 Welcome, I'm Dr. Ryan McCoy from the Bend Tissue Engineering Research Group based at the Royal College of Surgeons and Islands which is headed by Professor Fogel O'Brien. We would like to spend the next few minutes describing a methodology termed with those of operation that we feel will be of benefit to the tissue engineering community. Tissue engineering approaches to developing functional substitutes are highly complex, multivariate systems where many aspects of the biomaterials, cell sources or the bioregulatory factors may be controlled in an effort to enhance tissue formation. The choices made with respect to these input variables will ultimately determine the type of tissue produced, with the success being judged based on multiple performance criteria reflecting both the quantity and quality of the fabricated tissue. Managing the trade-offs between these different performance criteria as a function of the culture conditions is a major challenge for researchers. One methodology capable of helping researchers to manage the trade-offs in these performance criteria and identify optimal operating ranges is termed Windows of Operation. In this paper, we aim to introduce the tissue engineering community to the Windows of Operation methodology through its conceptual application to a perfusion bioreactor case study. We use perfusion bioreactor systems to enhance the in vitro cultivation of barygram substitutes. Two key variables that have been shown to influence the successful outcome of such strategies include the mean pore size of the highly porous scaffolds the cells are seated onto and the flow rate at which media is perfused through these constructs. The challenge for researchers in optimizing these variables can be better appreciated once the relationship between pore size, cell morphology and flow rate is better understood. In highly porous scaffolds, cells can attach to one of two morphology types, flat akin to TD monolayer culture or bridging where the cell spans the void space of the pore. As the mean pore size decreases, it is easier for cells to bridge the void space and therefore more cells adapt to bridging morphology type. The attachment type becomes important when flow profusion is applied to the cell seeder scaffold. Flow profusion creates shear stresses in the scaffold that are sensed by the cells. Translation of these physical forces to biological signal is termed mechanical transduction where cytoskeletal deformation is one of the primary mechanisms. At low flow rates, flat cells will experience limited deformation and hence mechanical stimulation whilst the bridging cells, behaving like a sail in the wind, experience much larger deformations and therefore mechanical stimulations. As these shear stresses increase further, the flat cells will begin to experience larger deformations whilst the bridging cells will begin to exceed the maximum deformation levels and detach from the scaffold. Windows of operation plots are two-dimensional maps showing regions of feasible operating ranges. To identify a window of operation, firstly a control plot for an output variable of interest, such as cell detachment, needs to be created with respect to the input variables of interest, for example in this study, pore size and shear stress. This control plot identifies areas for which specific operating conditions will lead to performance criteria above or below specified levels. Similar plots can then be made for other output variables of interest, such as the fold change in COX-2 gene expression. The researcher can then simplify the plots by defining a maximum or minimum limit desirable for each output. When the control plots are then overlaid, a feasible window of operation representing the operating conditions for pore size and shear stress which reflect the desired performance criteria will be revealed. In this paper, we studied the effect of a range of pore sizes and shear stresses and implemented a Windows of operation approach to determine the optimal operating conditions based on cell detachment levels and fold change in COX-2 gene expression. Setting performance criteria to a maximum detachment level of 20%, we achieve a suitable operating region defined by the light grey colour. Similarly, setting a minimum increase in gene expression of COX-2 to 45 fold gives us a feasible operating region defined by the dark grey region. Overlaying these plots, we are then able to determine that a small feasible window of operation, as identified by the black region, exists that allow both criteria to be met simultaneously. Having identified an optimal operating region, we then use Windows of operation to study how the size of this feasible operating region changed as the performance criteria were relaxed. Firstly, we increased the maximum cell detachment level to 25%. We then lowered the minimum change in gene expression to 25 fold. Finally, we increased the maximum cell detachment again to 40%. As we can see from these figures, relaxing these constraints yields increasingly larger windows of operation. We would like to thank you for taking the time to watch our video abstract and invite you to read the full manuscript on the Biotechnology and Bioengineering website.