Share Email Print
cover

Proceedings Paper

Autofluorescence multiphoton microscopy for quality control of human vascular tissue constructs (Conference Presentation)
Author(s): Daniel A. Gil; Gaurav Kaushik; Elizabeth Torr; Elizabeth S. Berge; Cheryl Soref; Peyton Uhl; Gianluca Fontana; Jessica Antosiewicz-Bourget; Collin Edington; Michael P. Schwartz; Linda G. Griffith; James A. Thomson; William T. Daly; William L. Murphy; Melissa C. Skala
cover GOOD NEWS! Your organization subscribes to the SPIE Digital Library. You may be able to download this paper for free. Check Access

Paper Abstract

Engineered tissues offer great promise as engrafted therapies and in vitro models, but these tissues require a vascular network to retain viability at large scales. Significant efforts are focused on optimizing these in vitro vascular constructs, yet current evaluation methods require fixation and immunostaining. These destructive evaluation methods alter vascular network morphology, and cannot non-invasively monitor vascular assembly over time. Here, we demonstrate that autofluorescence multiphoton microscopy (MPM) can quantitatively assess the morphology of living 3D vascular networks without fixation, labels, or dyes. Autofluorescence MPM was used to non-invasively monitor the effect of culture conditions on 3D vascular network formation. Human embryonic stem (ES) cell-derived endothelial cells and primary human pericytes cultured in polyethylene glycol (PEG) hydrogels self-assembled into 3D vascular networks. Autofluorescence MPM of the metabolic co-enzyme NAD(P)H (excitation/emission wavelengths of 750 nm/400-460 nm) was used to quantify morphological parameters at day 6 of culture. Specifically, vessel diameter, vascular density, branch point density, and integration of endothelial cells into the network were quantified. Dynamic culture conditions (flow at 1μL/sec) led to vascular networks with higher mean vessel diameter compared to static culture (p<0.05). Standard immunohistochemistry found that vascular networks were positive for markers of endothelial cells, pericytes, and tight junctions. Scanning electron micrographs confirmed vessel lumen formation with pericytes wrapped around vessels. Dye transit of FITC-dextran through the network confirmed leaky endothelial barrier function. Our results demonstrate that autofluorescence MPM can non-invasively evaluate in vitro 3D vascular networks, and could be used for quality control of engineered tissues.

Paper Details

Date Published: 14 March 2018
PDF
Proc. SPIE 10497, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XVI, 104970L (14 March 2018); doi: 10.1117/12.2290562
Show Author Affiliations
Daniel A. Gil, Univ. of Wisconsin-Madison (United States)
Gaurav Kaushik, Univ. of Wisconsin-Madison (United States)
Elizabeth Torr, Univ. of Wisconsin-Madison (United States)
Elizabeth S. Berge, Morgridge Institute for Research (United States)
Univ. of Wisconsin-Madison (United States)
Cheryl Soref, Univ. of Wisconsin-Madison (United States)
Peyton Uhl, Univ. of Wisconsin-Madison (United States)
Gianluca Fontana, Univ. of Wisconsin-Madison (United States)
Jessica Antosiewicz-Bourget, Morgridge Institute for Research (United States)
Collin Edington, Massachusetts Institute of Technology (United States)
Michael P. Schwartz, Univ. of Wisconsin-Madison (United States)
Linda G. Griffith, Massachusetts Institute of Technology (United States)
James A. Thomson, Morgridge Institute for Research (United States)
William T. Daly, Univ. of Wisconsin-Madison (United States)
William L. Murphy, Univ. of Wisconsin-Madison (United States)
Melissa C. Skala, Morgridge Institute for Research (United States)
Univ. of Wisconsin-Madison (United States)


Published in SPIE Proceedings Vol. 10497:
Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XVI
Daniel L. Farkas; Dan V. Nicolau; Robert C. Leif, Editor(s)

© SPIE. Terms of Use
Back to Top