Incorporating a ECM hydrogel into an in vitro microvasculature system – Part II and update
This is just an update for the experiments Tejas and I have been working on for the past two weeks:
First, we tried incorporating 20 μl of Geltrex (10 mg/ml, diluted with DMEM from stock concentration: i.e. 15.6 mg/ml) in the back of a non-porous chip with 0.02 μm blue fluorescent nano-spheres (1:10 dilution from stock concentration). These are a couple of pictures of how this arrangement looks like under normal light (top panel) and a UV light lamp (bottom panel):
Non-porous chip with Geltrex seeded with fluorescent nano-beads on the back side.
Last time, we attempted to do a Z-scan in a system where we put fluorescent beads diluted in water on the top (flat) side of a non-porous chip and fluorescent beads of a different color/size suspended in Geltrex on the other side (bottom or edged side). We tried this set up again last week:
Top and back sides of a non-porous chip using fluorescent beads for visualization.
In the first few frames, a monolayer of fluorescent beads can be seen in one of the windows of the chip and then, in later frames, different Z-planes from the back of the chip come into focus as we manually move through the gel.
Update:
Last week we tried a system in which we used fMLP to asses whether we could see neutrophil migration through an ECM mimetic hydrogel (15.6 mg/ml Geltrex, polymerized at 37ºC for 40 minutes) incorporated at the back of a chip. For this test, we used chips without membranes that were set on a PDMS oval gasket containing fMLP (0.1 μM) or PBS 1x in the case of the control. On the flat side of the chip we put another gasket to accomodate the neutrophils at the beginning of the experiment.
After 1.5 h, we performed the following Z-scans for the control and the setup containing the chemoattractant.
Control:
fMLP (0.1 μM):
The first image was taken, in both cases, in a Z-plane that is near the level of the chip windows. Later Z-planes were taken as we went through the gel towards to back of the chip and then back again towards the front side. In the control experiment, most of the neutrophils were located near the window level of the chip. The neutrophils, in the arrangement containing fMLP, were observed towards a middle slice within the gel in the back of the chip.