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

Abstract

Source: Wadosky, K. M., et al. Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer. 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.

Protocol

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 — about 8-10 weeks of age. Specific ages of mice can vary amongst stud…

Representative Results

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 …

Materials

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