Microfabricated devices are useful tools for manipulating and interrogating large numbers of single cells in a rapid and cost-effective manner, but connecting these systems to the existing platforms used in routine high-throughput screening of libraries of cells remains challenging. Methods to sort individual cells of interest from custom microscale devices to standardized culture dishes in an efficient and automated manner without affecting the viability of the cells are critical. Combining a commercially available instrument for colony picking (CellCelector, AVISO GmbH) and a customized software module, we have established an optimized process for the automated retrieval of individual antibody-producing cells, secreting desirable antibodies, from dense arrays of subnanoliter containers. The selection of cells for retrieval is guided by data obtained from a high-throughput, single-cell screening method called microengraving. Using this system, 100 clones from a mixed population of two cell lines secreting different antibodies (12CA5 and HYB099-01) were sorted with 100% accuracy (50 clones of each) in approximately 2 h, and the cells retained viability.

Objectives/Hypothesis To develop a vocal fold scarring model using an ablative laser in the rabbit as a platform for testing bioengineered therapies for missing or damaged lamina propria. Study Design Prospective controlled animal study. Methods An optimal laser energy level was first determined by assessing the depths of vocal fold injury created by a Holmium:YAG laser at various energy levels on fresh cadaveric rabbit larynges. The selected energy level was then used to create controlled unilateral injuries in vocal folds of New Zealand white rabbits, with the contralateral folds serving as uninjured controls. After 4 weeks, the larynges were harvested and subjected to excised-larynx phonation with high-speed imaging and immunohistochemical staining for collagen types I and III, elastin, and hyaluronic acid (HA) with quantitative histological analysis. Results 1.8 joules produced full-thickness injury of the lamina propria without extensive muscle injury. After 4 weeks, the injured vocal folds vibrated with reduced amplitude (P = 0.036) in excised-larynx phonation compared to normal vocal folds. The injured vocal folds contained a higher relative density of collagen type I (P = 0.004), higher elastin (P = 0.022), and lower HA (P = 0.030) compared to normal controls. Collagen type III was unchanged. Conclusions With its potential for higher precision of injury, this laser vocal fold scarring model may serve as an alternative to scarring produced by cold instruments for studying the effects of vocal fold lamina propria bioengineered therapies. Level of Evidence N/A.

One of the primary mechanisms to vary one's vocal frequency is through vocal fold length changes. As stress and deformation are linked to each other, it is hypothesized that the anisotropy in the biomechanical properties of the vocal fold tissue would affect the phonation characteristics. A biomechanical model of vibrational frequency rise during vocal fold elongation is developed which combines an advanced biomechanical characterization protocol of the vocal fold tissue with continuum beam models. Biomechanical response of the tissue is related to a microstructurally informed, anisotropic, nonlinear hyperelastic constitutive model. A microstructural characteristic (the dispersion of collagen) was represented through a statistical orientation function acquired from a second harmonic generation image of the vocal ligament. Continuum models of vibration were constructed based upon Euler-Bernoulli and Timoshenko beam theories, and applied to the study of the vibration of a vocal ligament specimen. From the natural frequency predictions in dependence of elongation, two competing processes in frequency control emerged, i.e., the applied tension raises the frequency while simultaneously shear deformation lowers the frequency. Shear becomes much more substantial at higher modes of vibration and for highly anisotropic tissues. The analysis was developed as a case study based on a human vocal ligament specimen.

The vocal folds are known to be mechanically anisotropic due to the microstructural arrangement of fibrous proteins such as collagen and elastin in the lamina propria. Even though this has been known for many years, the biomechanical anisotropic properties have rarely been experimentally studied. We propose that an indentation procedure can be used with uniaxial tension in order to obtain an estimate of the biomechanical anisotropy within a single specimen. Experiments were performed on the lamina propria of three male and three female human vocal folds dissected from excised larynges. Two experiments were conducted: each specimen was subjected to cyclic uniaxial tensile loading in the longitudinal (i.e. anterior-posterior) direction, and then to cyclic indentation loading in the transverse (i.e. medial-lateral) direction. The indentation experiment was modeled as contact on a transversely isotropic half-space using the Barnett-Lothe tensors. The longitudinal elastic modulus EL was computed from the tensile test, and the transverse elastic modulus ET and longitudinal shear modulus GL were obtained by inverse analysis of the indentation force-displacement response. It was discovered that the average of EL/ET was 14 for the vocal ligament and 39 for the vocal fold cover specimens. Also, the average of EL/GL, a parameter important for models of phonation, was 28 for the vocal ligament and 54 for the vocal fold cover specimens. These measurements of anisotropy could contribute to more accurate models of fundamental frequency regulation and provide potentially better insights into the mechanics of vocal fold vibration.

Traditionally, viscoelastic shear properties of human vocal fold tissues have been described by the linear viscoelastic moduli G' and G” at small strain amplitudes. However, once the mechanical behavior becomes nonlinear these moduli are no longer sufficient for viscoelastic characterization. MITlaos, a rheological framework developed for better describing such behavior, was used to characterize the nonlinear viscoelastic properties of the vocal fold lamina propria when subjected to large-amplitude oscillatory shear (LAOS). MITlaos involved Fourier transform processing to convert raw stress–strain signals into a filtered/smoothed total stress signal based on the odd-integer harmonic components, with nonlinear viscoelastic parameters simultaneously derived. Rheometric testing of vocal fold cover specimens was performed with increasing strain amplitudes using a controlled-strain, simple-shear rheometer. Smoothed Lissajous–Bowditch curves generated from MITlaos were plotted in Pipkin space, and the nonlinear analysis was summarized by variations of rheological fingerprints. Pipkin diagrams served as a geometric means for identifying the onset of nonlinear behavior, and any distortions due to noise. Results showed that the human vocal fold cover when subjected to LAOS demonstrated intracycle strain stiffening and intercycle strain softening with increasing strain amplitude, as well as shear thinning with increasing strain-rate amplitude. [Work supported by NIH.]