Fig 1.
a) Schematic representation of SEAM architecture composed of PMMA housings with embedded magnets, PDMS channels, and a removable cell culture insert. b) Images of the removable culture inserts including porous track etched (top) and soft self-assembled (bottom) culture regions. Scale bar = 1 mm. c) Assembled SEAM culture module with top and bottom microfluidic channels filled with red and blue dyes to demonstrate distinct fluidic compartments within a magnetically sealed architecture.
Fig 2.
a) Schematic and assembled seeding module composed of top and bottom housing layers and a magnetically sealed culture insert. In the assembled module (right), the bottom channel was filled with yellow dye and the open access cell addition region filled with blue dye to aid visualization. b) Workflow (steps 1–4) to establish co-cultured tissue interfaces. The desired cell number was added to the culture region of the insert and allowed to attach. The insert was then decoupled from the PMMA housings, flipped over, resealed, and seeded with a second population. c) Representative image of co-cultured primary human alveolar epithelium stained for occludin (green) and primary human microvascular endothelium stained for VE-cadherin (red) both with nuclear counterstain (blue). Scale bars = 40 μm.
Fig 3.
a) Layers comprising the gravity fed perfusion module. b) Side view of connected perfusion and culture modules. The fluidic circuit (inlet reservoir, aa, through the culture module to the outlet reservoir, bb) was gently primed with medium using a syringe. c) Once filled, surface tension kept the medium from spilling out of the reservoirs, and the difference in hydrostatic pressure ΔP induced fluid flow from inlet to outlet reservoirs.
Table 1.
Differential height vs. predicted flow rate and shear stress.
Fig 4.
SEAM experimental workflow options.
a) Cells are seeded directly on the culture region of the desired insert at a known density using the seeding module. b) The culture insert with attached cells is transferred to the culture platform for long-term perfusion culture and exposure studies. c) The insert can then be directly transferred for off chip lysis and nucleic acid isolation or d) mounted on a slide for imaging. Cells can also be imaged within the platform using a long working distance objective. e) The modular approach enables a combination of imaging and gene level responses to be easily explored.
Fig 5.
SEAM readouts after LPS stimulation.
a) To explore SEAM readouts that mimic vascular inflammation, human microvasular endothelial cells were seeded and transferred to the coupled culture and micro-perfusion modules. b) After apical simulation with LPS for 4 hours, cells were and stained in-channel and the membrane was transferred to a glass slide to explore ICAM1 surface expression after exposure to stimulation. Scale bar = 40 μm. c) To demonstrate rapid and robust RNA isolation, inserts were removed from the architecture and transferred to off-chip RNA isolation. Statistically significant (p<0.001) differences in mRNA expression were observed in ICAM1, CXCL1, CXCL2, and CCL2 with VFW (endothelial marker) remaining constant. SEAM is able to replicate both gene expression and differential surface protein responses to LPS stimulation with an easy to use workflow.
Fig 6.
a) Incorporation of self-assembled membrane into laminated carrier insert. Young’s modulus of membrane is similar to brain tissue. b) After 3 days in culture on the SA membrane, separate membranes were stained for MAP2 (dendridic marker, scale bar = 40 μm) and processed for gene expression analysis. Gene expression of Neurodap1, NCAM1, TrkA, and c-fos, on the soft self-assembled substrate were closer to rat brain gene expression (dashed horizontal line) than those cultured on rigid substrates.