Generation of Microtumors: An In Vitro Culture Technique to Culture 3D Microtumors to Analyze Response to Drug Treatment

Abstract

Source: Gilbert, A. N. et al. Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing. J. Vis. Exp. (2016)

In this video, we generate microtumors from patient-derived tumor cells using 3D human biogel. These microtumors closely resemble the in vivo tumor microenvironment and serve as potential platforms for drug testing.

Protocol

1. Microtumor Generation Preparation of Reagents Prepare complete NBM by adding the following to one 500 mL bottle of Neurobasal Media (NBM) to prepare complete NBM: 10 mL B-27 supplement without vitamin A, 5 mL N2 supplement, 100 µL EGF, 100 µL Fibroblast Growth Factor (FGF)-Basic, 5 mL amphotericin B, 0.5 mL gentamycin, 5 mL L-glutamine and prepare neutralized High Density human biogel (HuBiogel) (HDHG at 3 mg/mL) per internal protocol. Similar…

Materials

Collagenase-I	Sigma-Aldrich	CO130
Neurobasal-A	Life Technologies	10888-022
N-2 Supplement	Life Technologies	17502-048	1x final concentration
B-27 Supplement w/o Vitamin A	Life Technologies	12587-010	1x final concentration
Recombinant Human FGF-basic	Life Technologies	PHG0266	10 ng/mL final concentration
L-Glutamine	Corning Cellgro Mediatech	25-005-CI	2 mM final concentration
Gentamicin	Life Technologies	15750-060	50 ng/mL final concentration
Trypan Blue	Pierce ThermoScientific	15250-061
High Density Hubiogel	Vivo Biosciences	HDHG-5
DMSO	Fisher	BP231	For dissolution of calcein AM & compounds
Dulbecco's Phosphate-Buffered Saline with Ca/Mg	Corning Cellgro Mediatech	20-030-CV	Diluted to 1x with MiliQ ultrapure water (for pre-fixation wash)
10% Neutral Buffered Formalin	Protocol	032-060
100 mm glass Petri dishes	Fisher	08-748D