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

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

PMID32690957

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

I9278

Sigma-Aldrich

Insulin solution human, sterile-filtered, BioXtra, suitable for cell culture

C2139

Sigma-Aldrich

Collagenase from Clostridium histolyticum, suitable for release of rat epididymal adipocytes and hepatocytes (for methodology see Type II and Type IV), Type VIII, 0.5-5.0 FALGPA units/mg solid, ≥125 CDU/mg solid

S2770

Sigma-Aldrich

Sodium hydroxide solution, 1.0 N, BioReagent, suitable for cell culture

D8662

Sigma-Aldrich

Dulbecco′s Phosphate Buffered Saline, With MgCl₂ and CaCl₂, liquid, sterile-filtered, suitable for cell culture

F0291

Sigma-Aldrich

hBFGF, FGF-Basic, recombinant, expressed in E. coli, suitable for cell culture

H0396

Sigma-Aldrich

Hydrocortisone-Water Soluble, BioReagent, suitable for cell culture

C8052

Sigma-Aldrich

Cholera Toxin from Vibrio cholerae, ≥90% (SDS-PAGE), lyophilized powder

A9576

Sigma-Aldrich

Bovine Serum Albumin solution, 30% in DPBS, sterile-filtered, BioXtra, suitable for cell culture

P4333

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

D6546

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

E9644

Sigma-Aldrich

hEGF, EGF, recombinant, expressed in E. coli, lyophilized powder, suitable for cell culture

D2429

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

20-188

Millipore

RIPA Lysis Buffer, 10X, 100 mL RIPA Lysis Buffer, 10X for Immunoprecipitation & Western Blotting.

11836153001

Roche

cOmplete^™, Mini Protease Inhibitor Cocktail, Tablets provided in a glass vial

W4128

Sigma-Aldrich

Williams′ Medium E, With sodium bicarbonate, without L-glutamine, liquid, sterile-filtered, suitable for cell culture

F0926

Sigma-Aldrich

Fetal Bovine Serum, USDA approved, sterile-filtered, suitable for cell culture