Fig 1.
Mesenchymal stem cell homing mimicked on a microfluidic device.
(a) Theoretical schematic of peripheral MSC homing process. (b) Illustration of the microfluidic device and stem cell homing on-chip.
Fig 2.
Extravasation of MSCs under a gradient of homing factor.
(a) An image of the PDMS-based microfluidic device. (b) The microfluidic device set up for cell seeding. Micropipette tips were used as a media reservoir. (c) A FITC-dextran gradient within the collagen matrix on a microfluidic device with endothelial monolayers (blue squares) embedded in cell channels. Red arrows indicate the direction of MSC migration due to an SDF-1α gradient. The graph indicates the fluorescence intensity of dextran within the chip. (d) A microscopic image of MSC extravasation and migration through the collagen matrix on a microfluidic device. (e) RFP-tagged endothelial cells are used to distinguish migrating MSCs. (f) MSC extravasation. The white arrow indicates MSCs, and red arrow indicates endothelial cells.
Fig 3.
Confocal microscopic images of endothelial monolayer formed within the channel of the microfluidic device.
(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.
Fig 4.
3D confocal images of MSCs migrating transendothelially.
(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.
Fig 5.
Numbers of extravasating and directionally migrating MSCs in the direction of the SDF-1α gradient under different conditions.
(a) Control group; no inhibitor and no SDF-1α gradient. *p>0.05 (b) MSCs under SDF-1α gradient *p<0.05 (c) MSCs under AMD3100 (CXCR4 antagonist) treatment. *p>0.05 (d) MSCs under Y-27632 (Rho-ROCK inhibitor) treatment *p<0.05. (e) MSCs under NSC23766 (RAC inhibitor) treatment *p<0.05. (f-j) Graph of numbers of extravasated MSCs moving toward and away from the SDF-1α gradient. Y-axis represents numbers of cells, and X-axis represents number of days. White arrows indicate the extravasated MSCs. Paired t-test was applied for number of MSCs migrating on each side for each conditions with n = 8.
Fig 6.
Migration distance of MSCs over the duration of the experiment.
Blue dots in the collagen matrix indicate the locations of nuclei of MSCs (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.
Fig 7.
Morphology of F-actin-stained MSCs under different conditions (branch counting).
(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.
Fig 8.
Illustration of extravasation and directional migration of MSCs in different conditions.
(a) Morphology of a normal MSC. (b) An MSC under SDF-1α gradient. (c) An MSC exposed to Y-27632, a Rho-ROCK inhibitor. (d) An MSC exposed to AMD3100, a CXCR4 antagonist. (e) MSC exposed to NSC23766, a Rac inhibitor. (f) Migration of MSCs due to the chemokine effect of SDF-1α. Blue color represents the number of stem cells migrating in response to the homing factor.