Journal: Stem Cell Reports

Article Title: Long-term adherence of human brain cells in vitro is enhanced by charged amine-based plasma polymer coatings

doi: 10.1016/j.stemcr.2022.01.013

Figure Lengend Snippet: Neuronal cells detach from standard glass surfaces sooner than from tissue culture polystyrene (A) Schematic of the cell culture microenvironment and modifications tested to improve neuronal attachment. (B) Human neural progenitor cells (NPCs) were pre-matured in BrainPhys + supplements for 0–2 weeks, replated on test surfaces, and further matured for up to 27 weeks in BrainPhys + supplements before phenotypic analyses. Before replating on test surfaces, post-mitotic precursors were sorted for DAPI exclusion at 2 weeks (see Experimental procedures). (C and D) Live neuronal cultures under phase contrast at 4× and 20× magnification, respectively. Detachment was quantified from 4× magnification phase contrast images by tracing regions with no cells (yellow) and calculating their combined area as a fraction of the total field of view area for each well. (E) Detachment on standard glass across multiple coating solutions: poly- l -ornithine (PLO; n = 4), PLO-laminin (Lam) (n = 6), Matrigel (n = 2), 24 h treatment of laminin (n = 6), 4 h treatment of laminin (n = 2), uncoated (n = 6), and poly- l -lysine (PLL; n = 2); n replicates from 2 independent experiments, means ± SEMs represented. (F) Cell detachment on standard glass compared to plastic (TCPS) both with PLO-Lam. Means ± SEMs from n = 5 biologically independent experiments, 2–6 replicates per experiment. (G and H) Spontaneous multi-electrode array (MEA) recordings of hPSC-derived neuronal cultures showing network burst activity and synchrony over 15–17 weeks on TCPS plates pre-coated with PLO-Lam. Means ± SEMs data from 16 replicate wells, each well containing 16 electrodes.

Article Snippet: Human neurons cultured on DAP, AAM, or glass coverslips were transferred into a recording chamber and perfused with artificial cerebrospinal fluid (ACSF) or BrainPhys Imaging (cat. no. 05796, STEMCELL Technologies; ) at RT (21°C–23°C).

Techniques: Cell Culture, Derivative Assay, Activity Assay

Journal: Stem Cell Reports

Article Title: Long-term adherence of human brain cells in vitro is enhanced by charged amine-based plasma polymer coatings

doi: 10.1016/j.stemcr.2022.01.013

Figure Lengend Snippet: DAP-LAM-treated glass surfaces support calcium imaging and optogenetic applications in human neuronal models in vitro (A–E) Calcium imaging recordings from hPSC-derived midbrain neurons after 10 weeks in maturation medium on glass-DAP with PLO-Lam. A total of 838 active regions of interest (ROIs) analyzed from 2 coverslips across 9 fields of view at the soma. (A) Example of an “active” neuronal population following Fluo-4-AM calcium dye incubation. White circles represent analyzed ROIs. (B) Example raster plots and calcium traces of active neurons showing spontaneous calcium events recorded over 4 min on glass-DAP in BrainPhys Imaging. (C) Mean % of active cells on glass-DAP with or without tetrodotoxin (TTX). Cells considered active if ΔF/F 0 > 5% from baseline. (D) Example traces of calcium events categorized into spontaneous calcium spikes (fast rising phase) and calcium waves (slow rising phases). (E) Proportion of active cells and their event types on glass-DAP following TTX perfusion. (F–J) Optogenetic responses of neurons on glass-DAP. Mature neurons (type 5, n = 9 across 4 coverslips) were patch-clamped and subject to 10 × 5 ms flashes of blue (475 nm) LED light of various intensities. (F) Example images of live neurons expressing the optogene synapsin:ChETA-YFP (left) and filled with rhodamine (right). (G) Typical optogenetic responses from the same patch-clamped neuron to blue LED stimuli at 0.1, 0.2, and 0.4 mW of power. Top: APs evoked by channelrhodopsin 2 (ChR2) membrane depolarization in current-clamp. Bottom: ChR2-mediated currents in voltage-clamp, held at −70 mV (H) Total spike success rate (%) of each neuron across 10 sweeps. Spikes >−10 mV in amplitude were included. (I and J) Maximum amplitudes of ChR2-mediated currents (I) and (J) of APs evoked by ChR2-mediated membrane depolarization. (B) and (H)–(J) Means ± SEMs. (B) p values calculated with 2-tailed paired Wilcoxon and (H)–(J) 2-tailed parametric unpaired tests, ns for p > 0.05.

Article Snippet: Human neurons cultured on DAP, AAM, or glass coverslips were transferred into a recording chamber and perfused with artificial cerebrospinal fluid (ACSF) or BrainPhys Imaging (cat. no. 05796, STEMCELL Technologies; ) at RT (21°C–23°C).

Techniques: Imaging, In Vitro, Derivative Assay, Incubation, Expressing, Membrane