Wound-on-Chip: A New Device for Studying Inflammation Phase of Wound Healing and Drug Discovery

Abstract

Considerable progress has been made in the field of microfluidics to enable the development of complex systems for modelling human skin. Multicellular culture systems incorporating fibroblasts or keratinocytes, vascular endothelial cells, and immune cells, as well as 3D culture systems, have been described. However, to date all models have lacked one or more of these features. In this thesis, a human skin wound-on-chip cell culture model was constructed successfully to simulate inflammation during the wound healing process. Inflammation was induced via TNF-α or by adding M1 and M2 macrophages as an immune cell to the system. The current wound-on-chip model facilitates the culturing of different cells types that play essential roles in wound healing to mimic the natural wound microenvironment. First, the microfluidic platform including dermal fibroblasts and endothelial cells was used to study the interaction of these cell types during the wound healing. Dermal fibroblasts and HUVECs were used as predominant cells in skin dermal tissue and Matrigel was used to mimic extracellular matrix (ECM). Co-culturing of HUVECs and dermal fibroblasts was led to the formation of vascular structure inside the Matrigel. The inflammation in this model was induced by treating the fibroblast layer with a TNF-α solution. The proposed model successfully simulated skin inflammation. TNF-α induce inflammation was investigated by analysing the pro-inflammatory cytokines level (IL-12p70, TNF, IL-10, IL-6, IL-1β and IL-8). Moreover, the model demonstrated the prevention of inflammation by application of a drug. The data of endothelial cell-cell junction staining with VE-Cadherin shows the difference in vascular structure formation in the presence of TNF-α and treatment with Dexamethasone. The endothelial cell-cell junctions were re-constructed by the application of Dexamethasone to the TNF-α treated model. The expression of pro-inflammatory cytokines and chemokine was dramatically decreased, indicating the recovery of skin inflammation. Another feature of proposed in vitro wound-on-chip model is that it is possible to culture the immune cells in the system; To study the immune responses and cellular interactions between HUVECs, dermal fibroblasts and macrophages as it happened during the wound healing in the human body. HUVECs formed a vascular structure in 3D channels whereas fibroblasts and macrophages established a largely 2D monolayer. Simulating inflammatory condition in the presence of macrophages was confirmed by measuring the expression level of pro-inflammatory cytokines. The current wound-on-chip model shows the formation of the vascular structure by HUVECs through the Matrigel as an ECM. Presence of M1 macrophages as an inflammatory type was led to having less vascularization. Whereas, M2 macrophages as an anti-inflammatory type helped the HUVECs to form a mature vascular structure. The anti-inflammatory effect of Dexamethasone was studied as a proof-of-concept. To conclude, the findings suggest that this microfluidic wound-on-chip model co-culturing dermal fibroblasts, endothelial cells and macrophages can mimic the wound microenvironment as well as the physiology of the human skin. This model has broad implications for modelling human disease as well as for screening of novel pharmacological agents (such as anti-inflammatory drugs) for future pre-clinical assessment. In the end, the wound-on-chip model was used to study the anti-inflammatory effect of Centella erecta (C.E.) and Propolis to validate the model as a successful tool for drug screening. As per previous experiments, the inflammation was modelled by adding TNF-α to the wound-on-chip device and also, by incorporating immune cells (M1 and M2 macrophages). After establishing the inflammation model on-chip, to simulate the anti-inflammation process, C.E. and Propolis extracts were applied to the fibroblasts layers. The expression level of pro-inflammatory cytokines, as well as the role of these natural products on endothelial cell-cell junctions, were studied. The data suggest that this in vitro skin wound-on-chip model could potentially be a powerful tool in pre-clinical assessments for pharmaceutical industries.