title: Characterization of metabolic changes associated with the functional development of 3D engineered tissues by non-invasive, dynamic measurement of individual cell redox ratios.
abstract: Non-invasive approaches to assess tissue function could improve significantly current methods to diagnose diseases and optimize engineered tissues. In this study, we describe a two-photon excited fluorescence microscopy approach that relies entirely on endogenous fluorophores to dynamically quantify functional metabolic readouts from individual cells within three-dimensional engineered tissues undergoing adipogenic differentiation over six months. Specifically, we employ an automated approach to analyze 3D image volumes and extract a redox ratio of metabolic cofactors. We identify a decrease in redox ratio over the first two months of culture that is associated with stem cell differentiation and lipogenesis. In addition, we demonstrate that the presence of endothelial cells facilitate greater cell numbers deeper within the engineered tissues. Since traditional assessments of engineered tissue structure and function are destructive and logistically intensive, this non-destructive, label-free approach offers a potentially powerful high-content characterization tool for optimizing tissue engineering protocols and assessing engineered tissue implants.
authors.affiliation: Department of Biomedical Engineering, Tufts University, 4 Colby Street, Medford, MA 02155, USA.
authors.forename: Kyle P
authors.initials: KP
authors.lastname: Quinn
authors.forename: Evangelia
authors.initials: E
authors.lastname: Bellas
authors.forename: Nikolaos
authors.initials: N
authors.lastname: Fourligas
authors.forename: Kyongbum
authors.initials: K
authors.lastname: Lee
authors.forename: David L
authors.initials: DL
authors.lastname: Kaplan
authors.forename: Irene
authors.initials: I
authors.lastname: Georgakoudi
mesh_terms: D000273:Adipose Tissue / Q000166:cytology; D001391:Azo Compounds / Q000378:metabolism; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D018920:Coculture Techniques; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted; D050356:Lipid Metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D036641:Microscopy, Fluorescence, Multiphoton; D055806:Microvessels / Q000166:cytology; D008954:Models, Biological; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D013194:Staining and Labeling; D013997:Time Factors; D023822:Tissue Engineering / Q000379:methods*
chemical_list: D001391:Azo Compounds; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; C011049:oil red O