WO2019164473A1

WO2019164473A1 - Method for visualization of 3-d tissue cultures

Info

Publication number: WO2019164473A1
Application number: PCT/US2018/018730
Authority: WO
Inventors: Thomas Steven VILLANI JR.; Graeme Patrick GARDNER; Nicholas Ingram Crider
Original assignee: Visikol Inc.
Priority date: 2018-02-20
Filing date: 2018-02-20
Publication date: 2019-08-29

Abstract

Provided are compositions and methods for visualizing a 3-dimensional tissue culture.

Description

METHOD FOR VISUALIZATION OF 3-D TISSUE CULTURES

FIELD OF THE INVENTION

[0001] The present application relates generally to histology, tissue culture, and in vitro drug screening, and more specifically to the visualization of 3-dimensional tissue cultures.

BACKGROUND

[0002] The efficacy of experimental potentially therapeutic drugs is often measured using in vitro methods. Until recently, the majority of in vitro drug screening was conducted on monolayer cell cultures. Two-dimensional layers of cells are exposed to a compound of interest and evaluated through imaging, flow cytometry, or spectrophotometric assays (Haycock (2011). 3D Cell Culture: Methods and Protocols 1-15.).

[0003] While this approach is still commonly used today in drug discovery, there are notable shortcomings in the ability of the results of these simple and inexpensive assays to translate in vivo. Accurate in vivo correlation is crucial for in vitro models since failure to eliminate unsuccessful candidate compounds in early screens results in failure in more expensive animal/human trials. The complex three-dimensional environment in vivo causes differences in response compared to monolayer cell cultures since monolayer models do not possess the diverse array of cells, chemistries, and biochemical environments present in tissues (Justice et al. (2009). Drug Discovery today: 14( 1), 102-107).

[0004] Because of the well-known limitations with traditional monolayer cell models, in vitro 3D culture models have been adapted since they have been shown more closely to mimic the environment of in vivo tissues more accurately (Pampaloni et al. (2007). Molecular Cell Biology: 5(10), 839). Such 3-D cultures include spheroids, organoids, microtissues, and the like. Due to the rapid adoption of these models, traditional imaging and histological- based approaches have been used with in vitro assays as well as others performing 3D cell culturing characterization These modalities include wide-field microscopy, spectroscopic techniques ( e.g . fluorescence, luminescence), classical histological sectioning and staining, or confocal microscopy. [0005] Unfortunately, there are major limitations to the current approaches used to assay 3-D cell cultures for drug discovery. Firstly, wide-field microscopy is limited to visualizing only the surface layer of cells, with limited ability to differentiate between interior and exterior cells. Spectroscopic techniques offer no ability to discriminate cell-by-cell, allowing for only population-level measurements, however they are very rapid and amenable to high throughput automation.

[0006] Confocal microscopy allows for the capturing of z-stacks of in-focus images at the focal plane in spheroids. However, due to light scattering caused by the opacity of spheroids, only cells near the surface are detectable. Currently, histological sectioning and staining is the only method available to image the interior cells of 3-D cultures, but this suffers from cumbersome manual processing that is not amenable to high throughput analysis.

[0007] The advantage of confocal microscopy is that the pinhole and confocal lens eliminate out-of-focus light from the detector, yielding a significant increase in sharpness and clarity of the images obtained. Confocal microscopy applied to thicker specimens (>20 pm) requires special considerations since optical limitations due to light scattering in opaque tissues limits imaging to the surface layers only.

[0008] Spheroids have been visualized with a clearing technique (ScaleA2 i.e.

SCALEVIEW-A2) prior to confocal imaging increased the depth of imaging to 100 pL

(hitps:;7wvvw.perkine¾mer.com/PPFs/downioads/ MiciOtissue-C ores. pdf). However,

there are several limitations to the Scale clearing agent such as slow clearing and the swelling and weakening of the spheroid, causing risk of damage during pipetting (Richardson et al. (2015). Cell: 162(2). 246-25.). Since 2013, many tissue clearing techniques have been developed for whole tissues, but these techniques suffer from one or more of these issues: 1) long incubation times; 2) cumbersome processing steps not amenable to automation or high throughput; 3) loss of structural integrity or damage to spheroid; and 4) chemical

incompatibility with polystyrene well-plates and/or non-adherent coatings.

[0009] One of the major hurdles in using non-aqueous, solvent-based tissue clearing methods (e.g. uDISCO, iDISCO, 3DISCO, BABB, Visikol® HISTO™) is the chemical compatibility of the strongly lipophilic solvents used in the clearing process, with the containers used to hold the sample. These strongly lipophilic chemicals (e.g. benzyl alcohol, dibenzyl ether, phenyl ether, benzyl benzoate, etc.) are not compatible with the typical plastics used to construct the well-plates (e.g. polystyrene, polypropylene, cyclic olefin copolymer) that spheroids are grown in for high-throughput experiments. Furthermore, some of these methods use organic solvents (e.g. THF) that can degrade the optical clarity of the non-adherent coatings used in well-plates for spheroid growth. Since a significant portion of the marketplace utilizes non-adherent coatings for the culture of 3D cell cultures, a clearing agent compatible with non-adherent coatings is vital.

[0010] In addition to material compatibility, an additional challenge in using the present non-aqueous, solvent-based tissue clearing methods is the need for at least one dehydration step usually with methanol, ethanol, or tetrahydrofuran (THF) to remove the water remaining in the tissue from the aqueous permeabilization, staining, and washing steps, prior to treatment with the hydrophobic clearing agent. This allows the hydrophobic clearing reagent to penetrate the tissue, replacing the dehydration solution, and producing transparency in the tissue through refractive index matching. (Tainaka et al, (2016) Ann Rev. Cell Develop. Biol. 32 (2016):713-741.)

[0011] Also, since many clearing agent solutions have high relative densities compared to the dehydration solution, the 3-D cultures float in the well when the agent is applied. These floating tissues can easily be aspirated by robotic liquid handlers, thus making it challenging to use clearing methods in automated, high-throughput environments. Furthermore, floating tissues are challenging to image in automated high-content confocal instruments since, when floating, they can stick to the sides of the well and once equilibrated, they come to rest in random positions in the well, interfering with the automated focusing and object detection systems integrated into the instrument.

[0012] Thus, there is an unmet need for a rapid tissue culture clearing technique resulting in optical clarity useful for visualization of 3-D cultures and for in vitro assays that is compatible with high-throughput analysis and does not risk damage to the spheroids or well- plates during automated pipetting operations, and which does not cause flotation and dislocation of spheroid in the well being cleared for examination. SUMMARY

[0013] It has been discovered that 3-D tissue culture s can be quickly visualized and without damage to the tissue using a method which excludes a dehydration step just before the specimen is contacted with a clearing formulation effective for clearing the 3-D specimen that had previously been in an aqueous environment. Since this method excludes a dehydration step prior to clearing which had previously to now been required for clearing 3-D cultures with non-aqueous clearing agents, the overall processing time is reduced, making the assay more amenable to high-throughput automation.

[0014] In addition, the method includes the use of a clearing agent which is compatible with the materials typically used in microplate construction. It has a lower proportion of strongly lipophilic solvents relative to other clearing formulations, and thus it does not interact with and degrade the polystyrene well-plates typically used in high-content confocal imaging studies. Also, since the density of the clearing solution relative to the aqueous solution with which the culture specimen is in contact just prior to the clearing step is negligible, this prevents the cultures from floating in the well when the clearing agent is applied, improving the method’s compatibility with automated pipetting and imaging instruments. Additionally, the method allows for 3-D culture growth/formation, clearing, and imaging to be carried out in the same well-plate, thereby eliminating time-consuming transfer steps, and greatly improving the convenience of the method. Also, the clearing solution used in the method is compatible with the hydrophilic, ultra-low attachment surfaces that are used to promote spheroid formation in well-plates.

[0015] Thus, the method allows for the rapid, high-throughput, compatible, clearing of 3-D tissue cultures without damage to the culture or microplate.

[0016] In one aspect, the disclosure provides a method of optically clearing a 3-D tissue culture specimen, comprising: contacting the 3-D specimen with a non-aqueous clearing agent for a time sufficient to clear the specimen for optical evaluation, the clearing solution having a density of from about 0.9 g/ml to about 1.2 g/ml, the 3-D specimen not being subjected to a dehydration step directly before being contacted with the clearing agent. [0017] In some embodiments, the 3-D specimen had been in contact with an penultimate aqueous solution from which it was removed just before being contacted with the clearing agent, the 3-D specimen not being subjected to a dehydration step after being removed from the penultimate aqueous solution. In some embodiments, the density differential between the clearing agent and the penultimate aqueous solution in which the specimen was in contact before the clearing step is less than 0.1 g/ml.

[0018] In certain embodiments, the clearing solution comprises 2,2,2-trichloroethanol (TCE), benzyl alcohol, polyethylene glycol (PEG), and methanol. In particular embodiments, the clearing agent comprises about 20% to about 40% TCE, about 20% to about 40% benzyl alcohol. In some embodiments, clearing agent further comprises from 0% to about 40% methanol/ethanol and from about 10% to about 40% PEG/(ethylene glycol (EG)/propylene glycol (PG)/Glycerol.

[0019] In particular embodiments, the 3-D specimen is a spheroid, organoid, microtissue, or organ-on-a-chip.

BRIEF DESCRIPTION OF THE FIGURES

[0020] The foregoing and other objects of the present disclosure, the various features thereof, as well as the invention itself may be more fully understood from the following description, when read together with the accompanying drawings in which:

[0021] FIG. 1 is a diagrammatic representation of the method for visualization of a 3- D culture according to the disclosure;

[0022] FIG. 2 is a representation of microscopic image obtained with an upright wide- field light microscope at lOx magnification of an unstained HepG2 spheroid optically cleared with the prior art BABB method;

[0023] FIG. 3 is a photographic representation of a confocal image z-stack, showing a 3-D tissue culture expressing dsRed fluorescent protein (red) and Cerulean fluorescent protein (blue);

[0024] FIG. 4 is a is a photographic representation of a confocal image z-stack, showing a spheroid stained with SYTOX green; and

[0025] FIG. 5 is a microscopic image obtained with ThermoFisher CX7 LZR high- content analysis system running in wide-field mode showing a 3D tissue culture specimen stained with Ki-67.

DESCRIPTION

[0026] The issued U.S. patents, allowed applications, published foreign applications, and references that are cited herein are hereby incorporated by reference in their entirety to the same extent as if each was specifically and individually indicated to be incorporated by reference. Patent and scientific literature referred to herein establishes knowledge that is available to those of skill in the art.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0028] The term“3-D tissue culture” refers to two or more layers of single or multiple cell types in culture; whether adherent to a scaffold, matrix, or other substrate, or to each other in the form of a spheroid in solution, or bound onto a channel, surface, or container.

Additionally, free floating tissue sections, whether synthetically grown or derived from whole tissues are considered“3-D tissue culture”.

[0029] A“specimen” of the 3-D tissue culture can be the entire culture or a portion thereof which retains the two or more layers of single or multiple cell types and may also retain a 3-D structures.

[0030] The term“dehydrated” refers to a state wherein there is no water present. A “dehydration step” refers to contacting the specimen with a solution that removes water from the specimen. Such a solution includes, but is not limited to, ethanol, methanol,

tetrahydrofuran (THF), and combinations thereof.

[0031] The term“penultimate solution” refers to the last an aqueous solution with which the 3-D tissue culture specimen was in contact just or immediately before it was contacted with the clearing agent. [0032] The present disclosure provides a method of optical visualization of a 3-D tissue culture specimen which enables in vitro assays utilizing these tissues. The method comprises contacting the specimen which had previously been in contact with an aqueous solution with a non-aqueous clearing solution, thereby enabling visualization of a component of the tissue culture without the loss of optical clarity. This method excludes a dehydration step just before the specimen is contacted with the clearing solution.

[0033] In the past, when a non-aqueous clearing agent was used, a dehydration step was required before the tissues were cleared and visualized. This is because the staining step is performed in an aqueous solution, and any water remaining in the clearing solution will reduce optical clarity because the immiscible water droplets cause light scattering in the clearing agent. Thus, the remaining water both in the tissue itself and the container must be removed to ensure that the non-aqueous clearing agent can effectively produce optical clarity in the tissue. This dehydration is usually accomplished by washing the tissues with an anhydrous, intermediately polar alcohol solution like methanol or ethanol. Once the water has been removed, the tissue is ready for optical clearing with a clearing agent ( e.g . Benzyl Alcohol/Benzyl Benzoate (BABB), or Visikol® HISTO-l™), and after optical clarity is achieved, the tissue can be imaged with a microscope (e.g. wide-field, confocal, light-sheet, or two photon).

[0034] The use of confocal microscopy for thicker specimens (>20 pm) requires special considerations since optical limitations due to light scattering in opaque tissues limits imaging depth. As the depth increases, there is a noticeable“eclipsing” of the inner layers due to light scattering reducing the detectable signal. The net effect introduces a bias in the data obtained from confocal imaging since only the outermost layers of cells are detected, and the outer layers of cells experience higher proliferation rates and increased exposure to test compounds (Selby et a/. (2017) Adv. Life Sci. R&D 22(5): 473-483). The eclipsing effect is sometimes not apparent in publications since it is common practice with confocal microscopy to utilize z-projections of spheroids.

[0035] The present method bypasses such problems by utilizing a clearing agent that solubilize a greater amount of water when compared to currently known non-aqueous clearing agents. Thus, this method eliminates the need for a dehydration step to remove excess water just before the specimen is contacted with the clearing

[0036] A useful clearing agent may comprise about 20% to about 40% 2,2,2- trichloroethanol (TCE), about 20% to about 40% Benzyl Alcohol (BA), about 10% to about 40% PEG/EG/PG/Glycerol, and from 0% to about 40% MeOH/EtOH. The density of a clearing agent useful in the present method is 0.9.1 g/ml and 1.2 g/ml. Density can be measured using a pycnometer.

[0037] Representative amounts of certain components in useful representative clearing agents and their densities according to the disclosure are listed below in Table 1.

Table 1

[0038] Clearing agents solutions with density between 0.9.1 g/ml and 1.2 g/ml dislocated the spheroids from the center of the well the least, while also ranking high in the qualitative assessment of optical clarity. Heretofore, clearing agents known in the art have densities greater than about 1.2 g/ml, such as HISATO-M, and as such as not useful in the present method.

[0039] 3-D tissue cultures useful for various in vitro assays include, but are not limited to, spheroids, organoids, microtissues, and organ-on-a chip. can. These 3-D cultures can be commercially obtained or produced by variety of techniques (see, e.g. Przyborski (2017) Technology Platforms for 3D Cell Culture: A Users Guide. John Wiley & Sons Ltd. ISBN: 9781118851500) including, but not limited to, aggregate based technologies (e.g. hanging drop, specialty patterned surfaces, ultra-low attachment surfaces), and hydrogels (e.g.

Matrigel®, HyStem®), and scaffolds (e.g. Alvetex®, CelluSponge™, Mimetix®).

[0040] Typically, the specimen is grown in aqueous growth and/or maintenance media which is removed before the specimen is contacted with the clearing agent.

[0041] After being removed from the aqueous solution, the specimen is then contacted with a clearing solution which enables optical visualization of a component within the specimen. For example, if the specimen has been genetically engineered to express endogenous fluorescence (such as GFP, YFP, RFP, mCherry, dsRed, Cerulean), after being subjected to clearing the fluorescent protein becomes visible (see EXAMPLE 1).

Additionally, if a cell viability stain (such as calcein AM, Cl2-resazurin, FUN 1, CFDA AM) that fluoresces upon uptake into live cells, the stain remaining in the cells becomes visible after being subjected to the clearing agent.

[0042] Optionally, after the medium is removed, the specimen is washed with a buffer solution which optionally contains a detergent or surfactant such as, but not limited to, Triton X-100, SDS, etc. The specimen may also have been in contact with other aqueous solutions such as, but not limited to, permeabilization, fixative, and/or staining solutions.

[0043] For example, once the tissue cultures have been grown, they optionally can be fixed with chemical crosslinking fixatives like formaldehyde, paraformaldehyde, or gluteraldehyde, and/or with precipitative fixatives like methanol or ethanol. [0044] Before staining, the tissue culture specimen optionally may be permeabilized to allow the staining molecules to penetrate evenly throughout the tissue (which may or may not have been previously fixed). Permeabilization uses various chemicals either alone or combined in aqueous solution such as alcohols ( e.g . methanol, ethanol), polar solvents (e.g. DMSO, acetone, THF), and surfactants (e.g. SDS, Triton X-100, Tween 20) to create pores in the tissues, allowing the staining molecules to pass through.

[0045] Optionally, the specimen is stained with optically active (usually fluorescent or colored in the visible spectrum) molecules used in biochemistry to study features within the tissue. When tissues stained with these molecules (e.g. fluorescent dyes, inks, fluorescently labeled antibodies, or other optically active stains and markers used in the biological sciences such as lectins), are submerged in the formulation they become optically transparent which allows the tissues to be visualized in their entirety with optical microscopy technique

[0046] Some of these pre-clearing steps involve the use of solutions that may first require the use of a dehydration step for efficacy. However, after their use, the need to dehydrate the specimen does not exist, and so the method purposefully excludes such a penultimate dehydration step before the clearing step.

[0047] Note that in the present method, the density differential between the solutions used in a penultimate (aqueous) step and the clearing step is less than < 0.1. A density differential outside this range is not contemplated by the present method.

[0048] Two known clearing methods ((BABB clearing (Smyrek et al. (2017) Biomed.

Opt. Express: 8, 484-499); see FIG. 7) and Visikol HISTO-M

(http://protocol.visikol.com/example/nci2l70-organoid-ki67) were compared with an embodiment of the present method (EXAMPLE 2) using the clearing agent prepared in EXAMPLE 1. The results summarized below in Table 2. Table 2

[0049] As shown above, the density differential between solutions used in a penultimate (aqueous) step and the clearing step) of previously known methods is greater than that of the present method A density differential of < 0.1 prevents the specimen from floating when the clearing agent is applied, allowing the specimen to be imaged immediately without equilibration. This combined with the exclusion of a dehydration step just before the clearing step, results in the present method which is rapid, highly compatible and with automated liquid handling and imaging systems, and that results in superior optical clarity.

[0050] Reference will now be made to specific examples illustrating the disclosure. It is to be understood that the examples are provided to illustrate exemplary embodiments and that no limitation to the scope of the disclosure is intended thereby. EXAMPLES

EXAMPLE 1

Preparation of an Exemplary Clearing Agent

[0051] A clearing formulation was produced by mixing the following commercially obtainable components in a single vessel at room temperature (RT) for 10 min: 25 mL 2,2,2- trichloroethanol (TCE); 33 mL benzyl alcohol; 32 mL PEG200; and 10 mL methanol

[0052] The resulting solution had a density of 1.1110 g/mL as measured by a pycnometer, and a Refractive Index (nD/20) of 1.48506 as measured by an automatic refractometer (Model: j357, Rudolph Research Analytical, Hackettstown, NJ).

EXAMPLE 2

Spheroid Dislocation Test

[0053] The clearing formulae listed in Table 1, above, were screened on fixed HepG2 (Sigma- Aldrich, St. Louis, MO) spheroids stored one spheroid per well in phosphate buffered saline (PBS) in a 96-well round bottom plate (Coming Cat# 4520, Coming, NY). After visually confirming the presence of a spheroid in the well, the PBS solution was manually aspirated out of the well with observation under an upright wide-field light microscope with a lOx objective to ensure the spheroid remained in the well after the solution was removed. Then, 100 pL of a clearing formula from Table 1 was added to a well for a 5 min.

equilibration period. The well was then observed under the same microscope to determine the location of the spheroid, and the optical clarity was qualitatively assessed.

[0054] The results are shown above in Table 1. EXAMPLE 3

Visualization of Cultured Spheroids Expressing Fluorescent Protein

[0055] A Human Islet Microtissue (Catalog# MT-0-002-01, InSphero, Schlieren, Switzerland) expressing dsRed fluorescent protein and Cerulean fluorescent protein was stored in a 4% PFA solution. The PFA solution was removed from the well containing the spheroid, and 100 pL of the clearing solution in Example 1 was introduced into the well. After 5 min, the spheroid was imaged using a ThermoFisher CX7 LZR high-content analysis system (Pittsburgh, PA).

[0056] FIG. 3 is a representative confocal z-projection of a spheroid captured by the instrument.

EXAMPLE 4

Visualization of Nuclear Stained Cultured Spheroids

[0057] 100 pL of a solution of live HepG2 cells (Sigma- Aldrich, St. Louis, MO) at a concentration of 1,500 cells per 100 pL in Dulbecco's Modified Eagle Medium (Gibco, Gaithersburg, MD) was seeded into each well of a Coming 96-well, round-bottom, ultra-low attachment well-plate (Coming Cat# 4520, Coming, NY) and cultured into spheroids per the procedure described by Sirenko et al. (Assay Drug Dev. Technol (2015) 13(7):402-414. doi: l0.l089/adt.20l5.655).

[0058] After this period, the DMEM growth medium was removed from the wells and replaced with 100 pL of 4% paraformaldehyde (PFA) in water. After 10 min, the PFA solution was removed from the wells and replaced with 100 pL PBS with 1% Triton X-100 solution.

[0059] After 10 min, the solution was removed from the wells and replaced with 100 pL of a 1 :500 dilution of SYTOX green nuclear label (Cat#: S7020, ThermoFisher Eugene, OR) in PBS with 1% Triton X-100. After 10 min, the label solution was removed from the wells and replaced with 100 pL PBS with 1% Triton X-100. [0060] After 10 min, the solution was removed from the wells and replaced with 100 pL of the clearing solution made in Example 1 until clear (about 5 min).

[0061] The spheroids were then imaged using a ThermoFisher CX7 LZR high-content analysis system (Pittsburgh, PA).

[0062] FIG. 4 is a representative confocal z-projection of a spheroid captured by the instrument.

EXAMPLE 5

Visualization of Cultured Spheroids

[0063] 100 pL of a solution of live HepG2 cells (Sigma- Aldrich, St. Louis, MO) at a concentration of 1,500 cells per 100 pL in Dulbecco's Modified Eagle Medium (Gibco, Gaithersburg, MD) was seeded into each well of a Coming 96-well, round-bottom, ultra-low attachment well-plate (Coming Cat# 4520, Coming, NY) and cultured into spheroids per the procedure described by Sirenko et al, supra.

[0064] After this period, the growth medium was then removed from the wells and replaced with 100 pL of 4% paraformaldehyde (PFA) in DI water for 10 min. The PFA solution was then removed and replaced with 100 pL PBS for 5 min. This step was repeated.

[0065] After 2 min, the solution was removed and replaced with 100 pL l00%_methanol for 2 min, after which the solution was removed and replaced with 100 pL 20% DMSO in methanol for 2 min. The solution was then removed and replaced with 100 pL 100% methanol for 2 min, after which the solution was removed and replaced with 100 pL 1% TritonTM X-100 in PBS.

[0066] After 2 min, the solution was removed and replaced with 100 pL Visikol HISTO Penetration Buffer (0.2% v/v Triton™ X-100, 0.3 M glycine, and 20% v/v DMSO in PBS) and shaken gently for 15 min. The buffer solution was removed and replaced with 100 pL Visikol HISTO Blocking Buffer (0.2% v/v Triton™ X-100, 6% v/v donkey semm, and 10% v/v DMSO in PBS) and shaken gently at RT for 15 min. [0067] The buffer solution was then removed and replaced with 100 pL Thermo Ki67

Ab-4, rabbit polyclonal antibody, (catalog # RB-1510-PO) (ThermoFisher Eugene, OR) in Visikol HISTO Antibody Buffer (0.2% v/v Tween™ 20, 10 pg/mL Heparin, 3% v/v donkey serum, and 5% v/v DMSO in PBS) at a 1 : 100 dilution. Tissues were then incubated for 30 min at RT with gentle shaking in the dark. The antibody solution was removed and replaced with 100 pL Visikol HISTO lx Washing Buffer (0.2% v/v Tween™ 20, 10 pg/mL Heparin in PBS) and shaken gently for 10 min. This washing step was repeated 2 times. The washing buffer was removed and replaced with 100 pL Invitrogen Goat Anti-Rab IgG AlexaFluor Plus 488 highly cross-absorbed (cat # A32731) (ThermoFisher Eugene, OR)_in Visikol HISTO Antibody Buffer (0.2% v/v Tween™ 20, 10 pg/mL Heparin, 3% v/v donkey serum, and 5% v/v dimethylsulfoxide (DMSO) in PBS) at a 1 :200 dilution factor. Tissues were then incubated for 30 min at RT in the dark. The antibody solution was removed and replaced with 100 pL Visikol HISTO lx washing buffer for 10 min. This step was repeated 2 times.

[0068] The washing buffer was removed and replaced with 100 pL of the clearing solution made in Example 1 until the spheroids are clear (about 5 min).

[0069] The spheroids were then imaged using a ThermoFisher CX7 LZR high-content analysis system. The results are shown in FIG. 5.

EQUIVALENTS

[0070] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific composition and procedures described herein. Such equivalents are considered to be within the scope of this disclosure, and are covered by the following claims.

Claims

1. A method of optically clearing a 3-dimensional (3-D) tissue culture specimen, comprising:

contacting the 3-D specimen with a non-aqueous clearing agent for a time sufficient to clear the specimen for optical evaluation, the clearing solution having a density of from about 0.9 g/ml to about 1.2 g/ml,

the 3-D specimen not being subjected to a dehydration step directly before being contacted with the clearing solution.

2. The method of claim 1, wherein the 3-D specimen had been in contact with an penultimate aqueous solution from which it was removed just before being contacted with the clearing agent, the 3-D specimen not being subjected to a dehydration step after being removed from the penultimate aqueous solution.

3. The method of claim 2, wherein the density differential between the clearing agent and the penultimate aqueous solution in which the specimen was in contact before the clearing step is less than 0.1 g/ml.

4. The method of claim 1, wherein the clearing agent comprises 2,2,2-trichloroethanol (TCE), benzyl alcohol, polyethylene glycol (PEG), and methanol.

5. The method of claim 4, wherein the clearing agent comprises about 20% to about 40% TCE, about 20% to about 40% benzyl alcohol.

6. The method of claim, 5, wherein the clear agent further comprises from 0% to about 40% methanol/ethanol and from about 10% to about 40% PEG/ethylene glycol

(EG)/propylene glycol (PG)/Glycerol.

7. The method of claim 1, wherein the 3-D specimen is a spheroid, organoid, microtissue, or organ-on-a-chip.