human cortical neuronal cells hcn 2  (ATCC)

Journal: Pharmaceutics

Article Title: A Human Brain-Chip for Modeling Brain Pathologies and Screening Blood–Brain Barrier Crossing Therapeutic Strategies

doi: 10.3390/pharmaceutics16101314

Figure Lengend Snippet: Characterization of the human cortical Brain-Chip. ( A ) Representative confocal images showing the hiPSC-derived microvascular endothelial-like cells (iBMECs) attached to the porous membrane (vascular channel). ( i ) Immunostaining against the tight junction marker ZO-1. Stack of Z-series for the vascular channel (left) and high magnification optical section of ZO-1 staining (right) are shown. ( ii ) Immunostaining against the brain microvascular endothelial cell marker GLUT1 (stack of Z-series). ( B ) Confocal images of astrocytes (GFAP) with pericytes (NG2) ( i ) and neurons (MAP2) with microglia (Iba1 and CD68) ( ii ) attached to the porous membrane in the brain channel. Confocal images (stack of Z-series) of the entire brain channel ( top ) and high-magnification confocal optical sections ( bottom ). All cell types were present and uniformly distributed along the entire brain channel. ( C ) ( i ): Confocal micrograph (stack of z-series) showing immunofluorescence staining against GFAP (astrocytes) and MAP2 (neurons) coupled with phase contrast for visualization of the porous membrane. ( ii ): Digital 3D reconstruction of z-series image stacks showing the Brain-Chip from the side. The interrupted line indicates the location of the porous membrane separating the brain from the vascular channel. The nuclear staining (Hoechst) on the vascular side indicates the iBMECs. A GFAP signal is detected in the vascular side (arrows). Arrows in both images indicate the astrocytic end-feet passing through the 7 μm pores extending into the vascular channel. ( D ) Schematic representation of the experimental design and averaged data from quantitative barrier function analysis via apparent permeability (Papp) to 3 kDa fluorescent dextran crossing through the vascular channel to the brain channel on Days 1 through 6 in microfluidics. Chips with and without iBMECs were examined (N = 6 chips/group). Each data point represents an individual chip. Graph: mean ± SEM. Shaded box: range of Papp values shown in animal models. ( E ) Confocal images showing GABAergic (VGAT) and glutamatergic neurons (VGLUT1) in the brain channel of the chip. All stainings were performed in Brain-Chips after six days in microfluidics. ( F ) Examination of functional connectivity between GABAergic and glutamatergic neurons using pharmacology and extracellular glutamate measurements. The experimental design (top) and extracellular glutamate quantification (mean ± SEM) for the indicated time points and treatments are shown. N = 3 chips/group, *** p < 0.001, two-way ANOVA and post hoc Tukey’s test.

Article Snippet: Commercial human iPSC-derived cortical glutamatergic and GABAergic neurons and human primary astrocytes were purchased from NeuCyte (Mountain View, CA, USA; SynFire ® Co-Culture kit; Cat.# 1010-7.5).

Techniques: Derivative Assay, Membrane, Immunostaining, Marker, Staining, Immunofluorescence, Permeability, Functional Assay

Journal: Pharmaceutics

Article Title: A Human Brain-Chip for Modeling Brain Pathologies and Screening Blood–Brain Barrier Crossing Therapeutic Strategies

doi: 10.3390/pharmaceutics16101314

Figure Lengend Snippet: TNFα-induced neuroinflammation and BBB disruption in the Brain-Chip. ( A ) Outline of the experimental design. Beginning on Day 2 in microfluidics, 100 ng/mL of TNFα were dosed in the brain channel and replenished 24 h later. Chips dosed with PBS were used as the control. Immunocytochemistry and extracellular glutamate measurements were performed on Day 4. Effluents were collected daily from Day 1 to Day 4 for the BBB permeability assay (Days 1–4) and cytokines/chemokines analysis (Days 2–4). ( B ) Representative confocal images of microglia (CD68) and neurons (MAP2) ( i ) and astrocytes ( ii ). ( iii ) Averaged data (mean ± SEM) for the number of CD68+ cells and MAP2 intensity. TNFα treatment increases the numbers of CD68-positive cells, indicative of microglial reactivity. The signal intensity of the neuronal dendritic marker MAP2 is decreased, suggesting neuronal dysfunction. High-resolution stacks of z-series from brain channel areas (50% coverage of the channel) were analyzed for each chip. N = 3 chips/treatment. Confocal images of all chips used for MAP and CD68 analysis can be found in . The morphology of reactive astrocytes upon TNFα exposure changes from a polygonal state to a more elongated state (see for additional chips and images). ( C ) Averaged data (mean ± SEM) of extracellular glutamate measurements in brain effluents collected at the end of the experiment (day 4). N = 3 chips/group. Red asterisks: comparison with TNFα-treated chips without microglia. Black asterisks: comparison with the respective control. ( D ) Apparent permeability (Papp) of the barrier across days (mean ± SEM). Papp on Day 2 was measured immediately before TNFα perfusion. Chips with microglia, N = 4; chips without microglia, N = 3. ( B – D ) * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, Student’s t -test ( B ) and one-way ( C ) or two-way ( D ) ANOVA with post hoc Tukey’s test (significantly different compared with all other groups).

Article Snippet: Commercial human iPSC-derived cortical glutamatergic and GABAergic neurons and human primary astrocytes were purchased from NeuCyte (Mountain View, CA, USA; SynFire ® Co-Culture kit; Cat.# 1010-7.5).

Techniques: Disruption, Control, Immunocytochemistry, Permeability, Marker, Comparison

Journal: Pharmaceutics

Article Title: A Human Brain-Chip for Modeling Brain Pathologies and Screening Blood–Brain Barrier Crossing Therapeutic Strategies

doi: 10.3390/pharmaceutics16101314

Figure Lengend Snippet: TNFα-induced secretion of cytokines and chemokines and contribution of microglia. Longitudinal analysis of cytokines and chemokines in brain channel effluents collected at the indicated time points. Effluent collection on Day 2 was performed immediately prior to TNFα dosing (baseline levels of cytokines and chemokines). Brain-Chips with and without microglia were examined to determine their contribution to the observed inflammatory responses. All other cell types (astrocytes, pericytes, glutamatergic and GABAergic neurons, brain microvascular endothelial-like cells) were present in the chips. Brain-Chips (with and without microglia) treated with PBS were used as controls. Graphs: averaged data (mean ± SEM) from 4 chips for each group. Microglial-specific responses are indicated by the blue rectangles.

Article Snippet: Commercial human iPSC-derived cortical glutamatergic and GABAergic neurons and human primary astrocytes were purchased from NeuCyte (Mountain View, CA, USA; SynFire ® Co-Culture kit; Cat.# 1010-7.5).

Techniques: