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Organotypic culture assays for murine and human primary and metastatic-site tumors.

Nature protocols (2020-07-22)
Veena Padmanaban, Eloise M Grasset, Neil M Neumann, Andrew K Fraser, Elodie Henriet, William Matsui, Phuoc T Tran, Kevin J Cheung, Dan Georgess, Andrew J Ewald
ABSTRACT

Cancer invasion and metastasis are challenging to study in vivo since they occur deep inside the body over extended time periods. Organotypic 3D culture of fresh tumor tissue enables convenient real-time imaging, genetic and microenvironmental manipulation and molecular analysis. Here, we provide detailed protocols to isolate and culture heterogenous organoids from murine and human primary and metastatic site tumors. The time required to isolate organoids can vary based on the tissue and organ type but typically takes <7 h. We describe a suite of assays that model specific aspects of metastasis, including proliferation, survival, invasion, dissemination and colony formation. We also specify comprehensive protocols for downstream applications of organotypic cultures that will allow users to (i) test the role of specific genes in regulating various cellular processes, (ii) distinguish the contributions of several microenvironmental factors and (iii) test the effects of novel therapeutics.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Triton X-100, laboratory grade
Sigma-Aldrich
Penicillin-Streptomycin, Solution stabilized, with 10,000 units penicillin and 10 mg streptomycin/mL, 0.1 μm filtered, BioReagent, suitable for cell culture
Sigma-Aldrich
Insulin solution human, sterile-filtered, BioXtra, suitable for cell culture
Sigma-Aldrich
Glycerol, ≥99.5%
Roche
cOmplete, Mini Protease Inhibitor Cocktail, Tablets provided in a glass vial
Sigma-Aldrich
Dulbecco′s Phosphate Buffered Saline, With MgCl2 and CaCl2, liquid, sterile-filtered, suitable for cell culture
Sigma-Aldrich
Dulbecco′s Modified Eagle′s Medium - high glucose, With 4500 mg/L glucose, sodium pyruvate, and sodium bicarbonate, without L-glutamine, liquid, sterile-filtered, suitable for cell culture
Sigma-Aldrich
Bovine Serum Albumin solution, 30% in DPBS, sterile-filtered, BioXtra, suitable for cell culture
Sigma-Aldrich
Sodium hydroxide solution, 1.0 N, BioReagent, suitable for cell culture
Sigma-Aldrich
Cholera Toxin from Vibrio cholerae, ≥90% (SDS-PAGE), lyophilized powder
Sigma-Aldrich
hEGF, EGF, recombinant, expressed in E. coli, lyophilized powder, suitable for cell culture
Sigma-Aldrich
Ethylenediaminetetraacetic acid, anhydrous, BioUltra, ≥99% (titration)
Sigma-Aldrich
hBFGF, FGF-Basic, recombinant, expressed in E. coli, suitable for cell culture
Sigma-Aldrich
Collagenase from Clostridium histolyticum, release of rat epididymal adipocytes and hepatocytes tested (for methodology see Type II and Type IV), Type VIII, 0.5-5.0 FALGPA units/mg solid, ≥125 CDU/mg solid
Sigma-Aldrich
Deoxyribonuclease I from bovine pancreas, Standardized vial containing 2,000 Kunitz units of DNase I (D4527), vial of ≥0.25 mg total protein
Sigma-Aldrich
Hydrocortisone-Water Soluble, BioReagent, suitable for cell culture
Sigma-Aldrich
Williams′ Medium E, With sodium bicarbonate, without L-glutamine, liquid, sterile-filtered, suitable for cell culture
Sigma-Aldrich
Dulbecco′s Modified Eagle′s Medium - low glucose, 10 ×, With 1000 mg/L glucose (1x), without L-glutamine, sodium bicarbonate, and folic acid, liquid, sterile-filtered, suitable for cell culture