG. 3 is an image showing the results of an analysis of the Hoechst / PI ratio using a Thunder imager, in which the human-derived cancer organoid sample prepared in 2-1 of Experimental Example 2 was analyzed before being treated with temozolomide. [Figure 4] FIG. 4 is an image showing the results of an analysis of the Hoechst / PI ratio using a Thunder imager, in which the human-derived cancer organoid sample prepared in 2-1 of Experimental Example 2 was treated with temozolomide and then analyzed.

[0037] [Diagram 5] Figure 5 shows images of samples in an ibidi 8-well chamber viewed with the naked eye before and after cancer organoid clearing. [Figure 6]Figure 6 shows the results of analysis of DAPI, green fluorescence, and red fluorescence signals at 5X and 20X objectives of a Macro Laser Light Sheet Illumination Imaging System confocal microscope, analyzed to obtain 3D live images of cancer organoids that were cleared in a process that included pretreatment with 1X PBS.

[0038] [Figure 7] Figure 7 shows the results of analysis of DAPI, green fluorescence, and red fluorescence signals with 5X and 20X objective lenses of a macro laser light sheet illumination imaging system confocal microscope, an analysis that confirmed that 3D live images of cleared cancer organoids can be obtained in a process that includes pretreatment with 1X PBS, and that live images of cancer organoids at depth can be obtained. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] Best Mode The present invention will be described in detail below. On the other hand, the aspects of the present invention may be variously modified in many different forms, and the scope of the present invention is not limited to the aspects described below. In addition, the aspects of the present invention are provided to enable those skilled in the art to more clearly understand the present invention.

[0040] Furthermore, unless the context clearly indicates otherwise, as used herein, the terms "comprises," "comprising," "includes," and / or "including" specify the presence of other elements but do not exclude the presence of other elements.

[0041] One aspect of the present invention provides a method for pre-treating a biological sample having a size of 1 mm or less to make it transparent, the method comprising treating a fixed biological sample having a size of 1 mm or less with a pre-treatment solution comprising phosphate-buffered saline (PBS).

[0042] In this case, the biological sample can be spheroid or organoid.In addition, the spheroid or organoid can be formed from cells derived from brain, blood vessel, liver, lung, kidney, pancreas, stomach or intestine. The biological sample, being a spheroid or organoid, may have an average diameter in the range of 0.05 mm to 1 mm.

[0043] By way of example of average diameter of a biological sample, the biological sample may have an average diameter in the range of 0.05 mm to 1 mm, 0.10 mm to 1 mm, 0.15 mm to 1 mm, 0.20 mm to 1 mm, 0.25 mm to 1 mm, 0.30 mm to 1 mm, 0.35 mm to 1 mm, 0.40 mm to 1 mm, 0.45 mm to 1 mm, 0.50 mm to 1 mm, 0.55 mm to 1 mm, 0.60 mm to 1 mm, 0.65 mm to 1 mm, 0.70 mm to 1 mm, 0.75 mm to 1 mm, or 0.80 mm to 1 mm.

[0044] The present invention was derived to overcome the problem that when a biological sample having an average diameter smaller than that of general biological tissue is cleared, the biological sample floats on the surface of the pretreatment solution during the pretreatment process. When the object to be cleared is a biological sample having a size within the above range, the effects demonstrated in the present invention, typically the effect of preventing structural damage during the pretreatment process, can be achieved.

[0045] The fixed biological sample having a size of 1 mm or less may be a biological sample fixed with at least one selected from the group consisting of paraformaldehyde, ethylene glycol diglycidyl ether, dipropylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, glycerol polyglycidyl ether, glutaraldehyde, and polyacrylamide. Preferably, in an exemplary embodiment of the present invention, paraformaldehyde is used.

[0046] Another aspect of the present invention provides a method for clearing a biological sample having a size of 1 mm or less, the method comprising: pretreating a fixed biological sample having a size of 1 mm or less with a pretreatment solution comprising phosphate buffered saline (PBS); and contacting the resulting pretreated biological sample with a clearing agent comprising a compound represented by formula 1, an optical isomer of the compound represented by formula 1, a hydrate of the compound represented by formula 1, or a salt of the compound represented by formula 1.

[0047] [Formula 1] [ka] In formula 1, R 1 and R 2 are each independently a straight chain or branched alkyl group having 1 to 10 carbon atoms, and p, q, and r each independently represent an integer ranging from 0 to 10.

[0048] Preferably, R 1 and R 2 are each independently a straight chain or branched alkyl group having 1 to 5 carbon atoms, and p, q, and r each independently represent an integer ranging from 0 to 5. More preferably, R 1 and R 2 are each independently methyl; p, q, and r are each independently an integer of 1.

[0049] Most preferably, the compound represented by formula 1 is a compound represented by formula 2 or a hydrate of the compound represented by formula 2. [Formula 2] [ka]

[0050] The clearing agent may contain the CHAPS compound represented by formula 2 or a hydrate of the compound represented by formula 2 in a concentration range of 2% w / v (% weight / volume) to 55% w / v (% weight / volume). Preferably, the clearing agent contains CHAPS in a concentration range of 4% w / v to 50% w / v. In this case, the solution for providing the concentration in the above range may be a simulated body fluid used in the art. More specifically, distilled water, phosphate buffered saline (PBS), Tris buffer solution (TBS), or the like may be used. However, the solution for providing the concentration is not limited thereto. When the clearing agent contains the CHAPS compound in a concentration of less than 2% w / v, the clearing rate of the biological tissue may be significantly slowed. In addition, when the clearing agent contains the CHAPS compound in a concentration higher than 55% w / v, the CHAPS compound may not be completely dissolved in the clearing agent.

[0051] In addition, the clearing agent may further comprise at least one selected from the group consisting of 3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate (CHAPSO), sucrose, fructose, glycerol, diatrizoic acid, Triton X-100, Tween 20, 2,2'-thiodiethanol, iohexol, and chloral hydrate. Preferably, the clearing agent further comprises urea. The above-mentioned components play a role in accelerating the clearing of biological samples by controlling osmotic pressure.

[0052] The clearing agent may contain the above-mentioned substance that accelerates the clearing of the biological sample in a concentration range of 5% w / v to 80% w / v, 5% w / v to 75% w / v, 10% w / v to 70% w / v, 5% w / v to 50% w / v, or 35% w / v to 60% w / v. In this case, when the clearing agent contains the substance at a concentration of less than 5% w / v, the clearing rate of the biological tissue may be slowed. In addition, when the clearing agent contains the substance at a concentration higher than 80% w / v, crystallization may occur or the material may not dissolve in the solution.

[0053] As a specific example, when urea is used as a substance for accelerating the clearing of a biological sample, the clearing agent may contain urea in a concentration range of 10% w / v to 70% w / v, and preferably in a concentration range of 20% w / v to 60% w / v. In addition, the concentration of the material for accelerating the clearing of a biological sample may be appropriately adjusted to the preferred concentration range of the CHAPS compound.

[0054] The method for making biological tissue transparent may be performed in a temperature range of 4°C to 50°C, 10°C to 50°C, 12°C to 48°C, 14°C to 46°C, 16°C to 44°C, 18°C ​​to 42°C, 20°C to 40°C, 24°C to 39°C, 28°C to 38°C, 30°C to 37°C, or 33°C to 34°C.

[0055] Furthermore, after pretreatment of the biological sample and prior to clearing the biological sample by contacting the biological sample with a clearing agent, the clearing method may further comprise treating the biological sample with agarose. Since the biological sample is further treated with agarose, damage to the spheroids or organoids can be minimized and the structure of the spheroids or organoids can be maintained.

[0056] A further aspect of the present invention provides a clearing pretreatment composition for a biological sample having a size of 1 mm or less, the composition comprising phosphate buffered saline (PBS).

[0057] Yet another aspect of the present invention provides a clearing kit for a biological sample having a size of 1 mm or less, the kit comprising: a clearing pretreatment composition for a biological sample having a size of 1 mm or less, the composition comprising phosphate buffered saline (PBS); and a clearing agent comprising a compound represented by formula 1, an optical isomer of the compound represented by formula 1, a hydrate of the compound represented by formula 1, or a salt of the compound represented by formula 1.

[0058] [Formula 1] [ka] In formula 1, R 1 and R 2 are each independently a straight chain or branched alkyl group having 1 to 10 carbon atoms, and p, q, and r each independently represent an integer ranging from 0 to 10.

[0059] In this case, the clarifying agent may further comprise at least one selected from the group consisting of 3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate (CHAPSO), sucrose, fructose, glycerol, diatrizoic acid, Triton X-100, Tween 20, 2,2'-thiodiethanol, iohexol, and chloral hydrate. Preferably, the clarifying agent further comprises urea.

[0060] Another aspect of the present invention provides a method for improving the clearing efficiency of a biological sample having a size of 1 mm or less, the method comprising treating a fixed biological sample having a size of 1 mm or less with a pretreatment solution comprising phosphate buffered saline (PBS).

[0061] Yet a further aspect of the present invention provides a method for reducing damage to a biological sample having a size of 1 mm or less during a process of clearing the biological sample, the method comprising treating a fixed biological sample having a size of 1 mm or less with a pretreatment solution comprising phosphate buffered saline (PBS).

[0062] According to one aspect of the present invention, the transparent method can solve the problem that tissue floats up when small sample, which is spheroid or organoid with a size of 1 mm or less, is transparentized, and the problem that tissue is damaged can be solved.In addition, the transparent method has the effect that researchers can confirm the location of the sample.

[0063] Furthermore, by applying high-throughput imaging and clearing techniques to the three-dimensional image of living cells, a protocol can be established that allows researchers to easily screen the efficacy and toxicity of new drugs.By applying this clearing process to human-derived cancer organoids, the clearing method simultaneously enables relatively precise analysis and evaluation, which can be effectively used for identifying the causes of various diseases, treating them, and also evaluating the efficacy and toxicity of new drugs.

[0064] Mode for invention The present invention will now be described in detail with reference to embodiments and experimental examples. However, the following embodiments and examples are intended to merely illustrate exemplary embodiments of the present invention, and the scope of the present invention is not limited to the following embodiments and examples.

[0065] <Experimental Example 1> Comparison of transparency levels in mouse brains depending on the type of pretreatment solution The following experiment was conducted to compare the transparency levels of mouse brains according to the type of pretreatment solution (1X PBS or 40% w / v sucrose), in which all animal experiments described herein were conducted in accordance with the guidelines of the Animal Resources Committee of the Korea Institute of Toxicology (approval number RS17003).

[0066] 1-1: Fixed First, adult mice (8 weeks old) were anesthetized with the inhalation anesthetic isoflurane at a flow rate of 1 cc / min and perfused transcardially with 50 mL of cold 1× PBS followed by cold 4% paraformaldehyde (PFA). The organs were then extracted, immersed in 4% PFA (aqueous solution), and incubated for 24 hours at a temperature of 4° C. In this case, the range of the cold temperature is not particularly limited and may be in the range of 0° C. to 10° C.

[0067] 1-2: Pre-processing Next, the above samples (organs) were pretreated by incubating them in 1X PBS or 40% sucrose solution for 24 hours at temperatures ranging from 0°C to 10°C. In this case, 1X PBS has a pH of about 7.4 and 10 mM PO4 3- The sucrose solution was phosphate buffered saline (PBS) with a salt concentration of 137 mM NaCl, and 2.7 mM KCl. In addition, the concentration unit of the 40% sucrose solution was % w / v (% weight / volume), and an aqueous solution was used as the sucrose solution.

[0068] 1-3: Transparency To clear each of the fully pretreated samples, an aqueous mixture of 20% w / v CHAPS, 50% w / v urea, and 50 mM sodium azide was mixed at 100 rpm for 5 hours at a temperature of 35° C. and incubated for 48 hours, where CHAPS is a compound represented by Formula 1 herein or a hydrate of the compound represented by Formula 1.

[0069] 1-4:Result The transparency levels of mouse brains depending on the type of pretreatment solution were compared through the fluorescence levels. In this case, the difference in the loss of green fluorescent protein (GFP) fluorescence between the two pretreatment solutions in the process of brain tissue clearing of genetically modified fluorescent mice was compared through fluorescent images. The results are shown in Figure 1.

[0070] FIG. 1 is an image showing the comparative results of transparency and fluorescence levels for mouse brains according to the type of pretreatment solution. As shown in FIG. 1, it was confirmed that the 1× PBS pretreatment solution used as the pretreatment solution for mouse brain clearing consequently induced clearing well enough to replace the 40% w / v (% weight / volume) sucrose pretreatment solution disclosed in patent document KR 10-2017-0105551.

[0071] As described above, when spheroids or organoids having a size of 1 mm or less were pretreated with 40% w / v (% weight / volume) sucrose solution disclosed in patent document KR 10-2017-0105551 to be cleared, the spheroids or organoids floated on the surface of the pretreatment solution due to density difference. As a result, the fragile structure of the sample, which is a spheroid or organoid, was damaged, so that the internal structure of the sample could not be meaningfully observed even after clearing. On the other hand, the density difference between the 1X PBS pretreatment solution and the spheroid or organoid was slight, and thus the damage to the structure of the sample could be minimized. Therefore, it was confirmed through this experiment that the 1X PBS pretreatment solution was effectively used in the clearing process of spheroids or organoids.

[0072] <Experimental Example 2> Immunostaining and clearing of human cancer organoids To evaluate whether the inclusion of a pretreatment process with 1X phosphate-buffered saline (PBS) actually induced better immunostaining and clearing of cancer organoids, the following experiment was performed.

[0073] 2-1: Preparation of human-derived organoids To prepare human-derived organoids, tissues from glioblastoma patients were dissociated into single cells and mixed with 20 μL of Matrigel on a paraffin film at the College of Medicine, Seoul National University. When cultured in an incubator at a temperature of 37 °C, the samples formed a hemispherical shape with a size of 1 mm, which was jelly-like. The samples were then placed in neuronal culture medium and grown for 4 days. After 4 days, the samples were cultured on an orbital shaker with shaking at a speed ranging from 60 RPM to 80 RPM (Figure 2). Figure 2 is an image of an organoid sample cultured in neuronal culture medium in the preparation process of human-derived organoids in 2-1 of Experimental Example 2. When the medium was refreshed once every 3 days of growth, human-derived cancer organoids were prepared after 4 weeks.

[0074] 2-2: Observation of organoids without clearing Cancer organoid samples were transferred to an ibidi 8-well chamber and images of organoids were taken in 3D on a Leica Thunder microscope before and after treatment of the organoids with temozolomide, a drug used to treat brain tumors. Routine nuclear staining for 3D fluorescence images was processed with Hoechst (blue) and PI (red), used as dead cell markers. The Hoechst / PI ratios before and after treatment of the organoids are shown in Figures 3 and 4 using a Thunder imager.

[0075] FIG. 3 is an image showing the results of an analysis of the Hoechst / PI ratio using a Thunder imager, in which the human-derived cancer organoid sample prepared in 2-1 of Experimental Example 2 was analyzed before being treated with temozolomide. FIG. 4 is an image showing the results of an analysis of the Hoechst / PI ratio using a Thunder imager, in which the human-derived cancer organoid sample prepared in 2-1 of Experimental Example 2 was treated with temozolomide and then analyzed.

[0076] By looking at the Hoechst / PI ratio of the human cancer organoid samples analyzed by the Thunder imager before treating the samples with temozolomide, it was confirmed that almost no dead cells were found (Figure 3). On the other hand, by looking at the Hoechst / PI ratio of the human cancer organoid samples analyzed by the Thunder imager after treating the samples with temozolomide (Figure 4), it was confirmed that the number of dead cells increased compared to that before treating the samples with temozolomide.

[0077] 2-3: Cancer organoid clearing method including pretreatment process with 1X PBS When the amount of cell death induced by temozolomide in cancer organoids increased, the use of tissue clearing techniques was required for more detailed and accurate analysis of the samples. In addition, unlike the clearing of large biological tissues such as mouse brains, a pretreatment process with 1X phosphate-buffered saline (PBS) was essential to smoothly induce organoid clearing.

[0078] The clearing process for cancer organoids was as follows. A.Fixed First, human-derived cancer organoids were treated with temozolomide, which is used to treat brain tumors, and incubated for 36 hours. After 36 hours of incubation, PBS was added to the human-derived cancer organoids to remove the culture medium. After removing the PBS, the human-derived cancer organoids were treated with 4% paraformaldehyde (PFA) at a temperature of 4°C for 1 hour to fix the organoids.

[0079] B. Pretreatment The fixed organoids were transferred to 1X PBS and placed on an orbital shaker at 4°C for 24 hours. The cancer organoids were transferred to an ibidi 8-well chamber. Then, the solution of 0.8% agarose was cooled to 60°C and poured into the chamber containing the cancer organoids to fix the organoids. In this case, agarose plays a role in minimizing damage to the cancer organoids and maintaining their structure.

[0080] C. Transparent The organoids were treated with a clearing solution for living tissue (40% w / v CHAPS and 40% w / v urea) and incubated at a temperature of 37° C. for 24 hours at a speed of 100 rpm. After incubation, the solution was refreshed with distilled water three times for 2 hours to remove the clearing solution for living tissue. The organoids were once again treated with a clearing solution for living tissue (40% w / v CHAPS and 40% w / v urea) and incubated at a temperature of 37° C. for 24 hours at a speed of 100 rpm. After incubation, the solution was refreshed with distilled water three times for 2 hours to remove the clearing solution for living tissue. Figure 5 shows images of samples in an ibidi 8-well chamber viewed with the naked eye before and after cancer organoid clearing.

[0081] Next, to ensure space for the antibody to penetrate into the cancer organoid tissue, the organoid was treated with a treatment solution for tissue infiltration (0.2% Triton X-100, 10% DMSO, and 0.1X PBS) at a temperature of 4°C for 4 hours. After incubation, the solution was refreshed with distilled water for 2 hours three times to remove the treatment solution for tissue infiltration. The primary antibody was diluted 1:100 in a solution containing 0.1X PBS, 0.01% sodium azide, and 0.1% Tween 20, and the cancer organoid sample was treated with the solution and incubated at a temperature of 4°C for 2 days. The primary antibody was then removed from the cancer organoid tissue by refreshing with 0.1X PBS solution three times for 3 hours.

[0082] The secondary antibody was diluted 1:100 in a solution containing 0.1X PBS, 0.01% sodium azide, and 0.1% Tween 20, and the cancer organoid samples were treated with the solution and incubated at a temperature of 4°C for 2 days. Finally, the secondary antibody was removed from the cancer organoid tissue by refreshing with 0.1X PBS solution three times for 3 hours. For nuclear staining, the samples were treated with DAPI (sigma catalog number D 9542) for 1 hour. Residual staining reagent was removed with distilled water. The cancer organoid samples were placed in a clearing solution for biological tissues (40% w / v CHAPS and 40% w / v urea) and incubated at a temperature of 37°C for 24 hours at a speed of 100 rpm.

[0083] As shown in the above results, when the clearing technique of the present invention was applied to human-derived cancer organoids, it was confirmed that the researchers were able to easily prepare samples for clearing of cancer organoids and at the same time make the samples transparent.

[0084] <Experimental Example 3> Observation of the cellular structure and distribution of organoids, and analysis of organoids at depth through immunostaining of cleared organoids In order to analyze the cell distribution and structure of the human-derived cancer organoids that have been cleared through the processes A to C that constitute 2-3 of Experimental Example 2, the cancer organoids were treated with Vimentin antibody, which is used as a marker for mesenchymal cells. In addition, because the cancer organoids were brain tumor samples, Tuj1 antibody, which is used as a marker for neurons, was treated to analyze the distribution of neurons.

[0085] To obtain three-dimensional live images, DAPI, green fluorescence, and red fluorescence signals were analyzed with 5X and 20X objectives of a macro laser light sheet illumination imaging system confocal microscope (Figure 6). Figure 6 shows the results of analysis of DAPI, green fluorescence, and red fluorescence signals with 5X and 20X objectives of a macro laser light sheet illumination imaging system confocal microscope, which were analyzed to obtain three-dimensional live images of cancer organoids that were cleared in a process that included pretreatment with 1X PBS.

[0086] The shape of each mesenchymal cell, as well as the shape of neurons and nuclei, was clearly shown in three dimensions. Furthermore, it was confirmed that it was possible to obtain images of cancer organoids at depth (Figure 7). Figure 7 shows the results of analysis of DAPI, green fluorescence, and red fluorescence signals at 5X and 20X objective lenses of a macro laser light sheet illumination imaging system confocal microscope, which was used to obtain three-dimensional live images of cancer organoids that had been cleared in a process that included pretreatment with 1X PBS, and confirmed that it was possible to obtain live images of cancer organoids at depth.

[0087] Moreover, to demonstrate the importance of the three-dimensional configuration, Imaris software was used to enable quantification of the number of cells per unit area (Figure 7). With Imaris software, drug efficacy and toxicity can be quantified and analyzed in three dimensions during drug development, resulting in a much more objective and quantitative assessment than two-dimensional analysis.

[0088] Therefore, the clearing method of the present invention, which includes pretreatment with phosphate buffered saline (PBS), can easily and quickly make spheroids or organoids transparent, which can be useful for imaging of spheroids or organoids.In addition, the clearing method can be effectively used for identifying the causes of various diseases, treating them, and predicting the efficacy and toxicity of drugs.Furthermore, the clearing method can be used by being incorporated into various medical devices, and can be applied to manufacture kits that can be effectively used as in vitro diagnostic devices.

[0089] Industrial Applicability The clearing pretreatment method of the biological sample of 1 mm or less size, which is spheroid or organoid, according to the present invention uses phosphate buffered saline (PBS) as pretreatment solution instead of the existing sucrose solution, thus solving the problem that spheroid or organoid floats on the water surface due to density difference, which causes damage to the structure of the sample.Therefore, it has the effect that spheroid or organoid can be made transparent while maintaining its original shape, and thus it is possible to image deep inside.

Claims

1. A method for pre-treating a biological sample having a size of 1 mm or less to make it transparent, comprising the steps of: Pretreating a fixed biological sample having a size of 1 mm or less by incubating it with a pretreatment solution containing phosphate buffered saline (PBS) at 0-10°C; and Clarifying the resulting pretreated biological sample by contacting it with a clarifying agent comprising the compound 3-[(3-cholamidopropyl)-dimethylammonio]-propanesulfonate (CHAPS) represented by formula 2 or a hydrate thereof in an amount of 2-55 w / v % and urea in an amount of 10-70 w / v %; The method comprising: [Formula 2] 【Chemistry 1】

2. The method of claim 1, wherein the biological sample is a spheroid or an organoid.

3. The method of claim 2, wherein the spheroids or organoids are formed from cells derived from the brain, blood vessels, liver, lung, kidney, pancreas, stomach, or intestine.

4. The method of claim 1, wherein the biological sample has an average diameter in the range of 0.05 mm to 1 mm.

5. 2. The method of claim 1, wherein the fixed biological sample having a size of 1 mm or less is a biological sample fixed with at least one selected from the group consisting of paraformaldehyde, ethylene glycol diglycidyl ether, dipropylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, glycerol polyglycidyl ether, glutaraldehyde, and polyacrylamide.

6. 10. The method of claim 1, further comprising treating the biological sample with agarose after pre-treatment of the biological sample and prior to clearing the biological sample by contacting the biological sample with a clearing agent.

7. A method for improving transparency efficiency of a biological specimen having a size of 1 mm or less, comprising: Pretreating a fixed biological sample having a size of 1 mm or less by incubating it in a pretreatment solution containing phosphate buffered saline (PBS) at 0-10°C; Clarifying the resulting pretreated biological sample by contacting it with a clarifying agent comprising the compound 3-[(3-cholamidopropyl)-dimethylammonio]-propanesulfonate (CHAPS) represented by formula 2 or a hydrate thereof in an amount of 2-55 w / v % and urea in an amount of 10-70 w / v %; The method comprising: [Formula 2] 【Chemistry 2】

8. 1. A method for reducing damage to a biological specimen having a size of 1 mm or less during a process of clearing the biological specimen, comprising: Pretreating a fixed biological sample having a size of 1 mm or less by incubating it in a pretreatment solution containing phosphate buffered saline (PBS) at 0-10°C; Clarifying the resulting pretreated biological sample by contacting it with a clarifying agent comprising the compound 3-[(3-cholamidopropyl)-dimethylammonio]-propanesulfonate (CHAPS) represented by formula 2 or a hydrate thereof in an amount of 2-55 w / v % and urea in an amount of 10-70 w / v %; The method comprising: [Formula 2] 【Chemistry 3】

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