Recovering 3D Structures from Matrigel

3D structures were recovered from Matrigel using Recovery Solution (BD, USA) according to the manufacturer's instructions as described previously [17 (link), 58 (link)]. Briefly, 3D cultures were first washed with ice-cold PBS, and then the Matrigel containing the 3D structures was removed from the well, transferred to 15 mL tube containing the pre-chilled Recovery Solution (1 mL per well), incubated on ice for 45min with intermittent mixing and then centrifuged at 1000 rpm for 10 min at 4°C. The supernatant containing the dissolved Matrigel was discarded and the 3D structures were washed once with PBS. To make single-cell suspension of recovered 3D structures, cells were trypsinized using trypsin-EDTA (0.25%, Invitrogen).