Abstract
BackgroundThe engineering of functional tissues is a complex multi-stage process, the success of which depends on the careful control of culture conditions and ultimately tissue maturation. To enable the efficient optimization of tissue development protocols, techniques suitable for monitoring the effects of added stimuli and induced tissue changes are needed.Methodology/Principal FindingsHere, we present the quantitative use of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) as a noninvasive means to monitor the differentiation of human mesenchymal stem cells (hMSCs) using entirely endogenous sources of contrast. We demonstrate that the individual fluorescence contribution from the intrinsic cellular fluorophores NAD(P)H, flavoproteins and lipofuscin can be extracted from TPEF images and monitored dynamically from the same cell population over time. Using the redox ratio, calculated from the contributions of NAD(P)H and flavoproteins, we identify distinct patterns in the evolution of the metabolic activity of hMSCs maintained in either propagation, osteogenic or adipogenic differentiation media. The differentiation of these cells is mirrored by changes in cell morphology apparent in high resolution TPEF images and by the detection of collagen production via SHG imaging. Finally, we find dramatic increases in lipofuscin levels in hMSCs maintained at 20% oxygen vs. those in 5% oxygen, establishing the use of this chromophore as a potential biomarker for oxidative stress.Conclusions/SignificanceIn this study we demonstrate that it is possible to monitor the metabolic activity, morphology, ECM production and oxidative stress of hMSCs in a non-invasive manner. This is accomplished using generally available multiphoton microscopy equipment and simple data analysis techniques, such that the method can widely adopted by laboratories with a diversity of comparable equipment. This method therefore represents a powerful tool, which enables researchers to monitor engineered tissues and optimize culture conditions in a near real time manner.
Highlights
The goal of tissue engineering is the development of functional tissue equivalents for the repair or replacement of that lost to damage or disease [1]
Based on multispectral analysis approaches, we demonstrate that the major endogenous chromophores that contribute to the observed two-photon excited fluorescence (TPEF) signal from human mesenchymal stem cells (hMSCs) include NAD(P)H, flavoroteins and lipofuscin
Identification of intrinsic cellular fluorophores To identify the intrinsic cellular fluorophores excited in the hMSCs at 755 nm and 860 nm, TPEF emission spectra were collected from 400 nm to 700 nm for each excitation wavelength (Figure 1A)
Summary
The goal of tissue engineering is the development of functional tissue equivalents for the repair or replacement of that lost to damage or disease [1]. Tissues are constructed via a complex process in which non-terminally differentiated cells, such as human mesenchymal stem cells (hMSCs), are combined with a biomaterial scaffold and induced to differentiate into a functional tissue via a variety of physical and/or chemical stimuli. Design and optimization of culture protocols requires close monitoring of multiple tissue characteristics during the development of the engineered tissue. The engineering of functional tissues is a complex multi-stage process, the success of which depends on the careful control of culture conditions and tissue maturation. To enable the efficient optimization of tissue development protocols, techniques suitable for monitoring the effects of added stimuli and induced tissue changes are needed
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