Name: handling and assessment of human primary prostate organoid culture
Uploaded: 2019-01-17T15:00:00
Duration: 8 min 45 s
Description: Watch this Scientific Journal Video about Handling and Assessment of Human Primary Prostate Organoid Culture at JoVE.com

We thank the UIC Biorespository members, Dr. Klara Valyi-Nagy, and Alex Susma, as well as the urologists, Drs. Michael Abern, Daniel Moreira, and Simone Crivallero, for facilitation of tissue acquisition for the primary cell cultures. We thank the UIC Urology patients for donating their tissue to research. This work was funded, in part, by the Department of Defense Prostate Cancer Research Program Health Disparities Idea Award PC121923 (Nonn) and the UIC Center for Clinical and Translation Science Pre-doctoral Education for Clinical and Translational Scientists (PECTS) Program (McCray and Richards) and by the National Institutes of Health&#39;s National Cancer Institute, Grant Numbers U54CA202995, U54CA202997, and U54CA203000, known as the Chicago Health Equity Collaborative (Nonn and Richards). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the Department of Defense.

<ol>
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The authors have nothing to disclose.

Organotypic culture is an exciting new method for recapitulating tissue with the convenience of an in vitro environment. Currently, labs grow organoids from many types of tissues for various endpoints. The methods described in this paper summarize useful endpoints and highlight new techniques to fully characterize 3D primary prostate cell cultures.
There are a variety of media recipes for cultivating human primary prostate cell organoids5,6,13. While all achieve viable and comparable results, KSFM-based media13 uses minimal additives so it is described here. Additionally, papers have been published using multiple concentrations for matrix gel, from 10% to 75% to culture prostate organoids5,6,13. Because matrix gel is an expensive reagent, and 33% matrix gel has been shown to be sufficient to cultivate long-term (2-3 weeks), viable, unclumped organoids13, this is the recommended concentration. However, matrix gel can vary in protein concentration between lot numbers, so this should be taken into account when plating. It is also recommended to purchase matrix gel in bulk for use across multiple experiments to reduce inconsistency between lots. Other labs have published different formats for plating matrix gel, such as droplets instead of coating a 96-well plate well5. Both methods form viable organoids, but the format described here allows for cells to be plated more sparsely, which has been shown to promote expansion of cells into larger organoids13. However, plating density should be optimized based off patient-specific formation efficiency. Matrix gel is available in many formats including growth factor-reduced, phenol red-free, high concentration, etc. Growth factor-reduced is recommended for defined culture conditions, and phenol red-free is recommended when working with cells that express GFP or in experiments that involve z-stack image capture. It is critical that any matrix gel plating steps be performed with ice-cold reagents, as matrix gel solidifies quickly at room temperature.
Organoids grown under different experimental treatments may result in changes to shape, so bright-field imaging is widely used to study observed morphological phenotypes. Nevertheless, recording and quantifying area or shape is a challenge for two reasons: 1) selection bias during image capture and 2) applying a two-dimensional parameter such as area to a three-dimensional sample. One strategy of image capture is to record a random field and measure a predetermined number of organoids in that field, but this can create bias during selection, and all organoids in the selected field may not be in focus. Whole-well imaging optimized for the 96-well microplate format eliminates this sampling bias by collecting the entire organoid population of interest. However, depending on the microscope objectives on hand, multiple fields may need to be collected and tiled in order to obtain a whole-well image. Some labs have described measuring area from a single z-plane12, but to capture all organoids in focus, it is recommended to collect and stack multiple planes into a single EDF-image13. In some cases, a meniscus in the matrix gel may cause vignetting in the image, and background correction methods may need to be applied using image analysis software. Exact volume is ideally the most appropriate measurement for organoid size; however, this is difficult to precisely obtain, even with multiple z-stack images. Other useful morphological dimensions, such as circularity and maximum/minimum ratio, can easily be calculated from all the organoids in a whole-well EDF image. Together, these methods overcome sampling bias challenges and enable measurement of 2D parameters from 3D objects in a 3D space.
Formalin fixation and paraffin embedding of organoids is a common method to obtain hematoxylin and eosin (H&#38;E), immunohistochemical (IHC), and immunofluorescent (IF) staining for visualization and analysis6,11,20. However, publications that have described this technique lack comprehensive details on the embedding process. Additionally, locating organoids within the paraffin block can be challenging. Some labs pre-stain organoids with trypan blue prior to embedding to aid in location during sectioning11. The method described here incorporates the use of a histological dye for orientation of the organoid/histology gelplug during embedding to promote efficiency of sectioning. H&#38;E slides facilitate examination of necrosis, nuclear texture, and proliferation, and thus it should be a mandatory endpoint for organoid culture to ensure that cells are healthy throughout the sphere. A strength of organoid culture is that samples are comprised of a heterogeneous mixture of cells that better resemble in vivo patient tissue. In the prostate, for example, both basal and luminal cells are present in prostate organoids5, while cells grown in 2D lack luminal differentiation8. To assess organoid differentiation, it is recommended that researchers assess basal markers such as CK5 and p63 and luminal markers such as androgen receptors and CK88. Any other experimental proteins of interest can also be studied using these staining techniques.
Although FFPE sections allow visualization of cells residing in the inner compartment via histologic methods (H&#38;E, IF, IHC), images are limited to cross sections, and organoid shape may be altered by the embedding process. Whole-mount staining enables an organoid to be stained and observed in situ, preserving morphological phenotypes and permitting images of protein localization. Whole-mounting is a tool utilized to stain whole-tissue or whole-animal specimens, such as the zebrafish or mouse embryo, and is easily adapted for organoids. The technique described here was modified from the procedure by Mah&#233;et al.11, which details the initial growth and culture of gastrointestinal organoids directly on a chamber slide for staining, and requires fixation of the entire parent culture. The method outlined here involves the transfer of a single organoid (or organoids) of interest onto a chamber slide at the time of staining. This enables the selection of individual organoids for whole-mount analysis in an ongoing experiment without fixation of the parent culture. When performing whole-mount staining, it is necessary to optimize permeabilization and the incubation time for primary and secondary antibodies to ensure penetration throughout the specimen (anywhere from one or more days is recommended). Once imaged via confocal microscopy, 3D renderings can be produced to enable visualization and localization of a specific protein and calculate the number of positively stained cells present in a sample. Flow cytometry is a useful endpoint to quantify populations of basal, luminal, or stem cells5,14. To do this, cells are recovered from the matrix gel and gently dissociated for staining. Users should carefully select appropriate markers for separation based on the current literature. For human primary prostate epithelial cells, CD26 and CD49f are suitable luminal and basal markers, respectively5,21.
In summary, this protocol details human primary prostate organoid growth, collection, and experimental endpoints. Of note, the mounting technique described can be applied to a variety of other assays that are normally employed for 2D cells, such as fluorescent probe-based experiments looking at proliferation19, apoptosis, and subcellular organelle stains. Additionally, the collection and dissociation method described here could be utilized in preparation for single-cell RNA sequencing18. Collectively, this demonstrates the possibility for a variety of novel, high-fidelity endpoints that researchers can optimize and explore in the future.

Organoids are a valuable tool to study organogenesis and disease. They provide a less expensive alternative to animal models, and patient-derived organoids are evolving as a strategy for personalized medicine1,2,3. This three-dimensional (3D) culture system involves seeding stem or progenitor cells (harvested from tissue or induced pluripotent stem cells) into a gel of extracellular matrix components (matrix gel)4. The cells proliferate and differentiate, resulting in organotypic structures that recapitulate the cellular hierarchy and morphology of the organ of interest&#39;s functional unit. Organoids have been grown using cells originating from a variety of organs, including the salivary gland, stomach, intestine, liver, prostate, lung, and brain4. Although there are numerous protocols describing steps to establish prostate organoids5,6, it is challenging to find sufficiently detailed methods on how to achieve quantitative endpoints from organoids. This paper summarizes methods developed for human primary prostate cells and details a series of suggested endpoints to assess the organoid phenotypes. These techniques were optimized for prostate organoids and may be applicable to other 3D cell cultures.
Prostate organoid cultures have recently emerged as a valuable in vitro model that lacks the limitations of monolayer cultures using established immortalized cell lines. Benign prostate epithelium and prostate cancer is challenging to model in vitro using immortalized cell lines. The number of benign cell lines is limited, and all have undergone transformation with oncogenes7. Primary prostate epithelial cells in monolayers do not differentiate into luminal cells and lack androgen receptors8. The majority of prostate cancer cell lines do not have functional androgen receptors, a significant mediator of early disease state, and lack key genetic changes that have been found in patient tumors9. Prostate organoid cultures can be grown easily from benign epithelium and are valuable in studying stem cell properties, progenitor cells, differentiation, and effects of experimental changes in the microenvironment4,5. Prostate cancer organoids can be grown as part of a precision medicine approach to modeling patient disease and response to therapies10.
This protocol has been compiled from existing protocols that use various cell types, but it has been optimized here for use in human primary prostate cells. It compliments protocols outlined by Sawyers, Clevers, and Shen5,6 that describe growth, passaging, bright-field imaging, cryopreservation, and RNA and DNA isolation of prostate mouse and human organoids. The whole-mount protocol is modified from Mah&#233;&#160;et al.11, who used gastrointestinal epithelial organoids and describes live imaging and frozen and paraffin embedding. Borten et al.12 described the analysis of breast, colon, and colorectal cancer organoid morphology from bright-field imaging. Additionally, Richards et al.13 used a method for the morphologic assessment of prostate organoids. Finally, Hu et al.14 described a method of adhering prostate organoids to a chamber slide overnight prior to the immunofluorescent staining to observe single cells and dispersion of spheres for flow cytometry analysis.
The goal of this protocol is to demonstrate in sufficient detail these technically challenging methods as one protocol, including cell seeding; media and matrix gel maintenance; collection of cells for flow cytometry; RNA, DNA, and protein extraction; morphologic assessment from bright-field z-stack analysis; embedding, processing and sectioning for histological staining; and whole-mounting for immunofluorescence or fluorescent probe assays. The biological relevance and interpretation of these various endpoints will vary among experimental design and antibodies used for analysis. Through utilization of this protocol, users should feel prepared to set up an experiment with a toolkit of endpoints.
Human primary prostate epithelial (PrE) cells were established in the lab by the protocol described by Peehl15,16 and maintained to a limited number of passages (up to four) as previously described17, but they are also available from commercial vendors. Hepatocyte serum-free media-based6, KSFM-based13, and R-spondin 1-conditioned5 media are all published as having successfully produced organoids. KSFM-based requires the fewest number of additives, so it is described here.

<table>
	<tbody>
		<tr>
			<td>Cells of interest</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Keratinocyte-SFM (KSFM)</td>
			<td>ThermoFisher Scientific</td>
			<td>17005042</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, charcoal stripped, USDA-approved regions (FBS)</td>
			<td>ThermoFisher Scientific</td>
			<td>12676029</td>
			<td></td>
		</tr>
		<tr>
			<td>5&#945;-Dihydrotestosterone (DHT)</td>
			<td>Sigma-Aldrich</td>
			<td>D-073-1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Flat bottom, polystyrene 96-well cell culture treated plate</td>
			<td>ThermoFisher Scientific</td>
			<td>161093</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel (matrix gel)</td>
			<td>Corning</td>
			<td>Various</td>
			<td>Matrix-gel: growth Factor Reduced, Phenol-red free, etc. depending on application</td>
		</tr>
		<tr>
			<td>Ice Bucket</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL micro-centrifuge tubes or 15 mL conical</td>
			<td>ThermoFisher Scientific</td>
			<td>05-408-130, 339650</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase (neutral protease)</td>
			<td>STEMCELL Technologies</td>
			<td>7923</td>
			<td>neutral protease</td>
		</tr>
		<tr>
			<td>Hanks&#39;&#160;balanced salt solution&#160;(HBSS)</td>
			<td>ThermoFisher Scientific</td>
			<td>14025076</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express (cell dissociation enzymes)</td>
			<td>ThermoFisher Scientific</td>
			<td>12605036</td>
			<td>Cell dissociation enzymes (trypsin may also work, depending on cell type, but TrypLE is more gentle and recommended for primary cells)</td>
		</tr>
		<tr>
			<td>TRIzol</td>
			<td>ThermoFisher Scientific</td>
			<td>15596018</td>
			<td>suggested RNA extraction solution</td>
		</tr>
		<tr>
			<td>RIPA Lysis and Extraction Buffer</td>
			<td>ThermoFisher Scientific</td>
			<td>89900</td>
			<td>suggested protein extraction solution</td>
		</tr>
		<tr>
			<td>DNAzol</td>
			<td>ThermoFisher Scientific</td>
			<td>10503027</td>
			<td>suggested DNA extraction solution</td>
		</tr>
		<tr>
			<td>Organoids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flat bottom, polystyrene 96-well cell culture treated plate</td>
			<td>ThermoFisher Scientific</td>
			<td>161093</td>
			<td></td>
		</tr>
		<tr>
			<td>Brightfield microscope with camera capabilities</td>
			<td></td>
			<td></td>
			<td>ex. &#8206;EVOS FL Auto Imaging System</td>
		</tr>
		<tr>
			<td>Photo analysis software</td>
			<td></td>
			<td></td>
			<td>ex. Photoshop, CELLESTE, ImageJ, MorphoLibJ, CellProfiler</td>
		</tr>
		<tr>
			<td>Graphing software</td>
			<td></td>
			<td></td>
			<td>ex. Graphpad, Excel, etc</td>
		</tr>
		<tr>
			<td>Organoids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ice pack</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Masking tape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips (1000 &#956;L)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>STEMCELL Technologies</td>
			<td>7923</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9045</td>
			<td></td>
		</tr>
		<tr>
			<td>HistoGel (histology-gel)</td>
			<td>ThermoFisher Scientific</td>
			<td>HG-4000-012</td>
			<td></td>
		</tr>
		<tr>
			<td>Pencil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#34;Plunger&#34; from 1 cc insulin syringe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Casette</td>
			<td>Thomas Scientific</td>
			<td>1202D72</td>
			<td></td>
		</tr>
		<tr>
			<td>Container to hold fixative</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10% neutral buffered formalin (NBF)</td>
			<td>Sigma-Aldrich</td>
			<td>HT501128</td>
			<td></td>
		</tr>
		<tr>
			<td>Histology pen, xylene and EtOH-resistant</td>
			<td>Sigma-Aldrich</td>
			<td>Z648191-12EA</td>
			<td>STATMARK pen</td>
		</tr>
		<tr>
			<td>Ethanol (histologic grade)</td>
			<td>Fisher Scientific</td>
			<td>A405P-4</td>
			<td>For fixation and graded dilutions during processing</td>
		</tr>
		<tr>
			<td>Xylenes</td>
			<td>Sigma-Aldrich</td>
			<td>214736-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Deionized water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Leica</td>
			<td>various</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue processor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding workstation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Economy Tissue Float Bath</td>
			<td>Daigger</td>
			<td>EF4575E XH-1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome blades</td>
			<td>Ted Pella</td>
			<td>27243</td>
			<td></td>
		</tr>
		<tr>
			<td>Positively charged microscope slides</td>
			<td>Thomas Scientific</td>
			<td>1158B91</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory Oven</td>
			<td>ThermoFisher Scientific</td>
			<td>PR305225G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin and Eosin Stain Kit</td>
			<td>Vector Laboratories</td>
			<td>H-3502</td>
			<td>Suggested H&#38;E staining kit</td>
		</tr>
		<tr>
			<td>ABC Peroxidase Standard Staining Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>32020</td>
			<td>Suggested immunohistochemistry staining kit</td>
		</tr>
		<tr>
			<td>Androgen Receptor Primary Antibody (AR)</td>
			<td>Cell Signaling Technology</td>
			<td>5153S</td>
			<td>Suggested primary antibody for IHC</td>
		</tr>
		<tr>
			<td>FFPE, sectioned organoid sample baked on a slide</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (histologic grade)</td>
			<td>Fisher Scientific</td>
			<td>A405P-4</td>
			<td>dilutions should be performed using deinoized water</td>
		</tr>
		<tr>
			<td>Xylenes</td>
			<td>Sigma-Aldrich</td>
			<td>214736-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Deionized water (DI H2O)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antigen retrieval solution</td>
			<td>Sigma-Aldrich, Abcam</td>
			<td>C9999, ab93684</td>
			<td></td>
		</tr>
		<tr>
			<td>1x Phosphate Buffered Saline - PBS</td>
			<td>ThermoFisher Scientific</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Horse Serum</td>
			<td>thermoFisher Scientific</td>
			<td>31874</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovin Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A2058</td>
			<td></td>
		</tr>
		<tr>
			<td>Counterstain (DAPI, Hoescht)</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td>suggested for storage but not required</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Coplin</td>
			<td>Fisher Scientific</td>
			<td>19-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Staining rack</td>
			<td>IHC World</td>
			<td>M905-12DGY</td>
			<td></td>
		</tr>
		<tr>
			<td>Staining dish</td>
			<td>IHC World</td>
			<td>M900-12B</td>
			<td></td>
		</tr>
		<tr>
			<td>Decloaking chamber</td>
			<td>Biocare Medical</td>
			<td>DC2012</td>
			<td></td>
		</tr>
		<tr>
			<td>Humidity chamber</td>
			<td>Thomas Scientific</td>
			<td>1219D68</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrophobic barrier pen</td>
			<td>Vector Laboratories</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytokeratin 8/18 primary antibody (CK8)</td>
			<td>ARP American Research Products</td>
			<td>03-GP11</td>
			<td>suggested primary antibody for IF</td>
		</tr>
		<tr>
			<td>p63-alpha antibody (p63)</td>
			<td>Cell Signaling Technology</td>
			<td>4892S</td>
			<td>suggested primary antibody for IF</td>
		</tr>
		<tr>
			<td>Keratin 5 Polyclonal Antibody (CK5)</td>
			<td>Biolegend</td>
			<td>905501</td>
			<td>suggested primary antibody for IF</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit secondary</td>
			<td>ThermoFisher Scientific</td>
			<td>A21245</td>
			<td>suggested secondary antibody for p63 or CK5 detection (do not use at same time)</td>
		</tr>
		<tr>
			<td>Goat anti-guinea pig secondary</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11075</td>
			<td>suggested secondary antibody for CK8 detection</td>
		</tr>
		<tr>
			<td>Microscope cover glass</td>
			<td>Globe Scientific</td>
			<td>1414-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-fade mounting media</td>
			<td>ThermoFisher Scientific</td>
			<td>S36972</td>
			<td></td>
		</tr>
		<tr>
			<td>Organoids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips (1000 &#956;L)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips (200 &#956;L)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>8-well chamber slide</td>
			<td>ThermoFisher Scientific</td>
			<td>154534PK</td>
			<td>It is also possible to use a confocal dish, depends on preference of user</td>
		</tr>
		<tr>
			<td>Cell-Tak</td>
			<td>Corning</td>
			<td>CB40240</td>
			<td>optional adherent reagent</td>
		</tr>
		<tr>
			<td>1x Phosphate Buffered Saline (PBS)</td>
			<td>ThermoFisher Scientific</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>4% paraformaldehye (PFA)</td>
			<td>Biotium</td>
			<td>22023</td>
			<td>other fixatives such as methanol or formalin can be used</td>
		</tr>
		<tr>
			<td>50mM NH4Cl</td>
			<td>sigma-Aldrich</td>
			<td>254134</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton&#8482; X-100 (non-ionic detergent)</td>
			<td>sigma-Aldrich</td>
			<td>X100-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Horse Serum (NHS)</td>
			<td>thermoFisher Scientific</td>
			<td>31874</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovin Serum Albumin (BSA)</td>
			<td>sigma-Aldrich</td>
			<td>A2058</td>
			<td></td>
		</tr>
		<tr>
			<td>Counterstain (DAPI, Hoescht)</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>Counterstain (phalloidin)</td>
			<td>thermoFisher Scientific</td>
			<td>A22287</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>sigma-Aldrich</td>
			<td>S2002</td>
			<td>suggested for storage but not required</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cytokeratin 8/18 primary antibody (CK8)</td>
			<td>ARP American Research Products</td>
			<td>03-GP11</td>
			<td>suggested primary antibody for IF</td>
		</tr>
		<tr>
			<td>p63-alpha antibody (p63)</td>
			<td>Cell Signaling Technology</td>
			<td>4892S</td>
			<td>suggested primary antibody for IF</td>
		</tr>
		<tr>
			<td>Keratin 5 Polyclonal Antibody (CK5)</td>
			<td>Biolegend</td>
			<td>905501</td>
			<td>suggested primary antibody for IF</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit secondary</td>
			<td>ThermoFisher Scientific</td>
			<td>A21245</td>
			<td>suggested secondary antibody for p63 or CK5 detection (do not use at same time)</td>
		</tr>
		<tr>
			<td>Goat anti-guinea pig secondary</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11075</td>
			<td>suggested secondary antibody for CK8 detection</td>
		</tr>
		<tr>
			<td>Visikol HISTO-M</td>
			<td>Visikol</td>
			<td>various</td>
			<td>optional clearing agent</td>
		</tr>
		<tr>
			<td>Organoids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips (1000 &#956;L)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips (200 &#956;L)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>8-well chamber slide</td>
			<td>ThermoFisher Scientific</td>
			<td>154534PK</td>
			<td>It is also possible to use a confocal dish, depends on preference of user</td>
		</tr>
		<tr>
			<td>Cell-Tak</td>
			<td>Corning</td>
			<td>CB40240</td>
			<td></td>
		</tr>
		<tr>
			<td>1x Phosphate Buffered Saline - PBS</td>
			<td>ThermoFisher Scientific</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell assay of interest</td>
			<td>Various</td>
			<td>Various</td>
			<td>Click-iT EdU Alexa Fluor proliferation assay (fluorescently-labelled EdU proliferation kit), Image-iT Lipid Peroxidation Kit, etc) and fluorescent probes/dyes (ex. HCS mitochondrial Health Kit, CellMask, LIVE/DEAD Viability assays, CellROX reagents, etc)</td>
		</tr>
		<tr>
			<td>Organoids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ice bucket</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL eppendorf tubes or 15 mL conical</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>STEMCELL Technologies</td>
			<td>7923</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express (cell dissociation enzymes)</td>
			<td>ThermoFisher Scientific</td>
			<td>12605036</td>
			<td>Cell dissociation enzymes (trypsin may also work, depending on cell type, but TrypLE is more gentle and recommended for primary cells)</td>
		</tr>
		<tr>
			<td>Hanks&#39;&#160;balanced salt solution&#160;(HBSS)</td>
			<td>ThermoFisher Scientific</td>
			<td>14175079</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Tube with Cell Strainer Snap Cap</td>
			<td>Fisher Scientific</td>
			<td>08-771-23</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytokeratin 5 Antibody - FITC</td>
			<td>Millipore Sigma</td>
			<td>FCMAB291F</td>
			<td>Suggested flow antibody</td>
		</tr>
		<tr>
			<td>Cytokeratin 8 Antibody - Alexafluor 405</td>
			<td>Abcam</td>
			<td>ab210139</td>
			<td>Suggested flow antibody</td>
		</tr>
		<tr>
			<td>CD49f - Alexafluor 647</td>
			<td>BioLegend</td>
			<td>313609</td>
			<td>Suggested flow antibody</td>
		</tr>
		<tr>
			<td>CD26 - PE</td>
			<td>BioLegend</td>
			<td>320576</td>
			<td>Suggested flow antibody</td>
		</tr>
	</tbody>
</table>

handling and assessment of human primary prostate organoid culture

This paper describes a detailed protocol for three-dimensional (3D) culturing, handling, and evaluation of human primary prostate organoids. The process involves seeding of epithelial cells sparsely in a 3D matrix gel on a 96-well microplate with media changes to cultivate expansion into organoids. Morphology is then assessed by whole-well capturing of z-stack images. Compression of z-stacks creates a single in-focus image from which organoids are measured to quantify a variety of outputs, including circularity, roundness, and area.DNA, RNA, and protein can be collected from organoids recovered from the matrix gel. Cell populations of interest can be assessed by organoid dissociation and flow cytometry. Formalin-fixation-paraffin-embedding (FFPE) followed by sectioning is used for the histological assessment and antibody staining. Whole-mount immunofluorescent staining preserves organoid morphology and facilitates observation of protein localization in organoids in situ. Commercial assays that are traditionally used for 2D monolayer cells can be modified for 3D organoids. Used together, the techniques in this protocol provide a robust toolbox to quantify prostate organoid growth, morphologic characteristics, and expression of differentiation markers.

Upon successful culture of human primary prostate organoids, morphology and differentiation can be assessed using bright-field image analysis and FFPE and whole-mount staining techniques.
The process of bright-field image capture is illustrated in Figure 1A. Organoids are grown in a 3D matrix and dispersed across different focal planes. To observe organoids in focus across many planes, it is suggested to use an EDF image (Figure 1B, right) instead of a single z-plane (Figure 1B, left). In the lab, organoids grown under different experimental conditions may result in changes in morphology. Using area, circularity (defined by how similar the organoid is to a circle) and max/min radius (measure of length) give users an indication of organoid size and shape and provide a quantifiable readout of morphology. To emphasize the usefulness of these measures, representative images of organoids with similar area but differing morphology are shown in Figure 1C, in which a long organoid will be less circular and have a larger maximum/minimum ratio than a spherical organoid.
Embedding organoids for histology is a useful endpoint to observe the interior of samples and ensure that necrosis is not present within the core of an organoid. A workflow for this process and representative results of the unstained, sectioned samples under a bright-field microscope are provided in Figure 2A,B,C. Figure 3A shows a mishandled organoid (left) compared to properly handed organoids in Figure 3A (right) and Figure 3B. To determine the differentiation-state of the sample, it is recommended to look at basal cell markers (cytokeratin 5 and p63)8 along with luminal cell markers (cytokeratin 8)8. If it is desired to stain organoids in situ, whole-mount staining is a convenient alternative, and these representative results are provided in Figure 3C,D. 
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59051/59051fig1v2.jpg" /> 
Figure 1: Morphology assessment of organoids in 3D culture. (A) Image collection and compression workflow for human primary prostate organoids acquired by a transmitted light inverted microscope with a motorized X/Y scanning stage and companion software. (B) Representative images of a single z-stack (left) vs. an EDF image (right) of a whole-well sample of human primary prostate organoids, showing that more organoids are in focus when multiple z-stacks are combined into a single projected image (scale bar = 1000 &#181;m). (C) Representative images for area, circularity, and max/min ratio of morphologically dissimilar human primary prostate organoids which have the same area (scale bar = 200 &#181;m). <a href="https://www.jove.com/files/ftp_upload/59051/59051fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59051/59051fig2.jpg" /> 
Figure 2: Organoid embedding workflow and representative sectioning results. (A) Human primary prostate organoid embedding workflow. (B) Representative image of an unstained, fresh slide containing human primary prostate organoids under a bright field microscope depicting agar (green arrow), histology gel (black arrow), bubble (blue arrow), organoids (red arrows) (scale bar = 1000 &#181;m). (C) Unstained freshly cut slide (left) vs. unstained dry slide (right), organoids (red arrows) appear translucent when dry (scale bar = 500 &#181;m) and are harder to discern under a bright-field microscope. <a href="https://www.jove.com/files/ftp_upload/59051/59051fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59051/59051fig3.jpg" /> 
Figure 3: Histological staining on sectioned and whole-mounted organoids. (A) H&#38;E staining of a broken human primary prostate organoid from mishandling (aggressive pipetting, wrong processing protocol, left) next to a well-handled organoid (right) (scale bar = 100 &#181;m). (B) Human primary prostate organoid that has been formalin-fixed, paraffin-embedded, sectioned, and stained with basal cytokeratin 5 or luminal cytokeratin 8 (top) or basal p63 and luminal cytokeratin 8 (bottom) and imaged with confocal microscope (scale bar = 100 &#181;m). (C) A whole-mounted human primary prostate organoid stained with basal cytokeratin 5 or luminal cytokeratin 8 and counterstained with phalloidin and DAPI, imaged with confocal microscope (scale bar = 100 &#181;m). (D) A whole-mounted human primary prostate cell organoid stained with fluorescently labeled EdU and counter stained with Hoescht, imaged with confocal microscope (scale bar = 500 &#181;m) <a href="https://www.jove.com/files/ftp_upload/59051/59051fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Handling and Assessment of Human Primary Prostate Organoid Culture at JoVE.com

Handling and Assessment of Human Primary Prostate Organoid Culture

Here, we present a protocol to guide human primary prostate organoid handling then suggest endpoints to assess phenotype. Seeding, culture maintenance, recovery from matrix gel, morphologic quantification, embedding and sectioning, FFPE sectioning, whole-mount staining, and application of commercial assays are described.

This paper describes a detailed protocol for three-dimensional (3D) culturing, handling, and evaluation of human primary prostate organoids. The process ...

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