Novel organoid culture system for assessing toxicity of nanomaterials

Abstract

The present invention relates to a liver organoid suspension culture method for assessing the safety and toxicity of nanomaterials and, more specifically, to a culture method in which nanomaterials can be introduced into cells constituting an organoid, wherein when a suspension culture system according to the present invention is used, the organoid culture method provides a reliable tool for assessing the stability of nanomaterials, is highly likely to be applicable to high-throughput nano safety screening, and can accelerate the research and development of nanomaterials in various fields.

Description

A novel organoid culture system for nanomaterial toxicity assessment

[0001] The present invention relates to a method for culturing organoids for evaluating the safety and toxicity of nanomaterials, and more particularly, to a culturing method by which nanomaterials can be introduced into cells constituting organoids.

[0002]

[0003] The rapid advancement of nanotechnology has led to the discovery of numerous nanomaterials, expanding into new areas. The potential applications of nanomaterials range from cosmetics and biopharmaceuticals to nanoelectronics and mechanical engineering. Consequently, advancements in nanotechnology have significantly increased public exposure to nanomaterials. This rapid expansion of nanotechnology for human use is limited by a lack of understanding of how the unique physicochemical properties of these materials affect biological systems, particularly toxicological mechanisms. As other studies have shown, understanding the scope of health and safety risks associated with nanomaterial exposure, advancing knowledge, and accelerating the advancement of nanotechnology are fundamental.

[0004] Two-dimensional (2D) cell-based assays have been considered the gold standard for in vitro nanotoxicity assessment due to their cost-effectiveness, reproducibility, and high throughput, despite the lack of reliability between in vitro and in vivo assays. Animal models are the gold standard in toxicology research, but in vivo experiments are expensive, low-throughput, time-consuming, labor-intensive, and subject to ethical constraints. Furthermore, the Environmental Protection Agency (EPA) has announced plans to eliminate mammalian toxicity testing by 2035. Organoids, self-organizing, multicellular 3D tissues, are considered a promising alternative to animal models. Organoid models offer several advantages over 2D cell models, including a relatively long lifespan and the complete absence of ethical concerns associated with animal testing. Furthermore, organoids exhibit a high degree of similarity to real organs, possessing both structural and functional characteristics. The significant cellular heterogeneity of organoids allows them to replicate the structural barriers and complexity of real organs.

[0005] Recent studies have demonstrated the potential of organoids as physiologically relevant models for assessing nanomaterial toxicity. Recent studies have reported the toxicity of silver nanoparticles in cerebral organoids and established a toxicity assessment of nanoparticles in colonic organoids. While these studies highlight the potential of organoids as a tool for assessing nanoparticle toxicity, limitations remain. One major limitation is the lack of confirmation of nanoparticle internalization by cells within organoids. Despite these studies reporting nanoparticle toxicity in organoid systems, they do not provide direct evidence of nanoparticle internalization and intracellular localization. This lack of evidence may raise concerns about the reliability and validity of reported toxicological results.

[0006] Another limitation of organoid-based toxicity assessment of nanoparticles is the lack of standardization in organoid preparation and handling. The extracellular matrix (ECM), a unique meshwork of structural proteins and glycosaminoglycans, serves as a physical framework for organoid 3D morphology and growth. Organoids are typically embedded and cultured on solidified ECM scaffolds to form dome-like structures. However, organoids grown on ECM domes exhibit size heterogeneity induced by spatiotemporal gradients in morphology, oxygen, and nutrients, making them unsuitable for in vitro screening assays. Furthermore, nanoparticles tend to aggregate in the ECM and exhibit inefficient cellular internalization due to the complex network and charged components inherent in the ECM.

[0007] The present invention reports the development of a novel organoid culture system for evaluating the safety and toxicity of nanomaterials. This system demonstrates that nanomaterials are internalized within organoids cultured using this system, overcoming limitations associated with the ECM, a barrier to nanomaterial accumulation. The protocol produced organoids of relatively uniform size and enabled the manipulation of organoid numbers suitable for standardization. Our results suggest that suspension culture of organoids provides an accurate and reliable platform for evaluating the safety of nanomaterials.

[0008]

[0009] The present invention relates to the construction of a novel organoid culture system for evaluating the stability and toxicity of nanomaterials.

[0010] The present invention aims to provide an accurate and reliable platform for evaluating the stability of nanomaterials by overcoming the limitations associated with the extracellular matrix (ECM), which is a barrier to nanomaterial internalization, producing organoids of uniform size and manipulating the number of organoids suitable for standardization, thereby providing a customized toxicity assessment tailored to each tissue or individual.

[0011]

[0012] The present invention is a novel organoid culture method for evaluating the stability and toxicity of nanomaterials. It is an organoid that uses a floating culture method rather than the existing adherent organoid, and its purpose is to overcome the limitations related to ECM and to control the number of organoids suitable for uniform size and standardization to produce an accurate and reliable platform.

[0013] As a specific example of the invention, the nanomaterial may be a nanoparticle that generates toxicity when reacting with living tissue, and specifically may be a metal, heavy metal, inorganic oxide, carbon-based nanomaterial, or polymer nanomaterial, but is not limited thereto.

[0014] As a specific example of the invention, the nanomaterial may be capable of causing changes in biological metabolism and immune responses when reacting with biological tissues, and specifically, may cause metabolic disorders or recovery of metabolic disorders, induce or alleviate inflammatory responses, induce or alleviate cancer, etc., but is not limited thereto.

[0015] As a specific example of the invention, the organoid may be formed through self-renewal, differentiation, and self-organization by selecting from a group of stem cells including adult stem cells, embryonic stem cells, and induced pluripotent stem cells.

[0016] As a specific example of the invention, the organoids may have functions that mimic the brain, gastrointestinal tract, intestine (small intestine, large intestine), stomach, thyroid, thymus, testis, prostate, liver, pancreas, epithelium, kidney, heart, retina, glioblastoma, etc., but are not limited thereto.

[0017]

[0018] The organoid culture method according to the present invention can provide an efficient analysis for nanomaterial-induced toxicity evaluation.

[0019] The organoid culture method according to the present invention is in a floating state, unlike the adherent organoid that uses only a portion of the organoid, so that a more accurate toxicity evaluation of nanomaterials can be performed by using the entire organoid.

[0020] The organoid culture method according to the present invention can help internalize nanoparticles by exhibiting uniform size and structural integrity, thereby enabling more accurate toxicity assessment of nanomaterials compared to existing organoid toxicity assessment platforms.

[0021] Figure 1 is a dark-field microscopy image of an ECM dome after exposure to gold nanoparticles (AuNPs). The following Figures, including Figure 1, 2, and 3, illustrate the limitations of an extracellular matrix (ECM)-embedded organoid culture system for safety assessment of existing nanomaterials.

[0022] Figure 2 is a representative bright-field microscopic image of mouse liver organoids (mLO) embedded and cultured in domes containing ECM of various concentrations.

[0023] Figure 3 is a dark field microscope image taken after exposing AuNPs to mLO embedded in ECM.

[0024] Figure 4 is a schematic diagram of a floating culture system for mLO, which is a drawing regarding preparation and optimization of a floating organoid culture system.

[0025] Figure 5 is about the optimization process of the floating organoid culture conditions. Figure 5A is the result of mLO embedded in an ECM dome and cultured for 3 days, transferred to an attachment plate and an ultra-low attachment plate, and then cultured in a floating manner, and confirmed under a microscope. Figure 5B is the result of monitoring using a live cell analysis system after embedded in an ECM dome and cultured for 3 to 5 days, and then converted to a floating manner.

[0026] Figure 6 is a fluorescence microscopy image of ECM-embedded mLO and floating mLO immunostained with differentiation marker proteins to confirm differentiation.

[0027] Figure 7A shows the results of qRT-PCR analysis of undifferentiated mLO, differentiated ECM-embedded mLO, and floating mLO, and Figure 7B is a heatmap representing the results of RNAseq analysis.

[0028] Figure 8 shows the results of changes in the size and size distribution of mLO in ECM-embedded and floating culture organoid systems, where Figure 8A shows automatic detection and quantification of mLO, and Figure 8B is a table of the size distribution of mLO based on the diameter and amount of organoids recognized in Figure 8A.

[0029] Figure 9 shows the results of a comparison regarding the introduction of nanomaterials into liver organoids according to cell culture methods. Figure 9A is a fluorescence microscope image of ECM-embedded and floating cultured organoids exposed to FITC-polystyrene, and Figure 9B is a fluorescence microscope image of the organoid paraffin section of Figure 9A.

[0030] Figure 10 shows the results of flow cytometry analysis of ECM-embedded and suspension-cultured organoids exposed to FITC-polystyrene.

[0031] Figure 11A is a dark-field microscopy image of organoids exposed to AuNPs, and Figure 11B is a dark-field microscopy image of suspension-cultured organoids over time after exposure to AuNPs.

[0032] Figure 12 shows the results of evaluating the cytotoxicity of mLO nanoparticles cultured in ECM-embedded and suspended forms. Fluorescence microscopy images of mLO stained with calcein / propidium iodide (PI) after exposure to AuNP and ZnO.

[0033] Figure 13A is a bar graph showing the survival / death ratio by quantifying the calcein / propidium iodide fluorescence intensity of Figure 12, and Figure 13B is the result of flow cytometry analysis measuring apoptosis.

[0034]

[0035] The present invention is a novel organoid culture method for evaluating the stability and toxicity of nanomaterials. It is an organoid that uses a suspension culture method rather than the existing ECM-embedded organoids, and its purpose is to overcome the limitations related to ECM and to create an accurate and reliable platform by controlling the number of organoids to be suitable for uniform size and standardization.

[0036] As a specific example of the invention, the nanomaterial may be a nanoparticle that generates toxicity when reacting with living tissue, and specifically may be a metal, heavy metal, inorganic oxide, carbon-based nanomaterial, or polymer nanomaterial, but is not limited thereto.

[0037] As an embodiment of the invention, the nanomaterial may be a metal, a heavy metal, an inorganic oxide, a carbon-based nanomaterial, a polymer nanomaterial, etc., and specifically, may be a PS NP (polystyrene nanoparticle), Au NP (gold nanoparticle), ZnO NP (zinc oxide nanoparticle), fine dust, a chemical nanomaterial, a cosmetic composition, and more specifically, a nanomaterial that is PS NP, Au NP, ZnO NP, but is not limited thereto.

[0038] As a specific example of the invention, the nanomaterial may be capable of causing changes in biological metabolism and immune responses when reacting with biological tissues, and specifically, may cause metabolic disorders or recovery of metabolic disorders, induce or alleviate inflammatory responses, and induce or improve cancer, but is not limited thereto.

[0039] As an embodiment of the invention, the nanomaterial may be, but is not limited to, an inflammatory response, an immune response, a metabolic disease, or a carcinogen.

[0040] In the present invention, organoids are three-dimensional cell aggregates formed through self-renewal and self-organization from adult stem cells, embryonic stem cells, and induced pluripotent stem cells, and contain specific cells of a model organ. They can closely replicate the physiological functions of the human body, and by constructing organ analogs from patient tissue, they enable genetic information-based disease modeling and drug screening. They are attracting attention as an alternative to gene therapy and organ transplantation.

[0041] As a specific example of the invention, the organoid may be formed through self-renewal, differentiation, and self-organization by selecting from a group of stem cells including adult stem cells, embryonic stem cells, and induced pluripotent stem cells.

[0042] As a specific example of the invention, the organoid may include, but is not limited to, the brain, gastrointestinal tract, intestine (small intestine, large intestine), stomach, thyroid, thymus, testis, prostate, liver, pancreas, epithelium, kidney, heart, retina, glioblastoma, etc.

[0043] In one specific embodiment of the invention, the ECM-embedded organoid cultures are routinely maintained in an organic culture medium, wherein the organoids are passaged every 5 to 10 days, specifically every 6 to 8 days, and more specifically every 7 days.

[0044] As one specific example of the invention, a method for producing spherical organoids uses a high concentration of ECM of 50% or more, specifically, the ECM may have a concentration of 50 to 100%, and more specifically, may have a concentration of 50% or more.

[0045] As one specific example of the invention, the method of dissociating the passaged organoids was to break the mLO into small pieces by repeated pipetting using a 1 ml syringe equipped with a 26G blunt needle.

[0046] The fragmented mLO was centrifuged and the supernatant was removed.

[0047] As a specific example of the invention, the centrifugation conditions may be centrifugation at 100 to 150 G, and specifically, centrifugation at 125 G.

[0048] As a specific example of the invention, the time condition of the centrifugation may be 3 to 7 minutes, specifically 4 to 6 minutes, and more specifically 5 minutes.

[0049] Afterwards, the centrifuged pellet was resuspended in 100% Matrigel, and the resulting cell suspension was dispensed in a dome shape into a 48-well plate, and then placed in a 37°C incubator for 1 hour to solidify the Matrigel.

[0050] The above Matrigel is composed of a dissolved basement membrane matrix secreted by mouse sarcoma cells, which is a protein structure such as laminin, nidogen, collagen, and heparan sulfate protein oglycan, and is used for tissue culture. It may also contain TGF-β, EGF, and various growth factors.

[0051] In one specific example of the invention, the amount of the cell suspension dispensed into a well plate may vary depending on the size of the plate, but may be 10 to 50 μl, specifically 20 to 30 μl, and more specifically 25 μl.

[0052] Organoid culture medium was added to the hardened Matrigel in the incubator, and the medium was replaced every 2 to 3 days.

[0053] Afterwards, the Matrigel organoids cultured for 3 days are used for suspension culture.

[0054] The purpose of the present invention is to evaluate the toxicity of nanomaterials based on organoids cultured in suspension.

[0055] As one specific example of the invention, the method for culturing the organoid suspension is as follows. A) a step of washing the matrigel (100%) containing the organoid with PBS; B) a step of adding a cooled cell recovery solution to the well and dissolving it by pipetting; C) a step of incubating for about 30 minutes until the matrigel matrix is ​​completely dissolved and centrifuging it; D) a step of suspending it in an organoid culture medium containing 5% matrigel; and E) a step of incubating it in an ultra-low attachment plate.

[0056] As a specific example of the invention, the centrifugation conditions may be centrifugation at 100 to 150 G, and specifically, centrifugation at 125 G.

[0057] As a specific example of the invention, the time condition of the centrifugation may be 1 to 3 minutes, and specifically 1 to 2 minutes.

[0058] As a specific example of the invention, the Matrigel contained in the organoid culture medium may be 1 to 10%, specifically 3 to 8%, and more specifically 5%.

[0059] As a specific example of the invention, the medium replacement cycle in the incubation may be 1 to 5 days, specifically 2 to 4 days, and more specifically, may be replaced every 2 or 3 days.

[0060] Afterwards, the suspension-cultured organoids were changed to differentiation medium to differentiate them, and the medium was continuously replaced until differentiation was complete.

[0061] The composition of the above differentiation medium is 10 mM HEPES, 2 mM Glutamax, 1 mM N-acetylcysteine, 1% penicillin / streptomycin, 1x B-27, 50 ng / ml EGF (Epidermal Growth Factor), 100 ng / ml FGF10 (Fibroblast Growth Factor), 10 nM recombinant human [Leu15]-gastrin I, 50 nM A83-01, 10 μM DAPT, but is not limited thereto, and may vary depending on the type of organoid.

[0062] As a specific example of the invention, the differentiation medium is replaced daily, and the maximum culture period before the anti-inflammatory agent is administered may be 10 to 15 days, specifically 11 to 13 days, and more specifically, the differentiation medium may be replaced daily for up to 12 days to differentiate the organoids.

[0063] As a specific example of the invention, the anti-inflammatory agent may be at least one selected from the group consisting of steroids, non-steroids, immunospecific anti-inflammatory agents, herbal extracts, and antioxidants, and specifically, may be a steroid anti-inflammatory agent.

[0064] As a specific example of the invention, the steroid anti-inflammatory agent may be prednisone, dexamethasone, triamcinolone, betamethasone, beclomethasone, flunisolide, fluticasone, specifically, betamethasone, beclomethasone, dexamethasone, and more specifically, dexamethasone.

[0065] As a specific example of the invention, the period of treating the differentiated organoid with an anti-inflammatory agent may be 1 to 5 days, specifically 2 to 4 days, and more specifically 3 days.

[0066] Using the above-mentioned floating culture organoids, the differentiation of the organoids was confirmed through quantitative analysis such as immunostaining, qRT-PCR, and RNAseq, and the cell penetration ability of the nanoparticles was confirmed based on the analyzed organoids.

[0067]

[0068] As one embodiment of the invention, the present invention may provide an organoid culture kit for evaluating nanomaterial toxicity.

[0069] As one specific example of the invention, the culture kit may be a kit for culturing organoids using a floating culture method.

[0070] As a specific example of the invention, the nanomaterial whose toxicity is confirmed through the culture kit may be a metal, a heavy metal, an inorganic oxide, a carbon-based nanomaterial, or a polymer nanomaterial, but is not limited thereto.

[0071] As a specific example of the invention, the nanomaterials for which toxicity is confirmed through the culture kit may be metals, heavy metals, inorganic oxides, carbon-based nanomaterials, polymer nanomaterials, etc., and specifically, may be PS NPs (polystyrene nanoparticles), Au NPs (gold nanoparticles), ZnO NPs (zinc oxide nanoparticles), fine dust, chemical nanomaterials, cosmetic compositions, and more specifically, nanomaterials such as PS NPs, Au NPs, and ZnO NPs, but are not limited thereto.

[0072] As one specific example of the invention, the organoids available through the kit may be three-dimensional cell aggregates formed through self-renewal and self-organization from adult stem cells, embryonic stem cells, and induced pluripotent stem cells.

[0073] As a specific example of the invention, organoids that can be used through the kit may include, but are not limited to, brain, gastrointestinal tract, intestine (small intestine, large intestine), stomach, lingual, thyroid, thymus, testis, prostate, liver, pancreas, epithelium, kidney, heart, retina, glioblastoma, etc.

[0074]

[0075] Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are provided for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0076]

[0077]

[0078] Example 1. Experimental methods and materials

[0079] 1-1. Experimental materials

[0080] Collagenase (C9407), DNase I (DN25), [Leu15]-gastrin 1 (G9145), nicotinamide (N0636), N-acetylcysteine ​​(A0737), PBS (D8537), paraformaldehyde (158127), Triton X-100 (T878787), bovine serum albumin (A9418), calcein-AM (C1430), propidium iodide (P4170), DAPI (D9564), dexamethasone (D4902), and DAPT (D5942) were purchased from Sigma-Aldrich (MO, USA).

[0081] Matrigel (356231) and Cell Recovery solution (354253) were purchased from Corning (NY, USA).

[0082] High-glucose Dulbecco's modified Eagle's medium (DMEM) (31966-021), Advanced DMEM F / 12 (12634-010), B-27 (12587-010), HEPES (15630-056), Glutamax (35050-068), fetal bovine serum (26140-079), penicillin / streptomycin (15140-122), Dispase II (17105-041), and trypsin / EDTA (25200-056) were purchased from GIBCO (MA, USA). Recombinant murine EGF (315-09), murine Noggin (250-38), and murine HGF (315-23) were purchased from PeproTech (NJ, USA).

[0083] A83-01(2939) was purchased from Tocris Bioscience (Bristol, UK).

[0084] The cell division detection kit (556547) was purchased from BD Biosciences. TRIzol (15596026) and SYBR Green PCR Master Mix (4309155) were purchased from Thermo Fisher Scientific.

[0085] PrimeScript 1st strand cDNA synthesis kit (6110A) was purchased from TaKaRa.

[0086] FITC-labeled polystyrene nanoparticles (DCFG-L002) were purchased from CD Bioparticle (NY, USA).

[0087] AuNP (EM.GC50) was purchased from BBI solution (Cardiff, UK).

[0088] Zinc oxide (52504965) was purchased from BASF (Ludwigshafen, Germany).

[0089] Anti-HNF4α (sc8987) was purchased from Santa Cruz Biotechnology (CA, USA), anti-EpCAM (17-5791-80) was purchased from eBioscience (CA, USA), and fluorescent (Alexa Fluor 488 or 546)-conjugated anti-mouse and rabbit immunoglobulins were purchased from Invitrogen Life Technologies (A11001 and A11035).

[0090]

[0091] 1-2. Experimental animals

[0092] Mice were maintained in accordance with the policies of the CHA University Institutional Animal Care and Use Committee (IACUC). All experiments were conducted to minimize animal suffering. Male C57Bl / 6 mice were obtained from Orient Bio (Seongnam, Korea) and maintained in the animal facility of the CHA University Institutional Animal Care and Use Committee under a 12-h light / dark cycle with free access to food and water. This experiment was approved and conducted in accordance with the regulations and guidelines of the CHA University Institutional Animal Care and Use Committee.

[0093]

[0094] 1-3. Manufacturing and generation of liver organoids

[0095] Mouse LO has been previously developed. Mouse liver tissue was isolated and washed with ice-cold wash buffer (high-glucose DMEM supplemented with 1% fetal bovine serum and 1% penicillin / streptomycin). The tissue was then incubated in digestion buffer (0.125 mg / ml dispase II and collagenase, 0.1 mg / ml DNase I in wash medium) at 37°C for 40 min with vigorous shaking. Tubular structures and other hepatocytes were obtained from the supernatant. The supernatant was collected and centrifuged at 200 g for 10 min. The pellet was mixed with Matrigel at a 1:1 ratio. The cells were seeded in organic culture medium as described below. Advenced DMEM F / 12 was supplemented with 10 mM HEPES, 2 mM Glutamax, 1 mM N-acetylcysteine, 1x B-27, 5% R-spondin conditioned medium, 50 ng / ml EGF, 100 ng / ml nogg, 50 ng / ml murine HGF, 200 ng / ml FGF10, 10 mM nicotinamide, and 10 nM gastrin.

[0096]

[0097] 1-4. ECM-embedded organoid cultures

[0098] Mouse LO were routinely maintained in organoid culture medium (Advanced DMEM F / 12 supplemented with 10 mM HEPES, 2 mM Glutamax, 1 mM N-acetylcysteine, 1x B-27, 5% R-sulfondin conditioned medium, 50 ng / ml EGF, 100 ng / ml noggin, 50 ng / ml murine HGF, 200 ng / ml FGF10, 10 mM nicotinamide, and 10 nM gastrin). Organoids were passaged every 7 days; 1) mLO were fragmented into small pieces by repeated pipetting using a 1 ml syringe fitted with a 26 G blunt needle. 2) Cells were centrifuged at 125 G for 5 min at 4°C and the supernatant was removed. 3) The cell pellet was resuspended in 100% Matrigel, and the cell suspension (25 μl) was dropped into the center of a well of a pre-warmed 48-well plate to form a dome shape. 4) The plate was placed in a 37°C incubator for 1 h to solidify the Matrigel. 5) The Matrigel dome was then immersed in organoid culture medium, and the medium was replaced every 2–3 days. 6) Organoid growth and morphology were observed using an Olympus IX71S1F-3 / DP70 inverted microscope, an EVOS XL core imaging system, and an IncuCyte time-lapse microscopy system.

[0099]

[0100] 1-5. Suspension culture

[0101] For suspension culture, organoid-containing Matrigel (100%) was washed with PBS, and pre-chilled Cell Recovery solution was added to the wells on day 3. The Matrigel dome was broken by gentle pipetting using a wide-bore 1000p tip. The organoid and Matrigel mixture was then incubated on ice for approximately 30 minutes until the Matrigel matrix was completely dissolved. After incubation, the mixture was centrifuged at 125 g for 1 minute. The pellet was washed three times with PBS containing 10% fetal bovine serum and suspended in organoid culture medium containing 5% Matrigel. The organoids were transferred and incubated in ultra-low attachment plates. The medium was changed every 2 or 3 days.

[0102]

[0103] 1-6. Organoid differentiation

[0104] Mouse LOs were seeded and maintained in organoid culture medium for 3 days. The medium was then changed to differentiation medium. Mouse liver differentiation medium was supplemented with 10 mM HEPES, 2 mM Glutamax, 1 mM N-acetylcysteine, 1% penicillin / streptomycin, 1x B-27, 50 ng / ml EGF, 100 ng / ml FGF10, 10 nM recombinant human [Leu15]-gastrin I, 50 nM A83-01, and 10 μM DAPT. Differentiation medium was changed daily for 12 days. From days 13 to 15, organoids were cultured in differentiation medium with 3 μM dexamethasone.

[0105]

[0106] 1-7. Immunostaining

[0107] Organoids were fixed with 2% paraformaldehyde for 30 min at room temperature. The fixed organoids were then permeabilized with 0.2% Triton X-100 for 15 min and blocked with 3% bovine serum albumin for 1 h. Organoids were incubated overnight at 4°C with primary antibodies (1:50; HNF4α and 1:100; EpCAM) and treated with fluorescently conjugated secondary antibodies (1:1000; Alexa Fluor 488 and 546) in the dark for 2 h. After each step, organoids were washed three times with PBS for 10 min each, and nuclei were stained with DAPI (1:1000 dilution). Organoids were mounted on confocal plates (coverslip-bottom culture dishes) containing PBS and observed under a confocal microscope (Olympus FV-3000) (Olympus, Tokyo, Japan).

[0108]

[0109] 1-8. Quantitative real-time polymerase chain reaction (qRT-PCR)

[0110] Liver organoids were extracted from Matrigel by pipetting and lysed in Cell Recovery solution. The organoid and Matrigel mixture was centrifuged at 125 g for 3 minutes, and the supernatant was removed. The pellet was washed three times with PBS containing 10% fetal bovine serum. RNA was extracted from the organoids using TRIzol solution according to the manufacturer's instructions. RNA was transcribed into complementary DNA using the PrimeScript 1st-strand cDNA synthesis kit according to the manufacturer's instructions. The expression of selected genes was analyzed using a real-time PCR system (QuantStudio 1) with SYBR Green chemistry.

[0111]

[0112] 1-9. Quantitative analysis

[0113] Images of organoids were obtained from the IncuCyte Time-lapse Microscopy System. Individual organoids within the images were identified and represented by purple boxes using OrgaQuant. To measure diameter, the x and y coordinates from the bounding boxes of the detected organoids were converted to meters, and the area of ​​each organoid was calculated. The organoid size distribution was then plotted against the counts and equivalent area diameter (Da).

[0114]

[0115] 1-10. Nanoparticle cellular uptake analysis; fluorescence microscopy

[0116] Mouse LOs were exposed to 50 μg / ml FITC-PS for 24 h. The organoids were fixed with 2% paraformaldehyde for 30 min at room temperature. The nuclei were stained with DAPI (1:1000 dilution). The organoids were mounted on confocal plates (coverslip-bottom culture dishes) containing PBS and observed using a confocal microscope (Olympus FV-3000).

[0117] For paraffin sections, organoids were fixed with 2% paraformaldehyde for 30 min at room temperature. The paraffin embedding schedule was as follows: 70% ethanol for 30 min; 80% ethanol for 30 min; 90% ethanol for 30 min; 100% ethanol for 30 min; xylene for 1 h, twice; Paraffin wax (This is done in a 60 ℃ heat block for 2 hours. This process is done in a 1.5 ml centrifuge tube. Next, cut 5 mm from the bottom of the tube containing the organoids. Place the end of the tube on an embedding mold and melt the paraffin wax. After melting the wax, transfer the organoids into the embedding mold. The trimmed paraffin block is cut at 10 μm. Serial sections can be obtained from the organoids. The sections are mounted on slides and baked on a 50 ℃ hot plate overnight. Deparaffinization and rehydration of the sections are as follows: Xylene for 5 minutes, twice; 100% ethanol for 3 minutes, twice; 95%, 80% and 70% ethanol for 1 minute each. Nuclei were then stained with DAPI (1:1000 dilution). Coverslips were mounted on slides with mounting medium.

[0118]

[0119] 1-11. Nanoparticle cellular uptake analysis; flow cytometry

[0120] Mouse LOs were exposed to 50 μg / ml FITC-PS for 24 h. To obtain single cells from organoids, the Matrigel was disrupted by pipetting and lysed with Cell Recovery solution. The organoid and Matrigel mixture was centrifuged at 125 g for 3 min, and the supernatant was removed. The organoid pellet was suspended and incubated with trypsin / EDTA at 37°C for 30 min. After incubation, the suspension was pipetted up and down with a 200 μl pipette tip. The pellet was washed three times with PBS containing 10% fetal calf serum and resuspended in the same buffer. Flow cytometry data were acquired using a CytoFLEX (Beckman Coulter) equipped with CytoExpert software (Beckman Coulter, CA, USA).

[0121]

[0122] 1-12. Nanoparticle cellular uptake analysis; dark-field microscopy

[0123] Organoids were exposed to 20 μg / ml gold nanoparticles (AuNPs, 50 nm) for 24 h. Images of nanoparticle cellular uptake in organoids were acquired using darkfield illumination. Two types of annular beams, called darkfield illumination, were used, generated by an enhanced darkfield illumination Abbe condenser (Cytova, AL, USA) and a reflective objective lens (LMFLLN10XBD, Olympus) with a 10 mm working distance. Darkfield images were captured by a CMOS camera (ORX-10GS-51S5C, Oryx, Teledyne) (CA, USA) at a resolution of 2048 x 2048 pixels and a pixel width of 3.45 μm. The magnification of the dark field images was 10× using an objective lens (RMS10X-PF, LMPLFLN10XBD, Olympus). For z-stack scanning, a nano z-positioning stage (MZS500-E Z-axis stage, Thorlabs, NJ, USA) was used to capture depth-of-focus images of organoids. For time-lapse scanning, organoids were captured every 10 s for 1 h after AuNP treatment.

[0124]

[0125] 1-13. Calcein / PI double staining analysis

[0126] Mouse LOs were exposed to 20 μg / ml AuNPs and 20 μg / ml zinc oxide (ZnO) for 48 h. To detect live / dead cells, organoids were stained with 1 μM calcein-AM and 10 μg / ml propidium iodide (PI) for 30 min at 37°C. After culture, organoids were washed three times with PBS and observed using a confocal microscope (Olympus FV-3000).

[0127]

[0128] 1-14. Apoptosis assay

[0129] Mouse LOs were exposed to 20 μg / ml AuNPs and 20 μg / ml zinc oxide (ZnO) for 48 h. Matrigel was disaggregated by pipetting and resuspended in Cell Recovery solution. The organoid-Matrigel mixture was centrifuged at 125 g for 3 min, and the supernatant was removed. The organoid pellet was suspended and incubated with trypsin / EDTA at 37°C for 30 min. After incubation, the suspension was pipetted up and down using a 200 μl pipette tip. The pellet was washed with 10% fetal bovine serum (PBS) and resuspended in a staining buffer containing fluorescein-5-isothiocyanate, ANXA5, and propidium iodide provided in the apoptosis detection kit according to the manufacturer's instructions. Flow cytometry data were obtained using CytoFLEX (Beckman Coulter, USA) equipped with CytoExpert software (Beckman Coulter, CA).

[0130]

[0131] 1-15. Statistical Analysis

[0132] Statistical analysis was performed using Sigma Plot 15.0. Replicate data are expressed as mean ± standard deviation (SD). Significant differences between two nonparametric groups were statistically analyzed using the Mann-Whitney U-test. One-way analysis of variance was used for multiple comparisons. A p-value <0.05 was considered statistically significant.

[0133]

[0134] Example 2. Confirmation of ECM penetration of nanoparticles (NPs)

[0135] In the present invention, to determine whether nanoparticles penetrate existing organoids, we first examined whether nanoparticles (NPs) penetrated ECM domes. The ECM domes were exposed to AuNPs, which could be monitored using dark-field microscopy. The ECM domes were scanned along the z-axis at a step size of 0.375 μm, and 3D images were reconstructed from the z-stack images (Fig. 1). The 3D reconstruction results showed that AuNPs were located on the surface of the ECM dome and did not reach the inner region of the ECM dome. To test whether decreasing the ECM concentration could improve nanoparticle penetration into cells, organoids were cultured in domes containing various concentrations of ECM (Fig. 2). Mouse liver organoids (MLO) were selected because the liver, a major detoxification center in the human body, is where nanomaterials accumulate after inhalation, ingestion, and dermal penetration.

[0136] When high concentrations of ECM (>50%) were used in the domes, spherical organoids were generated, whereas cells did not form 3D structures and attached to the wells when low concentrations of ECM (25%) were used. Next, to determine whether the ECM scaffold acts as a barrier to nanoparticles, we investigated the cellular uptake of nanoparticles in ECM-embedded organoids. Domes were prepared with various concentrations of ECM, and nanoparticle penetration into cells was measured (Fig. 3).

[0137] Mouse LOs were exposed to AuNPs for 24 h, which could be monitored using dark-field microscopy. Time-lapse imaging revealed that while AuNPs moved freely in the culture medium, they were trapped and immobilized on the surface of the ECM dome even at a 50% concentration of ECM. Because AuNPs were trapped on the surface of the ECM dome, they could not penetrate the inner region of the ECM dome where the organoids were located. These results indicate that AuNPs did not penetrate the ECM dome and were not internalized by the cells (Figure 3).

[0138]

[0139] Example 3. Construction of a cell internalization system

[0140] To overcome the limitations of these organoid culture systems, we developed a system that achieves largely uniform organoids and efficient cellular internalization of nanoparticles. A schematic illustration of this modified approach is shown in Figure 4. Our previous results (Figure 2) demonstrated that when using high concentrations of ECM (>50%) in the initial stages of the culture method to form 3D-structured organoids, organoid fragments inevitably became embedded and cultured within domes. Therefore, we designed a culture method to isolate mLOs from ECM domes (100%) after organoid formation.

[0141] The ECM domes containing the formed mLOs were detached by gentle pipetting using a wide-bore 1000P pipette tip and lysed using cell recovery solution. The mLOs isolated from the ECM were then transferred to ultra-low attachment plates containing medium containing a low concentration of ECM. The optimal conditions for the suspension culture system were determined to maintain 3D spherical organoid structures (Fig. 5A, B). After detaching mLOs from the ECM domes, they were placed on various types of culture plates and analyzed under various concentration conditions of media containing ECM. When mLOs isolated from the ECM were transferred to culture plates for adherent cells, the organoids adhered intensively to the well surface and exhibited a flat disc shape (Fig. 5A). In contrast, when mLOs isolated from the ECM were transferred to ultra-low attachment plates, they maintained their 3D spherical structures, except when culture medium supplemented with a low concentration of ECM (1%) was used (Fig. 5A).

[0142] Next, we examined whether the timing of transitioning ECM-embedded organoids to suspension culture was critical for maintaining their 3D structure (Fig. 5B). Because organoids, which are composed of a 3D structure of a single cell layer, are hollow inside, they can be easily damaged by extracellular physical forces resulting from pipetting and centrifugation. Therefore, organoids were separated from the ECM at different culture time points (days 3, 4, and 5) and then transitioned to suspension culture. Mouse LOs transitioned on days 4 and 5 were observed to rupture and collapse into a 2D structure. In contrast, mLOs transitioned to suspension culture on day 3 maintained their 3D spherical shape. Considering all the results, the optimal suspension culture system for maintaining the 3D spherical structure of mLOs was selected: transferring mLOs cultured on day 3 to ultra-low attachment plates containing medium containing 5% ECM.

[0143] To investigate whether mLOs cultured in suspension preserved hepatic differentiation capacity, we analyzed the expression levels of marker genes or proteins of differentiated livers (Figs. 6 and 7A). Immunostaining demonstrated expression of hepatocyte markers HNF-4α and EpCAM in differentiated mLOs cultured using both ECM-embedded and suspension systems (Fig. 6). Consistent with these results, gene expression analysis demonstrated substantial upregulation of hepatocyte markers in both ECM-embedded and suspension-cultured mLOs after differentiation (Figs. 7A and 7B).

[0144] These results indicate that the floating-cultured organoids exhibit hepatocyte-like properties and maintain a 3D spherical structure without ECM.

[0145]

[0146] Example 4. Identification of individual organoids

[0147] Organoids cultured in ECM domes generally exhibit size heterogeneity, with organoids at the center of the dome tending to be smaller than those at the edges. This size heterogeneity in organoids cultured embedded in ECM domes is attributed to the spatiotemporal concentration gradients of morphogens (particularly Wnt3a), oxygen, and nutrients. This is because the edges of the dome, where the ECM is thinner, have a better supply of morphogens, oxygen, and nutrients. However, mLOs in suspension cultures transformed on day 3 and evaluated on day 6 exhibit relatively uniform sizes (Figures 5A, 5B). We hypothesized that the suspension culture method provides a similar level of morphogens, oxygen, and nutrients to all organoids, resulting in relatively low size heterogeneity.

[0148] To measure and quantify organoid size, OrgaQuant software was used to identify individual organoids in images recorded on day 6 (see purple box in Figure 8A). The size distribution of organoids in the suspension culture system exhibited a narrower peak than that of ECM-embedded organoids (Figure 8B). These results indicate that the suspension culture system produces more uniformly sized organoids compared to the ECM-embedded organoid system.

[0149]

[0150] Example 5. Investigation of the internalization efficiency of nanoparticles in suspension cultured organoids.

[0151] Next, we investigated the internalization efficiency of nanoparticles in both ECM-embedded organoids and suspension-cultured organoids to determine whether the suspension culture system could be used to assess the safety of nanomaterials.

[0152] To monitor cellular internalization of nanoparticles, mLO was treated with green fluorescent FITC-labeled polystyrene nanoparticles (FITC-PS NPs, 50 nm) (Figs. 9A, 9B, and 10). Suspension-cultured organoids exhibited a significantly stronger FITC fluorescence signal than ECM-embedded organoids. To quantify nanoparticle internalization, flow cytometry was used to measure cells exhibiting FITC fluorescence; organoids were exposed to FITC-polystyrene nanoparticles and then dissociated into single cells using trypsin / EDTA.

[0153] FITC fluorescence was observed in most (more than 99.9%) of the cells constituting the suspension-cultured organoids 1 hour after exposure to FITC-polystyrene nanoparticles, whereas in ECM-embedded organoids, only 2.78% of cells exhibited FITC fluorescence under the same conditions, increasing to more than 70% only after 6 hours of exposure. Next, we examined the cellular internalization of AuNPs by scanning the entire organoids using dark-field microscopy (Figs. 11A, B). Similar to previous results (Figs. 9A, 9B, 10), AuNPs were immobilized on the surface of the ECM dome and were not internalized into the cells. In contrast, in the suspension-cultured organoids, internalization of AuNPs was confirmed using time-lapse scanning (Fig. 11). These results indicate that the suspension culture method is more beneficial for the internalization of nanoparticles than the ECM-embedded organoid culture method.

[0154]

[0155] Example 6. Organoid-based toxicity assessment of nanoparticles

[0156] Finally, to assess the organoid-based toxicity of the nanoparticles, we performed viability / dead cell assays and apoptosis assays in mLO cells after exposure to the nanoparticles (Figs. 12 and 13). Gold nanoparticles (AuNPs) and zinc oxide nanoparticles (ZnO NPs), widely used in many different fields, were selected for the organoid-based toxicity experiments. Numerous studies have reported that AuNPs do not cause acute toxicity, although their properties vary depending on factors such as size, shape, charge, and the presence of ligands. We selected spherical AuNPs, which have been reported to be non-toxic both in vitro and in vivo, as a negative control.

[0157] Meanwhile, ZnO NPs are known to induce toxicity in various cell types through in vitro and in vivo experiments. Therefore, ZnO NPs were selected as a positive control for cytotoxicity. Organoids were exposed to ZnO NPs and AuNPs for 48 h, and cytotoxicity was measured using calcein and propidium iodide (PI) staining (Figures 12 and 13A). A relatively weak red fluorescence signal was observed in both ECM-embedded and suspension-cultured organoids that were not exposed to nanoparticles, indicating dead cells. When organoids were exposed to ZnO NPs, the red fluorescence signal significantly increased in suspension-cultured organoids, whereas it remained unchanged in ECM-embedded organoids. In contrast, when organoids were exposed to AuNPs, a weak red fluorescence signal was observed in both ECM-embedded and suspension-cultured organoids. This indicates that cytotoxicity occurred only in suspension-cultured organoids exposed to ZnO NPs. To further investigate the cell death induced by ZnO NPs, we monitored apoptosis using flow cytometry (Fig. 13B).

[0158] AuNPs did not increase apoptosis in either ECM-embedded or suspension-cultured organoids. However, when organoids were exposed to ZnO NPs, apoptosis was not observed in ECM-embedded organoids, but was significantly increased in suspension-cultured organoids. The difference in cytotoxicity between cell cultures despite exposure to the same concentration of ZnO NPs appears to be due to nanoparticle internalization. Previous results have shown that nanoparticle internalization was rarely observed in ECM-embedded organoids, but was observed in suspension-cultured organoids, suggesting that the cytotoxicity results induced by ZnO NPs are reliable for suspension-cultured organoids. In ECM-embedded organoids, exposure to either ZnO NPs or AuNPs did not significantly affect cytotoxicity, which could lead to the misconception that both nanoparticles are safe regardless of the presence or absence of nanoparticles, due to the low intracellular penetration of nanoparticles. Collectively, the results of these studies suggest that suspension-cultured organoid systems are promising tools for assessing the safety and toxicity of nanomaterials.

[0159]

[0160] In conclusion, we developed a novel organoid culture method that can be used to evaluate the safety and toxicity of nanomaterials. Specifically, organoids isolated from ECM and cultured in suspension on ultra-low attachment plates exhibited 3D spherical structures with relatively uniform sizes while maintaining differentiation potential comparable to conventional ECM-based culture methods. Furthermore, the suspension culture method is more conducive to nanoparticle internalization, which facilitates the system's utility for evaluating nanoparticle cytotoxicity, as evidenced by the insensitivity of ECM-embedded organoids to ZnO NP exposure, which induces cell death in suspended organoids.

[0161] Therefore, the suspended culture system provides a reliable tool for assessing the safety of nanomaterials and has the potential to be utilized in high-throughput nanosafety screening. This safety assessment system can accelerate research and development of nanomaterials in various fields by ensuring toxicity monitoring and the safe use of nanomaterials.

[0162]

[0163] The present invention has been described above, focusing on preferred embodiments thereof. Those skilled in the art will appreciate that the present invention can be implemented in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered illustrative rather than limiting. The scope of the present invention is set forth in the claims, not the foregoing description, and all differences within the scope equivalent thereto should be construed as being encompassed by the present invention.

[0164]

[0165] The present invention relates to a method for floating culture of liver organoids for evaluating the safety and toxicity of nanomaterials. When the floating culture system of the present invention is used, it provides a reliable tool for evaluating the stability of nanomaterials, has high applicability for high-throughput nano-safety screening, and can accelerate research and development of nanomaterials in various fields, so it is an invention with industrial applicability.

Claims

1. A method for suspension culture of cells for evaluating the toxicity of nanomaterials; (a) a step of producing an organoid from an adult stem cell, an embryonic stem cell, or an induced pluripotent stem cell; (b) a step of culturing the produced organoid by embedding it in an extracellular matrix (ECM); (c) a step of resuspending the organoids cultured in ECM in matrigel; (d) a step of washing and decomposing the Matrigel containing organoids to obtain only the organoids; (e) a step of suspending the separated organoid in an organoid culture medium containing Matrigel at a lower concentration than the Matrigel of (c); (f) incubating the suspended organoids in an ultra-low attachment plate; and (g) a step of differentiating the incubated organoid; A method for suspension culture of cells containing .

2. In claim 1, The above organoid mimics at least one function selected from the group consisting of brain, gastrointestinal tract, small intestine, large intestine, stomach, larynx, thyroid, thymus, testis, prostate, liver, pancreas, epithelium, kidney, heart, retina, and glioblastoma. Cell suspension culture method.

3. In claim 1, The ECM of the above step (b) is used having a concentration of 50 to 100%. Cell suspension culture method.

4. In claim 1, The matrigel of the organoid culture medium containing the low concentration of matrigel in the above step (e) uses matrigel having a concentration of 1 to 10%. Cell suspension culture method.

5. In claim 1, The medium replacement cycle in the incubation of the above step (f) is 1 to 5 days. Cell suspension culture method.

6. In claim 1, The differentiation stage of the organoids in step (g) above (1) A step of exchanging the medium of the suspended cultured organoids with a differentiation medium; (2) Step of exchanging the differentiation medium daily; and (3) Step of adding an anti-inflammatory agent to the differentiation medium; A cell suspension culture method comprising:

7. In claim 6, The differentiation medium in step (2) above is replaced daily for 10 to 15 days. Cell suspension culture method.

8. In claim 6, The anti-inflammatory agent of the above step (3) is at least one selected from the group consisting of steroids, non-steroids, immune-specific anti-inflammatory agents, herbal extracts, and antioxidants. Cell suspension culture method.

9. In claim 6, The period of administering the anti-inflammatory agent in step (3) above is 1 to 5 days. Cell suspension culture method.

10. A floating culture organoid for evaluating the toxicity of nanomaterials produced by the method of claim 1.

11. A kit for evaluating the toxicity of a nanomaterial comprising the floating culture organoid of claim 10.

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