Design of a pulsatile flow facility to evaluate thrombogenic potential of implantable cardiac devices.

Due to expensive nature of clinical trials, implantable cardiac devices should first be extensively characterized in vitro. Prosthetic heart valves (PHVs), an important class of these devices, have been shown to be associated with thromboembolic complications. Although various in vitro systems have been designed to quantify blood-cell damage and platelet activation caused by nonphysiological hemodynamic shear stresses in these PHVs, very few systems attempt to characterize both blood damage and fluid dynamics aspects of PHVs in the same test system. Various numerical modeling methodologies are also evolving to simulate the structural mechanics, fluid mechanics, and blood damage aspects of these devices. This article presents a completely hemocompatible small-volume test-platform that can be used for thrombogenicity studies and experimental fluid mechanics characterization. Using a programmable piston pump to drive freshly drawn human blood inside a cylindrical column, the presented system can simulate various physiological and pathophysiological conditions in testing PHVs. The system includes a modular device-mounting chamber, and in this presented case, a 23 mm St. Jude Medical (SJM) Regents® mechanical heart valve (MHV) in aortic position was used as the test device. The system was validated for its capability to quantify blood damage by measuring blood damage induced by the tester itself (using freshly drawn whole human blood). Blood damage levels were ascertained through clinically relevant assays on human blood while fluid dynamics were characterized using time-resolved particle image velocimetry (PIV) using a blood-mimicking fluid. Blood damage induced by the tester itself, assessed through Thrombin-anti-Thrombin (TAT), Prothrombin factor 1.2 (PF1.2), and hemolysis (Drabkins assay), was within clinically accepted levels. The hydrodynamic performance of the tester showed consistent, repeatable physiological pressure and flow conditions. In addition, the system contains proximity sensors to accurately capture leaflet motion during the entire cardiac cycle. The PIV results showed skewing of the leakage jet, caused by the asymmetric closing of the two leaflets. All these results are critical to characterizing the blood damage and fluid dynamics characteristics of the SJM Regents® MHV, proving the utility of this tester as a precise system for assessing the hemodynamics and thrombogenicity for various PHVs.

Some aspects of aerodynamic flow control using synthetic-jet actuation.

Aerodynamic flow control effected by interactions of surface-mounted synthetic (zero net mass flux) jet actuators with a local cross flow is reviewed. These jets are formed by the advection and interactions of trains of discrete vortical structures that are formed entirely from the fluid of the embedding flow system, and thus transfer momentum to the cross flow without net mass injection across the flow boundary. Traditional approaches to active flow control have focused, to a large extent, on control of separation on stalled aerofoils by means of quasi-steady actuation within two distinct regimes that are characterized by the actuation time scales. When the characteristic actuation period is commensurate with the time scale of the inherent instabilities of the base flow, the jets can effect significant quasi-steady global modifications on spatial scales that are one to two orders of magnitude larger than the scale of the jets. However, when the actuation frequency is sufficiently high to be decoupled from global instabilities of the base flow, changes in the aerodynamic forces are attained by leveraging the generation and regulation of 'trapped' vorticity concentrations near the surface to alter its aerodynamic shape. Some examples of the utility of this approach for aerodynamic flow control of separated flows on bluff bodies and fully attached flows on lifting surfaces are also discussed.

Reduction of procoagulant potential of b-datum leakage jet flow in bileaflet mechanical heart valves via application of vortex generator arrays.

Current designs of bileaflet mechanical heart valves put patients at an increased risk of thromboembolism. In particular, regurgitant flow through the b-datum line is associated with nonphysiologic flow characteristics such as elevated shear stresses, regions of recirculation, and increased mixing, all of which may promote thrombus formation. We have previously shown that passive flow control in the form of vortex generators mounted on the downstream leaflet surfaces can effectively diminish turbulent stresses. The objective of the current work is thus to determine the effect of vortex generators on the thromboembolic potential of the b-datum line leakage jet and to correlate that effect with the vortex generator-induced changes to the flow structure. Flow experiments were performed using a steady model of the transient b-datum line jet. These experiments encompassed flow visualization to gain an overall picture of the flow system, particle image velocimetry to quantify the flow field in detail, and in vitro experiments with human blood to quantify thrombus formation in response to the applied passive flow control. Thrombus formation was quantified over time by an assay for thrombin-antithrombin III (TAT III). In comparing results with and without vortex generators, significantly lower mean TAT III levels were observed at one time point for the case with vortex generators. Also, the TAT III growth rate of the case with vortex generators was significantly lower. While no differences in jet spreading were found with and without vortex generators, lower peak turbulent stresses were observed for the case with vortex generators. The results thus demonstrate the potential of applying passive flow control to cardiovascular hardware in order to mitigate the hemodynamic factors leading to thrombus formation.

A microperfused incubator for tissue mimetic 3D cultures.

High density, three-dimensional (3D) cultures present physical similarities to in vivo tissue and are invaluable tools for pre-clinical therapeutic discoveries and development of tissue engineered constructs. Unfortunately, the use of dense cultures is hindered by intra-culture transport limits allowing just a few layer thick cultures for reproducible studies. In order to overcome diffusion limits in intra-culture nutrient and gas availability, a simple scalable microfluidic perfusion platform was developed and validated. A novel perfusion approach maintained laminar flow of nutrients through the culture to meet metabolic need, while removing depleted medium and catabolites. Velocity distributions and 3D flow patterns were measured using microscopic particle image velocimetry. The effectiveness of forced convection laminar perfusion was confirmed by culturing 700 microm thick neural-astrocytic (1:1) constructs at cell density approaching that of the brain (50,000 cells/mm(3)). At the optimized flow rate of the nutrient medium, the culture viability reached 90% through the full construct thickness at 2 days of perfusion while unperfused controls exhibited widespread cell death. The membrane aerated perfusion platform was integrated within a miniature, imaging accessible enclosure enabling temperature and gas control of the culture environment. Temperature measurements demonstrated fast feedback response to environmental changes resulting in the maintenance of the physiological temperature within 37 +/- 0.2 degrees C. Reproducible culturing of tissue equivalents within dynamically controlled environments will provide higher fidelity to in vivo function in an in vitro accessible format for cell-based assays and regenerative medicine.

Culturing thick brain slices: an interstitial 3D microperfusion system for enhanced viability.

Brain slice preparations are well-established models for a wide spectrum of in vitro investigations in the neuroscience discipline. However, these investigations are limited to acute preparations or thin organotypic culture preparations due to the lack of a successful method that allows culturing of thick organotypic brain slices. Thick brain slice cultures suffer necrosis due to ischemia deep in the tissue resulting from a destroyed circulatory system and subsequent diffusion-limited supply of nutrients and oxygen. Although thin organotypic brain slice cultures can be successfully cultured using a well-established roller-tube method (a monolayer organotypic culture) (Gahwiler B H. Organotypic monolayer cultures of nervous tissue. J Neurosci Methods. 1981; 4: 329-342) or a membrane-insert method (up to 1-4 cell layers, <150 microm) (Stoppini L, Buchs PA, Muller D. A simple method for organotypic cultures of neural tissue. J Neurosci Methods 1991; 37: 173-182), these methods fail to support thick tissue preparations. A few perfusion methods (using submerged or interface/microfluidic chambers) have been reported to enhance the longevity (up to few hours) of acute slice preparations (up to 600 microm thick) (Hass HL, Schaerer B, Vosmansky M. A simple perfusion chamber for study of nervous tissue slices in vitro. J Neurosci Methods 1979; 1: 323-325; Nicoll RA, Alger BE. A simple chamber for recording from submerged brain slices. J Neurosci Methods 1981; 4: 153-156; Passeraub PA, Almeida AC, Thakor NV. Design, microfabrication and characterization of a microfluidic chamber for the perfusion of brain tissue slices. J Biomed Dev 2003; 5: 147-155). Here, we report a unique interstitial microfluidic perfusion technique to culture thick (700 microm) organotypic brain slices. The design of the custom-made microperfusion chamber facilitates laminar, interstitial perfusion of oxygenated nutrient medium throughout the tissue thickness with concomitant removal of depleted medium and catabolites. We examined the utility of this perfusion method to enhance the viability of the thick organotypic brain slice cultures after 2 days and 5 days in vitro (DIV). We investigated the range of amenable flow rates that enhance the viability of 700 microm thick organotypic brain slices compared to the unperfused control cultures. Our perfusion method allows up to 84.6% viability (p<0.01) and up to 700 microm thickness, even after 5 DIV. Our results also confirm that these cultures are functionally active and have their in vivo cyto-architecture preserved. Prolonged viability of thick organotypic brain slice cultures will benefit scientists investigating network properties of intact organotypic neuronal networks in a reliable and repeatable manner.

Passive flow control of bileaflet mechanical heart valve leakage flow.

Blood damage and platelet activation are inherent problems with present day mechanical heart valve designs. We investigate the approach of passive flow control applied to bileaflet mechanical heart valve (BMHV) flows as a means of optimizing leakage flow hemodynamics at length scales relevant to blood damage and platelet activation. Rectangular and hemispherical vortex generator (VG) arrays were mounted on the downstream surfaces of a 25 mm St. Jude Medical valve adjacent to the b-datum leaflet edge (central line where the two leaflets touch in closed position). The effect of VGs on the flow structure emanating from the b-datum line under both pulsatile and steady flow conditions was measured using high resolution particle image velocimetry technique. The VGs were seen to spatially disperse and dissipate the coherent leakage jet structure emanating from the b-datum line. This resulted in a significant diminution of turbulence stresses, particularly with the rectangular VG configuration. This study shows that passive flow control techniques deployed on BHMVs is potentially beneficial as significant control of flow at small length scales may be achieved without altering large scale designs of the valve.

MEMS-based fabrication and microfluidic analysis of three-dimensional perfusion systems.

This paper describes fabrication and fluidic characterization of 3D microperfusion systems that could extend the viability of high-density 3D cultures in vitro. High-aspect ratio towers serve as 3D scaffolds to support the cultures and contain injection sites for interstitial delivery of nutrients, drugs, and other reagents. Hollow and solid-top tower arrays with laser ablated side-ports were fabricated using SU-8. Appropriate sizing of fluidic ports improves the control of agent delivery. Microfluidic perfusion can be used to continuously deliver equal amount of nutrients through all ports, or more media can be delivered at some ports than the others, thus allowing spatial control of steady concentration gradients throughout the culture thickness. The induced 3D flow around towers was validated using micro particle image velocimetry. Based on experimental data, the flow rates from the characteristic ports were found to follow the analytical predictions.

Microfluidic engineered high cell density three-dimensional neural cultures.

Three-dimensional (3D) neural cultures with cells distributed throughout a thick, bioactive protein scaffold may better represent neurobiological phenomena than planar correlates lacking matrix support. Neural cells in vivo interact within a complex, multicellular environment with tightly coupled 3D cell-cell/cell-matrix interactions; however, thick 3D neural cultures at cell densities approaching that of brain rapidly decay, presumably due to diffusion limited interstitial mass transport. To address this issue, we have developed a novel perfusion platform that utilizes forced intercellular convection to enhance mass transport. First, we demonstrated that in thick (>500 microm) 3D neural cultures supported by passive diffusion, cell densities 90% viability in both neuronal cultures and neuronal-astrocytic co-cultures. This work demonstrates the utility of forced interstitial convection in improving the survival of high cell density 3D engineered neural constructs and may aid in the development of novel tissue-engineered systems reconstituting 3D cell-cell/cell-matrix interactions.

Active 3-D microscaffold system with fluid perfusion for culturing in vitro neuronal networks.

This work demonstrated the design, fabrication, packaging, and characterization of an active microscaffold system with fluid perfusion/nutrient delivery functionalities for culturing in vitro neuronal networks from dissociated hippocampal rat pup neurons. The active microscaffold consisted of an 8 x 8 array of hollow, microfabricated, SU-8 towers (1.0 mm or 1.5 mm in height), with integrated, horizontal, SU-8 cross-members that connect adjacent towers, thus forming a 3-D grid that is conducive to branching, growth, and increased network formation of dissociated hippocampal neurons. Each microtower in the microscaffold system contained a hollow channel and multiple fluid ports for media delivery and perfusion of nutrients to the in vitro neuronal network growing within the microscaffold system. Additionally, there were two exposed Au electrodes on the outer wall of each microtower at varying heights (with insulated leads running within the microtower walls), which will later allow for integration of electrical stimulation/recording functionalities into the active microscaffold system. However, characterization of the stimulation/recording electrodes was not included in the scope of this paper. Design, fabrication, fluid packaging, and characterization of the active microscaffold system were performed. Furthermore, use of the active microscaffold system was demonstrated by culturing primary hippocampal embryonic rat pup neurons, and characterizing cell viability within the microscaffold system.

Enhanced performance of a filter-sensor system.

In this paper are addressed two important, but seemingly unrelated issues: long term performance of a gas sensing array and performance of an air purification unit. It is shown that when considered together, the system can be regarded as a "smart filter". The enhancement is achieved by periodic differential sampling and measurement of the "upstream" and "downstream" gases of a filter. The correctly functioning filter supplies the "zero gas" from the downstream for the continuous sensor baseline correction. A key element in this scheme is the synthetic jet that delivers well-defined pulses of the two gases. The deterioration of the performance of the "smart filter" can be diagnosed from the response pattern of the sensor. The approach has been demonstrated on removal/sensing of ammonia gas from air.

High cell density three-dimensional neural co-cultures require continuous medium perfusion for survival.

Three-dimensional (3-D) models of neural cell culture may provide researchers with a more physiologically-relevant setting to study neurobiological phenomena than traditional two-dimensional (2-D) culture models. However, in the development of thick (>500 microm) 3-D cultures, diffusion limited mass transport necessitated the use of cell densities much lower than those found in the central nervous system (CNS). The goal of this study was to evaluate the effects of continuous medium perfusion on the survival of thick, 3-D neuronal-astrocytic co-cultures at cell densities closer to those found in brain tissue. At the cell density and thickness used for these studies, 10(4) cells/mm(3) and 500-750 microm, respectively, non-perfused cultures exhibited widespread cellular/matrix degradation and cell death. However, co-cultures perfused at relatively high rates (2.5-11.0 microL/min, corresponding to 6-27 medium exchanges/day) demonstrated decreased degradation and enhanced viability compared to non-perfused co-cultures. Furthermore, the highest perfusion rate evaluated, 11.0 microL/min, resulted in >90% cell viability and maintenance of culture thickness. Next generation 3-D neural cultures, with cell types and densities better approximating the CNS, may provide enhanced model fidelity and be valuable in the mechanistic study of cell growth, interactions, and the responses to chemical or mechanical perturbations.