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.