PT  - JOURNAL ARTICLE
AU  - Eline Huethorst
AU  - Marie FA Cutiongco
AU  - Fraser A Campbell
AU  - Anwer Saeed
AU  - Rachel Love
AU  - Paul M Reynolds
AU  - Matthew J Dalby
AU  - Nikolaj Gadegaard
TI  - Customizable, engineered substrates for rapid screening of cellular cues
AID  - 10.1101/693598
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 693598
4099  - http://biorxiv.org/content/early/2019/07/05/693598.short
4100  - http://biorxiv.org/content/early/2019/07/05/693598.full
AB  - Biophysical cues robustly direct cell responses and are thus important tools for in vitro and translational biomedical applications. High throughput platforms exploring substrates with varying physical properties are therefore valuable, however, currently existing platforms are limited in throughput, the biomaterials used, the capability to segregate between different cues and the assessment of dynamic cellular responses. Here we present a multiwell array (3×8) using a substrate engineered with patterns that present topography or rigidity cues welded to a bottomless plate with a 96-well format. Both the patterns on the engineered substrate and the well plate format can be easily customized, permitting systematic and efficient screening of biophysical cues. Here, we demonstrate three multiwell arrays patterned with a variety of topographical and mechanical cues (nano-grooves, soft pillars and nano pillars) tested with three different cell types. Using the multiwell array, we were able to measure cell functionality using analytical modalities such as live microscopy, qPCR and fluorescent immunochemistry. Cardiomyocytes cultured on 5µm grooves showed less variation in electrophysiology and contractile function. Nanopillars with 127 nm height, 100 nm diameter and 300 nm pitch showed improved chondrogenic maintenance from matrix deposition and chondrogenic gene expression. High aspect ratio pillars with an elastic shear modulus of 16 kPa mimicking the cortical bone altered cell adhesion, morphology, and increased expression of osteogenic genes. We have demonstrated the bespoke, controlled and high-throughput properties of the multiwell array that are currently unparalleled in the field today.AbbreviationsACANaggrecanAPD50action potential duration at 50% of the amplitudeCD50contraction duration at 50% of the amplitudeCHF3/Artrifluoromethane / argonCOL1Acollagen type 1aCOL2Acollagen type 2aDMEMdulbecco’s modified Eagle’s mediumEBLelectron beam lithographyfpsframes per secondIPAisopropyl alcoholMIBKmethyl isobutyl ketoneNiCrnichromeNILnanoimprint lithographyNMPN-Methyl-2-pyrrolidoneOCNosteocalcinOPNosteopontinqPCRquantitative polymerase chain reactionRIEreactive ion etchingRUNX2Runx family transcription factor 2SDstandard deviationSEMscanning electron microscopeSF6 / C4F8sulfur hexafluoride / octafluorocyclobutaneSOX9SRY-box 9Tcontractioncontraction timeTrelaxationrelaxation time.