Jelena Vukasinovic

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 <or=5.0 x 10(3) cells mm(-3) were required for survival. In 3D neuronal and neuronal-astrocytic co-cultures with increased cell density (10(4) cells mm(-3)), continuous medium perfusion at 2.0-11.0 microL min(-1) improved viability compared to non-perfused cultures (p < 0.01), which exhibited widespread cell death and matrix degradation. In perfused cultures, survival was dependent on proximity to the perfusion source at 2.00-6.25 microL min(-1) (p < 0.05); however, at perfusion rates of 10.0-11.0 microL min(-1) survival did not depend on the distance from the perfusion source, and resulted in a preservation of cell density with >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.

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.

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 Device for Long-Term Perfusion, Imaging, and Electrical Interfacing of Brain Tissue In vitro

Distributed microelectrode array (MEA) recordings from consistent, viable, ≥500 μm thick tissue preparations over time periods from days to weeks may aid in studying a wide range of problems in neurobiology that require in vivo-like organotypic morphology. Existing tools for electrically interfacing with organotypic slices do not address necrosis that inevitably occurs within thick slices with limited diffusion of nutrients and gas, and limited removal of waste. We developed an integrated device that enables long-term maintenance of thick, functionally active, brain tissue models using interstitial perfusion and distributed recordings from thick sections of explanted tissue on a perforated multi-electrode array. This novel device allows for automated culturing, in situ imaging, and extracellular multi-electrode interfacing with brain slices, 3-D cell cultures, and potentially other tissue culture models. The device is economical, easy to assemble, and integrable with standard electrophysiology tools. We found that convective perfusion through the culture thickness provided a functional benefit to the preparations as firing rates were generally higher in perfused cultures compared to their respective unperfused controls. This work is a step toward the development of integrated tools for days-long experiments with more consistent, healthier, thicker, and functionally more active tissue cultures with built-in distributed electrophysiological recording and stimulation functionality. The results may be useful for the study of normal processes, pathological conditions, and drug screening strategies currently hindered by the limitations of acute (a few hours long) brain slice preparations.

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.

Spot-Cooling by Confined, Impinging Synthetic Jet

Spot-cooling of discrete electronic packages mounted on a printed wiring board (PWB) is achieved by the impingement of an axisymmetric synthetic jet when the jet actuator is attached to one board and cools target integrated circuit (IC) on the opposite board. Present work demonstrates that even when the jet outflow is confined between two closely-spaced boards, the jet entrains ample volume flow rate of cooler ambient air, induces effective cooling by strong mixing near the heated surface, and ultimately dispenses the heated air to ambient. The cooling performance of the jet module is investigated experimentally in a scaled up model that enables high-resolution thermal and flow measurements. The test setup comprises of two circular parallel plates (D = 158.8mm) where one plate contains an integrated jet actuator and the opposite plate includes a target heater (dh = 86mm). The spacing between the plates is variable between D/12 and D/3. The flow within the gap is mapped using particle image velocimetry (PIV). It is found that confined jet impingement induces a countercurrent radial flow within the gap that includes a layer of cool ambient air entrained along the actuator plane and a layer of heated air that is dispensed along the target surface. Particle pathlines demonstrate significant mixing between the countercurrent streams and strong entrainment into the vortex rings that synthesize the jet. Heat transfer coefficient attains a local maximum away from the stagnation point that can be attributed to strong vorticity diffusion into the thermal boundary layer and enhanced mixing that accompanies the vortex ring impingement. Although the jet Reynolds number does not exceed 3300, the stagnation heat transfer coefficient is about 90 W/m2 K for H/D = 0.32.© 2003 ASME

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 (>500mum) 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, 104 cells/mm3 and 500-750mum, respectively, non-perfused cultures exhibited widespread cellular/matrix degradation and cell death. However, co-cultures perfused at relatively high rates (2.5-11.0muL/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, 11.0muL/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

A Fully Functional, Packaged Active Microscaffold System with Fluid Perfusion and Electrical Stimulation/Recording Functionalities for 3-D Neuronal Culture Studies

This paper and presentation, for the first time, demonstrates a fully functional, packaged active microscaffold system with fluid perfusion and electrical stimulation/recording functionalities for 3-D neuronal culture studies. The fluid perfusion capabilities of the active microscaffold serve as an artificial circulatory system to enable 3-D growth and proliferation of re-aggregate neuronal cultures. Increased cell survival on microscaffolds with nutrient perfusion at 14 and 21 days in vitro (DIV) is presented. Additionally, initial characterization of the electrical stimulation/recording functionality of the microscaffold system is presented.

Flow Through a Micro-Bioreactor in a Neural Interface System

A micro-bioreactor is developed for in vitro, controlled, growth of three-dimensional dissociated neural cultures that enables simultaneous, multipoint, electrical and fluidic interfacing, monitoring and recording of the response signals that are relevant to the studies of traumatic injury. Present experiments focus on the microfluidic system that is used to control the spatial concentration of nutrients and stimuli in the incubated tissue. The three-dimensional cellular environment in a micro-bioreactor is controlled by means of convective and diffusive fluidic processes to improve the neural cell survival rate, direct the cell growth and examine the network formation. To achieve global and local manipulation of the critical cell functions, flow within the reactor is induced from arrays of micro-machined nozzles in planar surfaces and within microscale hydrogel scaffolding. The flow and concentration fields within reactor are analyzed using microscale particle image velocimetry (PIV) and fluorescence. The flow within 25 μm thick layers between microfabricated structures is investigated using image-processing algorithms that are developed to improve spatial resolution by excluding out-of-focus particles. Mixing induced by delivery of stimuli/nutrients and waste extraction is considered by following the time-evolution and spatial propagration of the mixing front between the liquids based on the intensity of reflected light that is previously calibrated with concentration.Copyright