Fig. 3. 2-D Time-lapse imaging of spheroid-derived cells in 2-D monolayer culture.
(A) Schematic illustrating an eGFP-positive NHKc spheroid plated into 2-D monolayer culture as part of the epidermal spheroid re-playing assay. (B-E) Monitoring eGFP-labelled spheroid-derived (SD) cells moving from a spheroid over the course of 12 days in monolayer 2-D culture. (F) SDEGFP cells positioned along the edge of monolayer sheets by day 15. (G) SD-NHKc located along the edge of monolayer sheets were immunostained against DAPI (blue) and antibodies against (G1) P63 (red), (G2) K14 (green), and (G3) overlaid image. (H) A single SDGFP cell forming a dividing colony after 10 days in 2-D adherent culture. (I) Immunostaining of a SD-NHKc colony with antibodies against K14 (green), P63 (red), and DAPI (blue). (J) Colony renewal visible by Giemsa staining 20 days after a single spheroid is plated back into 2-D monolayer culture. Black arrows depict the radial migratory patterns of SD cells as they travel from (a’) the adherent spheroid to (b’-c’) open spaces throughout the dish. (J1) Configuration mapping of progressively expanding epidermal sheets from the attached spheroid. Contour lines delineate each new sheet layer. Orange arrow indicates the location of the 2-D attached spheroid. (K) Bright-field microscopic image of an adherent spheroid 15 h after attachment in 2-D culture. (K1) Magnified image depicting epidermal sheets forming underneath the adherent spheroid. (L-L1) Automated image of eGFP-tagged SD cells (pointed by white arrows) after > 10 days in monolayer 2-D culture. The white circle demarks the original location of the 2D attached spheroid. (M) Mean cell size of SD-NHKc at passage 3 and their mass cultured counterparts at corresponding passages as quantified by a Nikon TMS phase microscope using Infinity 1 Analyze Software. (N) Low density growth assay assessing the cumulative population doublings of adherent SD-NHKc and their monolayer mass cultures over the course of 20 days. (O) Survival time of SD-NHKc cells and their corresponding mass cultures in monolayer cultures by weeks.