Fluorescent imaging of endothelial cells in bioengineered blood vessels: the impact of crosslinking of the scaffold

Abstract

Fluorescent imaging is a useful tool tomonitor and evaluate bioengineered tissues and organs. However, autofluorescence emitted from the scaffold can be comparable or even overwhelm signals generated by fluorescently labelled cells and biomarkers. Using standard fluorescent microscopy techniques, a simple and easy-to-measure signal to noise ratio metric was developed, which can facilitate the selection of fluorescent biomarkers and the respective biomaterials for tissue engineering studies. Endothelial cells (MS1) expressing green-fluorescent protein and red fluorescent protein (mKate) were seeded on poly (epsilon-caprolactone)–collagen hybrid scaffolds that were prepared by crosslinking with glutaraldehyde, genipin and ethyl(dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide. All scaffolds had comparable mechanical properties, which could meet the requirements for vascular graft applications. ethyl(dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide crosslinked scaffolds had a high signal to noise ratio value because of its low autofluorescence in green and red channels. Genipin crosslinked scaffolds had a high signal to noise ratio only in the green channel, while glutaraldehyde crosslinked scaffolds had a low signal to noise ratio in both green and red channels. The signal to noise ratio was independent of the exposure time. The data show that although similar in their mechanical properties and ability to support cell growth, scaffolds crosslinked with different agents have significant differences in causing autofluorescence of the scaffolds. This result indicates that scaffold’s preparation method may have a significant impact on direct imaging of fluorescently labelled cells on scaffolds used for tissue engineering.

Efflux Pump Substrates Shuttled to Cytosolic or Vesicular Compartments Exhibit Different Permeability in a Quantitative Human Blood–Brain Barrier Model

“…Issues with monolayer confluence were attributed to the moving of or loss of the COL1 hydrogels on the glass surface. Conjugation of COL1 to EDC/NHS-activated glass addressed these issues and introduced no additional autofluorescence, similar to findings using EDC/NHS to cross-link COL1 hydrogels …”

“…Conjugation of COL1 to EDC/NHSactivated glass addressed these issues and introduced no additional autofluorescence, similar to findings using EDC/ NHS to cross-link COL1 hydrogels. 29 Quantifying Large and Small Molecule Permeability of hiPSC-BMECs on COL1 Hydrogels. In addition to the presence of relevant BMEC proteins, a BBB-like phenotype is confirmed by comparing the cellular permeability of benchmark molecules across a monolayer of cells to relevant reports.…”

Fiber‐based fluorescence lifetime imaging of recellularization processes on vascular tissue constructs

“…Although successful at monitoring cellular dynamics in 3D in vitro experiments, the regulatory barriers to the use of eGFP‐labeled cells in humans limit the in vivo applications of this approach. Other studies explored how to generate collagenous scaffolds with low autofluorescence levels, by using different crosslinking agents , but these alterations may impact cellular activity.…”

Imaging Cell Viability on Non-transparent Scaffolds — Using the Example of a Novel Knitted Titanium Implant

“…The combination of these two techniques has the major advantage that it can be easily transferred to other cell types, considering that incubation time might have to be adapted to the cell type of interest. However, care has to be taken when transferring this method to other nontransparent scaffolds, e.g., collagen based scaffolds which generally exhibit a strong green auto-fluorescence 17 . In this case other fluorescent tags might be used.…”