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).