High pressure flow cytometric sorting damages sperm

Sexing sperm by high-speed flow cytometry subjects them to high pressure. The routine operating pressure of the MoFlo SX flow cytometer for sperm sorting for commercial production has been 50 pounds/square inch (psi), with a standard 70 microm standard nozzle tip. It was hypothesized that lowering the sorting pressure could reduce sperm damage. Therefore, a series of experiments using semen from six bulls, sorted with three MoFlo SX sorters, was conducted to determine optimal pressure. An additional experiment was done with stallion spermatozoa. In Experiment 1, sorting at 30 psi compared to 50 psi with the 70 microm nozzle tip increased sperm motility post-thaw at 30 min and 2h from 40.5 to 48.0% and 30.0 to 40.2%, respectively (P<0.05). In Experiment 2, 49, 43, 37, 31, and 25 psi resulted in 24.2, 32.8, 35.6, 37.5, and 39.8% progressively motile spermatozoa post-thaw (P<0.05). In Experiment 3, 3 pressures (50, 40, 30 psi)x2 sorting methods were further evaluated. At 50, 40, and 30 psi, respective mean sperm motilities at 30 min were 44.8, 48.6, and 49.6% (P<0.05), and percentage of live spermatozoa were 51.7, 55.7, and 57.8% (P<0.05). The improvement of post-sort sperm quality with lowered pressure was also evident in stallion spermatozoa. After sorting at 30, 40 and 50 psi were 40.6, 34.5 and 30.1% motile spermatozoa (P<0.1), and were 76.7, 72.5 and 67.8% (P<0.05) live spermatozoa (determined by SYBR-14/propidium iodide staining). In Experiment 4 sorter performance was evaluated with two pressures (40 and 50 psi)x2 staining concentrations of bovine spermatozoa (75 x 10(6) and 100 x 10(6)mL(-1)). Lowering pressure to 40 psi did not lower sort rate and purity when compared to 50 psi (P>0.05), and higher sperm concentration during staining increased sort rate (P<0.05). In conclusion, lowering pressure of the MoFlo SX flow cytometer for sperm sorting from 50 psi (standard pressure) to 40 psi clearly improved sperm quality without a significant decrease in sorter performance.     

An approach to the simulation of fluid-structure interaction in the aortic valve

A pair of finite element models has been employed to study the interaction of blood flow with the operation of the aortic valve. A three-dimensional model of the left ventricle with applied wall displacements has been used to generate data for the spatially and time-varying blood velocity profile across the aortic aperture. These data have been used as the inlet loading conditions in a three-dimensional model of the aortic valve and its surrounding structures. Both models involve fluid-structure interaction and simulate the cardiac cycle as a dynamic event. Confidence in the models was obtained by comparison with data obtained in a pulse duplicator. The results show a circulatory flow being generated in the ventricle which produces a substantially axial flow through the aortic aperture. The aortic valve behaves in an essentially symmetric way under the action of this flow, so that the pressure difference across the leaflets is approximately uniform. This work supports the use of spatially uniform but temporally variable pressure distributions across the leaflets in dry or structural models of aortic valves. The study is a major advance through its use of truly three-dimensional geometry, spatially non-uniform loading conditions for the valve leaflets and the successful modelling of progressive contact of the leaflets in a fluid environment.     

Time-resolved DPIV analysis of vortex dynamics in a left ventricular model through bileaflet mechanical and porcine heart valve prostheses

The performance of the heart after a mitral valve replacement operation greatly depends on the flow character downstream of the valve. The design and implanting orientation of valves may considerably affect the flow development. A study of the hemodynamics of two orientations, anatomical and anti-anatomical, of the St. Jude Medical (SJM) bileaflet valve are presented and compared with those of the SJM Biocor porcine valve, which served also to represent the natural valve. We document the velocity field in a flexible, transparent (LV) using time-resolved digital particle image velocimetry (TRDPIV). Vortex formation and vortex interaction are two important physical phenomena that dominate the filling and emptying of the ventricle. For the three configurations, the following effects were examined: mitral valve inlet jet asymmetry, survival of vortical structures upstream of the aortic valve, vortex-induced velocities and redirection of theflow in abidance of the Biot-Savart law, domain segmentation, resonant times of vortical structures, and regions of stagnantflow. The presence of three distinct flow patterns, for the three configurations, was identified by the location of vortical structures and level of coherence corresponding to a significant variation in the turbulence level distribution inside the LV. The adverse effect of these observations could potentially compromise the efficiency of the LV and result in flow patterns that deviate from those in the natural heart.     

Micropatterned cell cultures on elastic membranes as an in vitro model of myocardium

We describe here a new in vitro protocol for structuring cardiac cell cultures to mimic important aspects of the in vivo ventricular myocardial phenotype by controlling the location and mechanical environment of cultured cells. Microlithography is used to engineer microstructured silicon metal wafers. Those are used to fabricate either microgrooved silicone membranes or silicone molds for microfluidic application of extracellular matrix proteins onto elastic membranes (involving flow control at micrometer resolution). The physically or microfluidically structured membranes serve as a cell culture growth substrate that supports cell alignment and allows the application of stretch. The latter is achieved with a stretching device that can deliver isotropic or anisotropic stretch. Neonatal ventricular cardiomyocytes, grown on these micropatterned membranes, develop an in vivo-like morphology with regular sarcomeric patterns. The entire process from fabrication of the micropatterned silicon metal wafers to casting of silicone molds, microfluidic patterning and cell isolation and seeding takes approximately 7 days.     

Using a CFD model to understand the fluid dynamics promoting E. coli breakage in a high-pressure homogenizer

A Computational Fluid Dynamic (CFD) model of flow in a high-pressure homogenizing valve (APV Gaulin model 30CD) was developed with the Fluent software. The 2D model consists of an unstructured hexagonal mesh, dense in the regions of high gradients. The flow (single-phase) was modeled as laminar upstream of and in the channel (gap) and turbulent downstream of the channel exit. Applying a realizable kappa-epsilon turbulence model, the CFD model accurately predicted the effect of gap space on fluid dynamic conditions upstream (inlet pressure and pressure gradient) and downstream (impact pressure) of the channel for a valve with a standard (CD-0) impact distance (0.25 mm) and a 1 cP fluid. This CFD model was then used to estimate the magnitude of the fluid dynamic parameters (except cavitation effects) presumed to be responsible for cell breakage, as a function of gap space, impact distance and fluid viscosity. The CFD models predicted that for a given volumetric flowrate the upstream fluid conditions (inlet pressure gradient, maximum channel strain rate) and the maximum energy dissipation rate in the post-gap jet depend only on the gap space and the fluid viscosity and not on the impact distance. The impact pressure however depends on the gap spacing, the fluid viscosity and especially the impact distance. Experimental results indicate that higher inlet pressures are required to break cells, if the impact distance is increased. By conducting experiments to isolate individual cell breakage mechanisms for a single pass, threshold values were identified for breaking Escherichia coli cells: pressure gradient, 1.2 x 10(12) Pa/m; energy dissipation rate, 1.0 x 10(10) m(3)/s(2); and impact pressure, 160 psig. By isolating the wall impact as the sole mechanism responsible for breaking the E. coli cells between 3000 and 6000 psig inlet pressure, a relationship between E. coli cell breakage rate and maximum wall impact pressure was established (eq 5).     

Empirical torsional potential functions from protein structure data. Phi- and psi-potentials for non-glycyl amino acid residues.

The torsional potential functions Vt(phi) and Vt(psi) around single bonds N--C alpha and C alpha--C, which can be used in conformational studies of oligopeptides, polypeptides and proteins, have been derived, using crystal structure data of 22 globular proteins, fitting the observed distribution in the (phi, psi)-plane with the value of Vtot(phi, psi), using the Boltzmann distribution. The averaged torsional potential functions, obtained from various amino acid residues in L-configuration, are Vt(phi) = 1.0 cos (phi + 60 degrees); Vt(psi) = 0.5 cos (psi + 60 degrees) - 1.0 cos (2 psi + 30 degrees) - 0.5 cos (3 psi + 30 degrees). The dipeptide energy maps Vtot(phi, psi) obtained using these functions, instead of the normally accepted torsional functions, were found to explain various observations, such as the absence of the left-handed alpha helix and the C7 conformation, and the relatively high density of points near the line psi = 0 degrees. These functions derived from observational data on protein structures, will, it is hoped, explain various previously unexplained facts in polypeptide conformation.     

Automatic bio-sampling chips integrated with micro-pumps and micro-valves for disease detection

The present study reports a microfluidic system using the concept of membrane-movement to design and fabricate micro-pneumatic valves and pumps to form a bio-sensing diagnostic chip. The automatic bio-sampling system includes a micro-diagnostic chip fabricated by using MEMS (micro-electro-mechanical systems) technology and an automatic platform comprising of a control circuit, a compressed air source and several electromagnetic valve switches. The control circuit is used to regulate the electromagnetic valve switches, causing thin PDMS membranes to deflect pneumatically by the compressed air and generate valving and pumping effects. The micro-diagnostic chip allows for the quick detection of diseases. Compared to large-scale systems, the new microfluidic system uses smaller amounts of samples and reagents and performs fast diagnosis in an automated format. Instead of using traditional pneumatic micro-pumps, the current study adopts a new design called "spider-web" micro-pumps to increase the pumping rate, and more importantly, improve the uniformity of flow rates inside multiple micro-channels. Experimental data show that for disease diagnosis, the bio-sensing chips integrated with the micro-pneumatic valves and the peristaltic micro-pumps could successfully perform diagnosis tests. Small amounts of samples and reagents could be injected into the diagnosis chips using the micro-pumps and the micro-pneumatic valves could effectively control the movement of the samples and reagents. In order to demonstrate the functionality of the developed device, detection of hepatitis C virus (HCV) and syphilis has been performed using the bio-sampling chips. Experimental data show that fluorescence signals from the microfluidic system were comparable to the ones using conventional testing methods. The developed chip could be easily extended for multiple disease detection. The automatic bio-sensing chips could provide a useful tool for fast disease detection and be crucial for a micro-total-analysis system.     

Analysis of blood flow through a model of the human arterial system under periodic body acceleration

The human system may be subjected to a body acceleration deliberated for example by making subjects lie down on vibrating tables or more frequently unintentionally, for example during travel in water and land or in air and space. The present study is concerned with the effects of externally imposed body accelerations on blood flow in a branched system of arteries. A finite-element model of flow in the arterial system subject to periodic body accelerations is presented. Computational results on the flow rates through selected arteries and the corresponding inlet and outlet pressures under different conditions (magnitude, frequency and direction) of applied acceleration are presented.     

Ion channels in boar sperm plasma membranes: characterization of a cation selective channel.

Plasma membranes isolated from cauda epididymal and ejaculated boar sperm were inserted into planar lipid bilayers and examined for the presence of ion channels. Channel fusion was frequently observed; the most prominent was a nonselective cation channel which conducted K, Na, Cs, Ca, and Ba. Channel opening did not show a strict dependence on voltage but was partially blocked by verapamil, nitrendipine, and ruthenium red. A channel with these characteristics was observed when plasma membranes were isolated by high-pressure nitrogen cavitation (650 psi, 78% sperm head plasma membranes) or at very low nitrogen pressures (50 psi, 90% sperm head plasma membranes), suggesting that this channel may be present in the plasma membrane overlying the sperm head.     

Parametric analysis of a novel semi-circular microfluidic CD-ELISA valve

CD-ELISA uses the microfluidic ranking method and centrifugal force to control the testing solution as it flows into the reaction region. The most challenging part of CD-ELISA is controlling the flow process for different biological testing solutions, i.e. the controlling sequence for the microfluidic channel valves. The microfluidic channel valve is therefore the most important fluid channel structure for CD-ELISA. In this study, we propose a valve design suitable for a wide range rotational speeds which can be applied for mass production (molding). Together with supporting experiments, simulation based on two-phase flow theory is used in this study, and the feasibility of this novel valve design is confirmed. Influencing design factors for the microfluidic channel valves in CD-ELISA are investigated, including various shapes of the arc, distance d, radius r, the location of the center of the circle, and the contact angle. From both the experimental results and the simulated results, it is evident that the narrowest channel width and the contact angle are the primary factors influencing valve burst frequency. These can be used as the main controlling factors during the design.     

Relation of branching angles to optimality for four cost principles

The literature has suggested that branching angles depend on some principle of optimality. Most often cited are the minimization of lumen surface, volume, power and drag. The predicted angles depend on the principle applied, chi and alpha. Assuming flow o r chi, chi can be determined from r chi 0 = r chi 1 + r chi 2 when the radii of the parent (r0) major (r1) and minor (r2) daughters are known. The term alpha = r2/r1. Using different values for chi and alpha, we present graphs for the major and minor branching angles theta 1 and theta 2 and psi = theta 1 + theta 2 for each of the four optimization principles. Because psi is almost independent of alpha for values of chi and alpha found in 198 junctions taken from a human pulmonary artery, we are able to produce a plot of psi versus chi for each of the four principles on one graph. A junction can be provisionally classified as optimizing for a given principle if, knowing chi, the psi obs - psi pred is least for that principle. We find that this nomographic classification agrees almost perfectly with a previous classification based on a more exacting measure, the percent cost index I, where I = observed cost/minimum cost. We explain why this is to be expected in most but not all cases. First we generate a contoured percent cost surface of c = I - 100 around the optimally located junction, J, and superimpose a surface of equal angular deviations a = psi pred-psi obs. We find that c increases and a usually increases with distance from J as the actual junction moves along a straight line away from J. We then produce a plot of c versus a for two competing principles. A comparison of the principles demonstrates that, for most cases, a is smaller for the principle which has the smaller c value.     

STUDIES ON THE MICROTUBULES IN HELIOZOA : III. A Pressure Analysis of the Role of These Structures in the Formation and Maintenance of the Axopodia of Actinosphaerium nucleofilum (Barrett)

Electron microscope preparations were made of specimens of Actinosphaerium nucleofilum fixed in glutaraldehyde before, during, and after exposure to high pressures (4,000 to 8,000 psi). A study of this material showed that, although other organelles were relatively stable, the microtubular elements of the axopodia and cytosome became unstable under pressure. Their rapid disintegration under pressure was correlated with beading and retraction of the axopodia. Moreover, after the release of pressure, microtubules reappeared as soon as, or sooner than the reextension of the axopodia. The rate of disintegration increased as the pressure was raised. At 4,000 psi, few if any tubules remained after 10 min, whereas at 6,000 and 8,000 psi the disintegration was much more rapid. Some adaptational reorganization of the microtubules and axopodia occurred while relatively low pressures were maintained. This was accompanied by an actual elongation of the axopodia in specimens maintained for 20 min at 4,000 psi, but was confined to knoblike axopodial remnants in animals kept at 6,000 psi. No regeneration of tubules or axopodia occurred at 8,000 psi. The presence of fibers and a finely fibrillar material in pressurized animals suggests that these may be derivatives of microtubular disintegration. This evidence, though purely morphological, is consistent with the hypothesis that microtubules play an important role not only in maintaining the formstability of the axopodia, but also in the active process by which the axopodia reextend themselves after retraction.     

Micropumps, microvalves, and micromixers within PCR microfluidic chips: Advances and trends

This review surveys the advances of microvalves, micropumps, and micromixers within PCR microfluidic chips over the past ten years. First, the types of microvalves in PCR chips are discussed, including active and passive microvalves. The active microvalves are subdivided into mechanical (thermopneumatic and shape memory alloy), non-mechanical (hydrogel, sol-gel, paraffin, and ice), and external (modular built-in, pneumatic, and non-pneumatic) microvalves. The passive microvalves also include mechanical (in-line polymerized gel and passive plug) and non-mechanical (hydrophobic) microvalves. The review then discusses mechanical (piezoelectric, pneumatic, and thermopneumatic) and non-mechanical (electrokinetic, magnetohydrodynamic, electrochemical, acoustic-wave, surface tension and capillary, and ferrofluidic magnetic) micropumps in PCR chips. Next, different micromixers within PCR chips are presented, including passive (Y/T-type flow, recirculation flow, and drop) and active (electrokinetically-driven, acoustically-driven, magnetohydrodynamical-driven, microvalves/pumps) micromixers. Finally, general discussions on microvalves, micropumps, and micromixers for PCR chips are given. The microvalve/micropump/micromixers allow high levels of PCR chip integration and analytical throughput.     

Transmission of poly(dT60) single-stranded DNA through polycarbonate track-etched ultrafiltration membranes

The objective of this study was to examine membrane filtration of a single stranded DNA (ssDNA) with 60 thymine nucleotides, and to elucidate the variables controlling its transmission across track-etched porous membranes. Dead end filtration measurements were performed using different pore size membranes (10, 15, and 30 nm) at different transmembrane pressures in solutions with ionic strength ranging from 0 to 1000 mM NaCl. The diffusivity of the ssDNA was determined using fluorescence recovery after photobleaching, yielding hydrodynamic radii ranging from 1.6 to 2.8 nm, with values decreasing with increasing solution ionic strength. Despite the small ssDNA/membrane pore size, nearly 100% rejection was observed for measurements performed with the 10 and 15 nm pore size membranes under low-ionic strength conditions. These high rejections can be attributed to strong repulsive electrostatic ssDNA-membrane interactions. With increasing ionic strength, electrostatic interactions as well as the effective size of the ssDNA decreases and the flexibility of the ssDNA increases, leading to a reduction in ssDNA rejection. A design of experiments approach was used to plan filtration experiments that adequately covered the variable space with a manageable number of experiments. The results yielded an empirical expression relating ssDNA rejection to pore size, solution ionic strength and transmembrane pressure. There was evidence of flow induced elongation at high-transmembrane pressures in the 30 nm pore size membranes, but not in the smaller pore size membranes. These results are consistent with critical flux estimates developed using a free draining model for the ssDNA.     

Brain slice stimulation using a microfluidic network and standard perfusion chamber

We have demonstrated the fabrication of a two-level microfluidic device that can be easily integrated with existing electrophysiology setups. The two-level microfluidic device is fabricated using a two-step standard negative resist lithography process. The first level contains microchannels with inlet and outlet ports at each end. The second level contains microscale circular holes located midway of the channel length and centered along with channel width. Passive pumping method is used to pump fluids from the inlet port to the outlet port. The microfluidic device is integrated with off-the-shelf perfusion chambers and allows seamless integration with the electrophysiology setup. The fluids introduced at the inlet ports flow through the microchannels towards the outlet ports and also escape through the circular openings located on top of the microchannels into the bath of the perfusion. Thus the bottom surface of the brain slice placed in the perfusion chamber bath and above the microfluidic device can be exposed with different neurotransmitters. The microscale thickness of the microfluidic device and the transparent nature of the materials [glass coverslip and PDMS (polydimethylsiloxane)] used to make the microfluidic device allow microscopy of the brain slice. The microfluidic device allows modulation (both spatial and temporal) of the chemical stimuli introduced to the brain slice microenvironments.     

The effect of high hydrostatic pressure and low temperature on lactic dehydrogenase and glutamic oxalacetic transaminase

LDH and GOT can be used with assurance as indicators of pressure-temperature effects in most regions of interest, specifically below 20,000 psi. LDH was susceptible to pressure deactivation at pressure levels below those tolerated by chymotrypsin, trypsin and alpha-amylase of Bacillus subtilis (17, 18, 20). Samples of LDH and GOT cooled to −20 °C were deactivated to the greatest extent by the application of pressure. The presence of glycerine and DMSO appeared to increase the sensitivity of GOT and LDH to pressure deactivation. When pressure was applied before cooling all pressures above 15,000 psi resulted in some deactivation of LDH and all pressures above 20,000 psi resulted in some deactivation of GOT.     

A new tool for probing of cell–cell communication: human embryonic germ cells inducing apoptosis of SKOV3 ovarian cancer cells on a microfluidic chip

A microfluidic device with unidirectional perfusion has been developed to observe the effect of human embryonic germ (hEG) cells on SKOV3 cells. The hEG and SKOV3 cells were seeded in the inlet and the outlet reservoirs separately, and co-cultured for 2 days. The medium was perfused unidirectionally from the inlet to the outlet. The growth inhibition of SKOV3 cells was monitored online and the apoptosis signals in SKOV3 culture area decreased along the flow of the medium. In conclusion, microfluidic chip is a potentially useful tool to investigate the effect of stem cells on cancer cells with intuitionistic cell-based screens.     

The effects of hydrostatic pressure upon the normal and narcotized nerve fiber

The properties of the giant axon of the squid Loligo pealii were studied at different hydrostatic pressures from 14.7 to 16,000 psi. At 4000 psi the resting potential, the membrane resistance, membrane capacity, the conduction velocity, the amplitude of the action potential, and the maximal change in the membrane impedance during activity were only slightly affected. At the same pressure the duration of the falling phase of the action potential was increased by about 40 to 60 per cent and the duration of the rising phase by about 20 to 35 per cent. The duration of the membrane impedance change during activity was increased by 50 to 100 per cent at 4000 psi. At pressures even slightly above atmospheric the threshold membrane current was appreciably reduced. At about 3000 to 7000 psi the fiber fired spontaneously. At pressures considerably above 5000 psi the membrane resistance decreased to about one-half to one-third the original value. The narcotizing effect upon the nerve fiber of 3 to 7 per cent ethanol was partly or almost completely opposed by low temperatures or high pressures.     

Probing concentration-dependent behavior of DNA-binding proteins on a single-molecule level illustrated by Rad51

Low throughput is an inherent problem associated with most single-molecule biophysical techniques. We have developed a versatile tool for high-throughput analysis of DNA and DNA-binding molecules by combining microfluidic and dense DNA arrays. We use an easy-to-process microfluidic flow channel system in which dense DNA arrays are prepared for simultaneous imaging of large amounts of DNA molecules with single-molecule resolution. The Y-shaped microfluidic design, where the two inlet channels can be controlled separately and precisely, enables the creation of a concentration gradient across the microfluidic channel as well as rapid and repeated addition and removal of substances from the measurement region. A DNA array stained with the fluorescent DNA-binding dye YOYO-1 in a gradient manner illustrates the method and serves as a proof of concept. We have applied the method to studies of the repair protein Rad51 and could directly probe the concentration-dependent DNA-binding behavior of human Rad51 (HsRad51). In the low-concentration regime used (100 nM HsRad51 and below), we detected binding to double-stranded DNA (dsDNA) without positive cooperativity.     

Planar microfluidic chamber for generation of stable and steep chemoattractant gradients

The extracellular availability of growth factors, hormones, chemokines, and neurotransmitters under gradient conditions is required for directional cellular responses such as migration, axonal pathfinding, and tissue patterning. These responses are, in turn, important in disease and developmental processes. This article addresses critical barriers toward devising a chemotaxis assay that is broadly applicable for different kinds of cancer cells through the design of a microfluidic chamber that produces a steep gradient of chemoattractant. Photolithography was used to create microchannels for chemoattractant delivery, flow diversion barriers/conduits, and small outlets in the form of apertures. The 1-μm apertures were made at the active surface by uncapping a thin (1.5 μm) layer of AZ1518. This process also created a vertical conduit that diverted the flow such that it occurred perpendicularly to the active, experimental surface where the gradients were measured. The other side of the vertical conduit opened to underlying 20-μm deep channels that carried microfluidic flows of tracer dyes/growth factors. Modeled data using computational fluid dynamics produced gradients that were steep along the horizontal, active surface. This simulation mirrors empirically derived gradients obtained from the flow analyses of fluorescent compounds. The open chamber contains a large buffer volume, which prevents chemoattractant saturation and permits easy cell and compound manipulation. The technique obviates the use of membranes or laminar flow that may hinder imaging, rinsing steps, cell seeding, and treatment. The utility of the chamber in the study of cell protrusion, an early step during chemotaxis, was demonstrated by growing cancer cells in the chamber, inducing a chemoattractant gradient using compressed air at 0.7 bar, and performing time-lapse microscopy. Breast cancer cells responded to the rapidly developed and stable gradient of epidermal growth factor by directing centroid positions toward the gradient and by forming a leading edge at a speed of 0.45 μm/min.