Fig. 1: Generation of sMOCS from HGSOC ascites.
From: Single cell-derived spheroids capture the self-renewing subpopulations of metastatic ovarian cancer
a Scheme illustrating the main steps of the method for generating single cell ovarian cancer spheroids (sMOCS) from HGSOC ascites. Ascitic fluid is centrifuged; the cell pellet is processed for the removal of red blood cells and dissociated as single cell suspension for monolayer culture of tumor cells (step 1); the remaining supernatant after the first centrifugation is processed in order to remove the residual fraction of cells and is used as supplement for growing and culturing sMOCS (step 2). Tumor cells from monolayer culture are dissociated, resuspended in specific culture media for growing single cell from HGSOC ascites and plated by limiting dilution at the density of 1 cell per well in a low-adhesion 96 well V bottom plate (step 3). sMOCS at first passage (P1) are observed after 8–12 days in culture as tridimensional structure of about 200–220 μm of diameter and then propagated through dissociation of a single spheroid in single cells. The single cells are resuspended in the described media and plated as previously indicated in order to obtain the next passages in culture (P2 and P3). The scheme illustrates that fresh ascites, monolayer culture of tumor cells, and sMOCS at different passages are then processed for scRNAseq to obtain their transcriptomic profile. b Bright field image analysis (x4magnification for 2D, scale bar 1000 μm; x40 magnification for sMOCS P1, scale bar 400 μm; x20 magnification for sMOCS P2 and P3, scale bar 200 μm).
