Preparing Organoids from Tumors: A 3D In Vitro Tumor Model
Published: April 30, 2023
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 |
Citar este artículo
Preparing Organoids from Tumors: A 3D In Vitro Tumor Model. J. Vis. Exp. (Pending Publication), e20065, doi: (2023).