of 3D Alvetex® 7 day cultures of HaCaT, co-culture and fibroblast cultures using the 42 minute (‘42bis’) SDS exposure. In an attempt to understand the mechanism behind the irritation and to see if the hypothetical dose-response curve is an accurate representation of the real life irritant-to-toxic responses, a method was developed to produce models more representative of the in vivo morphology needed for proper 3D skin model testing. Without raising the scaffold to the air-liquid interface the differentiation of the keratinocytes would be limited, resulting in impaired barrier function. The histology of HaCaT monocultures shows a 2-4 cell thick apical layer had developed. HaCaT migration and cell number increased with the addition of fibroblasts. The co-cultures had a thicker apical layer of cell thickness but with no obvious separation of HaCaTs and fibroblasts. The fibroblast monocultures had fibroblasts proliferated evenly across the Alvetex® scaffold except for at the apical surface where a 1 cell thick stratum of fibroblasts can be seen. ECM has been produced, evident by a higher background eosin staining but also that on taking 10µm sections, the scaffold does not disintegrate as the ECM is effectively gluing together the polystyrene fragments of the Alvetex®. A change in the SDS exposure protocol was made. The protocol was adopted as ‘42bis’ developed by [] Using the same SDS concentrations that achieved the well-defined SkinEthic™. 2D viability curves in section 6.1.1, the effect of 3D cultures on cell viability was assessed. Giving the resulting cell viability curves in Figure 64. At control; co-cultures had the greatest cell viability, followed by HaCaT keratinocytes and then fibroblasts by the MTT assay. HaCaTs
3D Alvetex® 7 day cultures of HaCaT, co-culture and fibroblast cultures using the 42 minute (‘42bis’) SDS exposure. In an attempt to understand the mechanism behind the irritation and to see if the hypothetical dose-response curve is an accurate representation of the real life irritant-to-toxic responses, a method was developed to produce models more representative of the in vivo morphology needed for proper 3D skin model testing. Without raising the scaffold to the air-liquid interface the differentiation of the keratinocytes would be limited, resulting in impaired barrier function. The histology of HaCaT monocultures shows a 2-4 cell thick apical layer had developed. HaCaT migration and cell number increased with the addition of fibroblasts. The co-cultures had a thicker apical layer of cell thickness but with no obvious separation of HaCaTs and fibroblasts. The fibroblast monocultures had fibroblasts proliferated evenly across the Alvetex® scaffold except for at the apical surface where a 1 cell thick stratum of fibroblasts can be seen. ECM has been produced, evident by a higher background eosin staining but also that on taking 10µm sections, the scaffold does not disintegrate as the ECM is effectively gluing together the polystyrene fragments of the Alvetex®. A change in the SDS exposure protocol was made. The ‘42bis’ protocol was adopted as developed by SkinEthic™. [] Using the same SDS concentrations that achieved the well-defined 2D viability curves in section 6.1.1, the effect of 3D cultures on cell viability was assessed. Giving the resulting cell viability curves in Figure 64. At control; co-cultures had the greatest cell viability, followed by HaCaT keratinocytes and then fibroblasts by the MTT assay. HaCaTs of
3D%20RECONSTRUCTED%20SKIN%20EQUIVALENT%20MODELS%20FOR%20IRRITANT%20TESTING.docx