The potential of label-free nonlinear optical molecular microscopy to non-invasively characterize the viability of engineered human tissue constructs

Abstract

Nonlinear optical molecular imaging and quantitative analytic methods were developed to non-invasively assess the viability of tissue-engineered constructs manufactured from primary human cells. Label-free optical measures of local tissue structure and biochemistry characterized morphologic and functional differences between controls and stressed constructs. Rigorous statistical analysis accounted for variability between human patients. Fluorescence intensity-based spatial assessment and metabolic sensing differentiated controls from thermally-stressed and from metabolically-stressed constructs. Fluorescence lifetime-based sensing differentiated controls from thermally-stressed constructs. Unlike traditional histological (found to be generally reliable, but destructive) and biochemical (non-invasive, but found to be unreliable) tissue analyses, label-free optical assessments had the advantages of being both non-invasive and reliable. Thus, such optical measures could serve as reliable manufacturing release criteria for cell-based tissue-engineered constructs prior to human implantation, thereby addressing a critical regulatory need in regenerative medicine.

Optical methods for quantitative and label-free sensing in living human tissues: principles, techniques, and applications

“…In particular, a variety of NLO techniques have been applied to address fundamental and applied studies in biology and medicine [86–89], as illustrated in Figure 10, including exciting applications of two-photon excitation fluorescence (TPEF) and second-harmonic generation (SHG) to label-free molecular sensing and imaging in clinical medicine [90, 91] and emerging in vivo applications of coherent anti-Stokes Raman scattering (CARS) [92]. …”

“…An illustration of using an NLO-based approach to characterize morphology and biochemistry in living engineered tissues is shown in Figure 11 [91]. While histological assessment would destroy the tissue construct, NLO microscopic imaging could be performed non-invasively via optical sectioning in 3-dimensions (3D) in the living tissue (left) under environmentally controlled and sterile conditions.…”

“…For these studies, cells and tissues were cultured from distinct primary human patient donors to intentionally introduce the intra-patient variability anticipated in clinical use [91]. Additionally, realistic stressing conditions anticipated during the biofabrication process were introduced to intentionally compromise cell viability.…”

“…Some DOS/DOT techniques, such as those involving streak cameras, can even enable near-ps scale temporal resolution to measure photon time-of-flight distributions in tissue [98, 99]. NLO methods provide microscopic spatial resolution for imaging of structures such as cells and collagen fibers in vivo [91]. Certain NLO techniques, such as those involving fluorescence lifetime, can also provide sub-ns temporal resolution to quantify the dynamic behavior of fluorescent molecules in living tissue [91].…”

“…NLO methods provide microscopic spatial resolution for imaging of structures such as cells and collagen fibers in vivo [91]. Certain NLO techniques, such as those involving fluorescence lifetime, can also provide sub-ns temporal resolution to quantify the dynamic behavior of fluorescent molecules in living tissue [91]. …”