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

Figure 2

Results from harvesting studies of NSCLC cell aggregates cultured in stirred tank bioreactors. Cells were cultured in BR-F/T using serum-free medium (SFM) and harvested at days 4 and 8 of culture using different cell dissociation protocols, namely, Standard protocol, Accutase+Collagenase III+DNase I (A+C+D), Trypsin 0.05%, and TrypLE Select. (a) Percentage of viable cells recovered after each dissociation protocol. Values were estimated by the ratio, where is the number of total cells recovered after dissociation protocol (determined by trypan blue exclusion assay) and is the number of total cells harvested from the bioreactor (determined by crystal violet nucleic stain assay) (error bars denote SD of 2 measurements). (b) Flow cytometry analysis of NSCLC culture after cell aggregate dissociation by A+C+D, trypsin 0.05, and TrypLE Select protocols: percentage of ALDH⁺ cells recovered at days 4 and 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. (c) Readhesion and expansion capacity of NSCLC cells harvested at days 4 and 8 using different dissociation protocols. Cells were dissociated and plated in collagen I-coated flasks and cultured for 4 days in static culture conditions using serum-containing medium. Left panel: fold increase in total cell concentration (estimated by crystal violet nucleic stain assay; error bars denote SD of 2 measurements). Right panel: percentage of ALDH⁺ cells determined after 4 days of culture (error bars denote SD of 2 measurements). (d) Number of tumor spheres per well generated by NSCLC cells derived from the inoculum and harvested from bioreactor culture at days 4 (left panel) and 8 (right panel) using different dissociation protocols. The number of tumor spheres was estimated up to five passages. Error bars denote SD of measurements from 4 wells. Statistical significance is indicated as follows: , , and .

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