Hybrid motility mechanism of sperm at viscoelastic-solid interface.

To fertilize eggs, sperm must pass through narrow, complex channels filled with viscoelastic fluids in the female reproductive tract. While it is known that the topography of the surfaces plays a role in guiding sperm movement, sperm have been thought of as swimmers, i.e., their motility comes solely from sperm interaction with the surrounding fluid, and therefore, the surfaces have no direct role in the motility mechanism itself. Here, we examined the role of solid surfaces in the movement of sperm in a highly viscoelastic medium. By visualizing the flagellum interaction with surfaces in a microfluidic device, we found that the flagellum stays close to the surface while the kinetic friction between the flagellum and the surface is in the direction of sperm movement, providing thrust. Additionally, the flow field generated by sperm suggests slippage between the viscoelastic fluid and the solid surface, deviating from the no-slip boundary typically used in standard fluid dynamics models. These observations point to hybrid motility mechanisms in sperm involving direct flagellum-surface interaction in addition to flagellum pushing the fluid. This finding signifies an evolutionary strategy of mammalian sperm crucial for their efficient migration through narrow, mucus-filled passages of the female reproductive tract.

Biological benefits of collective swimming of sperm in a viscoelastic fluid.

Collective swimming is evident in the sperm of several mammalian species. In bull (Bos taurus) sperm, high viscoelasticity of the surrounding fluid induces the sperm to form dynamic clusters. Sperm within the clusters swim closely together and align in the same direction, yet the clusters are dynamic because individual sperm swim into and out of them over time. As the fluid in part of the mammalian female reproductive tract contains mucus and, consequently, is highly viscoelastic, this mechanistic clustering likely happens in vivo. Nevertheless, it has been unclear whether clustering could provide any biological benefit. Here, using a microfluidic in vitro model with viscoelastic fluid, we found that the collective swimming of bull sperm in dynamic clusters provides specific biological benefits. In static viscoelastic fluid, clustering allowed sperm to swim in a more progressive manner. When the fluid was made to flow in the range of 2.43-4.05 1/sec shear rate, clustering enhanced the ability of sperm to swim upstream. We also found that the swimming characteristics of sperm in our viscoelastic fluid could not be fully explained by the hydrodynamic model that has been developed for sperm swimming in a low-viscosity, Newtonian fluid. Overall, we found that clustered sperm swam more oriented with each other in the absence of flow, were able to swim upstream under intermediate flows, and better withstood a strong flow than individual sperm. Our results indicate that the clustering of sperm can be beneficial to sperm migrating against an opposing flow of viscoelastic fluid within the female reproductive tract.

Co-Adaptation of Physical Attributes of the Mammalian Female Reproductive Tract and Sperm to Facilitate Fertilization.

The functions of the female reproductive tract not only encompass sperm migration, storage, and fertilization, but also support the transport and development of the fertilized egg through to the birth of offspring. Further, because the tract is open to the external environment, it must also provide protection against invasive pathogens. In biophysics, sperm are considered "pusher microswimmers", because they are propelled by pushing fluid behind them. This type of swimming by motile microorganisms promotes the tendency to swim along walls and upstream in gentle fluid flows. Thus, the architecture of the walls of the female tract, and the gentle flows created by cilia, can guide sperm migration. The viscoelasticity of the fluids in the tract, such as mucus secretions, also promotes the cooperative swimming of sperm that can improve fertilization success; at the same time, the mucus can also impede the invasion of pathogens. This review is focused on how the mammalian female reproductive tract and sperm interact physically to facilitate the movement of sperm to the site of fertilization. Knowledge of female/sperm interactions can not only explain how the female tract can physically guide sperm to the fertilization site, but can also be applied for the improvement of in vitro fertilization devices.

Computer-assisted beat-pattern analysis and the flagellar waveforms of bovine spermatozoa.

Obstructed by hurdles in information extraction, handling and processing, computer-assisted sperm analysis systems have typically not considered in detail the complex flagellar waveforms of spermatozoa, despite their defining role in cell motility. Recent developments in imaging techniques and data processing have produced significantly improved methods of waveform digitization. Here, we use these improvements to demonstrate that near-complete flagellar capture is realizable on the scale of hundreds of cells, and, further, that meaningful statistical comparisons of flagellar waveforms may be readily performed with widely available tools. Representing the advent of high-fidelity computer-assisted beat-pattern analysis, we show how such a statistical approach can distinguish between samples using complex flagellar beating patterns rather than crude summary statistics. Dimensionality-reduction techniques applied to entire samples also reveal qualitatively distinct components of the beat, and a novel data-driven methodology for the generation of representative synthetic waveform data is proposed.

Fluid viscoelasticity promotes collective swimming of sperm.

From flocking birds to swarming insects, interactions of organisms large and small lead to the emergence of collective dynamics. Here, we report striking collective swimming of bovine sperm in dynamic clusters, enabled by the viscoelasticity of the fluid. Sperm oriented in the same direction within each cluster, and cluster size and cell-cell alignment strength increased with viscoelasticity of the fluid. In contrast, sperm swam randomly and individually in Newtonian (nonelastic) fluids of low and high viscosity. Analysis of the fluid motion surrounding individual swimming sperm indicated that sperm-fluid interaction was facilitated by the elastic component of the fluid. In humans, as well as cattle, sperm are naturally deposited at the entrance to the cervix and must swim through viscoelastic cervical mucus and other mucoid secretions to reach the site of fertilization. Collective swimming induced by elasticity may thus facilitate sperm migration and contribute to successful fertilization. We note that almost all biological fluids (e.g. mucus and blood) are viscoelastic in nature, and this finding highlights the importance of fluid elasticity in biological function.

Dynamics of Bovine Sperm Interaction with Epithelium Differ Between Oviductal Isthmus and Ampulla.

In mammals, many sperm that reach the oviduct are held in a reservoir by binding to epithelium. To leave the reservoir, sperm detach from the epithelium; however, they may bind and detach again as they ascend into the ampulla toward oocytes. In order to elucidate the nature of binding interactions along the oviduct, we compared the effects of bursts of strong fluid flow (as would be caused by oviductal contractions), heparin, and hyperactivation on detachment of bovine sperm bound in vitro to epithelium on intact folds of isthmic and ampullar mucosa. Intact folds of oviductal mucosa were used to represent the strong attachments of epithelial cells to each other and to underlying connective tissue that exist in vivo. Effects of heparin on binding were tested because heparin binds to the Binder of SPerm (BSP) proteins that attach sperm to oviductal epithelium. Sperm bound by their heads to beating cilia on both isthmic and ampullar epithelia and could not be detached by strong bursts of fluid flow. Addition of heparin immediately detached sperm from isthmic epithelium but not ampullar epithelium. Addition of 4-aminopyridine immediately stimulated hyperactivation of sperm but did not detach them from isthmic or ampullar epithelium unless added with heparin. These observations indicate that the nature of binding of sperm to ampullar epithelium differs from that of binding to isthmic epithelium; specifically, sperm bound to isthmic epithelium can be detached by heparin alone, while sperm bound to ampullar epithelium requires both heparin and hyperactivation to detach from the epithelium.

Dynamic self-organization of microwell-aggregated cellular mixtures.

Cells with different cohesive properties self-assemble in a spatiotemporal and context-dependent manner. Previous studies on cell self-organization mainly focused on the spontaneous structural development within a short period of time during which the cell numbers remained constant. However the effect of cell proliferation over time on the self-organization of cells is largely unexplored. Here, we studied the spatiotemporal dynamics of self-organization of a co-culture of MDA-MB-231 and MCF10A cells seeded in a well defined space (i.e. non-adherent microfabricated wells). When cell-growth was chemically inhibited, high cohesive MCF10A cells formed a core surrounded by low cohesive MDA-MB-231 cells on the periphery, consistent with the differential adhesion hypothesis (DAH). Interestingly, this aggregate morphology was completely inverted when the cells were free to grow. At an initial seeding ratio of 1 : 1 (MDA-MB-231 : MCF10A), the fast growing MCF10A cells segregated in the periphery while the slow growing MDA-MB-231 cells stayed in the core. Another morphology developed at an inequal seeding ratio (4 : 1), that is, the cell mixtures developed a side-by-side aggregate morphology. We conclude that the cell self-organization depends not only on the cell cohesive properties but also on the cell seeding ratio and proliferation. Furthermore, by taking advantage of the cell self-organization, we purified human embryonic stem cells-derived pancreatic progenitors (hESCs-PPs) from co-cultured feeder cells without using any additional tools or labels.

Interstitial flows promote amoeboid over mesenchymal motility of breast cancer cells revealed by a three dimensional microfluidic model.

Malignant tumors are often associated with an elevated fluid pressure due to the abnormal growth of vascular vessels, and thus an increased interstitial flow out of the tumors. Recent in vitro works revealed that interstitial flows critically regulated tumor cell migration within a three dimensional biomatrix, and breast cancer cell migration behavior depended sensitively on the cell seeding density, chemokine availability and flow rates. In this paper, we focus on the role of interstitial flows in modulating the heterogeneity of cancer cell motility phenotype within a three dimensional biomatrix. Using a newly developed microfluidic model, we show that breast cancer cells (MDA-MB-231) embedded in a 3D type I collagen matrix exhibit both amoeboid and mesenchymal motility, and interstitial flows promote the cell population towards the amoeboid motility phenotype. Furthermore, the addition of exogenous adhesion molecules (fibronectin) within the extracellular matrix (type I collagen) partially rescues the mesenchymal phenotype in the presence of the flow. Quantitative analysis of cell tracks and cell shapes shows distinct differential migration characteristics of amoeboid and mesenchymal cells. Notably, the fastest moving cells belong to the subpopulation of amoeboid cells. Together, these findings highlight the important role of biophysical forces in modulating tumor cell migration heterogeneity and plasticity, as well as the suitability of microfluidic models in interrogating tumor cell dynamics at single-cell and subpopulation level.

An array microhabitat system for high throughput studies of microalgal growth under controlled nutrient gradients.

Microalgae have been increasingly recognized in the fields of environmental and biomedical engineering because of its use as base materials for biofuels or biomedical products, and also the urgent needs to control harmful algal blooms protecting water resources worldwide. Central to the theme is the growth rate of microalgae under the influences of various environmental cues including nutrients, pH, oxygen tension and light intensity. Current microalgal culture systems, e.g. raceway ponds or chemostats, are not designed for system parameter optimizations of cell growth. In this article, we present the development of an array microfluidic system for high throughput studies of microalgal growth under well defined environmental conditions. The microfluidic platform consists of an array of microhabitats flanked by two parallel side channels, all of which are patterned in a thin agarose gel membrane. The unique feature of the device is that each microhabitat is physically confined suitable for both motile and non-motile cell culture, and at the same time, the device is transparent and can be perfused through the two side channels amendable for precise environmental control of photosynthetic microorganisms. This microfluidic system is used to study the growth kinetics of a model microalgal strain, Chlamydomonas reinhardtii (C. reinhardtii), under ammonium (NH4Cl) concentration gradients. Experimental results show that C. reinhardtii follows Monod growth kinetics with a half-saturation constant of 1.2 ± 0.3 µM. This microfluidic platform provides a fast (~50 fold speed increase), cost effective (less reagents and human intervention) and quantitative technique for microalgal growth studies, in contrast to the current chemostat or batch cell culture system. It can be easily extended to investigate growth kinetics of other microorganisms under either single or co-culture setting.

Microgrooves and fluid flows provide preferential passageways for sperm over pathogen Tritrichomonas foetus.

Successful mammalian reproduction requires that sperm migrate through a long and convoluted female reproductive tract before reaching oocytes. For many years, fertility studies have focused on biochemical and physiological requirements of sperm. Here we show that the biophysical environment of the female reproductive tract critically guides sperm migration, while at the same time preventing the invasion of sexually transmitted pathogens. Using a microfluidic model, we demonstrate that a gentle fluid flow and microgrooves, typically found in the female reproductive tract, synergistically facilitate bull sperm migration toward the site of fertilization. In contrast, a flagellated sexually transmitted bovine pathogen, Tritrichomonas foetus, is swept downstream under the same conditions. We attribute the differential ability of sperm and T. foetus to swim against flow to the distinct motility types of sperm and T. foetus; specifically, sperm swim using a posterior flagellum and are near-surface swimmers, whereas T. foetus swims primarily via three anterior flagella and demonstrates much lower attraction to surfaces. This work highlights the importance of biophysical cues within the female reproductive tract in the reproductive process and provides insight into coevolution of males and females to promote fertilization while suppressing infection. Furthermore, the results provide previously unidentified directions for the development of in vitro fertilization devices and contraceptives.

Emergence of upstream swimming via a hydrodynamic transition.

We demonstrate that upstream swimming of sperm emerges via an orientation disorder-order transition. The order parameter, the average orientation of the sperm head against the flow, follows a 0.5 power law with the deviation from the critical flow shear rate (γ-γ_{c}). This transition is successfully explained by a hydrodynamic bifurcation theory, which extends the sperm upstream swimming to a broad class of near surface microswimmers that possess front-back asymmetry and circular motion.

Cooperative roles of biological flow and surface topography in guiding sperm migration revealed by a microfluidic model.

Successful reproduction in mammals requires sperm to swim against a fluid flow and through the long and complex female reproductive tract before reaching the egg in the oviduct. Millions of them do not make it. Despite their clinical importance, the roles played in sperm migration by the diverse biophysical and biochemical microenvironments within the reproductive tract are largely unknown. In this article, we present the development of a double layer microfluidic device that recreates two important biophysical environments within the female reproductive tract: fluid flow and surface topography. The unique feature of the device is that it enables one to study the cooperative roles of fluid flow and surface topography in guiding sperm migration. Using bull sperm as a model system, we found that microfluidic grooves embedded on a channel surface facilitate sperm migration against fluid flow. These findings suggest ways to design in vitro fertilization devices to treat infertility and to develop non-invasive contraceptives that use a microarchitectural design to entrap sperm.

SENSING DNA WITH ALTERNATING CURRENTS USING A NANOGAP SENSOR EMBEDDED IN A NANOCHANNEL DEVICE.

We report an integrated nanochannel/nanoelectrode sensor for the detection of DNA using alternating currents. We find that DNA can be detected using platinum as the metal for the detecting electrodes, with a signal to noise ratio exceeding 10. We argue that the signal is at least in part electrochemical in nature, thus holds the promise to yield a sequence-dependent signal. However, we also find that for large voltages, DNA attaches irreversibly to the driving electrodes.

A contact line pinning based microfluidic platform for modelling physiological flows.

This work introduces a contact line pinning based microfluidic platform for the generation of interstitial and intramural flows within a three dimensional (3D) microenvironment for cellular behaviour studies. A contact line pinning method was used to confine a natively derived biomatrix, collagen, in microfluidic channels without walls. By patterning collagen in designated wall-less channels, we demonstrated and validated the intramural flows through a microfluidic channel bounded by a monolayer of endothelial cells (mimic of a vascular vessel), as well as slow interstitial flows within a cell laden collagen matrix using the same microfluidic platform. The contact line pinning method ensured the generation of an engineered endothelial tube with straight walls, and spatially uniform interstitial fluid flows through the cell embedded 3D collagen matrix. Using this device, we demonstrated that the breast tumour cells' (MDA-MB-231 cell line) morphology and motility were modulated by the interstitial flows, and the motility of a sub-population of the cells was enhanced by the presence of the flow. The presented microfluidic platform provides a basic framework for studies of cellular behaviour including cell transmigration, growth, and adhesion under well controlled interstitial and intramural flows, and within a physiologically realistic 3D co-culture setting.

Universal protein fluctuations in populations of microorganisms.

The copy number of any protein fluctuates among cells in a population; characterizing and understanding these fluctuations is a fundamental problem in biophysics. We show here that protein distributions measured under a broad range of biological realizations collapse to a single non-gaussian curve under scaling by the first two moments. Moreover, in all experiments the variance is found to depend quadratically on the mean, showing that a single degree of freedom determines the entire distribution. Our results imply that protein fluctuations do not reflect any specific molecular or cellular mechanism, and suggest that some buffering process masks these details and induces universality.

Acceleration of emergence of bacterial antibiotic resistance in connected microenvironments.

The emergence of bacterial antibiotic resistance is a growing problem, yet the variables that influence the rate of emergence of resistance are not well understood. In a microfluidic device designed to mimic naturally occurring bacterial niches, resistance of Escherichia coli to the antibiotic ciprofloxacin developed within 10 hours. Resistance emerged with as few as 100 bacteria in the initial inoculation. Whole-genome sequencing of the resistant organisms revealed that four functional single-nucleotide polymorphisms attained fixation. Knowledge about the rapid emergence of antibiotic resistance in the heterogeneous conditions within the mammalian body may be helpful in understanding the emergence of drug resistance during cancer chemotherapy.

An introduction to micro-ecology patches.

Bacterial systems offer excellent tests of how well the general theoretical predictions of ecology dynamics do or do not in fact conform to reality. We believe that the basic rules that govern the cohabitation of competing species for limited resources are the same from bacteria to man, we just don't know the rules, and that fundamental studies of the games bacteria play will give fundamental insight into the vastly more complex systems we hope to attack later. In this tutorial review we discuss how simplified micro-ecologies constructed using tools of micro and nanofabrication techniques offer some idea of how physical principles and analysis can address the issue of complex ecology dynamics.

Upconverting nanophosphors for bioimaging.

Upconverting nanoparticles (UCNPs) when excited in the near-infrared (NIR) region display anti-Stokes emission whereby the emitted photon is higher in energy than the excitation energy. The material system achieves that by converting two or more infrared photons into visible photons. The use of the infrared confers benefits to bioimaging because of its deeper penetrating power in biological tissues and the lack of autofluorescence. We demonstrate here sub-10 nm, upconverting rare earth oxide UCNPs synthesized by a combustion method that can be stably suspended in water when amine modified. The amine modified UCNPs show specific surface immobilization onto patterned gold surfaces. Finally, the low toxicity of the UCNPs is verified by testing on the multi-cellular C. elegans nematode.

Complementary metal oxide semiconductor compatible fabrication and characterization of parylene-C covered nanofluidic channels with integrated nanoelectrodes.

Nanochannels offer a way to align and analyze long biopolymer molecules such as DNA with high precision at potentially single basepair resolution, especially if a means to detect biomolecules in nanochannels electronically can be developed. Integration of nanochannels with electronics will require the development of nanochannel fabrication procedures that will not damage sensitive electronics previously constructed on the device. We present here a near-room-temperature fabrication technology involving parylene-C conformal deposition that is compatible with complementary metal oxide semiconductor electronic devices and present an analysis of the initial impedance measurements of conformally parylene-C coated nanochannels with integrated gold nanoelectrodes.

Effects of different immersion media in multiphoton imaging of the epithelium and dermis of human skin.

In this work, we compared the performance of objectives with similar numerical aperture of 0.75 but different immersion media of air, water, glycerin, and oil in the imaging of human skin epithelium and dermis. In general, we found that the oil immersion objective recorded the strongest intensity at the same mechanical depth. We also characterized the focal shifts and found that with decreasing refractive index, the focal shift becomes increasingly more negative (for both the epithelium and dermis). In imaging the dermis, we estimated the image resolution at the depths of 18.8 and 30.2 microm, and found that the image resolution were comparable at these depths under the four types of immersion conditions. Our results demonstrate that by changing the immersion media, the main microscopic imaging effects are the recorded axial intensities and the focal shifts. The effects on the image resolution are negligible.