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: For most of the experiments, NPCs were differentiated into post-mitotic neuronal cultures for 2 weeks in NMM, then live cell (DAPI) sorted (BD FACSMelody) and replated at 1.58 × 10 5 –2.1 × 10 5 cells/cm on TCPS, coated glass coverslips, or standard glass coverslips, in NMM containing half-concentrations of growth factors ( , , and , detachment; , morphology; and , , and , electrophysiology).

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-treated glass surface supports human proliferative neuronal/glia progenitors, brain tumor cells, and post-mitotic mature neurons and astrocytes (A–D) Analysis of proliferative human brain cells on various substrates. Attachment determined by counting fixed nuclei (DAPI + ) over time. Means ± SEMs fold change in number of nuclei per well (normalized to day 0) calculated for 5–6 replicate wells/condition/time point (1 independent experiment). All of the surfaces were pre-coated with Matrigel. Proliferative human brain cells analyzed were (A) patient-derived glioblastoma tumor cells (GBMs), (B) hESC-derived glial precursor cells (GPCs), and (C) hESC-derived neural progenitor cells (NPCs). (D) Immunofluorescence staining of DNA (DAPI; blue) and cell soma (CellTracker, red) at latest time point (8 days for brain cells). See also Figure S3 . (E) Proliferation of non-neuronal human embryonic stem cells (H9, hESC) was measured as the fraction of area covered by colonies normalized to day 0 (n = 5–6 replicates from 1 independent experiment). (F) Immunofluorescence staining of DNA (DAPI; blue) and cell soma (CellTracker, red) at latest time point (5 days for stem cells). See also Figure S3 . (G and H) Cytotoxicity analysis of neuronal cultures. All of the surfaces were pre-coated with PLO-Lam. (G) Lactate dehydrogenase (LDH) relative concentrations measured in neuronal culture supernatant (n = 15 replicates from 5 independent experiments) to estimate cellular stress. (H) Without fixation, DNA marker DAPI is incorporated in the nuclei of dead or dying cells (DAPI + ), but not live cells (DAPI − ). Means ± SEMs proportions of live cells (DAPI − ). (I) Immunofluorescence staining of MAP2, GFAP, and synapsin at 10× magnification (top) and 40× magnification (bottom) in neuronal cultures after 9 weeks in maturation medium, on glass-DAP-LAM. (J) Live neuronal cultures on glass-DAP-LAM under phase contrast at 20× after 27 weeks in maturation medium. (K) FACS analysis of the proportion of neurons (Lv-synapsin-GFP) and astrocytes (Lv-GFAP-tdTomato) at 9 weeks in maturation medium on different surfaces. For (H) and (K), n = 3–5 replicates per condition from 2 independent experiments. p values calculated using Mann-Whitney tests, ns for p > 0.05.

Article Snippet: For most of the experiments, NPCs were differentiated into post-mitotic neuronal cultures for 2 weeks in NMM, then live cell (DAPI) sorted (BD FACSMelody) and replated at 1.58 × 10 5 –2.1 × 10 5 cells/cm on TCPS, coated glass coverslips, or standard glass coverslips, in NMM containing half-concentrations of growth factors ( , , and , detachment; , morphology; and , , and , electrophysiology).

Techniques: Derivative Assay, Immunofluorescence, Staining, Marker, MANN-WHITNEY