Despite the clinical need to avoid the use of xenobiotic agents and cells the use of an irradiated mouse fibroblast feeder layer in a mitogen rich media 
with 10% bovine serum as first used clinically in the early 1980’s remains the most commonly used methodology for rapid expansion of adult keratinocytes for clinical use to this day. The reason for this is simple. It is a robust and reliable methodology for culturing adult human skin cells often from small initial biopsies and so far it outperforms efforts to obtain a completely defined culture approach for human keratinocyte expansion. However with growing Pimozide concerns about the transmission of bovine spongiform encephalitis from the use of bovine serum it would be desirable to culture cells under completely defined culture conditions. Our previous research demonstrated that NHK could be expanded by co-culturing these cells with human dermal fibroblasts in Green’s media without foetal calf serum. We have also shown that human fibroblasts can perform as well as murine fibroblasts in supporting the expansion of keratinocytes and indeed keratinocytes expanded on fibroblasts in the absence of serum tended to show less differentiation than those expanded with serum which is another desirable property when expanding cells for clinical use. In the original Rheinwald and Green methodology the murine fibroblasts were lethally irradiated so that they could not expand in culture. However, empirical data from our laboratory shows that one can get expansion of keratinocytes on non-irradiated fibroblasts to satisfactory levels if one pays attention to the ratio between the fibroblasts and keratinocytes. Agent-based Benzethonium Chloride modeling is a computational approach that simulates the interactions of autonomous entities with each other and their local environment to predict higher level emergent patterns. Outputs of these models can be visual and easily accessible to biologists and models can be built more quickly and at lower cost than laboratory experiments, freeing resources for a more informed exploration of the hypothesis space. Our aim in this study was to take our recently established agent based model of keratinocyte colony expansion and extend it to look at the interactions between keratinocytes and fibroblasts to test hypotheses of how fibroblasts interact with keratinocytes to promote keratinocyte colony formation. Our approach was to use the extensive literature on keratinocyte/fibroblast interactions combined with in vitro experimentation to generate an initial rule set for defining fibroblast behaviour. This was then incorporated into the previous model for keratinocytes in monoculture and the model was adapted as necessary to simulate the macroscopic morphogenesis of NHKs and fibroblasts in vitro. The model was validated by comparison of the in virtuo model with in vitro multi-cellular behaviour of NHKs and HDFs both in single and co-culture conditions in Green’s medium in the presence and absence of serum. The robustness of the model to simulate the multicellular morphology of NHKs and various types of HDFs in co-culture was also tested by varying various properties of HDF. The model was then used to propose a range of hypotheses to explain the in vitro behaviour of these two cell types. Analysis of the model demonstrated that the proliferation and differentiation of NHK would be influenced by the initial ratio of HDF to NHK, the proliferation rate of the HDF and the timing of when HDF were introduced to NHKs. From these hypotheses we then focused on those which could be examined with an in virtuo/in vitro comparison. Thus, specifically, we looked at to what extent the ratio of fibroblasts to keratinocytes would promote colony forma.