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Animal procedures described here were performed with the approval of the Institutional Animal Care and Use Committee (IACUC) at the Department of Laboratory Animal Resources, Roswell Park Comprehensive Cancer Center, Buffalo, New York.
NOTE: Male mice to be dissected to isolate prostates or prostate tumors for generation of organoids should have at least reached the age of sexual maturity &#8212; about 8-10 weeks of age. Specific ages of mice can vary amongst studies. Some factors to consider when choosing age include age-dependent changes in prostate cell populations, age-dependent expression of specific promoter-driven Cre transgenes, and rate of prostate tumor progression in a particular GEMM.
NOTE: Figure 1 shows a pictorial description of the procedure for generation of tumor organoids.
1. Preparation
<ol>
	<li>Prepare mouse prostate organoid media according to Drost et al. with the following alterations. Use conditioned medium instead of recombinant proteins for Noggin and R-Spondin and use a final concentration of 1% (v/v), instead of 10%, for both Noggin- and R-Spondin-conditioned medium. 
	NOTE: HEK293 cells stably transfected with HA-mouse Noggin-Fc or HA-mouse Rspo1-Fc are used to produce Noggin- or R-Spondin- conditioned medium, respectively. These cell lines were a gift from the Calvin Kuo Laboratory at Stanford University.</li>
	<li>Prepare digestion solution in a 15 mL tube by diluting 20 mg/mL collagenase II with Advanced DMEM/F12(+++) media to a final concentration of 5 mg/mL. Add Y-27632 Rock Inhibitor to the collagenase II solution at a final concentration of 10 &#181;M. 
	NOTE: The ratio of digestion buffer to tissue is 1 mL to 50 mg, which we use according to Drost et al.</li>
</ol>
2. Mincing and Digestion of Tumor Tissue
<ol>
	<li>In the cell culture hood, place prostate tumor tissue in a sterile 10 cm culture dish, dissect and discard necrotic tissue.</li>
	<li>Mince the remaining prostate tumor tissue into 1 mm3 cubes by holding the tissue pieces with the sterile curved forceps and cutting with dissection scissors.</li>
	<li>Place the minced tumor pieces in the 15 mL tube with the digestion buffer by scooping them up with the curved side of the forceps. Digest the tumor tissue at 37 &#176;C with shaking for 1.5 to 2 h. Check digestion progress every 20 min. 
	NOTE: At this time, take out at least 2 mL of matrix from -20 &#176;C storage and thaw on ice. 1 mL aliquots of matrix will take approximately 3 h to thaw.</li>
	<li>After tissue digestion, centrifuge tube at 175 x g for 5 min at 4 &#176;C to form a cell pellet.</li>
	<li>Remove the supernatant, flick the tube to loosen the cell pellet, and resuspend the cell pellet in 1 mL of pre-warmed trypsin supplemented with 10 &#181;M Y-27632 Rock Inhibitor. Put the tube in a 37 &#176;C water bath for 5 min.</li>
	<li>After incubation, pipette up and down 5 times with a standard P1000 tip. Return the tube to 37 &#176;C water bath for another 5 min and repeat step 2.6. 
	NOTE: At this time in the procedure, warm a sterile 6-well cell culture dish by putting it into a 55 &#176;C incubator.</li>
</ol>
3. Counting Cells and Resuspension in Matrix
<ol>
	<li>Wash the cells by adding 9 mL of cold AdDMEM/F12(+++) and centrifuge the tube at 175 x g for 5 min at 4 &#176;C.</li>
	<li>After centrifugation, remove the supernatant, flick the tube to loosen the pellet, and wash the cells again by adding 10 mL of cold AdDMEM/F12(+++). Centrifuge the tube at 175 x g for 5 min at 4 &#176;C.</li>
	<li>After centrifugation, remove the supernatant, flick the tube to loosen the pellet, resuspend the cells in 1 mL of AdDMEM/F12(+++), and count the number of cells using a hemocytometer according to standard procedure.</li>
	<li>Seven-to-eight domes fit in one well of a 6-well dish when organoids are plated in matrix via a drop-wise fashion. Approximately 200 &#956;L of matrix will produce 7-8 domes. Decide how many wells of organoids are needed for future experimental purposes and calculate the volume of matrix required. Then, calculate the volume of cell-containing solution needed to have a final concentration of 1.0 x 106 cells/mL of matrix.</li>
	<li>After counting cells, centrifuge at 175 x g for 5 min at 4 &#176;C. Remove the supernatant, flick the tube to loosen the pellet, and resuspend the cells in volume of matrix calculated in step 3.4. 
	NOTE: Matrix remains in liquid form only at 4 &#176;C; keep matrix stock tubes and matrix-cell solutions on ice at all times.</li>
</ol>
4. Plating Matrix Domes and Application of Media
<ol>
	<li>Mix matrix-cell solution with a P200 pipet to evenly distribute the cells without introducing bubbles. Remove the 6-well culture dish from the 55 &#176;C incubator.</li>
	<li>Carefully pipette 200 &#956;L of matrix-cell solution and quickly drop the solution into a well to create domes.</li>
	<li>Repeat step 4.2. until volume of matrix-cell solution is spent. Allow the domes to solidify at room temperature for 2 min.</li>
	<li>Flip the 6-well dish upside-down and put the dish into a 37 &#176;C incubator to continue solidification for 20 min.</li>
	<li>After incubation, add 2 mL of mouse prostate organoid media to each well. Add synthetic androgen R1881 to each well for a final concentration of 1 nM and Y-27632 Rock Inhibitor to a final concentration of 10 &#956;M. Mix carefully and place the plate in a 37 &#176;C incubator for culturing. 
	NOTE: Organoid culture media needs to be supplemented with 10 &#181;M Y-27632 Rock Inhibitor for only 1 week after organoid generation.</li>
</ol>

<table><tbody><tr><td>0.25% Trypsin+2.21 mM EDTA</td><td>Sigma</td><td>25-053</td><td></td></tr><tr><td>A83-01</td><td>MedChemExpress</td><td>HY-10432</td><td></td></tr><tr><td>Advanced DMEM/F12+++</td><td>Gibco</td><td>12634</td><td></td></tr><tr><td>Analytical balance</td><td>Mettler Toledo</td><td>30216623</td><td></td></tr><tr><td>B27 (50x)</td><td>Gibco</td><td>17504044</td><td></td></tr><tr><td>Collagenase II</td><td>Gibco</td><td>17101015</td><td></td></tr><tr><td>EHS Sarcoma matrix, Pathclear Lot#19814A10</td><td>Manufactured by Trevigen</td><td>Requistitioned from the National Cancer Institute at the Frederick National Laboratory</td><td>Holder of grants from the National Cancer Institute can request matrix</td></tr><tr><td>HEPES (1 M)</td><td>Sigma</td><td>25-060</td><td></td></tr><tr><td>human recombinant Epidermal growth factor (EGF)</td><td>PeproTech</td><td>AF-100-15</td><td></td></tr><tr><td>L-glutamine (200 mM)</td><td>Sigma</td><td>25-005</td><td></td></tr><tr><td>N-Acetyl-L-Cysteine</td><td>Sigma</td><td>A9165</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma</td><td>P4333</td><td></td></tr><tr><td>Precision balance</td><td>Mettler Toledo</td><td>30216561</td><td></td></tr><tr><td>Single-edge carbon razor blade</td><td>Fisherbrand</td><td>12-640</td><td></td></tr><tr><td>Y-276632 (Rock Inhibitor)</td><td>APExBIO</td><td>A3008</td><td></td></tr></tbody></table>

preparing organoids from tumors: a 3d in vitro tumor model

Source: Wadosky, K. M., et al. <a href="https://www.jove.com/video/59710/" target="_blank">Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer</a>. J. Vis. Exp. (2019).
Organoids derived from tumors are a type of 3D cell culture model that can maintain the pathological features and organ-specific cell lineage of tumors. In the example protocol, we will see scientists generate organoids from a prostate tumor from a genetically engineered mouse model.

<img alt="Figure 1" src="/files/ftp_upload/20065/20065fig1.jpg" /> 
Figure 1: Flow chart of the protocol for generating prostate tumor organoids. After dissecting the prostate tumor, mince the tissue into 1 mm pieces. Digest the tumor pieces in collagenase, collect the cells, and digest in trypsin to obtain a single cell suspension. After counting cells, resuspend in volume of matrix required for a 1.0 x 106 cell/mL cell concentration. Plate domes in dish ...

Preparing Organoids from Tumors: A 3D In Vitro Tumor Model

Source: Wadosky, K. M., et al. Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer. J. Vis. Exp. (2019).
Organoids ...

Mince the tumor tissue into small pieces. Transfer the pieces into a tube with digestion buffer. Collagen fibers surrounding the cells get broken down, degrading the tissue&#39;s matrix. Then, pellet the digested pieces and discard most of the digestion buffer. Flick the tube to break up the pellet.
The solution now consists of various-sized cell clumps. Resuspend the cell clumps in a solution of trypsin and a ROCK inhibitor. The trypsin continues breaking down the tumor and the inhibitor prevents cell death. At regular intervals, pipette the solution to break up the clumps and disperse the cells. Then, resuspended the cells in matrix solution at the appropriate concentration.
Place drops of the solution in a culture plate to form matrix domes. Place the plates upside down in an incubator and allow the matrix to completely solidify. Then, add cell culture media and incubate, right side up. The matrix provides a scaffold to support three-dimensional growth of the organoid.

preparing-organoids-from-tumors-a-3d-in-vitro-tumor-model

Research

JoVE Encyclopedia of Experiments

Cancer Research

Prostate Cancer

Assays and techniques

1.3K ビュー. 出典:Wadosky, K. M., et al. 前立腺癌の遺伝子操作マウスモデルからの腫瘍オルガノイドの生成。J. Vis. Exp.(2019年)。 腫瘍由来のオルガノイドは、腫瘍の病理学的特徴と臓器特異的な細胞系統を維持できる3D細胞培養モデルの一種です。このプロトコルの例では、科学者が遺伝子操作マウスモデルから前立腺腫瘍からオルガノイドを生成する様子を見ていきます。 

ビデオ: 腫瘍からのオルガノイドの調製:3D In Vitro腫瘍モデル - 概念

Prostate Organoid Cultures as Tools to Translate Genotypes and Mutational Profiles to Pharmacological Responses

Presented here is a protocol to study pharmacodynamics, stem cell potential, and cancer differentiation in prostate epithelial organoids. Prostate organoids are androgen responsive, three-dimensional (3D) cultures grown in a defined medium that resembles the prostatic epithelium. Prostate organoids can be established from wild-type and genetically engineered mouse models, benign human tissue, and advanced prostate cancer. Importantly, patient derived organoids closely resemble tumors in genetics and in vivo tumor biology. Moreover, organoids can be genetically manipulated using CRISPR/Cas9 and shRNA systems. These controlled genetics make the organoid culture attractive as a platform for rapidly testing the effects of genotypes and mutational profiles on pharmacological responses. However, experimental protocols must be specifically adapted to the 3D nature of organoid cultures to obtain reproducible results. Described here are detailed protocols for performing seeding assays to determine organoid formation capacity. Subsequently, this report shows how to perform drug treatments and analyze pharmacological response via viability measurements, protein isolation, and RNA isolation. Finally, the protocol describes how to prepare organoids for xenografting and subsequent in vivo growth assays using subcutaneous grafting. These protocols yield highly reproducible data and are widely applicable to 3D culture systems.

Presented here is a protocol to study pharmacological responses in prostate epithelial organoids. Organoids closely resemble in vivo biology and recapitulate patient genetics, making them attractive model systems. Prostate organoids can be established from wildtype prostates, genetically engineered mouse models, benign human tissue, and advanced prostate cancer.

遺伝子型と突然変異プロファイルを薬理学的応答に変換するツールとしての前立腺オルガノイド培養

Presented here is a protocol to study pharmacodynamics, stem cell potential, and cancer differentiation in prostate epithelial organoids. Prostate ...

JoVE Journal

がん研究

Isolation and Characterization of Patient-derived Pancreatic Ductal Adenocarcinoma Organoid Models

Pancreatic ductal adenocarcinoma (PDAC) is amongst the most lethal malignancies. Recently, next-generation organoid culture methods enabling the 3-dimensional (3D) modeling of this disease have been described. Patient-derived organoid (PDO) models can be isolated from both surgical specimens as well as small biopsies and form rapidly in culture. Importantly, organoid models preserve the pathogenic genetic alterations detected in the patient&#39;s tumor and are predictive of the patient&#39;s treatment response, thus enabling translational studies. Here, we provide comprehensive protocols for adapting tissue culture workflow to study 3D, matrix embedded, organoid models. We detail methods and considerations for isolating and propagating primary PDAC organoids. Furthermore, we describe how bespoke organoid media is prepared and quality controlled in the laboratory. Finally, we describe assays for downstream characterization of the organoid models such as isolation of nucleic acids (DNA and RNA), and drug testing. Importantly we provide critical considerations for implementing organoid methodology in a research laboratory.

Patient-derived organoid cultures of pancreatic ductal adenocarcinoma are a rapidly established 3-dimensional model that represent epithelial tumor cell compartments with high fidelity, enabling translational research into this lethal malignancy. Here, we provide detailed methods to establish and propagate organoids as well as to perform relevant biological assays using these models.

患者由来膵管腺癌オルガノイドモデルの単離と特徴付け

Pancreatic ductal adenocarcinoma (PDAC) is amongst the most lethal malignancies. Recently, next-generation organoid culture methods enabling the ...

Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells

The sense of taste is mediated by taste buds on the tongue, which are composed of rapidly renewing taste receptor cells (TRCs). This continual turnover is powered by local progenitor cells and renders taste function prone to disruption by a multitude of medical treatments, which in turn severely impacts the quality of life. Thus, studying this process in the context of drug treatment is vital to understanding if and how taste progenitor function and TRC production are affected. Given the ethical concerns and limited availability of human taste tissue, mouse models, which have a taste system similar to humans, are commonly used. Compared to in vivo methods, which are time-consuming, expensive, and not amenable to high throughput studies, murine lingual organoids can enable experiments to be run rapidly with many replicates and fewer mice. Here, previously published protocols have been adapted and a standardized method for generating taste organoids from taste progenitor cells isolated from the circumvallate papilla (CVP) of adult mice is presented. Taste progenitor cells in the CVP express LGR5 and can be isolated via EGFP fluorescence-activated cell sorting (FACS) from mice carrying an Lgr5EGFP-IRES-CreERT2 allele. Sorted cells are plated onto a matrix gel-based 3D culture system and cultured for 12 days. Organoids expand for the first 6 days of the culture period via proliferation and then enter a differentiation phase, during which they generate all three taste cell types along with non-taste epithelial cells. Organoids can be harvested upon maturation at day 12 or at any time during the growth process for RNA expression and immunohistochemical analysis. Standardizing culture methods for production of lingual organoids from adult stem cells will improve reproducibility and advance lingual organoids as a powerful drug screening tool in the fight to help patients experiencing taste dysfunction.

The protocol presents a method for culturing and processing lingual organoids derived from taste stem cells isolated from the posterior taste papilla of adult mice.

成人マウス味幹細胞由来の舌下オルガノイドの生成と培養

The sense of taste is mediated by taste buds on the tongue, which are composed of rapidly renewing taste receptor cells (TRCs). This continual turnover is ...

Preparing Organoids from Tumors: A 3D In Vitro Tumor Model

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Preparing Organoids from Tumors: A 3D In Vitro Tumor Model