Prestoblue viability reagent reduction assay

Encapsulated and non-encapsulated spheroids were cultured for two weeks in shake flasks before proceeding to drug exposure. Subsequently, spheroids were distributed in 12-well plates and the PrestoBlue™ Viability Reagent reduction assay (Cat. #A13262, Life Technologies) was performed according to the manufacturer’s instruction. Subsequently, spheroids were exposed to the chemotherapeutic drugs SN-38 (the active metabolite of irinotecan) and docetaxel (Carbosynth, Comptom, UK), in a nano-range of concentration (0–100 nM). Both drugs were diluted in DMSO, which served as vehicle control. Cells were exposed to drugs for seven days, adding fresh medium with drugs after three to four days. Then, the resazurin reduction ability was measured again and each well was normalized to the initial measurement. Results are shown as the fold change in metabolic activity compared to the activity of cells exposed to the vehicle, which was set as 1. The encapsulated spheroids were: ES-2, -11: n = 2, ES-6, -12, -16: n = 3; non-encapsulated spheroids: ES-6, -12: n = 3 ES-16: n = 2. The analysis was complemented by FDA/TO-PRO^® 3 staining for visualization of live/dead cells by microscopy, as described above.

Viable cells were quantified by trypan blue exclusion, as described elsewhere (Abecasis et al., 2017 (link)). For viability assessment, the enzyme substrate fluorescein diacetate (FDA, Sigma–Aldrich) and the DNA dye propidium iodide (PI, Sigma–Aldrich) were used (Serra et al., 2011 (link); Silva et al., 2015 (link)). In this method, direct staining of the live aggregates was performed followed by observation at the fluorescence microscope (DMI6000, Leica, Wetzlar, Germany), as described elsewhere (Serra et al., 2011 (link)). Cells that accumulated the metabolized product of FDA were considered live, and cells stained with PI were considered dead.
For evaluation of metabolic activity, the reduction capacity of the cultures was measured by PrestoBlue^® Viability Reagent reduction assay (Life Technologies), according to the manufacturer’s instruction. PrestoBlue^® reagent is reduced by viable cells and becomes highly fluorescent. This color change can be detected using fluorescence measurements. Samples of 2 to 5 capsules were taken from suspension agitated cultures into a 96-well plate. The microencapsulated cells were incubated with PrestoBlue^® reagent for 3 h at 37°C. After this period, the supernatant was collected, and the fluorescence was read at 560-nm excitation and 590-nm emission in the microplate reader Infinite^®200 PRO (NanoQuant, Tecan Trading AG).

Metabolic activity was assessed through resazurin reduction by using the PrestoBlue™ Viability Reagent reduction assay (Cat. #A13262, Life Technologies), following the manufacturer’s instruction. The samples were incubated with the reagent for one hour at 37 °C and the fluorescence was read at 560 nm excitation and 590 nm emission in an Infinite^® 200 PRO microplate reader (NanoQuant, Tecan Trading AG, Männedorf, Switzerland). Metabolic activity was measured three to four days post-encapsulation and repeated once a week, for up to one month in culture. Results are shown as the fold change in metabolic activity relative to the first day of measurement (ES-6, -12, -16: n = 3, ES-2, -11: n = 2). The non-parametric Kruskal–Wallis test was performed for statistical analysis.