The study is funded by the German Cancer Research Center DKTK, Partner site Munich, a partnership between DKFZ and University Hospital LMU Munich. The study is also supported by the German Cancer Aid grant (#70113426 and #70113433). Paraffin embedding of tissue and organoids has been performed at the Core facility of the Institute of Anatomy, Faculty of Medicine, LMU Munich, Munich. Confocal Imaging has been performed at the Core facility Bioimaging at the Biomedical Center (BMC). The authors want to thank Simone Hofmann, Maria Fischer, Cornelia Herbst, Sabine Fink, and Martina Rahmeh, for technical help.

Tumor tissue specimens from ovarian cancer surgeries were collected and patient-derived organoids were generated in compliance with the Ethics Committee of LMU University (17-471), adhering to the existing applicable EU, national, and local regulations. Each patient involved in the study has consented in written form. When working with fresh tissue samples, Biosafety Level 2 safety permission and Laminar Flow cabinets are required. Given the potentially infectious nature of the tissue samples, which cannot be ruled out due to the lack of routine testing of relevant infectious diseases, it is necessary to ensure that institutional bio-safety regulations are strictly adhered to and that adequate personal protective equipment is available for the personnel conducting the experiments.
1. Preparations
<ol>
	<li>Medium preparation
	<ol>
		<li>Prepare the 2D and 3D media freshly once per week and store them at 4 &#176;C. 
		NOTE: The exact composition of the ingredients for each medium is listed in Table 1. Both ovarian cancer medium 1 (OCM1) and OCM2 can be additionally supplemented by HER1&#223; (final concentration: 50 ng/mL), resulting in four different conditions: OCM1, OCM1+HER1&#223;, OCM2, OCM2+HER1&#223;.</li>
		<li>Create stock solutions of the growth factor reagents and keep them at 20 &#176;C and of A83-01 and nicotinamide (storage at +4 &#176;C). Due to the temperature sensitivity of the growth factors, use thawed stock solutions immediately for medium preparation and avoid repeated thawing cycles by creating aliquots. 
		NOTE: Media preparation is a critical step that needs to be strictly controlled.</li>
		<li>Preparation of RSPO-1 conditioned medium
		<ol>
			<li>Thaw HA-R-Spondin1-Fc 293 T cells (storage at -80 &#176;C) quickly at 37 &#176;C. Wash them with 10 mL of basal culture medium, supplemented with 10% fetal calf serum (FCS), henceforth referred to as basal culture medium++.</li>
			<li>Resuspend the cell pellet in 12 mL of basal culture medium++ and seed it in a T75 flask.</li>
			<li>Split the cells in a ratio of 1:6 with a dissociation reagent containing trypsin (4 min at 37 &#176;C) when the cells reach confluence. Seed them into a new T75 flask.</li>
			<li>The next day, add phleomycin D1 (1,25 &#181;L/mL) to the medium.</li>
			<li>Split the cells in a ratio of 1:20 with trypsin (4 min at 37 &#176;C) when the cells reach confluence. Seed the cells into multiple T75 flasks (number of flasks according to the aimed final volume) in 30 mL of basal culture medium++ (containing 10 % FCS) supplemented with phleomycin D1.</li>
			<li>Start the conditioned media production when the cells reach approximately 50% of confluence: replace the medium with 40 mL of basal culture medium supplemented with 5% FCS without phleomycin D1.</li>
			<li>Collect the first supernatant after 3 days. To remove the debris, centrifuge for 10 min at 1,200 &#215; g. Keep the supernatant in a sterile container at 4 &#176;C.</li>
			<li>Add new basal culture medium supplemented with 5% FCS to the cells. Collect the second supernatant after 3-4 days. Centrifuge for 10 min at 1,200 &#215; g. Add the second supernatant to the first supernatant.</li>
			<li>Strain the conditioned medium by using a 0.2 &#181;m bottle top filter.</li>
			<li>For quality control and RSPO1-quantification, add the produced medium to a 293T cell line (293T WntR, 7xTcf-eGFP)14, stably transduced with GFP-plasmid carrying Wnt reporter18. 
			NOTE: Depending on the quantity and intensity of the GFP signal, the activity of RSPO1 as an agonist of the Wnt pathway is tested by the quantification of the Wnt reporter cell line.</li>
			<li>Keep aliquots of 15 mL for immediate use (within 1 week) at 4 &#176;C. For longer storage (6 months) keep the conditioned medium at -20 &#176;C.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Initiation of an ovarian cancer organoid culture
<ol>
	<li>Primary tissue isolation
	<ol>
		<li>Transport fresh and viable tissue preferably immediately after surgery and perform isolation within a maximum of 20 h after collection. Ensure proper transport condition in the tissue collection medium with a transport box equipped with cold packs at approximately 4 &#176;C.</li>
		<li>In the lab, prepare the necessary reagents and equipment to facilitate timely sample processing:
		<ol>
			<li>Thaw Basement Membrane Extract Type II matrix (BME 2 matrix) on ice (approximately 2 h).</li>
			<li>Prepare disposable scalpels, sterilized dissection tools (scissors and tweezers), and 400 &#181;m-sized filter inserts for 50 mL tubes.</li>
			<li>Prepare a container with a small amount of liquid nitrogen for snap freezing and a prewarmed water bath at 37 &#176;C. 
			NOTE: Work within a cell culture laminar flow hood.</li>
		</ol>
		</li>
		<li>Wash the fresh tissue in a Petri dish thoroughly in Phosphate-Buffered Saline (PBS) (without Ca++ and Mg++). Inside the Petri dish, fragment the tissue using the disposable scalpels and/or scissors into small, 3-5 mm sized pieces.</li>
		<li>Separate the obtained tissue into three sections for the following purposes:
		<ol>
			<li>Collect tissue in cryogenic tubes. Transfer the cryogenic tubes into the prepared container filled with liquid nitrogen for shock freezing.</li>
			<li>Collect tissue (2-3 mm) into a histological specimen container filled with formalin for fixation (24 h) and embed them in paraffin for staining purposes19,20.</li>
			<li>Further, homogenize the tissue before enzymatic digestion. Maximize the mechanical dissociation with a scalpel and transfer it into a 50 mL tissue tube. 
			NOTE: Make sure that the tissue is always soaked in PBS during homogenization to avoid drying out of the tissue.</li>
		</ol>
		</li>
		<li>Combine the homogenized tissue with a digestion mixture containing PBS (without Ca++ and Mg++), Collagenase I (stock solution 5 U/&#181;L, final concentration 1 U/&#181;L), and a selective ROCK1 and 2 inhibitor (3 &#181;M final concentration) (components listed in Table 1). Use 15 mL of digestion mixture in a 50 mL tube for each 2 cm3 of the tumor. 
		NOTE: Limited material (e.g., biopsy samples) requires only small amounts (approximately 2-3 mL) of digestion mixture to ensure enzymatic dissociation.</li>
		<li>For enzymatic dissociation, incubate the tube for 1.5 h in a water bath at 37 &#176;C. Conduct sporadic and vigorous vortexing (approximately every 20 min for 10-15 s) to support mechanic dissociation.</li>
		<li>After incubation, add 15 mL of cold basal culture medium++ to the tube. Centrifuge 5 min at 300 &#215; g.</li>
		<li>After removing the supernatant, add 5 mL of new basal culture medium++. Strain the cell suspension by using a 400 &#181;m filter into a fresh 50 mL tube.</li>
		<li>Centrifuge for 5 min at 300 &#215; g. Remove the supernatant and keep the cell pellet.</li>
		<li>For removal of erythrocytes, add 5 mL of Red Blood Cell Lysing (RBC) Buffer to the cell pellet. Incubate in a water bath for 5 min at 37 &#176;C.</li>
		<li>Add 5 mL of basal culture medium++ for lysis inactivation. Centrifuge for 5 min at 300 &#215; g. Add 3 mL of basal culture medium++ to the cell pellet.</li>
		<li>Count viable cells after 1:1 dilution with trypan blue stain solution (e.g., 10 &#181;L of the sample + 10 &#181;L of trypan blue stain solution) in an automatic cell counter or manually in a Neubauer Chamber.</li>
		<li>Calculate the number of cells needed for direct 3D seeding. For each tumor deposit, seed into a 48-well format plate at least 2-3 wells per ovarian cancer medium, which corresponds to a total of 8-12 wells in a seeding matrix (OCM1, OCM1+HER1&#223;, OCM2, OCM2+HER1&#223;). Calculate 30,000 cells for each well in a droplet of 25 &#181;L of BME Type 2 matrix. Optionally, choose a 24-well format with 50 &#181;L of BME 2 matrix and 50,000 seeded cells per well.</li>
		<li>Seed the remaining cells in 2D (see step 2.1.13).</li>
		<li>Pipette the amount of basal culture medium++ suspension that contains the total number of needed cells into a new tube and centrifuge for 5 min at 300 &#215; g. Remove the supernatant and keep the cell pellet.</li>
		<li>On the ice, add the total amount of cold BME 2 matrix (25 &#181;L for each well in a 48-well format plate; hence, 100 &#181;L for 4 wells) to the pellet and mix well to achieve an even distribution of cells per well by gently pipetting the pellet up and down without creating bubbles.</li>
		<li>Seed the cells in droplets of BME 2 matrix (25 &#181;L) to the prewarmed, empty 48-well plate. To achieve even distribution between the wells, gently and slowly move the pipette up and down (3-4x) inside the tube before transferring the BME 2 matrix droplet into each well to prevent cell precipitation during distribution.</li>
		<li>After incubating the plate at 37 &#176;C for at least 30 min to consolidate the droplets of the BME 2 matrix, pipette 250 &#181;L of each of the four ovarian cancer media (OCM1, OCM1+HER1&#223;, OCM2, OCM2+HER1&#223;) into the corresponding wells.</li>
		<li>In parallel to the direct 3D seeding procedure, preserve the remaining isolated cells by starting a 2D culture.
		<ol>
			<li>After centrifugation for 5 min at 300 &#215; g, add the pellet to the 2D medium. Select the format (T75 flask or T25 flask), depending on the number of cells available after 3D seeding.</li>
			<li>Incubate the flask for 3-5 days at 37 &#176;C for cell adhesion. Keep the same medium for the first 72 h.</li>
			<li>When the cells reach confluence, transfer the culture into the BME 2 matrix. Do not expand primary isolates in 2D culture by splitting as long-term propagation on 2D is detrimental to stemness potential.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Transfer from 2D culture to 3D culture
	<ol>
		<li>In the T75 or T25 flask, wash the monolayer 2x with 5 mL of PBS to detach the cells from the bottom surface. Incubate with 1 mL of trypsin for 10 min at 37 &#176;C.</li>
		<li>Add 5 mL of cold basal culture medium++ for inactivation of the dissociation reagent. Centrifuge for 5 min at 300 &#215; g.</li>
		<li>Add 3 mL of basal culture medium++ to the pellet. Count the cells as described in steps 2.1.12-2.1.13. Seed for the different media compositions (see Figure 1) as described in steps 2.1.16-2.1.18.</li>
	</ol>
	</li>
	<li>Evaluation of organoid growth
	<ol>
		<li>Image the wells at least once per week to document organoid growth. Make high-quality comparable images of 2D/3D culture and direct seeding plates by phase contrast microscope. Take care of consistency in magnification and use 4x for an overview of the wells (quantification) and 10x and 20x to document the morphology of organoids.</li>
		<li>Organize storage of pictures in folders dedicated to individual lines.</li>
		<li>Select the best organoid line for long-term cultivation by comparative assessment of organoid formation at the different seeding modalities (2D followed by 3D seeding versus direct 3D seeding) and different media compositions (OCM1, OCM1+ HER1&#223;, OCM2, and OCM2+ HER1&#223;).
		<ol>
			<li>On average, 14-21 days after isolation, evaluate the organoid-forming potential (quantity) as well as the size and cellular phenotype of organoids grown under different conditions. Look for the following features: absence of vacuoles, membrane blebbing or loss of adhesion, and rounding up of cells.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Long-term organoid cultivation
<ol>
	<li>Organoid passaging
	<ol>
		<li>Avoid splitting organoids too frequently and during the proliferative phase. Conduct mechanical and enzymatic digestion procedures with intervals of more than 10 days.</li>
		<li>To disrupt each BME 2 matrix droplet, add 250 &#181;L (48-well format) or 500 &#181;L (24-well format) of ice-cold basal culture medium++. Ensure complete dissolution of the droplet and pipette intermittently up and down and scrape the bottom of the plate. Transfer the cell suspension into a 15 mL tube and place it on ice. Add 1 mL of ice-cold basal culture medium++. 
		NOTE: The removal efficiency of the BME 2 matrix depends strongly on the medium temperature since the best dissolution is achieved below 4 &#176;C.</li>
		<li>Pool 2-3 technical replicate wells for even expansion . Centrifuge for 5 min at 300 &#215; g. Inspect the content of the suspension against the source of light to see if the BME 2 gel is still visible. Remove the supernatant and repeat washing with an ice-cold medium if the BME 2 matrix is still visible.</li>
		<li>Incubate with 1 mL of trypsin (stored at 20-25 &#176;C) in a water bath at 37 &#176;C for 7-10 min. Vortex sporadically for 10 s to enhance dissociation. Inspect the tube periodically visually to see if dissociation is progressing.</li>
		<li>Pull the cell suspension up and down with a syringe through a needle with a size ranging from 23 G to 27 G. Check if large cell clumps are still present and repeat the procedure if necessary. 
		NOTE: Fragmentation through a syringe is optional, and needle size depends on the required&#160;dissociation efficacy.&#160;A homogeneous cell suspension leads to even distribution among the wells.</li>
		<li>Add 1 mL of cold basal culture medium++ to inactivate the reaction.</li>
		<li>OPTIONAL: Count the cells as described in steps 2.1.12-2.1.13 and decide the splitting ratio to the parental passage (e.g., 1:3 wells).</li>
		<li>Centrifuge for 5 min at 300 &#215; g. Remove the supernatant.</li>
		<li>Conduct seeding as described in steps 2.1.16-2.1.18 and apply the respective optimal ovarian cancer organoid medium according to the already established long-term culture. Change the ovarian cancer medium every 3-4 days.</li>
		<li>In case of disruption of the BME 2 matrix droplet during medium change, consider reseeding without enzymatic digestion. To avoid disruption of the droplet, carefully conduct the medium change procedure without direct pipetting onto the BME 2 matrix droplet. Additionally, avoid leaving an excessive amount of residual basal culture medium++ on the pellet before dilution with the BME 2 matrix at step 2.1.16 as it might affect the fragility of the droplets.</li>
	</ol>
	</li>
	<li>Cryopreservation of organoids 
	NOTE: Conduct cryopreservation of the organoids during the proliferation phase in the first week after passage.
	<ol>
		<li>Disrupt the BME 2 matrix droplet with ice-cold basal culture medium++ according to step 3.1.2. After centrifugation and discarding the supernatant as described in step 3.1.3., work on ice and resuspend the cell pellet in 1 mL of ice-cold cryopreservation medium. Transfer the cell suspension to prelabeled 1.8 ml cryogenic tubes.</li>
		<li>Store the tubes in a precooled isopropyl alcohol-containing freezing container and place them at -80 &#176;C overnight.</li>
		<li>Keep the stock tubes at -80 &#176;C for a maximum of 2 months. Transfer to liquid nitrogen for long-term stable storage.</li>
	</ol>
	</li>
	<li>Thawing of organoid stocks
	<ol>
		<li>Prewarm 9 mL of basal culture medium++ in a 37 &#176;C water bath in a labeled 15 mL tube.</li>
		<li>Transfer the desired stock vial from the storage at -80 &#176;C or to liquid nitrogen into a transport container filled with liquid nitrogen.</li>
		<li>Transfer the cryotube to a water bath at 37 &#176;C and gently agitate it until the frozen material near the walls of the tube begins to thaw.</li>
		<li>Distribute the organoid suspension slowly into the labeled tube filled with 9 mL of medium. Gently shake the tube. Centrifuge for 5 min at 300 &#215; g and remove the supernatant.</li>
		<li>Conduct seeding as described in steps 2.1.16-2.1.18 and apply the respective optimal ovarian cancer organoid medium according to the already determined long-term culture conditions for the individual line.</li>
	</ol>
	</li>
	<li>Fixation of organoids
	<ol>
		<li>Perform organoid collection as described in steps 3.1.2-3.1.3.</li>
		<li>Add 3 mL of 4% paraformaldehyde (PFA) in PBS (pH 7.4) and incubate for 1 h at room temperature.</li>
		<li>Wash 2x with 5 mL of PBS, centrifuge for 3 min at 300 &#215; g, and remove the supernatant. Add 4 mL of fresh PBS to the pellet and keep it at 4 &#176;C until embedding. 
		NOTE: Fixed organoids are stable and storage at 4 &#176;C is possible for 1 month before embedding.</li>
		<li>Heat the histological gel (stored at -20 &#176;C) at 65 &#176;C until liquefied.</li>
		<li>Detect the fixed organoids settled and visible at the bottom of the tube. Remove the supernatant. In case of a disrupted cell pellet, conduct centrifugation for 3 min at 300 &#215; g and discard the supernatant PBS.</li>
		<li>Suspend the pellet in 100 &#181;L of warm gel by gently pipetting up and down. Transfer the droplet to a piece of sealing film and allow solidification to occur after ~15 min at room temperature.</li>
		<li>Move the fixed gel droplet to the tissue paraffin cassette. Conduct standard tissue embedding protocols19,20.</li>
		<li>Cut 5-10 &#181;m thick slices with a microsectioning cutting tool. Transfer the cuts onto histology slides. Dry the slides for 1 h at 65 &#176;C; keep them in a dry place.</li>
	</ol>
	</li>
	<li>Immunofluorescence staining of organoid sections
	<ol>
		<li>Move the prepared slides through glass trays with the following dilution series: 2 x 15 min clearing agent for histology; 15 min 100% ethanol; 1 min 100% ethanol; 10 min 96% ethanol; 5 min 70% ethanol; 5 min 50% ethanol.</li>
		<li>Add PBS and keep 5 min on the shaker at 100 rpm. Repeat the washing 2x.</li>
		<li>Add&#160;antigen&#160;retrieval&#160;solution (Tris(hydroxymethyl)aminomethane-Ethylenediaminetetraacetic acid (EDTA)-buffer [pH 9.0] or Citrate [pH 6.0]) to the slides that are placed in the thermostable chamber. In a steamer, keep the container with the slides for 30 min, followed by cool down to room temperature.</li>
		<li>Repeat step 3.5.2.</li>
		<li>Transfer the chamber containing the slides for 15 min into 1% permeabilization&#160;detergent solution (in PBS).</li>
		<li>Repeat step 3.5.2.</li>
		<li>Contour the location of organoids on the slides with an immunostaining wax pen to maintain the droplet during the following steps.</li>
		<li>Add on each slide 100 &#181;L of 10% serum in a dilution medium, depending on the species of the secondary antibody.</li>
		<li>Repeat step 3.5.2.</li>
		<li>Dilute the primary antibodies in a medium, leading to a final volume of 100 &#181;L. Keep at 4 &#176;C for at least 16 h in an incubation tray/ humidity chamber.</li>
		<li>Repeat step 3.5.2.</li>
		<li>Dilute the secondary antibodies in a medium, leading to 100 &#181;L droplets, and keep for 2 h at room temperature in an incubation tray.</li>
		<li>Repeat step 3.5.2.</li>
		<li>Add nuclear counterstain solution DAPI (4&#39;,6-Diamidin-2-phenylindol, Dihydrochloride) diluted in a dilution medium 1:1,000. Keep for 10 min at room temperature for incubation.</li>
		<li>Repeat step 3.5.2.</li>
		<li>Add mounting medium and cover with the coverslip. Secure the slides with transparent nail polish once dry (approximately after 8-12 h).</li>
		<li>Image with a confocal microscope or other fluorescence microscope. Make overview pictures as well as detailed images of subcellular structures that capture the main characteristics of organoid morphology.</li>
	</ol>
	</li>
</ol>

Standardizing Ovarian Cancer Organoid Biobanking

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+drug+screening+of+patient-derived+tumor+organoids+using+high-content+fluorescent+imaging.">Protocol for drug screening of patient-derived tumor organoids using high-content fluorescent imaging.</a> STAR Protoc. 3 (2), 101407 (2022).</li><li>Senkowski, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+platform+for+efficient+establishment+and+drug-response+profiling+of+high-grade+serous+ovarian+cancer+organoids.">A platform for efficient establishment and drug-response profiling of high-grade serous ovarian cancer organoids.</a> Dev Cell. 58 (12), 1106-1121 (2023).</li><li>LeSavage, B. L., Suhar, R. A., Broguiere, N., Lutolf, M. P., Heilshorn, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+cancer+organoids.">Next-generation cancer organoids.</a> Nat Mater. 21 (2), 143-159 (2022).</li><li>Hoffmann, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+expansion+of+high-grade+serous+ovarian+cancer+organoids+requires+a+low-Wnt+environment.">Stable expansion of high-grade serous ovarian cancer organoids requires a low-Wnt environment.</a> EMBO J. 39 (6), e104013 (2020).</li><li>Kessler, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Notch+and+Wnt+pathways+regulate+stemness+and+differentiation+in+human+fallopian+tube+organoids.">The Notch and Wnt pathways regulate stemness and differentiation in human fallopian tube organoids.</a> Nat Commun. 6, 8989 (2015).</li><li>Trillsch, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+to+optimize+the+biobanking+of+ovarian+cancer+organoids+by+accommodating+patient-specific+differences+in+stemness+potential.">Protocol to optimize the biobanking of ovarian cancer organoids by accommodating patient-specific differences in stemness potential.</a> STAR Protoc. 4 (3), 102484 (2023).</li><li>Maenhoudt, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+organoids+from+ovarian+cancer+as+experimental+and+preclinical+models.">Developing organoids from ovarian cancer as experimental and preclinical models.</a> Stem Cell Reports. 14 (4), 717-729 (2020).</li><li>Fuerer, C., Nusse, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+vectors+to+probe+and+manipulate+the+Wnt+signaling+pathway.">Lentiviral vectors to probe and manipulate the Wnt signaling pathway.</a> PLoS One. 5 (2), e9370 (2010).</li><li>. Leica ASP300S - Advanced smart processor vacuum tissue processor, instructions for use, V 2.1 Available from: <a target="_blank" href="https://www.leicabiosystems.com/sites/default/files/media_product-download/2022-01/Leica_ASP300S_IFU_2v1N_en.pdf">https://www.leicabiosystems.com/sites/default/files/media_product-download/2022-01/Leica_ASP300S_IFU_2v1N_en.pdf</a> (2021)</li><li>Thermo Scientific. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Microm EC350 Modular tissue embedding center Instruction manual. , (2009).</li><li>Nanki, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+ovarian+cancer+organoids+capture+the+genomic+profiles+of+primary+tumours+applicable+for+drug+sensitivity+and+resistance+testing.">Patient-derived ovarian cancer organoids capture the genomic profiles of primary tumours applicable for drug sensitivity and resistance testing.</a> Sci Rep. 10 (1), 12581 (2020).</li></ol>

M.K. is listed as an inventor on a patent related to a medium for ovarian cancer organoids. F.T. received research funding, advisory board, honoraria, and travel expenses from AstraZeneca, Clovis, Eisai, ImmunoGen, Medac, MSD, PharmaMar, Roche, SAGA diagnostics, and Tesaro/GSK. S.M. received research funding, advisory board, honorary or travel expenses: AbbVie, AstraZeneca, Clovis, Eisai, GlaxoSmithKline, Hubro, Medac, MSD, Novartis, Nykode, Olympus, PharmaMar, Pfizer, Roche, Sensor Kinesis, Teva, Tesaro.

The designed protocol addresses previous challenges of ovarian cancer organoid biobanking with regard to organoid formation and long-term passage potential and ensures the generation of fully expandable lines from the majority of solid tumor deposits. The surgical collection process of tumor samples to be used for organoid generation significantly impacts yield and expansion potential. Tumor tissue samples can be obtained during various procedures, including multi-visceral surgery, diagnostic laparoscopy, or biopsy. The ...

Clinical management of patients with epithelial ovarian cancer remains challenging due to its heterogeneous clinical presentation at advanced stages and high recurrence rates1. Improving our understanding of ovarian cancer development and biological behavior requires research approaches that address the patient-specific variability during the course of the disease, treatment response, and histopathological as well as molecular features2.
Biobanking, characterized by the systematic collection and long-term preservation of tumor samples derived from ovarian cancer patients along with their clinical information offers the preservation of a large patient cohort in different disease stages, including tumor samples from primary debulking surgeries, after neoadjuvant chemotherapy and from recurrent disease. It holds valuable potential for advancing cancer research serving as a resource of promising prognostic biomarkers and therapeutic targets3. However, conventional biobanking methods, such as formalin fixation and freezing, are not amenable to conducting functional studies on the original tumor samples due to the loss of viability and the disruption of the native three-dimensional tissue architecture4,5.
Studies of molecular mechanisms, in oncology and beyond, crucially depend on the use of appropriate experimental models that faithfully reflect the biology of the disease and maintain in vitro properties of the tissue observed in vivo. Patient-derived organoids, based on the preservation of the renewal potential, reproduce in the lab the original structure and function of the epithelium and allow testing in a patient-specific context. Therefore, they have emerged as highly promising tools for cancer research and personalized medicine, bridging the gap between clinical diversity and laboratory research6,7,8,9. Tailored therapeutic strategies based on individual drug responses of organoid lines and testing of the functional relevance of molecular profiles, can potentially be directly applied to patient care10,11. The possibility of long-term cultivation including patient-specific characteristics and the collection of relevant prospective clinical data over time holds great promise to identify novel prognostic and predictive factors involved in disease progression and resistance mechanisms3,9.
Nonetheless, building a biobank that includes organoids from different tumor samples requires a combination of strict adherence to complex methodology and setting up protocols for easy maintenance12. Process standardization ensures that the biobank can be established and maintained efficiently by trained staff even at high turnover, while at the same time adhering to the highest quality standards13. Several studies reported the successful generation of stable ovarian cancer organoid lines corresponding to the mutational and phenotypical profile of the original tumor with varying efficiency rates. Still, routine bio banking remains challenging in practice, particularly for long-term stable growth of lines, which is a prerequisite for large-scale expansion or successful genomic editing.
In particular, the issue of expandability remains vaguely defined in the field as organoids that show slow and limited growth potential are occasionally counted as established lines. As initially demonstrated by Hoffmann et al., a study whose principal findings provided the basis for this further developed protocol, optimal handling of ovarian cancer tissue requires a unique strategy to accommodate heterogeneity14. Phenotypic characterization of the organoids obtained by this method and close similarity with parental tumor tissue were confirmed by panel DNA sequencing and transcriptomics analysis of mature cultures (4-10 months of cultivation) demonstrating the stability of the model8,9,12,14.
In contrast to the paracrine environment that regulates the homeostasis in the healthy fallopian tubes, the epithelial layer, which likely yields high-grade serous ovarian cancer (HGSOC), cancer regeneration potential, and organoid formation capacity, is less dependent on exogenous Wnt supplementation. Moreover, active Bone Morphogenetic Protein (BMP) signaling, characterized by the absence of Noggin in organoid medium, proved to be beneficial for the establishment of long-term cultures from ovarian cancer solid tissue deposits14,15. During systematic biobanking of solid deposits of ovarian cancer, we have confirmed these findings and set up the pipeline, with details outlined in this protocol that ensures sustained long-term expansion in the majority of cases. We find that parallel testing of different media compositions and seeding modalities when working with primary isolates are essential to improve the establishment of long-term stable organoid lines and to increase yields enabling robust propagation and expansion to multi-well formats required for downstream experiments16.
Furthermore, the purity and quality of the samples collected during surgery are of crucial importance for the translational potential of ovarian cancer organoids in basic research and molecular diagnostics. The complexity of the clinical presentation of HGSOC requires close cooperation between the surgeons, oncologists, and the scientists in the lab to ensure that relevant material is correctly identified, transport conditions are kept constant, and organoid lines are generated with high efficiency representing the most important characteristics of the disease of each patient. This protocol provides a standardized but adaptable framework to capture the full potential of ovarian cancer organoids, considering the heterogeneity that characterizes ovarian cancer16,17. Notably, this protocol enables reliable biobanking of the broad spectrum of ovarian cancer clinical presentation, including different histological types (high-grade and low-grade ovarian cancer, LGSOC), different deposits from the same patients who exhibit differences in stemness regulation, tissues from surgeries in post neoadjuvant setting, biopsy material, and samples from surgeries in the recurrent phase of disease progression.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 Sterican 26 G</td>
			<td>Braun, Melsungen, Germany</td>
			<td>4657683</td>
			<td></td>
		</tr>
		<tr>
			<td>100 Sterican 27 G</td>
			<td>Braun, Melsungen, Germany</td>
			<td>4657705</td>
			<td></td>
		</tr>
		<tr>
			<td>293T HA Rspo1-Fc</td>
			<td>R&#38;D systems, Minneapolis, USA</td>
			<td>3710-001-01</td>
			<td>Alternative: R-Spondin1 expressing Cell line, Sigma-Aldrich, SC111</td>
		</tr>
		<tr>
			<td>A-83-01 (TGF-b RI Kinase inhibitor IV)</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>616454</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F-12 Medium&#160;</td>
			<td>Gibco, Thermo Scientific, Waltham, USA</td>
			<td>12634028</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-p53 antibody (DO1)</td>
			<td>Santa Cruz Biotechnology, Texas, USA</td>
			<td>sc-126</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PAX8 antibody</td>
			<td>Proteintech, Manchester, UK&#160;</td>
			<td>10336-1-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement (50x)</td>
			<td>Gibco, Thermo Scientific, Waltham, USA</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottle-top vacuum filter 0.2 &#181;m</td>
			<td>Corning, Berlin, Germany&#160;</td>
			<td>430049</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLSTAR cell culture flask, 175 cm2</td>
			<td>Greiner Bio-one, Kremsm&#252;nster, Austria</td>
			<td>661175</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLSTAR cell culture flask, 25 cm2</td>
			<td>Greiner Bio-one, Kremsm&#252;nster, Austria</td>
			<td>690160</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLSTAR cell culture flask, 75 cm2</td>
			<td>Greiner Bio-one, Kremsm&#252;nster, Austria</td>
			<td>658175</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase I</td>
			<td>Thermo Scientific, Waltham, USA</td>
			<td>17018029</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar 48-well Clear TC-treated&#160;</td>
			<td>Corning, Berlin, Germany&#160;</td>
			<td>3548</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryo SFM</td>
			<td>PromoCell &#8211; Human Centered Science, Heidelberg, Germany</td>
			<td>C-29912</td>
			<td></td>
		</tr>
		<tr>
			<td>Cultrex Reduced Growth Factor Basement Membrane Extract, Type 2, Pathclear</td>
			<td>R&#38;D systems, Minneapolis, USA</td>
			<td>3533-005-02</td>
			<td>Alternative: Matrigel, Growth Factor Reduced Basement membrane matrix&#160; Corning, 356231&#160;</td>
		</tr>
		<tr>
			<td>Cy5 AffiniPure Donkey Anti-Mouse IgG</td>
			<td>Jackson Immuno</td>
			<td>715-175-151</td>
			<td></td>
		</tr>
		<tr>
			<td>DAKO&#160; Citrate Buffer, pH 6.0, 10x Antigen Retriever</td>
			<td>Sigma-Aldrich, Merck, Darmstadt, Germany</td>
			<td>C9999-1000ML</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Scientific, Waltham, USA</td>
			<td>62248</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti rabbit Alexa Fluor Plus 555</td>
			<td>Thermo Scientific, Waltham, USA</td>
			<td>A32794</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-Goat IgG Alexa Fluor Plus 488</td>
			<td>Thermo Scientific, Waltham, USA</td>
			<td>A32814</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#180;s Phosphate-Buffered Saline&#160;</td>
			<td>Gibco, Thermo Scientific, Waltham, USA</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Epredia Richard-Allan Scientific HistoGel</td>
			<td>Thermo Scientific, Waltham, USA</td>
			<td>Epredia HG-4000-012</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 24-well Polystyrene&#160;</td>
			<td>Corning, Berlin, Germany&#160;</td>
			<td>351447</td>
			<td></td>
		</tr>
		<tr>
			<td>Feather scalpel&#160;</td>
			<td>Pfm medical, Cologne, Germany</td>
			<td>200130010</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco, Thermo Scientific, Waltham, USA</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin 37% acid free, stabilized</td>
			<td>Morphisto, Offenbach am Main, Germany</td>
			<td>1019205000</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco, Thermo Scientific, Waltham, USA</td>
			<td>35050038</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Gibco, Thermo Scientific, Waltham, USA</td>
			<td>156630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Human EpCAM/TROP-1 Antibody</td>
			<td>R&#38;D systems, Minneapolis, USA</td>
			<td>AF960</td>
			<td></td>
		</tr>
		<tr>
			<td>Human FGF10</td>
			<td>Peprotech, NJ, USA</td>
			<td>100-26</td>
			<td></td>
		</tr>
		<tr>
			<td>Human recombinant BMP2</td>
			<td>Gibco, Thermo Scientific, Waltham, USA</td>
			<td>PHC7146</td>
			<td></td>
		</tr>
		<tr>
			<td>Human recombinant EGF</td>
			<td>Gibco, Thermo Scientific, Waltham, USA</td>
			<td>PHG0311L</td>
			<td></td>
		</tr>
		<tr>
			<td>Human recombinant Heregulin beta-1</td>
			<td>Peprotech, NJ, USA</td>
			<td>100-03</td>
			<td></td>
		</tr>
		<tr>
			<td>LAS X core Software</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td>https://webshare.leica-microsystems.com/latest/core/widefield/</td>
		</tr>
		<tr>
			<td>Leica TCS SP8 X&#160;White Light Laser Confocal Microscope</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 Supplement (100x)</td>
			<td>Gibco, Thermo Scientific, Waltham, USA</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Aldrich, Merck, Darmstadt, Germany</td>
			<td>N0636</td>
			<td></td>
		</tr>
		<tr>
			<td>Omnifix 1 mL</td>
			<td>Braun, Melsungen, Germany</td>
			<td>3570519</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Omnilab, Munich, Germany</td>
			<td>5170002</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyd&#160;</td>
			<td>Morphisto, Offenbach am Main, Germany</td>
			<td>1176201000</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>Gibco, Thermo Scientific, Waltham, USA</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Sigma-Aldrich, Merck, Darmstadt, Germany</td>
			<td>P4333-100</td>
			<td></td>
		</tr>
		<tr>
			<td>PluriStrainer 400 &#181;m</td>
			<td>PluriSelect, Leipzig, Germany</td>
			<td>43-50400-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Primocin</td>
			<td>InvivoGen, Toulouse, France</td>
			<td>ant-pm-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Red Blood Cell Lysing Buffer</td>
			<td>Sigma-Aldrich, Merck, Darmstadt, Germany</td>
			<td>11814389001</td>
			<td></td>
		</tr>
		<tr>
			<td>Roticlear</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>A538.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgipath Paraplast</td>
			<td>Leica, Wetzlar, Germany</td>
			<td>39602012</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific Nunc Cryovials</td>
			<td>Thermo Scientific, Waltham, USA</td>
			<td>375418PK</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich, Merck, Darmstadt, Germany</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain</td>
			<td>Sigma-Aldrich, Merck, Darmstadt, Germany</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme&#160;</td>
			<td>Gibco, Thermo Scientific, Waltham, USA</td>
			<td>12604-013</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>PanReac AppliChem, Darmstadt, Germany</td>
			<td>A4974-0100</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>TOCRIS biotechne, Wiesbaden, Germany</td>
			<td>1254</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeocin</td>
			<td>Invitrogen, Thermo Scientific, Waltham, USA</td>
			<td>R25001</td>
			<td></td>
		</tr>
	</tbody>
</table>

strategy for biobanking of ovarian cancer organoids: addressing the interpatient heterogeneity across histological subtypes and disease stages

While the establishment of an ovarian cancer biobank from patient-derived organoids along with their clinical background information promises advances in research and patient care, standardization remains a challenge due to the heterogeneity of this lethal malignancy, combined with the inherent complexity of organoid technology. This adaptable protocol provides a systematic framework to realize the full potential of ovarian cancer organoids considering a patient-specific variability of progenitors. By implementing a structured experimental workflow to select optimal culture conditions and seeding methods, with parallel testing of direct 3D seeding versus a 2D/3D route, we obtain, in most cases, robust long-term expanding lines suitable for a broad range of downstream applications. 
Notably, the protocol has been tested and proven efficient in a great number of cases (N = 120) of highly heterogeneous starting material, including high-grade and low-grade ovarian cancer and stages of the disease with primary debulking, recurrent disease, and post-neoadjuvant surgical specimens. Within a low Wnt, high BMP exogenous signaling environment, we observed progenitors being differently susceptible to the activation of the Heregulin 1 &#223; (HER&#223;-1)-pathway, with HER&#223;-1 promoting organoid formation in some while inhibiting it in others. For a subset of the patient's samples, optimal organoid formation and long-term growth necessitate the addition of fibroblast growth factor 10 and R-Spondin 1 to the medium. 
Further, we highlight the critical steps of tissue digestion and progenitor isolation and point to examples where brief cultivation in 2D on plastic is beneficial for subsequent organoid formation in the Basement Membrane Extract type 2 matrix. Overall, optimal biobanking requires systematic testing of all main conditions in parallel to identify an adequate growth environment for individual lines. The protocol also describes the handling procedure for efficient embedding, sectioning, and staining to obtain high-resolution images of organoids, which is required for comprehensive phenotyping.

Author Spotlight: Advancing Personalized Medicine in Ovarian Cancer 

Strategy for Biobanking of Ovarian Cancer Organoids: Addressing the Interpatient Heterogeneity across Histological Subtypes and Disease Stages

The best clinical management in cutting-edge translational research for patients with advanced ovarian cancer has highest priority at our institution. With the biobanking of patient-derived organoids, we have achieved major progress towards individually-tailored management. This enables us to evaluate patient-specific characteristics and to identify predictive biomarkers.We have made significant advances in recent years in improving radical multivisceral surgery, chemotherapy, anti-angiogenic therapy, and also PARP inhibitor therapy in patients with advanced ovarian cancer and improved survival rates even with that. Now, the next big step would be to better predict which patient would benefit from which specific treatment. Currently, clinical treatment decisions are based on general histopathological features and on information on homologous recommendation deficiency or tested in paraffin-embedded tumor tissue.Biologic models that evaluate specific responses to chemotherapy and to targeted treatments have not yet been implemented in clinical routine. We have identified key characteristics of ovarian cancer tissue, specifically the culture requirements necessary to sustain its renewal potential in vitro, and use this knowledge to achieve robust long-term growth of organoid lines and establish biobank from samples at various stages of the disease. Our day-to-day experience in biobanking of ovarian cancer organoids effectively shows a remarkable heterogeneity in the biology of this malignancy, which could pave the way to new classification or provide the basis for new therapeutic solutions targeting STEMI's potential.

After initial tissue dissociation, filtration, and counting, cells are seeded in parallel directly in 3D format, as explained above, as well as the suspension in the flask for brief 2D expansion. In some cases, the transient 2D expansion positively influences the organoid formation, and the long-term line is successfully established via this route while comparative parallel 3D seeding can result in growth arrest (Figure 1). For each donor tissue that is processed, the cells are tested accord...

Watch this Scientific Journal Video about Author Spotlight: Advancing Personalized Medicine in Ovarian Cancer at JoVE.com

This protocol offers a systematic framework for the establishment of ovarian cancer organoids from different disease stages and addresses the challenges of patient-specific variability to increase yield and enable robust long-term expansion for subsequent applications. It includes detailed steps for tissue processing, seeding, adjusting media requirements, and immunofluorescence staining.

While the establishment of an ovarian cancer biobank from patient-derived organoids along with their clinical background information promises advances in ...

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Research

JoVE Journal

Cancer Research

Establishment of Ovarian Cancer Organoid Culture and its Evaluation

Ovarian Cancer Organoid Passaging and Cryopreservation for Long-Term Organoid Cultivation

Fixation of Ovarian Cancer Organoids for Long-Term Storage

1.8K Views.  University Hospital, Ludwig-Maximilian-University (LMU) Munich.  This protocol offers a systematic framework for the establishment of ovarian cancer organoids from different disease stages and addresses the challenges of patient-specific variability to increase yield and enable robust long-term expansion for subsequent applications. It includes detailed steps for tissue processing, seeding, adjusting media requirements, and immunofluorescence staining. 

Standardizing Ovarian Cancer Organoid Biobanking

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