Journal: bioRxiv

Article Title: NanoMEA: a versatile platform for high-throughput analysis of structure-function relationships in human stem cell-derived excitable cells and tissues

doi: 10.1101/453886

Figure Lengend Snippet: Design, fabrication, and characterization of the nanotopographically-patterned MEA devices. (a) High-throughput nanoMEA concept. Each well of the multiwell plate (i) supports independent cell cultures for high-throughput analysis. Within each well (ii), the electrode bed facilitates recording of field potentials generated by the overlying cells. Nanotopography (iii) is applied to each well (or a subset of wells) to promote cellular alignment and functional development. Captured signals are relayed, via an amplifier (iv), to a software program for subsequent analysis (v). (b) Low magnification image of multiwell MEA plate with nanotopography applied to each well. (c) Nafion nanotopography applied to a single well of a 48-well MEA plate. The presence of the nanoscale features causes light diffraction on the surface, giving the patterns a green-orange color in this image (white arrow). (d) SEM image of Nafion nanotopography. (e) NanoMEA fabrication schematic. Pristine wells are first treated with PEDOT to improve the sensitivity of the base electrode. A drop of Nafion resin is then applied to the substrate, and a PDMS mold is pressed into it. After overnight curing, the PDMS mold is removed to reveal Nafion topographic substrates underneath. (f) Percentage of MEA electrodes from which hPSC-CM signal detection could be clearly distinguished above background noise. (g) Quantification of noise recorded from bare, flat Nafion, and patterned Nafion electrodes with and without PEDOT treatment. Cells maintained on untreated and flat Nafion coated MEAs for 7 days were assessed for differences in: (h) depolarizing spike amplitude, (i) depolarizing spike slope, and (j) field potential duration corrected for beat period (FPDc). In all presented data, *p < 0.05.

Article Snippet: Commercially sourced (iCell) hPSC-CMs and hPSC-neurons were purchased from Cellular Dynamics International (CDI) and were stored, thawed, and maintained according to the manufacturer’s protocol.

Techniques: High Throughput Screening Assay, Generated, Functional Assay, Software

Journal: bioRxiv

Article Title: NanoMEA: a versatile platform for high-throughput analysis of structure-function relationships in human stem cell-derived excitable cells and tissues

doi: 10.1101/453886

Figure Lengend Snippet: Enhanced structural and functional properties of human cardiomyocytes on nanoMEAs. (a) Immunostained image of hPSC-CMs on flat Nafion substrates. Inset shows detail of the sarcomeric structures present. (b) Immunostained image of hPSC-CMs on nanotopographically-patterned Nafion substrates. Inset shows detail of the sarcomeric structures present. (c) Quantification of sarcomere length from patterned and unpatterned hPSC-CMs. (d) Quantification of z-band widths from patterned and unpatterned hPSC-CMs. (e) Histogram detailing frequency of F-actin fibers in cultured cardiomyocytes that possess a given angle of alignment relative to vertical. All images collected from patterned cultures were oriented so that the underlying pattern ran vertically. (f) Immunoblot results from unpatterned and patterned hPSC-CMs, detailing expression levels of β-myosin heavy chain (β-MyHC), cardiac troponin I (cTnI), and connexin 43 (Cx43), as well as GAPDH internal controls. (g) Densitometric analysis of band intensity, providing quantification of the changes in protein expression illustrated in (f). (h) Quantification of pixel intensity in images collected from hPSC-CM cultures stained with a primary antibody that targets Cx43. (i) Measurement of maximum diastolic potential (MDP) using whole cell patch clamp analysis of patterned and unpatterned hPSC-CMs. (j) Measurement of conduction velocity (CV) across hPSC-CM monolayers on flat and nanotopographically-patterned MEAs. Conduction was measured specifically in both the transverse (TCV) and longitudinal (LCV) orientations and underlying nanotopography was organized to run longitudinally. In all presented data, *p < 0.03, **p < 0.003, ***p < 0.001.

Article Snippet: Commercially sourced (iCell) hPSC-CMs and hPSC-neurons were purchased from Cellular Dynamics International (CDI) and were stored, thawed, and maintained according to the manufacturer’s protocol.

Techniques: Functional Assay, Cell Culture, Western Blot, Expressing, Staining, Patch Clamp

Journal: bioRxiv

Article Title: NanoMEA: a versatile platform for high-throughput analysis of structure-function relationships in human stem cell-derived excitable cells and tissues

doi: 10.1101/453886

Figure Lengend Snippet: PGC1α expression in hPSC-CMs maintained on flat and nanotopographically-patterned substrates. (a) Immunoblot results from unpatterned and patterned hPSC-CMs, detailing expression levels PGC1-α and GAPDH internal controls. (b) Densitometric analysis of band intensity, providing quantification of the changes in protein expression illustrated in (a).

Article Snippet: Commercially sourced (iCell) hPSC-CMs and hPSC-neurons were purchased from Cellular Dynamics International (CDI) and were stored, thawed, and maintained according to the manufacturer’s protocol.

Techniques: Expressing, Western Blot

Journal: bioRxiv

Article Title: NanoMEA: a versatile platform for high-throughput analysis of structure-function relationships in human stem cell-derived excitable cells and tissues

doi: 10.1101/453886

Figure Lengend Snippet: Effect of nanotopography on reactive oxygen species production in cultured hPSC-CMs. (a) Representative image from patterned cardiomyocyte culture stained with a superoxide detection reagent (red) following treatment with a reactive oxygen species (ROS) inhibitor (N-acetyl-L-cysteine; top) and an ROS inducer (Pyocyanin; bottom). (b) Representative image from patterned cardiomyocyte culture stained with an oxidative stress detection reagent (green) following treatment with an ROS inhibitor (top) and an ROS inducer (bottom). (c) Representative image from patterned cardiomyocyte culture stained with a nitric oxide (NO) detection reagent (blue) following treatment with an NO scavenger (c-PTIO; top) and an NO inducer (L-arginine; bottom). (d) Quantification of pixel intensity in images collected from cardiac cultures stained with a superoxide detection reagent. Samples analyzed were patterned cells treated with an ROS inhibitor (ROS-), patterned cells treated with an ROS inducer (ROS+), untreated unpatterned cells, and untreated patterned cells. Superoxide presence was significantly lower than all other groups in the ROS- samples, while it was significantly higher than all other groups examined in the ROS+ samples. No difference was observed between patterned and unpatterned samples. (e) Quantification of pixel intensity in images collected from cardiac cultures stained with an oxidative stress detection reagent. Samples analyzed were patterned cells treated with an ROS inhibitor (ROS-), patterned cells treated with an ROS inducer (ROS+), untreated unpatterned cells, and untreated patterned cells. ROS+ exhibited significantly higher values then all other groups, while ROS- was significantly lower than all other groups examined, with the exception of the patterned samples. No significant difference was observed between patterned and unpatterned samples. (f) Quantification of pixel intensity in images collected from cardiac cultures stained with an NO detection reagent. Samples analyzed were patterned cells treated with an NO scavenger (NO-), patterned cells treated with an NO inducer (NO+), untreated unpatterned cells, and untreated patterned cells. NO- was significantly lower than all other groups, while NO+ was significantly higher than all other groups examined. A significant difference was also observed between patterned and unpatterned samples. In all presented data, *p < 0.05.

Article Snippet: Commercially sourced (iCell) hPSC-CMs and hPSC-neurons were purchased from Cellular Dynamics International (CDI) and were stored, thawed, and maintained according to the manufacturer’s protocol.

Techniques: Cell Culture, Staining

Journal: bioRxiv

Article Title: NanoMEA: a versatile platform for high-throughput analysis of structure-function relationships in human stem cell-derived excitable cells and tissues

doi: 10.1101/453886

Figure Lengend Snippet: Effect of nanotopography on spatial distribution of connexin 43 proteins in cultured hPSC-CMs. (a) Representative immunostained image of hPSC-CMs on flat Nafion substrates showing random distribution of gap junctions. (b) Representative immunostained image of hPSC-CMs on patterned Nafion substrates showing more polar orientation of gap junctions. Yellow arrow indicates nanotopography orientation.

Article Snippet: Commercially sourced (iCell) hPSC-CMs and hPSC-neurons were purchased from Cellular Dynamics International (CDI) and were stored, thawed, and maintained according to the manufacturer’s protocol.

Techniques: Cell Culture

Journal: bioRxiv

Article Title: NanoMEA: a versatile platform for high-throughput analysis of structure-function relationships in human stem cell-derived excitable cells and tissues

doi: 10.1101/453886

Figure Lengend Snippet: Baseline electrophysiology in patterned and unpatterned hPSC-CM cultures. (a) Representative baseline field potential recording from hPSC-CM monolayers maintained on flat Nafion MEAs for 21 days. (b) Representative baseline field potential recording from hPSC-CM monolayers maintained on patterned Nafion MEAs for 21 days. (c) Comparison of beat interval variability metrics calculated from analysis of hPSC-CMs maintained on patterned and unpatterned MEAs for 21 days. Median difference in beat interval (ΔBI), mean ΔBI, and the standard deviation of the ΔBI were compared in order to provide quantification of baseline arrhythmic properties in line with previously published methods .

Article Snippet: Commercially sourced (iCell) hPSC-CMs and hPSC-neurons were purchased from Cellular Dynamics International (CDI) and were stored, thawed, and maintained according to the manufacturer’s protocol.

Techniques: Standard Deviation

Journal: bioRxiv

Article Title: NanoMEA: a versatile platform for high-throughput analysis of structure-function relationships in human stem cell-derived excitable cells and tissues

doi: 10.1101/453886

Figure Lengend Snippet: Electrophysiological response of patterned and unpatterned hPSC-CMs to treatment with verapamil. (a) Representative traces (averaged across 10 beats) recorded from hPSC-CM monolayers on flat MEAs and subjected to increasing doses of verapamil. (b) Representative traces recorded from hPSC-CM monolayers on nanoMEAs and subjected to increasing doses of verapamil. (c) Normalized dose response curve illustrating the effect for increasing concentrations of verapamil on the field potential durations corrected for beat rate (FPDc) of unpatterned and patterned hPSC-CMs. The R 2 values for the unpatterned and patterned cultures were 0.86 and 0.60, respectively.

Article Snippet: Commercially sourced (iCell) hPSC-CMs and hPSC-neurons were purchased from Cellular Dynamics International (CDI) and were stored, thawed, and maintained according to the manufacturer’s protocol.

Techniques:

Journal: bioRxiv

Article Title: NanoMEA: a versatile platform for high-throughput analysis of structure-function relationships in human stem cell-derived excitable cells and tissues

doi: 10.1101/453886

Figure Lengend Snippet: Electrophysiological response of patterned and unpatterned hPSC-CMs to treatment with bepridil and carbenoxolone. (a) Representative traces (averaged across 10 beats) recorded from hPSC-CM monolayers on flat MEAs and subjected to increasing doses of bepridil. (b) Representative traces recorded from hPSC-CM monolayers on nanoMEAs and subjected to increasing doses of bepridil. (c) Normalized dose response curve illustrating effect of increasing concentrations of bepridil on the FPDc of unpatterned and patterned hPSC-CMs. The R 2 values for the unpatterned and patterned cultures were 0.26 and 0.39, respectively. (d) Normalized dose response curves illustrating the effect of increasing concentrations of carbenoxolone on the conduction velocity of unpatterned hPSC-CM monolayers. Dose response curves were calculated from analysis of propagation speeds in both the transverse (TCV) and longitudinal (LCV) directions. The R 2 values for TCV and LCV curve fits were 0.23 and 0.11, respectively. (e) Normalized dose response curves illustrating the effect of increasing concentrations of carbenoxolone on the TCV and LCV of patterned hPSC-CM monolayers. The R 2 values for TCV and LCV curve fits were 0.21 and 0.28, respectively. (f) Normalized dose response curves illustrating the effect of increasing concentrations of carbenoxolone on the TCV of patterned and unpatterned hPSC-CMs. (g) Normalized dose response curves illustrating the effect of increasing concentrations of carbenoxolone on the LCV of patterned and unpatterned hPSC-CM monolayers.

Article Snippet: Commercially sourced (iCell) hPSC-CMs and hPSC-neurons were purchased from Cellular Dynamics International (CDI) and were stored, thawed, and maintained according to the manufacturer’s protocol.

Techniques: