221 Immune Cell Model for Burn Immune Reactions An important advantage of our FSE model over other models is the use of primary cells instead of cell lines, making these models more representative for the in vivo situation. In other studies, macrophages or T cells were studied by microscopy, but cells were not quantified or analyzed by flow cytometry after culture. A limitation of this study was the absence of keratinocyte and fibroblast markers in the flow cytometry panel. As we showed that an interplay occurs between immune cells and skin cells, it would be interesting to study the effect on keratinocytes and fibroblasts as well. This could give more information on the effect of monocytes or T cells on the healing process in a skin environment. In the current set-up, the effect of burn injury on studied monocyte and T cell markers appeared to be limited. It would be interesting to observe the effect of burn injury on monocytes and T cells for a longer period of time. Moreover, the effect on other monocyte/macrophage and T cell markers or cytokines could be studied. This model can also be used to study the effect of specific immune cell subsets on wound healing. Monocytes can be activated towards M1 or M2 macrophages and T cells can be skewed towards Tregs, Th1, Th2 or Th17 cells. Similarly, the addition of neutrophil subsets, so far still a technical and logistical challenge, would be of great interest to advance our knowledge on the effect of immune cells on burn wound healing [5,55]. However, culturing neutrophils in FSEs will be difficult due to the short lifespan neutrophils and inability to cryopreserve them [56]. Nevertheless, we anticipate that these immunocompetent models will be useful to study therapeutics that modulate inflammatory reactions in the burned skin. In this pilot study, we developed an FSE model that incorporated monocytes and T cells for the study of burn injury. With this model, we come another step closer to the development of more realistic skin models that will allow the study of both innate and adaptive immune reactions related to burn injury while avoiding the need for animal experimentation. MATERIALS AND METHODS Human skin samples Skin samples were obtained from adult patients who underwent abdominoplasty at the Red Cross Hospital in Beverwijk, Medical Clinic in Velsen or Spaarne Gasthuis in Haarlem. Samples from 17 different donors were used (donor age: 48±13 years; sex: 93% female). Consent for the use of these anonymized, post-operative residual tissue samples was received through an informed opt-out protocol, in accordance with the national guidelines (https://www.coreon.org/) and approved by the institutional privacy officers. Subjects were actively informed of this procedure and were able to easily withdraw at any 7
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