Investigating the Role of Surface Materials and Three Dimensional Architecture on <i>In Vitro</i> Differentiation of Porcine Monocyte-Derived Dendritic Cells

<div><p><i>In vitro</i> generation of dendritic-like cells through differentiation of peripheral blood monocytes is typically done using two-dimensional polystyrene culture plates. In the process of optimising cell culture techniques, engineers have developed fluidic micro-devises usually manufactured in materials other than polystyrene and applying three-dimensional structures more similar to the <i>in vivo</i> environment. Polydimethylsiloxane (PDMS) is an often used polymer for lab-on-a-chip devices but not much is known about the effect of changing the culture surface material from polystyrene to PDMS. In the present study the differentiation of porcine monocytes to monocyte-derived dendritic cells (moDCs) was investigated using CD172a^pos pig blood monocytes stimulated with GM-CSF and IL-4. Monocytes were cultured on surfaces made of two- and three-dimensional polystyrene as well as two- and three-dimensional PDMS and carbonised three-dimensional PDMS. Cells cultured conventionally (on two-dimensional polystyrene) differentiated into moDCs as expected. Interestingly, gene expression of a wide range of cytokines, chemokines, and pattern recognition receptors was influenced by culture surface material and architecture. Distinct clustering of cells, based on similar expression patterns of 46 genes of interest, was seen for cells isolated from two- and three-dimensional polystyrene as well as two- and three-dimensional PDMS. Changing the material from polystyrene to PDMS resulted in cells with expression patterns usually associated with macrophage expression (upregulation of <i>CD163</i> and downregulation of <i>CD1a</i>, <i>FLT3</i>, <i>LAMP3</i> and <i>BATF3</i>). However, this was purely based on gene expression level, and no functional assays were included in this study which would be necessary in order to classify the cells as being macrophages. When changing to three-dimensional culture the cells became increasingly activated in terms of <i>IL6</i>, <i>IL8</i>, <i>IL10</i> and <i>CCR5</i> gene expression. Further stimulation with LPS resulted in a slight increase in the expression of maturation markers (<i>SLA-DRB1</i>, <i>CD86</i> and <i>CD40</i>) as well as cytokines (<i>IL6</i>, <i>IL8</i>, <i>IL10</i> and <i>IL23A</i>) but the influence of the surfaces was unchanged. These findings highlights future challenges of combining and comparing data generated from microfluidic cell culture-devices made using alternative materials to data generated using conventional polystyrene plates used by most laboratories today.</p>