E-FLOAT: Extractable floating liquid gel-based organ-on-a-chip for airway tissue modeling under airflow

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Directional migration of mesenchymal stem cells under an SDF-1α gradient on a microfluidic device.

Homing of peripheral stem cells is regulated by one of the most representative homing factors, stromal cell-derived factor 1 alpha (SDF-1α), which specifically binds to the plasma membrane receptor CXCR4 of mesenchymal stem cells (MSCs) in order to initiate the signaling pathways that lead to directional migration and homing of stem cells. This complex homing process and directional migration of stem cells have been mimicked on a microfluidic device that is capable of generating a chemokine gradient within the collagen matrix and embedding endothelial cell (EC) monolayers to mimic blood vessels. On the microfluidic device, stem cells showed directional migration toward the higher concentration of SDF-1α, whereas treatment with the CXCR4 antagonist AMD3100 caused loss of directionality of stem cells. Furthermore, inhibition of stem cell's main migratory signaling pathways, Rho-ROCK and Rac pathways, caused blockage of actomyosin and lamellipodia formation, decreasing the migration distance but maintaining directionality. Stem cell homing regulated by SDF-1α caused directional migration of stem cells, while the migratory ability was affected by the activation of migration-related signaling pathways

Rapid fabrication of PMMA/PET-E/PMMA for thermoplastic microfluidic membrane devices

We present a method to rapidly fabricate a large number of thermoplastic microfluidic systems where horizontal microchannels are separated by a porous membrane for modelling biological barriers. We optimize and combine solvent bonding, milling and laser cutting to fabricate in excess of 100 devices in two days. The optimized protocol relies on a commercial solvent blend, retention grooves for solvent distribution, and force application to create strong, leak-free bonds. The devices are evaluated for suitability for modelling biological barriers by culturing endothelial cells and measuring permeability using a plate reader

3D confocal images of MSCs migrating transendothelially.

<p>(a-c) Red fluorescence shows the cytosol of RFP-tagged EC. Green fluorescence shows actin fibers of both MSCs and ECs. Cells without red fluorescence are MSCs. Blue are nuclei of the cells stained with DAPI. (d) Ortho view of transendothelial migration of MSCs. Blue and orange arrows indicate the side view range of the directions of movement. White arrows indicate the locations of nuclei of migrating MSCs.</p

Rapid assembly of PMMA microfluidic devices with PETE membranes for studying the endothelium

Biomicrofluidic devices and organ-on-a-chip (OOC) systems with integrated membranes are often fabricated from two different thermoplastic materials but bonding of such dissimilar thermoplastics remains challenging to manufacture at scale. Here, we present a method to bond poly(methylmethacrylate) layers to a polyethylene terephthalate porous membrane to create membrane-based microfluidic devices for biological barrier modeling. By combining milling, laser cutting and chlorocarbon-based solvent bonding supported by retention grooves, we achieved a fabrication rate of 36 devices in 5 h. Chlorocarbon-based solvent bonding resulted in bond strength of ~10 J/m2 and did not adversely affect the membrane pore structure or the channel cross-sectional shape. The bonded devices were found to support long term culture of human endothelial cells that developed expected morphology and cell-cell adhesion contacts as evidenced by immunofluorescent labeling of VE-cadherin. Barrier permeability was measured to be 3.38 × 106 cm/s for 10 kDa dextran using a sampling-based method compatible with mass spectrometry and scintillation techniques and was in agreement with literature. To validate the devices for cell migration experiments, THP-1 monocytes were introduced into devices with confluent endothelial monolayers. Monocytes adhered to and migrated through the endothelium. Activation of the endothelium with TNF-α prior to introducing monocytes significantly increased monocyte adhesion. Overall, the rapid device fabrication method achieved medium-volume production rates and was found to support both cell culture and experiments associated with measuring barrier and endothelial function. This fabrication method has potential to both accelerate biomicrofluidics and OOC research in the lab and accelerate development of commercialized microfluidic membrane devices

Morphology of F-actin-stained MSCs under different conditions (branch counting).

<p>(a) Morphology of MSCs without SDF-1α gradient. (b) Morphology of MSCs under the gradient of SDF-1α. (c) Morphology of MSCs under Y-27632 treatment (d) Morphology of MSCs under AMD3100 treatment (e) Morphology of MSCs under NSC23766 treatment (f) An average number of actin branches of MSCs counted with Image J. One-way ANOVA analysis was used for branch counting. *p<0.05 represents at least one group has inequality.</p

Migration distance of MSCs over the duration of the experiment.

<p><b>Blue dots in the collagen matrix indicate the locations of nuclei of MSCs</b> (a) Control (b) SDF-1α condition. *p>0.05 (c) AMD3100-treated condition. *p>0.05 (d) Y-27632-treated condition. *p<0.05 (e) NSC23766-treated condition. *p<0.05 (f) RFP-expressing endothelial monolayer confluent to the collagen matrix. RFP-expressing ECs are distinguishable from MSCs. (g) Average migration distance of individual MSCs toward or away from the SDF-1α gradient. Graph bars from top to bottom; Control, SDF-1α, AMD3100, Y-27632, NSC23766. Purple and red fluorescence represents endothelial cells. The monolayer near the collagen matrix is shown in purple because of the higher population of ECs along the Z-axis. Orange triangle represents the presence of SDF-1α gradient. F-test was used to compare the average migration of each conditions with the control group. *p<0.05 represents significant variance difference while *p>0.05 represents no significant variance difference. One-way ANOVA analysis for finding inequality group showed *p<0.05.</p

Confocal microscopic images of endothelial monolayer formed within the channel of the microfluidic device.

<p>(a) Side view of the endothelial monolayer confluent with collagen matrix. RFP represents the expression of VE-cadherin and blue represents DAPI. (b) Front view of the endothelial monolayer confluent with both the channel and the collagen matrix. GFP represents expression of actin fibers within the cells. (c) Overall view of the endothelial monolayer confluent within the channel of the microfluidic device. (d) Ortho view of the endothelial monolayer. The top image shows homogeneous confluence of cellular monolayer formed throughout the channel. Blue territory represents the PDMS posts.</p