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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.

SUBMITTER: Chen LC

PROVIDER: S-EPMC4106121 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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

Chen Leng-Chun LC Lloyd William R WR Kuo Shiuhyang S Kim Hyungjin Myra HM Marcelo Cynthia L CL Feinberg Stephen E SE Mycek Mary-Ann MA

Biomaterials 20140520 25

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 metabol ...[more]

PMID: 24854093

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Project description:SignificanceNeedle biopsy (NB) procedures are important for the initial diagnosis of many types of cancer. However, the possibility of NB specimens being unable to provide diagnostic information, (i.e., non-diagnostic sampling) and the time-consuming histological evaluation process can cause delays in diagnoses that affect patient care.AimWe aim to demonstrate the advantages of this label-free multimodal nonlinear optical imaging (NLOI) technique as a non-destructive point-of-procedure evaluation method for NB tissue cores, for the visualization and characterization of the tissue microenvironment.ApproachA portable, label-free, multimodal NLOI system combined second-harmonic generation (SHG) and third-harmonic generation and two- and three-photon autofluorescence (2PF, 3PF) microscopy. It was used for intraoperative imaging of fresh NB tissue cores acquired during canine cancer surgeries, which involved liver, lung, and mammary tumors as well as soft-tissue sarcoma; in total, eight canine patients were recruited. An added tissue culture chamber enabled the use of this NLOI system for longitudinal imaging of fresh NB tissue cores taken from an induced rat mammary tumor and healthy mouse livers.ResultsThe intraoperative NLOI system was used to assess fresh canine NB specimens during veterinary cancer surgeries. Histology-like morphological features were visualized by the combination of four NLOI modalities at the point-of-procedure. The NLOI results provided quantitative information on the tissue microenvironment such as the collagen fiber orientation using Fourier-domain SHG analysis and metabolic profiling by optical redox ratio (ORR) defined by 2PF/(2PF + 3PF). The analyses showed that the canine mammary tumor had more randomly oriented collagen fibers compared to the tumor margin, and hepatocarcinoma had a wider distribution of ORR with a lower mean value compared to the liver fibrosis and the normal-appearing liver. Moreover, the loss of metabolic information during tissue degradation of fresh murine NB specimens was shown by overall intensity decreases in all channels and an increase of mean ORR from 0.94 (standard deviation 0.099) to 0.97 (standard deviation 0.077) during 1-h longitudinal imaging of a rat mammary tumor NB specimen. The tissue response to staurosporine (STS), an apoptotic inducer, from fresh murine liver NB specimens was also observed. The mean ORR decreased from 0.86 to 0.74 in the first 40 min and then increased to 0.8 during the rest of the hour of imaging, compared to the imaging results without the addition of STS, which showed a continuous increase of ORR from 0.72 to 0.75.ConclusionsA label-free, multimodal NLOI platform reveals microstructural and metabolic information of the fresh NB cores during intraoperative cancer imaging. This system has been demonstrated on animal models to show its potential to provide a more comprehensive histological assessment and a better understanding of the unperturbed tumor microenvironment. Considering tissue degradation, or loss of viability upon fixation, this intraoperative NLOI system has the advantage of immediate assessment of freshly excised tissue specimens at the point of procedure.

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

Publications

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

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