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…
Declarações
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)
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).