Head and neck squamous cell carcinoma organoid and culture method, culture medium and application thereof

Abstract

The invention discloses a head and neck squamous cell carcinoma organ as well as a culture method, a culture medium and application thereof. The culture medium is prepared from the following components based on Advanced DMEM / F12: 1 percent of Antibiotic-Antibiotic, 1 percent of GlutaMAX, 1 percent of HEPES buffer solution, 50ng / ml of FGF-10, 50ng / ml of EGF, 1nM of bFGF, 500ng / ml of Wnt3a recombinant protein, 500ng / ml of Noggin recombinant protein, 0.1 mu g / ml of R-spondin-1 recombinant protein, 1mM of N-acetylcysteine, 10mM / ml of nicotinamide and 1x of B27 supplement. The culture medium and the culture method disclosed by the invention are particularly suitable for culturing head and neck squamous cell carcinoma organoid, and the obtained organoid has important significance for researching tumor generation, a unique invasive growth mechanism, prognosis, therapeutic targets, drug screening and the like, and has wide application prospects.

Description

Technical Field

[0001] The present invention relates to the field of medicine and biology, and in particular to a head and neck squamous cell carcinoma organoid and a culture method, culture medium and application thereof. Background Art

[0002] Squamous cell carcinoma (SCC) of the head and neck is an epithelial malignant tumor. It is the most common head and neck malignant tumor, accounting for 90% of all head and neck malignant tumors, and ranks sixth among systemic malignant tumors. At present, the main treatment methods are surgical resection, adjuvant chemoradiotherapy, targeted and immunotherapy drugs, etc. Although the methods are diverse, the overall survival rate of SCC patients has not been significantly improved in recent decades. Due to the lack of obvious early symptoms, most SCC patients are already in the middle and late stages when diagnosed. Its biological behavior is characterized by rapid local invasion, distant metastasis, high postoperative recurrence rate (50% recurrence rate after 2 years), and poor prognosis. In addition, the genome of head and neck SCC is complex, with many mutated genes, and each subtype has significant differences in clinical and molecular characteristics. Therefore, there is no targeted drug for head and neck SCC. Existing drugs for the treatment of head and neck SCC are either lacking in specificity, or have high drug toxicity, or drug resistance, etc., which makes treatment difficult, so the prognosis of patients is poor. How to use new technologies, new chemotherapeutic drugs, and even specific targeted drugs to conduct risk assessment for SCC patients and select appropriate treatment options is a problem that precision medicine needs to solve.

[0003] Organoids are 3D cell clusters that are differentiated and self-assembled from adult stem cells, embryonic stem cells, or induced pluripotent stem cells that are specific to human organs. They can reproduce certain characteristics and functions of tissues and organs. In 2013, organoids were named one of the top ten technologies of the year by Science magazine. They can be integrated with other biotechnologies, including but not limited to gene editing, single-cell genomics, and microfluidics, providing a way to study the pathogenesis, development process, drug development, evaluation of drug efficacy, new diagnostic and therapeutic technologies, etc. of diseases. Organoids of various tissue types have been successfully constructed in succession, but the success rate of organoid culture of SCC is low, the time and cost are long, and the sample requirements are high. As a result, the establishment of head and neck SCC organoids has rarely been reported, and the research on SCC has been hindered to a certain extent. Summary of the invention

[0004] In view of the defects of the prior art, the object of the present invention is to provide a culture medium, a culture method and an application of head and neck squamous cell carcinoma organoids.

[0005] The technical solution of the present invention is:

[0006] The first aspect of the present invention provides a culture medium for culturing head and neck squamous cell carcinoma organoids, the culture medium comprising Advanced DMEM / F12 and the following components based on Advanced DMEM / F12: 1% Antibiotic-Antimycotic, 1% GlutaMAX, 1% HEPES buffer, 50ng / ml FGF-10, 50ng / ml EGF, 1nM bFGF, 500ng / ml Wnt3a recombinant protein, 500ng / ml Noggin recombinant protein, 0.1μg / ml R-spondin-1 recombinant protein, 1mM N-acetylcysteine, 10mM / ml nicotinamide, and 1x B27 supplement.

[0007] A second aspect of the present invention provides a kit for culturing head and neck squamous cell carcinoma organoids, comprising the above-mentioned culture medium for culturing head and neck squamous cell carcinoma organoids.

[0008] A third aspect of the present invention provides a method for culturing head and neck squamous cell carcinoma organoids, wherein head and neck squamous cell carcinoma tumor cells are cultured using the above-mentioned head and neck squamous cell carcinoma organoid culture medium or head and neck squamous cell carcinoma organoid kit to obtain the head and neck squamous cell carcinoma organoids.

[0009] Furthermore, the present invention provides a method for culturing head and neck squamous cell carcinoma organoids, the specific steps of which are as follows:

[0010] (a) Tissue fragmentation

[0011] The head and neck squamous cell carcinoma tumor tissue was crushed to obtain tissue fragments;

[0012] (b) Tissue digestion

[0013] Digesting the tissue fragments with digestive enzymes to obtain a suspension of head and neck squamous cell carcinoma;

[0014] (c) Filtration

[0015] The head and neck squamous cell carcinoma suspension is filtered, the supernatant is discarded by centrifugation, the precipitate is resuspended to obtain a single cell suspension of head and neck squamous cell carcinoma, and the single cell suspension of head and neck squamous cell carcinoma is inoculated and cultured to obtain head and neck squamous cell carcinoma organoids.

[0016] Furthermore, the present invention provides a method for culturing head and neck squamous cell carcinoma organoids, wherein the digestive enzyme is trypsin.

[0017] Furthermore, the present invention provides a method for culturing head and neck squamous cell carcinoma organoids, wherein in step (c), culturing refers to culturing in an environment of 37° C. and a CO 2 concentration of 5%.

[0018] Furthermore, the present invention provides a method for culturing head and neck squamous cell carcinoma organoids, wherein in step (c), the medium is replaced every 2-3 days during the culture period.

[0019] Furthermore, the present invention provides a method for culturing head and neck squamous cell carcinoma organoids. In step (c), the head and neck squamous cell carcinoma suspension is filtered through 100 μm, 70 μm, and 40 μm cell filters in sequence.

[0020] A fourth aspect of the present invention provides a head and neck squamous cell carcinoma organoid, which is cultured by the above-mentioned head and neck squamous cell carcinoma organoid culture method.

[0021] The head and neck squamous cell carcinoma organoids provided by the present invention have at least the following applications:

[0022] 1) Study the pathogenesis of head and neck squamous cell carcinoma, and prepare or screen drugs for the treatment of head and neck squamous cell carcinoma;

[0023] 2) Using head and neck squamous cell carcinoma organoids to prepare drug evaluation or screening models;

[0024] 3) Explore new diagnostic and therapeutic technologies using head and neck squamous cell carcinoma organoids.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The head and neck squamous cell carcinoma organoids obtained using the culture medium and culture method provided by the present invention can be subcultured for multiple times, and the organoids obtained after subculture maintain the same morphology and properties as the primary head and neck squamous cell carcinoma organoids, meeting the needs of large-scale replication of head and neck squamous cell carcinoma organoids.

[0027] 2. The head and neck squamous cell carcinoma organoids obtained using the culture medium and culture method provided by the present invention have cell tissue morphology and expression of protein molecular markers that are highly consistent with clinical samples, and can effectively maintain cell tissue specificity.

[0028] 3. The head and neck squamous cell carcinoma organoids obtained using the culture medium and culture method provided by the present invention can be used to screen therapeutic drugs for head and neck squamous cell carcinoma, study the occurrence and development mechanism of head and neck squamous cell carcinoma, screen head and neck squamous cell carcinoma-related biomarkers, etc., which not only meets the needs of scientific research, but also provides useful options in clinical drug guidance. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0030] Figure 1 Schematic diagram of the passaging of head and neck squamous cell carcinoma organoids provided in an embodiment of the present invention (P1 refers to the first generation of head and neck squamous cell carcinoma organoids, P2 refers to the second generation of head and neck squamous cell carcinoma organoids, and P5 refers to the fifth generation of head and neck squamous cell carcinoma organoids);

[0031] Figure 2 HE staining images of the P4 generation head and neck squamous cell carcinoma organoids and the SCC parent tumor provided in the embodiments of the present invention (HE 20X, P4 refers to the fourth generation head and neck squamous cell carcinoma organoids, PDO refers to the P4 generation head and neck squamous cell carcinoma organoids; Parental refers to the SCC parent tumor);

[0032] Figure 3 IHC staining diagram of the P4 generation head and neck squamous cell carcinoma organoid and the SCC parent tumor provided in the embodiment of the present invention (IHC 20X, P4 refers to the fourth generation head and neck squamous cell carcinoma organoid, PDO refers to the P4 generation head and neck squamous cell carcinoma organoid; Parental refers to the SCC parent tumor);

[0033] Figure 4 FISH staining diagram of the P4 generation head and neck squamous cell carcinoma organoid and the SCC parent tumor provided in the embodiment of the present invention (FISH 100X, P4 refers to the fourth generation head and neck squamous cell carcinoma organoid, PDO refers to the P4 generation organoid; Parental refers to the SCC parent tumor);

[0034] Figure 5 HPV staining images of the P4 generation head and neck squamous cell carcinoma organoids and the SCC parental tumor provided in the embodiments of the present invention (CISH 20X, P4 refers to the fourth generation head and neck squamous cell carcinoma organoids, PDO refers to the P4 generation head and neck squamous cell carcinoma organoids; Parental refers to the SCC parental tumor). DETAILED DESCRIPTION

[0035] After a large number of experiments and in-depth research, the present invention provides an in vitro construction method for head and neck squamous cell carcinoma organoids. Human head and neck squamous cell carcinoma cells are extracted and induced to construct organoids. Their morphology is observed under a microscope. The tissue cell morphology and protein expression of the head and neck squamous cell carcinoma organoids are observed and verified through HE staining and immunohistochemistry to obtain organoids that are highly consistent with the tissue cell morphology and protein expression of the patient's tumor sample.

[0036] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0037] The present invention is described in detail below with reference to specific embodiments.

[0038] Example 1

[0039] The present embodiment provides a culture medium for culturing head and neck squamous cell carcinoma organoids, the culture medium comprising Advanced DMEM / F12, and the following components based on Advanced DMEM / F12: 1% Antibiotic-Antimycotic, 1% GlutaMAX, 1% HEPES buffer, 50ng / ml fibroblast growth factor (FGF-10), 50ng / ml EGF, 1nM bFGF, 500ng / ml Wnt3a recombinant protein, 500ng / ml Noggin recombinant protein, 0.1μg / ml R-spondin-1 recombinant protein, 1mM N-Acetylcysteine ​​(NAC), 10mM / ml nicotinamide, and 1x B27 supplement.

[0040] The culture medium provided in this embodiment achieves an optimized reduction in the amount of GlutaMAX used, and reduces the types and amounts of specific growth factors used, compared to the culture medium currently used to culture head and neck squamous cell carcinoma organoids, thereby effectively reducing the culture cost of SCC organoids. The formula of the culture medium provided in this embodiment has been obtained through extensive experimental verification, which means that subsequent researchers can use it directly without the need for time-consuming exploratory experiments, which significantly improves research efficiency and saves a lot of valuable time for experimenters.

[0041] Example 2

[0042] This embodiment provides a kit for culturing head and neck squamous cell carcinoma organoids, comprising the culture medium for culturing head and neck squamous cell carcinoma organoids provided in Example 1. The kit is based on the existing culture medium, which can effectively save the cost and time of the enterprise in the research and development process; based on the culture medium for culturing head and neck squamous cell carcinoma organoids provided in Example 1, combined with a scientific research and development process, the successful development and market launch of the head and neck squamous cell carcinoma organoid culture kit is ensured; the development of the kit not only reduces the cost of the enterprise, but also improves the research and development efficiency, and provides a powerful tool for the research of head and neck squamous cell carcinoma.

[0043] Example 3

[0044] This embodiment provides a method for culturing head and neck squamous cell carcinoma organoids. In this culturing method, the culture medium used is the culture medium for culturing head and neck squamous cell carcinoma organoids provided in Example 1.

[0045] Sample source: A 63-year-old male patient who underwent surgical resection in the Department of Oral and Maxillofacial Head and Neck Tumors of the Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine was diagnosed with SCC by postoperative pathology, with the primary lesion located in the left oropharynx. The patient signed an informed consent form.

[0046] (1) The specific training methods are as follows:

[0047] The head and neck squamous cell carcinoma tumor tissue was quickly placed in a centrifuge tube containing tissue preservation solution, the main components of which were DMEM + amphotericin B (500 ng / ml) + 5% penicillin-streptomycin (the 5% concentration was determined based on DMEM), and the centrifuge tube was placed in an ice box and transported to the laboratory;

[0048] In a biosafety cabinet, the head and neck squamous cell carcinoma tumor tissue was transferred to a culture dish, and the tissue was soaked in PBS containing 5% penicillin-streptomycin (the 5% concentration was determined based on PBS) for about 10 minutes, and then transferred to another PBS culture dish also containing 5% penicillin-streptomycin, and the capsule tissue was removed in the culture dish;

[0049] (a) Tissue fragmentation

[0050] Cut the head and neck squamous cell carcinoma tumor tissue into approximately 1 mm 3 Large and small tissue fragments;

[0051] (b) Tissue digestion

[0052] The tissue fragments were transferred to a centrifuge tube containing trypsin at a concentration of 0.13% (wt / vol) and digested in a shaker at 37°C for 1 h;

[0053] The digestion was terminated by adding DMEM medium containing serum to obtain a suspension of head and neck squamous cell carcinoma;

[0054] (c) Filtration

[0055] The suspension of head and neck squamous cell carcinoma was filtered using 100 μm, 70 μm, and 40 μm cell filters in sequence to obtain a single cell suspension of head and neck squamous cell carcinoma;

[0056] Count the cells using a counting plate and add an appropriate amount of culture medium to resuspend the cell pellet according to the counting results;

[0057] When inoculating, the resuspended cell pellet was mixed with matrix gel and inoculated in a 24-well plate. 40 μl of matrix gel was added to each well of the 24-well plate, and about 10 4 cells;

[0058] When culturing primary head and neck squamous cell carcinoma organoids, invert the inoculated 24-well plate and place it in a 37°C incubator for about 40 minutes. After the colloid solidifies, place the 24-well plate upright and add an appropriate amount of culture medium to each well for culture. During the culture period, replace the culture medium every 2-3 days.

[0059] (2) Subculture of head and neck squamous cell carcinoma organoids

[0060] During the culture of HNSCC organoids, observe the number of HNSCC organoids under a microscope as needed. When the number of HNSCC organoids is sufficient, perform subculturing. The subculturing steps are as follows:

[0061] Discard the original culture medium, melt the matrix gel with pre-cooled PBS, gently blow the mixture of matrix gel and head and neck squamous cell carcinoma organoids and transfer it to a centrifuge tube. After centrifugation, add PBS to wash, remove all the matrix gel, add trypsin to digest for 3 minutes, and add culture medium to terminate the digestion. Count the cells under the microscope, centrifuge and discard the supernatant, resuspend the precipitate with an appropriate amount of culture medium, and mix it with matrix gel (add 40 μl of matrix gel to each well of the 24-well plate, and about 10 4 The inoculated 24-well plate was inverted and placed in a 37°C incubator for about 40 minutes. After the colloid solidified, the 24-well plate was placed upright and an appropriate amount of culture medium was added to each well for culture.

[0062] During the culture period, the culture medium was replaced every 2-3 days.

[0063] (3) Morphological observation of head and neck squamous cell carcinoma organoids

[0064] At each stage of different growth densities, a stereo microscope was used to observe the gross morphology of the subcultured head and neck squamous cell carcinoma organoids, and photographs were taken to record them. The morphology of the head and neck squamous cell carcinoma organoids is spherical with continuous edges. After being subcultured to P2 (second-generation head and neck squamous cell carcinoma organoids) and P5 (fifth-generation head and neck squamous cell carcinoma organoids), the gross morphology remained unchanged, and the number of organoids increased after each subculture, and the spheroids showed a slow enlargement trend. P1 (first-generation head and neck squamous cell carcinoma organoids), P2, and P5 head and neck squamous cell carcinoma organoids can be seen. Figure 1 .

[0065] (4) Morphological identification of head and neck squamous cell carcinoma organoids

[0066] The head and neck squamous cell carcinoma organoids obtained after culture were processed using a modified agar encapsulation method:

[0067] Fixation: Carefully pipette the head and neck squamous cell carcinoma organoid tissue into a 24-well cell culture plate, add 10% neutral buffered formaldehyde, and fix at room temperature for 2-4 hours;

[0068] Pre-embedding: Preheat the frozen agar solution to a viscous state in advance. After the head and neck squamous cell carcinoma organoid tissue is fixed, use a 3ml pipette to suck out the head and neck squamous cell carcinoma organoid tissue and place it in a disposable plastic embedding mold. Use a pipette to suck out an appropriate amount of agar solution and slowly add it around the head and neck squamous cell carcinoma organoid tissue until the agar wraps all the head and neck squamous cell carcinoma organoid tissue; wait for the agar to solidify at room temperature, and put it into 75% alcohol, 85% alcohol, 95% alcohol, and 100% alcohol for dehydration together with the mold, each time for 5-10 minutes, to facilitate the agar block to be removed from the plastic embedding mold;

[0069] Dehydration: Place the agar block containing pre-embedded head and neck squamous cell carcinoma organoid tissue in the embedding box and immerse it in 75% alcohol for 30 minutes, 85% alcohol for 30 minutes, 95% alcohol for 30 minutes, 100% alcohol for 1 hour, and 100% alcohol for 1 hour.

[0070] Transparent and wax-immersed: xylene 30 min, xylene 30 min, paraffin 62°C 30 min, paraffin 62°C 1 h, paraffin 62°C 1 h;

[0071] Sectioning: Gently pick up the agar block after treatment with tweezers, embed it in paraffin, and slice it into 3μm sections;

[0072] Staining: Hematoxylin & Eosin (HE) staining, routine histological observation, and comparison with SCC parent tumor.

[0073] By observing the HE sections of the passaged head and neck squamous cell carcinoma organoids and their corresponding SCC parent tumor tissues, it was found that the passaged head and neck squamous cell carcinoma organoids retained the histopathological morphological characteristics of their corresponding SCC parent tumors, such as Figure 2 .

[0074] The immunohistochemistry (IHC) staining of relevant protein markers was performed on the Roche fully automated immunohistochemistry staining machine BenchMark ULTRA using the Envision two-step method. The results showed that the protein markers (CKH, CK7, and P63) were consistent with the IHC results of their corresponding SCC parental tumor tissues, such as Figure 3 .

[0075] Fluorescence in situ hybridization (FISH) was performed on the head and neck squamous cell carcinoma organoids: the paraffin-embedded head and neck squamous cell carcinoma organoids were sliced ​​into several 3μm sections and baked at 65℃ for 1-2h; dewaxed with xylene, hydrated with gradient alcohol, and the sections were placed in 90℃ distilled water for 30min. The head and neck squamous cell carcinoma organoids were digested in a water bath in 54℃ pepsin digestion solution for 20-30min, dehydrated with gradient alcohol and dried. Under light-proof conditions, 5-10μl of probe (EGFR) was added, and the coverslip was covered with rubber cement to seal the slide. The hybridizer was denatured at 76℃ for 10min and hybridized at 42℃ for 18h. After the incubation, the slides were removed, the rubber cement was removed, and the slides were immersed in a 2x SSC / 0.3% NP-40 (pH7.0-7.2) solution preheated to 68°C for 2 minutes. The sections were then immersed in a 2xSSC / 0.1% NP-40 solution at room temperature for 2 minutes, and finally placed in a 2x SSC buffer at room temperature for 2 minutes. Dehydrate with gradient alcohol, dry naturally in the dark, add 10μl DAPI counterstain solution to the corresponding area of ​​probe hybridization, cover with a coverslip, and stain at -20°C for 30 minutes, and observe the results under a fluorescence microscope. Tumor cells were searched under three objective lenses: 10x, 40x, and 100x oil objective, and the red and green signals in 20 cells were counted under a 100x oil objective. FISH results showed that the EGFR gene was not amplified in the P4 generation organoids (the fourth generation of head and neck squamous cell carcinoma organoids) and the SCC parent tumor (such as Figure 4 ), the two results are consistent.

[0076] Chromogenic in situ hybridization (CISH) detection technology was used to detect cases of HPV infection. PBS was used instead of the probe as a blank control, and a known positive SCC tumor tissue was used as a positive control. The sections were digested with pepsin at 37°C and the probe was added for hybridization overnight. After incubation with horseradish peroxidase-labeled digoxigenin primary antibody and DAB colorimetric solution was added to develop the color to observe the results. The results showed that HPV infection was not detected in the P4 generation organoids (the fourth generation of head and neck squamous cell carcinoma organoids) and the SCC parent tumors, and the results of the two were consistent (such as Figure 5 ).

[0077] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention. In the absence of conflict, the embodiments of the present application and the features in the embodiments can be combined with each other arbitrarily.

Claims

1. A culture medium for culturing head and neck squamous cell carcinoma organoids, characterized in that: The culture medium includes Advanced DMEM / F12, and the following components based on Advanced DMEM / F12: 1% Antibiotic-Antimycotic, 1% GlutaMAX, 1% HEPES buffer, 50ng / ml FGF-10, 50ng / ml EGF, 1nM bFGF, 500ng / ml Wnt3a recombinant protein, 500ng / ml Noggin recombinant protein, 0.1μg / ml R-spondin-1 recombinant protein, 1mM N-acetylcysteine, 10mM / ml nicotinamide, and 1x B27 supplement.

2. A kit for culturing head and neck squamous cell carcinoma organoids, characterized in that: A culture medium for culturing head and neck squamous cell carcinoma organoids according to claim 1.

3. A method for culturing head and neck squamous cell carcinoma organoids, characterized in that: The head and neck squamous cell carcinoma tumor cells are cultured using the head and neck squamous cell carcinoma organoid culture medium of claim 1 or the head and neck squamous cell carcinoma organoid kit of claim 2 to obtain the head and neck squamous cell carcinoma organoid.

4. The method for culturing head and neck squamous cell carcinoma organoids according to claim 3, characterized in that: The specific steps are as follows: (a) Tissue fragmentation The head and neck squamous cell carcinoma tumor tissue was crushed to obtain tissue fragments; (b) Tissue digestion Digesting the tissue fragments with digestive enzymes to obtain a suspension of head and neck squamous cell carcinoma; (c) Filtration The head and neck squamous cell carcinoma suspension is filtered, the supernatant is discarded by centrifugation, the precipitate is resuspended to obtain a single cell suspension of head and neck squamous cell carcinoma, and the single cell suspension of head and neck squamous cell carcinoma is inoculated and cultured to obtain head and neck squamous cell carcinoma organoids.

5. The method for culturing head and neck squamous cell carcinoma organoids according to claim 4, characterized in that: The digestive enzyme is trypsin.

6. The method for culturing head and neck squamous cell carcinoma organoids according to claim 4, characterized in that: In step (c), culturing refers to culturing at 37° C. and a CO 2 concentration of 5%.

7. The method for culturing head and neck squamous cell carcinoma organoids according to claim 4, characterized in that: In step (c), during the culture period, the medium is replaced every 2-3 days.

8. The method for culturing head and neck squamous cell carcinoma organoids according to claim 4, characterized in that: In step (c), the head and neck squamous cell carcinoma suspension is filtered through 100 μm, 70 μm, and 40 μm cell filters in sequence.

9. A head and neck squamous cell carcinoma organoid, characterized in that: The organoids are obtained by culturing the head and neck squamous cell carcinoma organoids according to any one of claims 3 to 8.

10. The head and neck squamous cell carcinoma organoid according to claim 9, characterized in that The organoid has at least the following applications: 1) Study the pathogenesis of head and neck squamous cell carcinoma, and prepare or screen drugs for the treatment of head and neck squamous cell carcinoma; 2) Using head and neck squamous cell carcinoma organoids to prepare drug evaluation or screening models; 3) Explore new diagnostic and therapeutic technologies using head and neck squamous cell carcinoma organoids.

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