Single-cell transcriptomic analysis of immune responses within a microfluidic skin equivalent
Microfluidic human skin equivalents (HSEs) were constructed by 3D printing and consisted of an endothelial lined vascular microchannel within a 3D dermal matrix embedded with fibroblasts.  Keratinocytes were cultured on the surface to mimic the epidermis.  Following exposure of the epidermis to lipopolysaccharide (LPS) and nigericin, human monocytes were injected into the vascular channel and migrated into the HSE.  Single-cell RNA sequencing was performed on control or treated HSEs after 1 or 6 days to analyse the inflammatory and immunological responses to LPS and nigericin activation.  These studies identified dynamic upregulation and resolution of inflammatory gene signatures in the tissue resident cells in response to treatment, alongside differentiation of monocytes into tissue resident macrophages. Overall design: HSEs were treated with 100 ng/ml LPS and 10 mM nigericin or left untreated (control), and primary human monocytes were injected into the microchannel.  Control and treated samples were collected at days 1 and 6 for RNA-seq analysis, and three HSEs were pooled for each condition/timepoint.   Monocytes were enriched 1:1 with resident cells by magnetic activated cell sorting for CD45.  Approximately 15,000 viable cells were sequenced per condition/timepoint.