3.5 Enzymatic digestion of agarose for spheroids recovery
We intended to evaluate the possibility to recover the spheroids from the hydrogel matrix. for further processing and/or other biological studies To assess the degradability of the A-C hydrogels, the blends with 0.25% and 0.125% agarose containing MCF-7 spheroids at different days of growth were incubated with β-Agarase from Pseudomonas atlantica (Malmqvist, 1978)(Fu & Kim, 2010). After O.N. incubation at 37 °C with 40 U Agarase, in the case of 0.25% blended hydrogels only the spheroids located in the outer layer of the hydrogel were recovered (upper panels of Figure 10S). Most of the hydrogel remained intact and the spheroids continued to growth within as in control hydrogels not treated with Agarase. On the other hand, in the case of the softest matrices, containing 0.125% agarose, all the tumoroids were recovered after Agarase treatment (lower panels of Figure 10S). Indeed, after O.N. treatment, the hydrogel was completely dissolved. Noteworthy, as shown in the stability test (Figure 2), the hydrogels with 0.125% agarose showed a spontaneous higher degradation than those with 0.25%. Thus, the addition of agarose boosted the degradation process leading to the complete dissolution of the softer matrix. The images of Figure 10S show the morphology of the spheroids collected after Agarase treatment in both types of hydrogels at different times of growth. Furthermore, the recovered tumoroids were fixed and their nuclei stained with DAPI to show their suitability for further processing and studies. As reported in Figure 9, some free individual cells, detached from the surface of the spheroids, can be observed, likely due to the centrifugation steps performed for the staining protocol. Nevertheless, the spheroids preserve their shape and their morphology.