IADR Abstract Archives
Morphological Characterization of Tumor Spheroids for Cell Viability Assays
Objectives: The objective of this assay was to characterize the size and morphology of head and neck carcinoma spheroids for cell viability assays.
Methods: To make low-adhesion plates, 60 μL of a 5 mg/mL solution of poly-HEMA in 95% ethanol was added to each well of untreated 96-well plates. They were dried for 24 hr, sterilized 40 min under ultraviolet light, and stored sealed at 4 ° C. The characterization was performed for the Cal-27 and HEp-2 line. For each line between 500 and 8,000 cells per well were seeded in a 96-well plate. The plate was centrifuged at 2730 rpm for 5 min and incubated for 24 hr at 37 °C. The spheroids were photographed and the average diameter was calculated according to the number of cells seeded. The test was repeated at 48 h.
Results: Cal-27 forms homogeneous and regular spheroids. HEp-2 forms irregular spheroids. For both lines the average diameter increased according to the initial number of cells seeded. The difference between size at 24 and 48 h was not statistically significant for the same number of cells. HEp-2 spheroids are larger in diameter than CAL-27. For Cal-27 the percentage of diameter variation for the same number of cells is 5% at 24 h and 10% at 48 h, for HEp-2 it was from 1 to 8% for both times.
Conclusions: Both lines formed multicellular spheroids, but the morphology and size differ. The ideal diameter of the spheroid for cytotoxicity assays is reported between 300 to 500 μm, which is achieved by seeding 4500 cells for the Cal-27 line and 500 for the HEp-2 line. It is recommended to start the tests on spheroids later of 24 h of formation due to the smallest size variation.
Methods: To make low-adhesion plates, 60 μL of a 5 mg/mL solution of poly-HEMA in 95% ethanol was added to each well of untreated 96-well plates. They were dried for 24 hr, sterilized 40 min under ultraviolet light, and stored sealed at 4 ° C. The characterization was performed for the Cal-27 and HEp-2 line. For each line between 500 and 8,000 cells per well were seeded in a 96-well plate. The plate was centrifuged at 2730 rpm for 5 min and incubated for 24 hr at 37 °C. The spheroids were photographed and the average diameter was calculated according to the number of cells seeded. The test was repeated at 48 h.
Results: Cal-27 forms homogeneous and regular spheroids. HEp-2 forms irregular spheroids. For both lines the average diameter increased according to the initial number of cells seeded. The difference between size at 24 and 48 h was not statistically significant for the same number of cells. HEp-2 spheroids are larger in diameter than CAL-27. For Cal-27 the percentage of diameter variation for the same number of cells is 5% at 24 h and 10% at 48 h, for HEp-2 it was from 1 to 8% for both times.
Conclusions: Both lines formed multicellular spheroids, but the morphology and size differ. The ideal diameter of the spheroid for cytotoxicity assays is reported between 300 to 500 μm, which is achieved by seeding 4500 cells for the Cal-27 line and 500 for the HEp-2 line. It is recommended to start the tests on spheroids later of 24 h of formation due to the smallest size variation.