Overview
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 biogel matrices can be used as well.
- Microtumor Production and Culture
- Obtain freshly dissociated PDX cells as single cell suspension in complete NBM, on ice.
- Mix the cell suspension with an equal volume of trypan blue solution (0.4% in PBS) and analyze using hemocytometer to determine cell number and viability by trypan blue exclusion. Remove volume necessary to generate 50,000 cells/microtumor where volume = (50,000 cells/tumor * # tumors)/(viable cell count / 1 mL) and place into a fresh conical tube.
- Concentrate cells by centrifugation at 150 x g, for 8 min, at room temperature. Discard the supernatant and resuspend the cell pellet with ice-cold HDHG solution in a final ratio of 1 part cells in FBS and 4 parts HDHG.
- Use an electronic multichannel pipette to dispense 10 µL per pin cell-HDHG mixture onto a 96-pin steel plate (with hydrophobic coating) to generate microtumors (2 mm beads each containing 50,000 cells).
- Place 3D tumor beads inside tissue culture incubator (37 °C, 5% CO₂, humidified) for 15 min to gelate the beads.
- After gelation, transfer the microtumors (10 µL) to a custom suspension culture chamber (50 mL) or large volume culture dish (10 cm) containing complete NBM using the electronic multi-channel pipette and custom pin-device.
- After 1–2 days in tissue culture incubator, transfer microtumors to 96-well culture plates containing 50 µL NBM/well using a wide-mouth dispensing pipette and perform various assay and analysis protocols.
- Drug Treatment and Maintenance of Microtumors
- Select final concentrations for drug testing.
NOTE: If the drug has known efficacy in 2D culture, select 2x the IC50 as the middle dose concentration and select 3-fold serial dilutions above and below for a total of 5 dose levels. For example, if the IC50 is 9 µM for the drug in 2D culture, select 2 µM, 6 µM, 18 µM, 54 µM, and 162 µM as final concentration for drug testing. - Prepare 2x drug dosing solution in complete NBM from dimethyl sulfoxide (DMSO) stock. Dilute dosing solution in 1% DMSO medium to prepare a 5 dose, 3-fold serial dilution.
- Add 50 µL of dosing solution to the microtumor well containing 50 µL in assay plates to achieve a final DMSO concentration of 0.5%. Repeat for each replicate (e.g., 4) at each drug dose determined in step 1.3.1.
- Maintain cultures in 37 °C, 5% CO₂, humidified tissue culture incubator for 1–14 days. Feed cultures twice weekly by refreshing media and drug solution as above.
- Select final concentrations for drug testing.
Subscription Required. Please recommend JoVE to your librarian.
Materials
Name | Company | Catalog Number | Comments |
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 |