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

Published: April 30, 2023

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…

Divulgaciones

The authors have nothing to disclose.

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

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Generation of Microtumors: An In Vitro Culture Technique to Culture 3D Microtumors to Analyze Response to Drug Treatment. J. Vis. Exp. (Pending Publication), e20691, doi: (2023).

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