Modulating patterned adhesion and repulsion of HEK 293 cells on microengineered parylene‐C/SiO₂ substrates

Abstract

This article describes high resolution patterning of HEK 293 cells on a construct of micropatterned parylene-C and silicon dioxide. Photolithographic patterning of parylene-C on silicon dioxide is an established and consistent process. Activation of patterns by immersion in serum has previously enabled patterning of murine hippocampal neurons and glia, as well as the human hNT cell line. Adapting this protocol we now illustrate high resolution patterning of the HEK 293 cell line. We explore hypotheses that patterning is mediated by transmembrane integrin interactions with differentially absorbed serum proteins, and also by etching the surface substrate with piranha solution. Using rationalized protein activation solutions in place of serum, we show that cell patterning can be modulated or even inverted. These cell-patterning findings assist our wider goal of engineering and interfacing functional neuronal networks via a silicon semiconductor platform.

Patterning human neuronal networks on photolithographically engineered silicon dioxide substrates functionalized with glial analogues

“…Parylene‐C is a biocompatible polymer used commercially to coat printed circuit boards. Photolithographic patterning of parylene‐C on silicon dioxide, followed by activation with serum, has enabled patterning of primary murine hippocampal cells, a human teratocarcinoma cell line, and the human embryonal kidney (HEK) 293 cell line . This straightforward and reliable technique is significantly simpler than some multistage protocols used for neuronal patterning.…”

“…Whilst parylene‐C has been used previously in the context of cell patterning and cell trapping, its use for neuronal patterning after serum activation is in its infancy. Exploration of the mechanisms underlying cell patterning suggests that both adhesive and repulsive components in serum interact to imbue each substrate with contrasting cytoadhesive or cytorepulsive characteristics, although these components are not yet characterized …”

“…Photolithographic patterning of parylene-C on silicon dioxide, followed by activation with serum, has enabled patterning of primary murine hippocampal cells, 7-10 a human teratocarcinoma cell line, 11,12 and the human embryonal kidney (HEK) 293 cell line. 13 This straightforward and reliable technique is significantly simpler than some multistage protocols used for neuronal patterning. Specifically, patterned parylene substrates are biologically stable and can be stored until needed (whereupon they are activated).…”

“…Exploration of the mechanisms underlying cell patterning suggests that both adhesive and repulsive components in serum interact to imbue each substrate with contrasting cytoadhesive or cytorepulsive characteristics, although these components are not yet characterized. 13 Although patterning primary murine hippocampal cells (which contain both neurons and glia) is effective, it remains unclear whether neurons in isolation are capable of patterning or whether glia adhere and (by close association) enable neurons to respect the underlying parylene geometry. The presence of glia amongst patterned neurons, though better reflecting the in vivo environment, may complicate downstream efforts to record from and stimulate individual neurons.…”

Cell Patterning on Photolithographically Defined Parylene-C: SiO₂ Substrates

“…Once prepared, activation of chips in fetal bovine serum enables a wide range of cell types to be patterned in culture. Our group has successfully patterned primary murine hippocampal cells [14][15][16] , the HEK 293 cell line 17 , the human neuron-like teratocarcinoma (hNT) cell line 18 , primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells. Figure 4 illustrates the potential to augment the patterning platform by using alternative activation solutions.…”