Madin Darby Canine Kidney (MDCK) cells can form a polarized epithelial layer in culture. We and others have observed that these layers are frequently characterized by extralayer cells, positioned above (apical to) the layer. One possible origin for extralayer cells is an initial failure in monolayer formation, meaning that cells “clumped” on top of each other during plating. Another possibility is that newly born division products fail to integrate into the extant layer.
To investigate the aetiology of misplaced cells, we have developed an analysis pipeline, including a suite of custom algorithms called Automated Layer Analysis (ALAn), to quantify aspects of MDCK cell layer architecture at different areas, ages, and cell densities. Using ALAn, we have determined that the number of extralayer cells correlates positively with layer density in a non-linear fashion. In addition, the layer becomes more organized over time, with cells settling into a layer by 24 hours, suggesting that these extra-layer cells are not a result of clumping during plating. We are now using this tool to investigate whether extralayer cells are a result of division, and whether or not misplaced cells can reintegrate into the developing monolayer to maintain tissue integrity.