Dynamic cell culture: a microfluidic function generator for live cell microscopy.

We present a microfluidic system for time-lapsed, live cell microscopy with the ability to control solution exchange via a dynamic flow controller. The application specific microfluidic plates are designed to maintain adherent and non-adherent cell types for multiple days with continuous medium perfusion. Upstream channels with flow controlled via custom software allow the delivery of unique exposure profiles to the cultured cells, such as square waves, step functions, ramps, etc.

Associating the mesoscale fiber organization of the tongue with local strain rate during swallowing.

The tongue is an intricately configured muscular organ that undergoes a stereotypical set of deformations during the course of normal human swallowing. In order to demonstrate quantitatively the relationship between 3D aligned lingual fiber organization and mechanics during swallowing, the tissue's myoarchitecture and strain rate were imaged before and during the propulsive phase of a 3.0ml water bolus swallow. Mesoscale fiber organization was imaged with high-resolution diffusion tensor imaging (DTI) and multi-voxel myofiber tracts generated along maximum diffusion vectors. Tissue compression/expansion was obtained via lingual pressure-gated phase-contrast (PC) MRI, a method which determines local strain rate as a function of the phase shift occurring along an applied gradient vector. The co-alignment of myofiber tract direction and the localized principal strain rate vectors was obtained by translating the strain rate tensor into the reference frame with the primary axis parallel to the maximum diffusion vector using Mohr's circle, resulting in the generation of fiber-aligned strain rate (FASR). DTI tractography displayed the complete fiber anatomy of the tongue, consisting of a core region of orthogonally aligned fibers encased within a longitudinal sheath, which merge with the externally connected styloglossus, hyoglossus, and genioglossus fibers. FASR images obtained in the mid-sagittal plane demonstrated that bolus propulsion was associated with prominent compressive strain aligned with the genioglossus muscle combined with expansive strain aligned with the verticalis and geniohyoid muscles. These data demonstrate that lingual deformation during swallowing involves complex interactions involving intrinsic and extrinsic muscles, whose contractility is directed by the alignment of mesoscale fiber tracts.

Resolving the three-dimensional myoarchitecture of bovine esophageal wall with diffusion spectrum imaging and tractography.

In order to determine the three-dimensional (3D) resolved muscular anatomy of the mammalian esophagus, we have examined its myoarchitecture with diffusion spectrum magnetic resonance imaging (DSI) and tractography. DSI measures diffusion displacement as a function of magnetic gradients of varied direction and intensity and displays the displacement profile as a 3D contour per voxel. In tractography, the orientation vectors of maximum diffusion/voxel are identified, and intervoxel associations are constructed by a streamline algorithm based on angular similarity in order to generate mesoscale myofiber tracts. We demonstrate that the proximal body of the esophagus consists of helically aligned crossing fiber populations that overlap between layers in the form of a "zipper" region along the length of the tissue. With increasingly distal position along the length of the tissue, helix angle and skeletal muscle prevalence are reduced such that fibers align themselves in the most distal location into distinct inner circular and outer longitudinal smooth muscle layers. We conclude that esophageal myoanatomy consists of crossing myofibers exhibiting a decreasing degree of helicity as a function of axial position and propose that this unique geometric construct provides a mechanism to resist distension and promote aboral flow.

Multiscale structural analysis of mouse lingual myoarchitecture employing diffusion spectrum magnetic resonance imaging and multiphoton microscopy.

The tongue consists of a complex, multiscale array of myofibers that comprise the anatomical underpinning of lingual mechanical function. 3-D myoarchitecture was imaged in mouse tongues with diffusion spectrum magnetic resonance imaging (DSI) at 9.4 T (b(max) 7000 smm, 150-microm isotropic voxels), a method that derives the preferential diffusion of water/voxel, and high-throughput (10 fps) two-photon microscope (TPM). Net fiber alignment was represented for each method in terms of the local maxima of an orientational distribution function (ODF) derived from the local diffusion (DSI) and 3-D structural autocorrelation (TPM), respectively. Mesoscale myofiber tracts were generated by alignment of the principal orientation vectors of the ODFs. These data revealed a consistent relationship between the properties of the respective ODFs and the virtual superimposition of the distributed mesoscale myofiber tracts. The identification of a mesoscale anatomical construct, which specifically links the microscopic and macroscopic spatial scales, provides a method for relating the orientation and distribution of cells and subcellular components with overall tissue morphology, thus contributing to the development of multiscale methods for mechanical analysis.

Anatomical basis of lingual hydrostatic deformation.

The mammalian tongue is believed to fall into a class of organs known as muscular hydrostats, organs for which muscle contraction both generates and provides the skeletal support for motion. We propose that the myoarchitecture of the tongue, consisting of intricate arrays of muscular fibers, forms the structural basis for hydrostatic deformation. Owing to the fact that maximal diffusion of the ubiquitous water molecule occurs orthogonal to the short axis of most fiber-type cells, diffusion-weighted magnetic resonance imaging (MRI) measurements can be used to derive information regarding 3-D fiber orientation in situ. Image data obtained in this manner suggest that the tongue consists of a complex juxtaposition of muscle fibers oriented in orthogonal arrays, which provide the basis for multidirectional contraction and isovolemic deformation. From a mechanical perspective, the lingual tissue may be considered as set of continuous coupled units of compression and expansion from which 3-D strain maps may be derived. Such functional data demonstrate that during physiological movements, such as protrusion, bending and swallowing, hydrostatic deformation occurs via synergistic contractions of orthogonally aligned intrinsic and extrinsic fibers. Lingual deformation can thus be represented in terms of models demonstrating that synergistic contraction of fibers at orthogonal or near-orthogonal directions to each other is a necessary condition for volume-conserving deformation. Evidence is provided in support of the supposition that hydrostatic deformation is based on the contraction of orthogonally aligned intramural fibers functioning as a mechanical continuum.

Three dimensional myoarchitecture of the human tongue determined in vivo by diffusion tensor imaging with tractography.

PURPOSE: To study the anatomical relationships involving the intrinsic and extrinsic myofiber populations of the human tongue employing diffusion tensor imaging (DTI) with tractography. MATERIALS AND METHODS: Images of the human tongue in vivo were obtained using a twice-refocused spin echo DTI pulse sequence at 1.5 T, isotropic 3 x 3 x 3 mm(3) voxels, b-value 500 seconds/mm(2), and 90 different diffusion sensitizing gradient directions. Multivoxel tracts were generated along the vectors, corresponding to the directions of maximal diffusion in each voxel. The data was visualized using custom fiber tracking software and images compared with known anatomy. RESULTS: DTI tractography depicts the complete three-dimensional (3D) myoarchitecture of the human tongue, specifically demonstrating the geometric relationships between the intrinsic and extrinsic myofiber populations. These results define the manner in which key extrinsic fiber populations merge with the longitudinally-, transversely-, and vertically-aligned intrinsic fibers. CONCLUSION: The current results display for the first time the use of DTI tractography in vivo to visualize the complete structural anatomy of the human tongue and allow us to consider fundamental structure-function relationships.

Microfluidic tissue model for live cell screening.

We have developed a microfluidic platform modeled after the physiologic microcirculation for multiplexed tissue-like culture and high-throughput analysis. Each microfabricated culture unit consisted of three functional components: a 50 microm wide cell culture pocket, an artificial endothelial barrier with 2 microm pores, and a nutrient transport channel. This configuration enabled a high density of cancer cells to be maintained for over 1 week in a solid tumor-like morphology when fed with continuous flow. The microfluidic chip contained 16 parallel units for "flow cell" based experiments where live cells were exposed to a soluble factor and analyzed via fluorescence microscopy or flow-through biochemistry. Each fluidically independent tissue unit contained approximately 500 cells fed with a continuous flow of 10 nL/min. As a demonstration, the toxicity profile of the anti-cancer drug paclitaxel was collected on HeLa cells cultured in the microfluidic format and compared with a 384-well dish for up to 5 days of continuous drug exposure.

Mechanical basis for lingual deformation during the propulsive phase of swallowing as determined by phase-contrast magnetic resonance imaging.

The tongue is an intricately configured muscular organ that undergoes a series of rapid shape changes intended to first configure and then transport the bolus from the oral cavity to the pharynx during swallowing. To assess the complex array of mechanical events occurring during the propulsive phase of swallowing, we employed tongue pressure-gated phase-contrast MRI to represent the tissue's local strain rate vectors. Validation of the capacity of phase-contrast MRI to represent local compressive and expansive strain rate was obtained by assessing deformation patterns induced by a synchronized mechanical plunger apparatus in a gelatinous material phantom. Physiological strain rate data were acquired in the sagittal and coronal orientations at 0, 200, 400, and 600 ms relative to the gating pulse during 2.5-ml water bolus swallows. This method demonstrated that the propulsive phase of swallowing is associated with a precisely organized series of compressive and expansive strain rate events. At the initiation of propulsion, bolus position resulted from obliquely aligned compressive and expansive strain, vertically aligned compressive strain and orthogonal expansion, and compressive strain aligned obliquely to the styloid process. Bolus reconfiguration and translocation resulted from a combination of compressive strain occurring in the middle and posterior tongue aligned obliquely between the anterior-inferior and the posterior-superior regions with commensurate orthogonal expansion, along with bidirectional contraction in the distribution of the transversus and verticalis muscle fibers. These data support the concept that propulsive lingual deformation is due to complex muscular interactions involving both extrinsic and intrinsic muscles.

Microfluidic System for Automated Cell-based Assays.

Microfluidic cell culture is a promising technology for applications in the drug screening industry. Key benefits include improved biological function, higher quality cell-based data, reduced reagent consumption, and lower cost. In this work, we demonstrate how a microfluidic cell culture design was adapted to be compatible with the standard 96-well plate format. Key design features include the elimination of tubing and connectors, the ability to maintain long term continuous perfusion cell culture using a passive gravity driven pump, and direct analysis on the outlet wells of the microfluidic plate. A single microfluidic culture plate contained 8 independent flow units, each with 10(4) cells at a flow rate of 50 µl/day (6 minute residence time). The cytotoxicity of the anti-cancer drug etoposide was measured on HeLa cells cultured in this format, using a commercial lactate dehydrogenase (LDH) plate reader assay. The integration of microfluidic cell culture methods with commercial automation capabilities offers an exciting opportunity for improved cell-based screening.