Optimization of 3D culture cell proliferation - part 4

Three major variables impact the 3D culture proliferation assay: limitations of detection, cell seeding density, and duration of 3D culture. While most established 3D culture protocols should be compatible with this assay, it may be helpful to empirically test and optimize these conditions before commencing pharmacological screening to promote assay sensitivity and reproducibility. This starts by developing a standard curve to determine the limitations for detection for each cell line. Cells should be serially diluted and seeded on ECM-coated stripwells before evaluating each well using the 3D culture cell proliferation assay. Absorbance should be evaluated at 1-, 2-, 3-, and 4-hr time points after adding substrate to determine the optimal incubation period. Background values are subtracted, data averaged, and standard curves plotted. The optimal curve is linear and has the lowest standard deviations. The standard curve will indicate what the highest number of detectable cells are based on the largest value within the linear range; it will also specify the lowest number of detectable cells in each well. These values provide the dynamic range for the assay. Since the ECM may affect cell proliferation rates, a growth curve may also be helpful in determining the optimal duration for 3D culture. Here, seeding densities may be used from an existing model or determined from the standard curve. When making a determination from the standard curve, sensitivity of this assay depends on the number of population doublings and overall absorbance, as a consequence of cell number. In the figure below, the seeding concentration for MDA-MB-231 cells in 3D BME culture is determined to allow for a 20-fold increase in cell number, which is just over five population doublings. The maximum value for the linear standard curve, 100,000 cells, is divided by 20 to give a seeding concentration of 5000 cells per well. Here, the cell doubling rate starts around two days and appears to increase with time, reaching saturation around day six. Since the MDA-MB-231 cells have a doubling rate of approximately 24 hr in traditional 2D cell culture, this represents an initial decrease in proliferation in the 3D BME culture model.

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