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

Protocol

1. Microtumor Generation

1. Preparation of Reagents
   1. 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.
2. Microtumor Production and Culture
   1. Obtain freshly dissociated PDX cells as single cell suspension in complete NBM, on ice.
   2. 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.
   3. 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.
   4. 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).
   5. Place 3D tumor beads inside tissue culture incubator (37 °C, 5% CO₂, humidified) for 15 min to gelate the beads.
   6. 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.
   7. 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.
3. Drug Treatment and Maintenance of Microtumors
   1. 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.
   2. 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.
   3. 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.
   4. 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.

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