Fig. 2. Spatiotemporal cell tracking of NHKc spheroids in 3-D suspension culture.

(A) Schematic illustrating 2-D monolayer NHKc cultures transfected with an eGFP reporter plasmid and employed for epidermal spheroid formation assay. (B) Manual image of eGFP-expressing SF-NHKc after 6 h in 3-D suspension culture. (B1) Automated image of eGFP-tagged NHKc suspensions (white dots) at 6 h. (C) Bright phase image of eGFP-expressing keratinocytes within a multicellular spheroid after 24 h in suspension culture. (D) eGFP-labeled cells are seen out of a spheroid after 36 h in suspension culture. (E) Fluorescent tracking of eGFP-labelled cells after 15 days in suspension culture. The proportion of eGFP-expressing cells in each spheroid compartment (i.e. center, edge, or outside) was quantified at distinct time points by ImageJ analysis. (F) Compact ring of cells generated from a single spheroid after 15 days in suspension. White contour lines delineate the outer edge of the spheroid (o); orange contour lines delineate the spheroid’s center (c) from the spheroid’s inner edge (e). (F1) Dense ring of cells immunostained with DAPI, antibodies against pan-tumor protein 63 targeting all TP63 isoforms (P63, red), and basal cytokeratin 14 (K14, green). Scale bar = 50 μM. (G) Expression levels of mRNAs encoding involucrin in cells within the dense floating ring compared to cells located outside the ring, relative to corresponding monolayer mass cultures as determined by Real-time RT-PCR. Data were normalized to GAPDH expression and reported as mean +/- standard deviation.