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J Tissue Eng Regen Med. 2016 Nov;10(11):955-966. doi: 10.1002/term.1876. Epub 2014 Feb 26.

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

Author information

1
Wake Forest Institute for Regenerative Medicine, Wake Forest Baptist Medical Center, Medical Center Boulevard, Winston-Salem, NC.
2
Virginia Tech-Wake Forest University School of Biomedical Engineering and Sciences, Virginia Tech, Blacksburg, VA, USA.
3
Bradley Department of Electrical and Computer Engineering, Wake Forest University School of Biomedical Engineering and Sciences, Virginia Tech, Blacksburg, VA, USA.

Abstract

Fluorescent imaging is a useful tool to monitor 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.

KEYWORDS:

autofluorescence; blood vessel; endothelial cells

PMID:
24616385
PMCID:
PMC4145050
DOI:
10.1002/term.1876
[Indexed for MEDLINE]
Free PMC Article

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