Scalable Culture Strategies for the Expansion of Patient-Derived Cancer Stem Cell Lines

Figure 1

Effect of bioreactor configuration and culture medium composition on the expansion of NSCLC cells. Cells were inoculated at 0.25 × 10⁶ cells/mL and cultured in a round-bottom bioreactor vessel equipped with a pitched 4-bladde impeller (BR-R/P4b) or in a flat-bottom bioreactor vessel equipped with a trapezoid-shaped paddle impeller (BR-F/T) using serum-containing medium (SCM) and serum-free medium (SFM). (a) Fluorescence microscopy images of NSCLC cultures at days 1, 4, and 8 of the three bioreactor experiments. Viability analysis of cultures stained with fluorescein diacetate (FDA—live cells, green) and propidium iodide (PI—dead cells, red). Scale bars: 100 μm. (b) Growth curve expressed in terms of cell number per volume of medium (determined by crystal violet nuclei stain assay; error bars denote SD of 3 measurements). (c) Aggregate size (average diameters of aggregates were determined by ImageJ software; error bars denote SD of measurements from 100 aggregates). (d) Flow cytometry analysis of NSCLC culture in bioreactors and in monolayer static systems: percentage of ALDH⁺ cells at day 8 of culture. The left panel shows the dot blot of ALDEFLUOR™ assay with an inhibitor (DEAB), and the right panel shows the dot blot without an inhibitor. The ALDH⁺ cell population is identified in green. (e) Fold increase in ALDH⁺ cells obtained at day 8 of culture in bioreactors and monolayer static culture systems in relation to the inoculum population.

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