Locomotion of bovine spermatozoa during the transition from individual cells to bundles.

Various locomotion strategies employed by microorganisms are observed in complex biological environments. Spermatozoa assemble into bundles to improve their swimming efficiency compared to individual cells. However, the dynamic mechanisms for the formation of sperm bundles have not been fully characterized. In this study, we numerically and experimentally investigate the locomotion of spermatozoa during the transition from individual cells to bundles of two cells. Three consecutive dynamic behaviors are found across the course of the transition: hydrodynamic attraction/repulsion, alignment, and synchronization. The hydrodynamic attraction/repulsion depends on the relative orientation and distance between spermatozoa as well as their flagellar wave patterns and phase shift. Once the heads are attached, we find a stable equilibrium of the rotational hydrodynamics resulting in the alignment of the heads. The synchronization results from the combined influence of hydrodynamic and mechanical cell-to-cell interactions. Additionally, we find that the flagellar beat is regulated by the interactions during the bundle formation, whereby spermatozoa can synchronize their beats to enhance their swimming velocity.

Flagellar Propulsion of Sperm Cells Against a Time-Periodic Interaction Force.

Sperm cells undergo complex interactions with external environments, such as a solid-boundary, fluid flow, as well as other cells before arriving at the fertilization site. The interaction with the oviductal epithelium, as a site of sperm storage, is one type of cell-to-cell interaction that serves as a selection mechanism. Abnormal sperm cells with poor swimming performance, the major cause of male infertility, are filtered out by this selection mechanism. In this study, collinear bundles, consisting of two sperm cells, generate propulsive thrusts along opposite directions and allow to observe the influence of cell-to-cell interaction on flagellar wave-patterns. The developed elasto-hydrodynamic model demonstrates that steric and adhesive forces lead to highly symmetrical wave-pattern and reduce the bending amplitude of the propagating wave. It is measured that the free cells exhibit a mean flagellar curvature of 6.4 ± 3.5 rad mm-1 and a bending amplitude of 13.8 ± 2.8 rad mm-1 . After forming the collinear bundle, the mean flagellar curvature and bending amplitude are decreased to 1.8 ± 1.1 and 9.6 ± 1.4 rad mm-1 , respectively. This study presents consistent theoretical and experimental results important for understanding the adaptive behavior of sperm cells to the external time-periodic force encountered during sperm-egg interaction.

Embedded 3D printing of dilute particle suspensions into dense complex tissue fibers using shear thinning xanthan baths.

In order to fabricate functional organoids and microtissues, a high cell density is generally required. As such, the placement of cell suspensions in molds or microwells to allow for cell concentration by sedimentation is the current standard for the production of organoids and microtissues. Even though molds offer some level of control over the shape of the resulting microtissue, this control is limited as microtissues tend to compact towards a sphere after sedimentation of the cells. 3D bioprinting on the other hand offers complete control over the shape of the resulting structure. Even though the printing of dense cell suspensions in the ink has been reported, extruding dense cellular suspensions is challenging and generally results in high shear stresses on the cells and a poor shape fidelity of the print. As such, additional materials such as hydrogels are added in the bioink to limit shear stresses, and to improve shape fidelity and resolution. The maximum cell concentration that can be incorporated in a hydrogel-based ink before the ink's rheological properties are compromised, is significantly lower than the concentration in a tissue equivalent. Additionally, the hydrogel components often interfere with cellular self-assembly processes. To circumvent these limitations, we report a simple and inexpensive xanthan bath based embedded printing method to 3D print dense functional linear tissues using dilute particle suspensions consisting of cells, spheroids, hydrogel beads, or combinations thereof. Using this method, we demonstrated the self-organization of functional cardiac tissue fibers with a layer of epicardial cells surrounding a body of cardiomyocytes.

Bundle formation of sperm: Influence of environmental factors.

Cooperative behaviour of sperm is one of the mechanisms that plays a role in sperm competition. It has been observed in several species that spermatozoa interact with each other to form agglomerates or bundles. In this study, we investigate the effect of physical and biochemical factors that will most likely promote bundle formation in bull sperm. These factors include fluid viscosity, swim-up process, post-thaw incubation time and media additives which promote capacitation. While viscosity does not seem to influence the degree of sperm bundling, swim-up, post-thaw migration time and suppressed capacitation increase the occurrence of sperm bundles. This leads to the conclusion that sperm bundling is a result of hydrodynamic and adhesive interactions between the cells which occurs frequently during prolonged incubation times.

An open-source automated magnetic optical density meter for analysis of suspensions of magnetic cells and particles.

We present a spectrophotometer (optical density meter) combined with electromagnets dedicated to the analysis of suspensions of magnetotactic bacteria. The instrument can also be applied to suspensions of other magnetic cells and magnetic particles. We have ensured that our system, called MagOD, can be easily reproduced by providing the source of the 3D prints for the housing, electronic designs, circuit board layouts, and microcontroller software. We compare the performance of our system to existing adapted commercial spectrophotometers. In addition, we demonstrate its use by analyzing the absorbance of magnetotactic bacteria as a function of their orientation with respect to the light path and their speed of reorientation after the field has been rotated by 90°. We continuously monitored the development of a culture of magnetotactic bacteria over a period of 5 days and measured the development of their velocity distribution over a period of one hour. Even though this dedicated spectrophotometer is relatively simple to construct and cost-effective, a range of magnetic field-dependent parameters can be extracted from suspensions of magnetotactic bacteria. Therefore, this instrument will help the magnetotactic research community to understand and apply this intriguing micro-organism.

Visualization of micro-agents and surroundings by real-time multicolor fluorescence microscopy.

Optical microscopy techniques are a popular choice for visualizing micro-agents. They generate images with relatively high spatiotemporal resolution but do not reveal encoded information for distinguishing micro-agents and surroundings. This study presents multicolor fluorescence microscopy for rendering color-coded identification of mobile micro-agents and dynamic surroundings by spectral unmixing. We report multicolor microscopy performance by visualizing the attachment of single and cluster micro-agents to cancer spheroids formed with HeLa cells as a proof-of-concept for targeted drug delivery demonstration. A microfluidic chip is developed to immobilize a single spheroid for the attachment, provide a stable environment for multicolor microscopy, and create a 3D tumor model. In order to confirm that multicolor microscopy is able to visualize micro-agents in vascularized environments, in vitro vasculature network formed with endothelial cells and ex ovo chicken chorioallantoic membrane are employed as experimental models. Full visualization of our models is achieved by sequential excitation of the fluorophores in a round-robin manner and synchronous individual image acquisition from three-different spectrum bands. We experimentally demonstrate that multicolor microscopy spectrally decomposes micro-agents, organic bodies (cancer spheroids and vasculatures), and surrounding media utilizing fluorophores with well-separated spectrum characteristics and allows image acquisition with 1280 [Formula: see text] 1024 pixels up to 15 frames per second. Our results display that real-time multicolor microscopy provides increased understanding by color-coded visualization regarding the tracking of micro-agents, morphology of organic bodies, and clear distinction of surrounding media.

Drug-Loaded IRONSperm clusters: modeling, wireless actuation, and ultrasound imaging.

Individual biohybrid microrobots have the potential to perform biomedicalin vivotasks such as remote-controlled drug and cell delivery and minimally invasive surgery. This work demonstrates the formation of biohybrid sperm-templated clusters under the influence of an external magnetic field and essential functionalities for wireless actuation and drug delivery. Ferromagnetic nanoparticles are electrostatically assembled around dead sperm cells, and the resulting nanoparticle-coated cells are magnetically assembled into three-dimensional biohybrid clusters. The aim of this clustering is threefold: First, to enable rolling locomotion on a nearby solid boundary using a rotating magnetic field; second, to allow for noninvasive localization; third, to load the cells inside the cluster with drugs for targeted therapy. A magneto-hydrodynamic model captures the rotational response of the clusters in a viscous fluid, and predicts an upper bound for their step-out frequency, which is independent of their volume or aspect ratio. Below the step-out frequency, the rolling velocity of the clusters increases nonlinearly with their perimeter and actuation frequency. During rolling locomotion, the clusters are localized using ultrasound images at a relatively large distance, which makes these biohybrid clusters promising for deep-tissue applications. Finally, we show that the estimated drug load scales with the number of cells in the cluster and can be retained for more than 10 h. The aggregation of microrobots enables them to collectively roll in a predictable way in response to an external rotating magnetic field, and enhances ultrasound detectability and drug loading capacity compared to the individual microrobots. The favorable features of biohybrid microrobot clusters place emphasis on the importance of the investigation and development of collective microrobots and their potential forin vivoapplications.

Serial imaging of micro-agents and cancer cell spheroids in a microfluidic channel using multicolor fluorescence microscopy.

Multicolor fluorescence microscopy is a powerful technique to fully visualize many biological phenomena by acquiring images from different spectrum channels. This study expands the scope of multicolor fluorescence microscopy by serial imaging of polystyrene micro-beads as surrogates for drug carriers, cancer spheroids formed using HeLa cells, and microfluidic channels. Three fluorophores with different spectral characteristics are utilized to perform multicolor microscopy. According to the spectrum analysis of the fluorophores, a multicolor widefield fluorescence microscope is developed. Spectral crosstalk is corrected by exciting the fluorophores in a round-robin manner and synchronous emitted light collection. To report the performance of the multicolor microscopy, a simplified 3D tumor model is created by placing beads and spheroids inside a channel filled with the cell culture medium is imaged at varying exposure times. As a representative case and a method for bio-hybrid drug carrier fabrication, a spheroid surface is coated with beads in a channel utilizing electrostatic forces under the guidance of multicolor microscopy. Our experiments show that multicolor fluorescence microscopy enables crosstalk-free and spectrally-different individual image acquisition of beads, spheroids, and channels with the minimum exposure time of 5.5 ms. The imaging technique has the potential to monitor drug carrier transportation to cancer cells in real-time.

Impact of Segmented Magnetization on the Flagellar Propulsion of Sperm-Templated Microrobots.

Technical design features for improving the way a passive elastic filament produces propulsive thrust can be understood by analyzing the deformation of sperm-templated microrobots with segmented magnetization. Magnetic nanoparticles are electrostatically self-assembled on bovine sperm cells with nonuniform surface charge, producing different categories of sperm-templated microrobots. Depending on the amount and location of the nanoparticles on each cellular segment, magnetoelastic and viscous forces determine the wave pattern of each category during flagellar motion. Passively propagating waves are induced along the length of these microrobots using external rotating magnetic fields and the resultant wave patterns are measured. The response of the microrobots to the external field reveals distinct flow fields, propulsive thrust, and frequency responses during flagellar propulsion. This work allows predictions for optimizing the design and propulsion of flexible magnetic microrobots with segmented magnetization.

Contactless acoustic micro/nano manipulation: a paradigm for next generation applications in life sciences.

Acoustic actuation techniques offer a promising tool for contactless manipulation of both synthetic and biological micro/nano agents that encompass different length scales. The traditional usage of sound waves has steadily progressed from mid-air manipulation of salt grains to sophisticated techniques that employ nanoparticle flow in microfluidic networks. State-of-the-art in microfabrication and instrumentation have further expanded the outreach of these actuation techniques to autonomous propulsion of micro-agents. In this review article, we provide a universal perspective of the known acoustic micromanipulation technologies in terms of their applications and governing physics. Hereby, we survey these technologies and classify them with regards to passive and active manipulation of agents. These manipulation methods account for both intelligent devices adept at dexterous non-contact handling of micro-agents, and acoustically induced mechanisms for self-propulsion of micro-robots. Moreover, owing to the clinical compliance of ultrasound, we provide future considerations of acoustic manipulation techniques to be fruitfully employed in biological applications that range from label-free drug testing to minimally invasive clinical interventions.

IRONSperm: Sperm-templated soft magnetic microrobots.

We develop biohybrid magnetic microrobots by electrostatic self-assembly of nonmotile sperm cells and magnetic nanoparticles. Incorporating a biological entity into microrobots entails many functional advantages beyond shape templating, such as the facile uptake of chemotherapeutic agents to achieve targeted drug delivery. We present a single-step electrostatic self-assembly technique to fabricate IRONSperms, soft magnetic microswimmers that emulate the motion of motile sperm cells. Our experiments and theoretical predictions show that the swimming speed of IRONSperms exceeds 0.2 body length/s (6.8 ± 4.1 µm/s) at an actuation frequency of 8 Hz and precision angle of 45°. We demonstrate that the nanoparticle coating increases the acoustic impedance of the sperm cells and enables localization of clusters of IRONSperm using ultrasound feedback. We also confirm the biocompatibility and drug loading ability of these microrobots, and their promise as biocompatible, controllable, and detectable biohybrid tools for in vivo targeted therapy.

Fabrication of Magnetic Molecularly Imprinted Beaded Fibers for Rosmarinic Acid.

The present study describes the fabrication of molecularly imprinted (MI) magnetic beaded fibers using electrospinning. Rosmarinic acid was selected as exemplary yet relevant template during molecular imprinting. A "design of experiments" methodology was used for optimizing the electrospinning process. Four factors, i.e., the concentration of the biodegradable polymer (polycaprolactone), the applied voltage, the flow rate, and the collector distance were varied in a central composite design. The production process was then optimized according to the suitability of the beaded fibers during microrobot fabrication, actuation, and drug release. The optimum average fiber diameter of MI beaded fibers was determined at 857 ± 390 nm with an average number of beads at 0.011 ± 0.002 per µm2. In vitro release profiles of the optimized MI beaded fibers revealed a lower burst rate and a more sustained release when compared to control fibers. Magnetic control of the MI beaded fibers was successfully tested by following selected waypoints along a star-shaped predefined trajectory. This study innovatively combines molecular imprinting technology with magnetic microrobots enabling targeted drug delivery systems that offer precise motion control via the magnetic response of microrobots along with selective uptake of a drug into the microrobot using MI beaded fibers in future.

Magnetic localization and control of helical robots for clearing superficial blood clots.

This work presents an approach for the localization and control of helical robots during removal of superficial blood clots inside in vitro and ex vivo models. The position of the helical robot is estimated using an array of Hall-effect sensors and precalculated magnetic field map of two synchronized rotating dipole fields. The estimated position is used to implement closed-loop motion control of the helical robot using the rotating dipole fields. We validate the localization accuracy by visual feedback and feature tracking inside the in vitro model. The experimental results show that the magnetic localization of a helical robot with diameter of 1 mm can achieve a mean absolute position error of 2.35 ± 0.4 mm (n = 20). The simultaneous localization and motion control of the helical robot enables propulsion toward a blood clot and clearing at an average removal rate of 0.67 ± 0.47 mm3/min. This method is used to localize the helical robot inside a rabbit aorta (ex vivo model), and the localization accuracy is validated using ultrasound feedback with a mean absolute position error of 2.6 mm.

Characterization of Flagellar Propulsion of Soft Microrobotic Sperm in a Viscous Heterogeneous Medium.

Several microorganisms swim by a beating flagellum more rapidly in solutions with gel-like structure than they do in low-viscosity mediums. In this work, we aim to model and investigate this behavior in low Reynolds numbers viscous heterogeneous medium using soft microrobotic sperm samples. The microrobots are actuated using external magnetic fields and the influence of immersed obstacles on the flagellar propulsion is investigated. We use the resistive-force theory to predict the deformation of the beating flagellum, and the method of regularized Stokeslets for computing Stokes flows around the microrobot and the immersed obstacles. Our analysis and experiments show that obstacles in the medium improves the propulsion even when the Sperm number is not optimal (S p ≠ 2.1). Experimental results also show propulsion enhancement for concentration range of 0-5% at relatively low actuation frequencies owing to the pressure gradient created by obstacles in close proximity to the beating flagellum. At relatively high actuation frequency, speed reduction is observed with the concentration of the obstacles.

Characterization of Helical Propulsion Inside In Vitro and Ex Vivo Models of a Rabbit Aorta.

In this work, the propulsion of a helical robot is experimentally characterized inside whole blood (in vitro model) and against the flowing streams of phosphate buffered saline (PBS) inside rabbit aorta (ex vivo model). The helical robot is magnetically actuated inside these models under the influence of rotating magnetic fields. The frequency response of the helical robot is characterized. Averaged speed is measured at actuation frequency of 8 Hz as 11.3 ± 0.52 (n = 5) and 7.45 ± 1.2 mm/s (n = 3) inside rabbit aorta and whole blood, respectively. The Speed of the robot inside rabbit aorta is characterized against flowing streams of PBS at flow rate of 90 ml/hr.

Controllable switching between planar and helical flagellar swimming of a soft robotic sperm.

Sperm cells undergo a wide variety of swimming patterns by a beating flagellum to maintain high speed regardless of the rheological and physical properties of the background fluid. In this work, we develop and control a soft robotic sperm that undergoes controllable switching between swimming modes like biological sperm cells. The soft robotic sperm consists of a magnetic head and an ultra-thin flexible flagellum, and is actuated using external magnetic fields. We observe that out-of-plane wobbling of the head results in helical wave propagation along the flagellum, whereas in-plane wobbling achieves planar wave propagation. Our theoretical predictions and experimental results show the ability of the soft robotic sperm to change its swimming speed by tuning the beating frequency of its flagellum and the propulsion pattern. The average speed of the soft robotic sperm increases by factors of 2 and 1.2 in fluids with viscosity of 1 Pa.s and 5 Pa.s at relatively low actuation frequencies, respectively, when they switch between planar to helical flagellar propulsion.

The Influence of Mechanical Rubbing on the Dissolution of Blood Clots.

Mechanical rubbing of blood clots is a potential minimally-invasive method for clearing clogged blood vessels. In this work, we investigate the influence of the interaction of the tip of a helical robot with blood clots. This interaction enables the dissolution of the blood clot and the release of the entrapped red blood cells and platelets from its three-dimensional fibrin fiber network. We analyze the pre- and post-conditions of the blood clots following 40 minutes of mechanical rubbing, under the influence of a rotating magnetic field in the frequency range of 20 Hz to 45 Hz. Our measurements show that the weight of the blood clots is decreased by 22.5 ± 11.1% at frequency of 25 Hz. We also validate the influence of mechanical rubbing using cell count and spectrophotometric analysis on phosphate buffered saline samples past the robot and the clot. The maximum cell count is measured as 654 ± 108 × 104 cells/m1 and 54 ± 12 × 104 cells/m1, whereas the absorbance is measured as 4. 35 × 10-6 mol and 1. 05 × 10-6 mol under the influence of mechanical rubbing and without mechanical rubbing, respectively.

An Investigation of the Sensing Capabilities of Magnetotactic Bacteria.

We investigate the sensing capabilities of magnetotactic bacteria (Magnetospirillum gryphiswaldense strain MSR1) to MCF-7 breast cancer cells. Cancer cells are allowed to grow inside a capillary tube with depth of 200 $\mu \mathrm {m}$ and motion of magnetotactic bacteria is investigated under the influence of oxygen gradient and geomagnetic field. The influence of cancer cells is modeled to predict the oxygen gradient within the capillary tube in three-dimensional space. Our experimental motion analysis and count of motile magnetotactic bacteria indicate that they migrate towards less-oxygenated regions within the vicinity of cancer cells. Bands of magnetotactic bacteria with average concentration of 18.8±2.0% are observed in close proximity to MCF-7 cells $(h = 20~ \mu \mathrm {m})$, whereas the concentration at proximity of $190~ \mu \mathrm {m}$ is 5.0 ± 6.8%.

Swimming Back and Forth Using Planar Flagellar Propulsion at Low Reynolds Numbers.

Peritrichously flagellated Escherichia coli swim back and forth by wrapping their flagella together in a helical bundle. However, other monotrichous bacteria cannot swim back and forth with a single flagellum and planar wave propagation. Quantifying this observation, a magnetically driven soft two-tailed microrobot capable of reversing its swimming direction without making a U-turn trajectory or actively modifying the direction of wave propagation is designed and developed. The microrobot contains magnetic microparticles within the polymer matrix of its head and consists of two collinear, unequal, and opposite ultrathin tails. It is driven and steered using a uniform magnetic field along the direction of motion with a sinusoidally varying orthogonal component. Distinct reversal frequencies that enable selective and independent excitation of the first or the second tail of the microrobot based on their tail length ratio are found. While the first tail provides a propulsive force below one of the reversal frequencies, the second is almost passive, and the net propulsive force achieves flagellated motion along one direction. On the other hand, the second tail achieves flagellated propulsion along the opposite direction above the reversal frequency.

Wireless magnetic-based closed-loop control of self-propelled microjets.

In this study, we demonstrate closed-loop motion control of self-propelled microjets under the influence of external magnetic fields. We control the orientation of the microjets using external magnetic torque, whereas the linear motion towards a reference position is accomplished by the thrust and pulling magnetic forces generated by the ejecting oxygen bubbles and field gradients, respectively. The magnetic dipole moment of the microjets is characterized using the U-turn technique, and its average is calculated to be 1.3x10⁻¹° A.m² at magnetic field and linear velocity of 2 mT and 100 µm/s, respectively. The characterized magnetic dipole moment is used in the realization of the magnetic force-current map of the microjets. This map in turn is used for the design of a closed-loop control system that does not depend on the exact dynamical model of the microjets and the accurate knowledge of the parameters of the magnetic system. The motion control characteristics in the transient- and steady-states depend on the concentration of the surrounding fluid (hydrogen peroxide solution) and the strength of the applied magnetic field. Our control system allows us to position microjets at an average velocity of 115 µm/s, and within an average region-of-convergence of 365 µm.