Cellular Manipulation Research Articles

Genetically Expressed RNA Strand Displacement for Cellular Manipulation.

Nucleic acid strand displacement is a pivotal concept in dynamic nucleic acid nanotechnologies, which has been extensively investigated and applied across various fields. Compared with DNA systems, the genetically expressed RNA strand displacement technology offers unique advantages for construction of genetic circuits in living cells, where RNA expression and modulation may be seamlessly integrated into the genomic network for long-term and stable regulations of diversified biological functionalities. This Concept paper provides an overview of previous efforts on developments of synthetic gene circuits through utilization of RNA strand displacement, including our endeavors in this field. Moreover, future prospects, potential applications and challenges of the genetically expressed RNA strand displacement technology are also discussed.

Programmable acoustic modular microrobots

The field of microrobotics has emerged as a promising area of research with significant applications in biomedicine, both in vitro and in vivo, such as targeted cargo delivery, microsurgery, and cellular manipulation. Microrobots actuated with multiple modalities have the potential for greater adaptability, robustness, and capability to perform various tasks. Modular units that can reconfigure into various shapes, create structures that may be difficult to fabricate as one whole unit, and be assembled on-site, could provide more versatility by assembly and disassembly of units on demand. Such multi-modal modular microrobots have the potential to address challenging applications. Here, we present a biocompatible cylindrical microrobot with a dome-shaped cavity. The microrobot is actuated by both magnetic and acoustic fields and forms modular microstructures of various shapes. We demonstrate the use of these microrobots for cellular manipulation by creating patterns on a surface.

Characterization and modulation of the unimolecular conformation of integrins with nanopore sensors

Integrins are a large family of heterodimeric mammalian transmembrane receptors that connect extracellular matrix (ECM) and the cytoskeleton. Integrin-mediated adhesion governs the cellular adhesion, mobility, and cell mechanobiology. Investigations on different integrins in cellular and the conformation change regulated by divalent metal ions and oligopeptides have been documented, they mainly adopted the cellular assays or surface manipulation to achieve the information of ensemble integrins with proteins and small molecules. This work presents the nanopore approach for the characterization of unimolecular integrin via resistive pulse sensing. The orientation selection in distinct nanopore in size and pH condition discloses the tiny disparity of the conformation and the local surface charge distribution. The divalent metal ions could efficiently modulate the conformation alteration of integrin, in which Ca2+ and Mn2+ could activate integrin αLβ2 to take larger physical dimension than Mg2+, and Ca2+ is superior to Mn2+ in the activation when integrin αLβ2 is treated in the mixed metal ions. The oligopeptide cRGD (cyclic Arg-Gly-Asp) and its derivatives cRGDfk and cRGDyk show specific binding with integrin αVβ3 and the complex of αVβ3-cRGD exhibits largest geometry upon Ca2+-mediation with characteristic nanopore translocation feature. This work provides a platform for unimolecular integrin sensing and the modulation of the local conformation transition regulated by divalent metal ions and oligopeptides, which will be valuable for the representation of integrin-related cellular behavior and bio-function.

Microfluidic Electroporation Arrays for Investigating Electroporation-Induced Cellular Rupture Dynamics.

Electroporation is pivotal in bioelectrochemistry for cellular manipulation, with prominent applications in drug delivery and cell membrane studies. A comprehensive understanding of pore generation requires an in-depth analysis of the critical pore size and the corresponding energy barrier at the onset of cell rupture. However, many studies have been limited to basic models such as artificial membranes or theoretical simulations. Challenging this paradigm, our study pioneers using a microfluidic electroporation chip array. This tool subjects live breast cancer cell species to a diverse spectrum of alternating current electric field conditions, driving electroporation-induced cell rupture. We conclusively determined the rupture voltages across varying applied voltage loading rates, enabling an unprecedented characterization of electric cell rupture dynamics encompassing critical pore radius and energy barrier. Further bolstering our investigation, we probed cells subjected to cholesterol depletion via methyl-β-cyclodextrin and revealed a strong correlation with electroporation. This work not only elucidates the dynamics of electric rupture in live cell membranes but also sets a robust foundation for future explorations into the mechanisms and energetics of live cell electroporation.

Signalling switches maintain intercellular communication in the vascular endothelium.

The single layer of cells lining all blood vessels, the endothelium, is a sophisticated signal co-ordination centre that controls a wide range of vascular functions including the regulation of blood pressure and blood flow. To co-ordinate activities, communication among cells is required for tissue level responses to emerge. While a significant form of communication occurs by the propagation of signals between cells, the mechanism of propagation in the intact endothelium is unresolved. Precision signal generation and targeted cellular manipulation was used in conjunction with high spatiotemporal mesoscale Ca2+ imaging in the endothelium of intact blood vessels. Multiple mechanisms maintain communication so that Ca2+ wave propagation occurs irrespective of the status of connectivity among cells. Between adjoining cells, regenerative IP3-induced IP3 production transmits Ca2+ signals and explains the propagated vasodilation that underlies the increased blood flow accompanying tissue activity. The inositide is itself sufficient to evoke regenerative phospholipase C-dependent Ca2+ waves across coupled cells. None of gap junctions, Ca2+ diffusion or the release of extracellular messengers is required to support this type of intercellular Ca2+ signalling. In contrast, when discontinuities exist between cells, ATP released as a diffusible extracellular messenger transmits Ca2+ signals across the discontinuity and drives propagated vasodilation. These results show that signalling switches underlie endothelial cell-to-cell signal transmission and reveal how communication is maintained in the face of endothelial damage. The findings provide a new framework for understanding wave propagation and cell signalling in the endothelium.

Entangling and disentangling mitotic chromosomes

The DNA topological challenges generated by cellular manipulation of extremely long DNA fibers remain poorly understood. In this issue of Molecular Cell, Hildebrand etal.1 describe how mitotic chromosomes are self entangled and that disentanglement requires TOP2 activity in late mitosis.

Modulation of bacterial membranes and cellular macromolecules by dimethyl sulfoxide: A dose-dependent study providing novel insights

Using Escherichia coli as a model, this manuscript delves into the intricate interactions between dimethyl sulfoxide (DMSO) and membranes, cellular macromolecules, and the effects on various aspects of bacterial physiology. Given DMSO's wide-ranging use as a solvent in microbiology, we investigate the impacts of both non-growth inhibitory (1.0 % and 2.5 % v/v) and slightly growth-inhibitory (5.0 % v/v) concentrations of DMSO. The results demonstrate that DMSO causes alterations in bacterial membrane potential, influences the electrochemical characteristics of the cell surface, and exerts substantial effects on the composition and structure of cellular biomolecules. Genome-wide gene expression data from DMSO-treated E. coli was used to further investigate and bolster the results. The findings of this study provide valuable insights into the complex relationship between DMSO and biological systems, with potential implications in drug delivery and cellular manipulation. However, it is essential to exercise caution when utilizing DMSO to enhance the solubility and delivery of bioactive compounds, as even at low concentrations, DMSO exerts non-inert effects on cellular macromolecules and processes.

Optothermal Microparticle Oscillator Induced by Marangoni and Thermal Convection.

The light-fueled microparticle oscillator, exemplifying sustained driving in a static light source, potentially holds applications in fundamental physics, cellular manipulation, fluid dynamics, and various other soft-matter systems. The challenges of photodamage due to laser focusing on particles and the control of the oscillation direction have always been two major issues for microparticle oscillators. Here, we present an optical-thermal method for achieving a 3D microparticle oscillator with a fixed direction by employing laser heating of the gold film surface. First, the microparticle oscillation without direction limitation is studied. The photothermal conversion originates from the laser heating of a gold film. The oscillation mechanism is the coordination of the forces exerted on the particles, including the thermal convective force, thermophoresis force, and gravity. Subsequently, the additional Marangoni convection force, generated by the temperature gradient on the surface of a microbubble, is utilized to control the oscillation direction of the microparticle. Finally, a dual-channel oscillation mode is achieved by utilizing two microbubbles. During the oscillation process, the microparticle is influenced by flow field forces and temperature gradient force, completely avoiding optical damage to the oscillating microparticle.

A putative design for the electromagnetic activation of split proteins for molecular and cellular manipulation.

The ability to manipulate cellular function using an external stimulus is a powerful strategy for studying complex biological phenomena. One approach to modulate the function of the cellular environment is split proteins. In this method, a biologically active protein or an enzyme is fragmented so that it reassembles only upon a specific stimulus. Although many tools are available to induce these systems, nature has provided other mechanisms to expand the split protein toolbox. Here, we show a novel method for reconstituting split proteins using magnetic stimulation. We found that the electromagnetic perceptive gene (EPG) changes conformation due to magnetic field stimulation. By fusing split fragments of a certain protein to both termini of the EPG, the fragments can be reassembled into a functional protein under magnetic stimulation due to conformational change. We show this effect with three separate split proteins: NanoLuc, APEX2, and herpes simplex virus type-1 thymidine kinase. Our results show, for the first time, that reconstitution of split proteins can be achieved only with magnetic fields. We anticipate that this study will be a starting point for future magnetically inducible split protein designs for cellular perturbation and manipulation. With this technology, we can help expand the toolbox of the split protein platform and allow better elucidation of complex biological systems.

Bioinspired microrobots and their biomedical applications.

Natural organisms and biological systems provide a rich source of inspiration for the development of bioinspired microrobots. These diminutive automatons, designed to emulate the intricate structures and functions of living entities, extend human capabilities across a spectrum of applications. This review endeavors to amalgamate and elucidate the underpinnings of such bioinspired microrobots design, traversing the interdisciplinary expanse of research. It delineates a spectrum of biomedical applications for bioinspired microrobots, encompassing targeted drug delivery, cellular manipulation, and minimally invasive surgical procedures, among others. Moreover, the current technical challenges and future directions of bioinspired microrobots in the biomedical field are discussed. The objective is to impart a holistic view to the readership, illuminating the significance of bioinspired microrobots in contemporary biomedicine and charting potential trajectories of innovation.

Challenges in Imaging Analyses of Biomolecular Condensates in Cells Infected with Influenza A Virus.

Biomolecular condensates are crucial compartments within cells, relying on their material properties for function. They form and persist through weak, transient interactions, often undetectable by classical biochemical approaches. Hence, microscopy-based techniques have been the most reliable methods to detail the molecular mechanisms controlling their formation, material properties, and alterations, including dissolution or phase transitions due to cellular manipulation and disease, and to search for novel therapeutic strategies targeting biomolecular condensates. However, technical challenges in microscopy-based analysis persist. This paper discusses imaging, data acquisition, and analytical methodologies' advantages, challenges, and limitations in determining biophysical parameters explaining biomolecular condensate formation, dissolution, and phase transitions. In addition, we mention how machine learning is increasingly important for efficient image analysis, teaching programs what a condensate should resemble, aiding in the correlation and interpretation of information from diverse data sources. Influenza A virus forms liquid viral inclusions in the infected cell cytosol that serve as model biomolecular condensates for this study. Our previous work showcased the possibility of hardening these liquid inclusions, potentially leading to novel antiviral strategies. This was established using a framework involving live cell imaging to measure dynamics, internal rearrangement capacity, coalescence, and relaxation time. Additionally, we integrated thermodynamic characteristics by analysing fixed images through Z-projections. The aforementioned paper laid the foundation for this subsequent technical paper, which explores how different modalities in data acquisition and processing impact the robustness of results to detect bona fide phase transitions by measuring thermodynamic traits in fixed cells. Using solely this approach would greatly simplify screening pipelines. For this, we tested how single focal plane images, Z-projections, or volumetric analyses of images stained with antibodies or live tagged proteins altered the quantification of thermodynamic measurements. Customizing methodologies for different biomolecular condensates through advanced bioimaging significantly contributes to biological research and potential therapeutic advancements.

A Two-Dimensional Transient Computational Multi-Physics Model for Analyzing Magnetic and Non-Magnetic Particle (Red Blood Cells and E. Coli Bacteria) Dynamics in a Traveling Wave Ferro-Magnetic Microfluidic Device for Potential Cell Separation and Sorting

Abstract This paper presents the theory and development, validation, and results of a transient computational multiphysics model for analyzing the magnetic field, particle dynamics, and capture efficiency of magnetic and nonmagnetic (e.g., Red Blood Cells and E. Coli bacteria) microparticles in a traveling wave ferromagnetic microfluidic device. This computational model demonstrates proof-of-concept of a method for greatly enhancing magnetic bioseparation in ferromicrofluidic systems using an array of copper conductive elements arranged in quadrature to create a periodic potential energy landscape. In contrast to previous works, our approach theoretically uses a microfluidic device with an electronic chip platform consisting of integrated copper electrodes that carry currents to generate programable magnetic field gradients locally. Alternating currents are applied to the electrodes in quadrature (using a 90 deg phase change from the neighboring electrode) to create a periodic magnetic field pattern that travels along the length of the microchannel. Our previous work evaluated magnetic and nonmagnetic particles in a static magnetic field within the same channel geometry. This work is a phase 2 study that expands on the previous work and analyzes the dynamics of magnetic and nonmagnetic entities characterized by material magnetic susceptibility in a transient magnetic field. This is an improvement over our previous work. The model, which is described in more detail in the methods section, combines a Eulerian-Lagrangian and two-way particle-fluid coupling CFD analysis with closed-form magnetic field analysis that is used to predict magnetic separation considering dominant magnetic and hydrodynamic forces similar to our previous works in magnetic drug targeting. The model was also validated with an experimental low frequency stationary flow study on separating nonmagnetic latex fluorescent particles in a water based ferrofluid. The results from the experimental study and the developed model demonstrate that the proposed device may potentially be used as an effective platform for microparticle and cellular manipulation and sorting. The developed multiphysics model could potentially be used as a design optimization tool for traveling wave ferromicrofluidic devices.

Magnetic Microrobots as a Platform for Cell Clean Up.

Mobile magnetic microrobots have been extensively used in a wide range of biomedical applications due to their numerous advantages. Magnetic microrobots in particular have been developed and shown great potential over the past two decades for the manipulation and migration of both single cells and cell aggregates. The efficient clearance of cell aggregates is crucial to prevent uncontrolled cell proliferation, tissue damage, and invasive surgeries, especially for those related to the vascular system. In this work, we showed cellular manipulation and mobility to achieve cellular clean up on Human Liver Cancer (HepG2) cells by using two types of untethered magnetic microrobots that are different in type and size. We performed the cellular clean up in the microchannel, which can demonstrate the closed working environment, and also on a glass slide to present an air-liquid interface. We showed that the microrobots could be able to move a cluster of cells in both conditions which could make them useful for sorting and separation applications. Furthermore, cell viability was assessed by using a trypan blue staining assay on HepG2 cells right after and 24 hours after microrobot actuation.

Rolling Helical Microrobots for Cell Patterning.

Microrobots, untethered miniature devices capable of performing tasks at the microscale, have gained significant attention in the fields of robotics and biomedicine. These devices hold immense potential for various industrial and scientific applications, including targeted drug delivery and cell manipulation. In this study, we present a novel magnetic rolling helical microrobot specifically designed for bio-compatible cell patterning. Our microrobot incorporates both open-loop and closed-loop control mechanisms, providing flexible, precise, and rapid control for various applications. Through experiments, we demonstrate the microrobot's ability to manipulate cells by pushing them while rolling and arranging cells into desired patterns. This result is particularly significant as it has implications for diverse biological applications such as tissue engineering and organoid development. Moreover, we showcase the effectiveness of our microrobot in a closed-loop control system, where it successfully follows a predetermined path from an origin to a destination. The combination of cellular manipulation capabilities and trajectory-tracking performance underlines the versatility and potential of our magnetic rolling helical microrobot. The ability to control and navigate the microrobot with high precision opens up new possibilities for advanced biomedical applications. These findings contribute to the growing body of knowledge in microbotics and pave the way for further research and development in the field.

Expanding the Abu Dhabi Bone Marrow Transplant Program (AD-BMT<sup>®</sup>) Scope with the Collection and Cryopreservation of Cord Blood Stem Cells

<i>Introduction:</i> The umbilical cord blood (UCB) is the blood from the placenta's blood vessels and the portion of the umbilical cord that remains attached to the newborn after the delivery when the cord is clamped and cut. UCB is rich in hematopoietic stem cells (HSC) and other stem cells (SC). Between 1974 and 1988, scientific knowledge and UCB application possibilities for human therapy opened a new significative era in cell therapy, using UCB HSC as an alternative source of SC for bone marrow transplant and the cure of patients with malignant hematological diseases. <i>Objective:</i> The possibility of having a UCB bank (UCB-B) in Abu Dhabi encouraged us to make this analysis. <i>Method:</i> A literature review from Google Scholar, PubMed, SciELO, Scopus, and other sources, about the increasing application of UCB-SC and storage in UCB-B was done to update about all the possibilities for translational medicine. <i>Results:</i> We prepared a UCB overview, including the pioneering use of UCB-SC for HSC transplantation, their biological composition, characteristics, and the UC-associated tissues like Wharton-Jelly and the human term placenta, UCB advantages, and disadvantages in HSC transplantation, the number of clinical trials applying UCB donation samples, and promising possibilities for regenerative medicine. The existence of different types of UCB-Bs, and the development of new specific cellular manipulation techniques. The UCB-B regulatory framework and some ethical issues were also included in the review. <i>Conclusions:</i> After 30 years of UCB-B, more than 100 public UCB-Bs worldwide, with millions of UCB units donated altruistically, more than 800,000 clinical-grade products are now available: a great source of transplantable SC and other cellular material for the development of new therapies. Even though the UCB as a source of HSC for transplantation has been recently statistical ranked in third place, after HSC apheresis collected and bone marrow aspirates, until now, more than 50,000 UCB HSC allogeneic transplants (HSC-Allo-T) have been performed worldwide in both children and adults to treat many different diseases, including hematologic, metabolic, immunologic, neoplastic, and neurologic disorders. A significant effort has been focused on enhancing engraftment to reduce the risk of infection and cost. <i>Recommendations:</i> To that end, we must understand in detail the molecular mechanisms controlling SC self-renewal that may lead to the development of ex-vivo systems for HSCs expansion, characterize the mechanisms regulating the homing of HSC and determine the relative place of UCB HSC-Allo-T, as compared to other sources.

Construction of Polymeric DNA Network and Application for Cell Manipulation

Comprehensive SummaryDeoxyribonucleic acid (DNA) is a biomacromolecule, as well as a polymeric material, whose sequences with different manipulative structures enable them to implement a series of functions, such as reorganization, target, and catalysis. Compared to existing traditional materials incapable of multifunctional integration, the polymeric DNA network is a form of material that can achieve functional integration while maintaining specific DNA properties. Furthermore, precise target enabled by DNA network is one of the most essential components of cellular manipulation. Hence, the DNA network is indispensable and irreplaceable to cell manipulation that it is a versatile tool for the understanding of basic laws of living life and treatments of diseases, such as cell isolation, cell delivery, and cell interference. Herein, the construction of polymeric DNA network is briefly introduced from the aspects of assembly modules, construction methods, and properties. Subsequently, the applications of DNA network in the field of cell manipulation, including cell isolation and encapsulation, intracellular inference and detection, and tumor therapy are summarized. Finally, we provide some prospects for further research and applications of DNA network for cell manipulation.

Design and Development of a Traveling Wave Ferro-Microfluidic Device and System Rig for Potential Magnetophoretic Cell Separation and Sorting in a Water-Based Ferrofluid.

The timely detection and diagnosis of diseases and accurate monitoring of specific genetic conditions require rapid and accurate separation, sorting, and direction of target cell types toward a sensor device surface. In that regard, cellular manipulation, separation, and sorting are progressively finding application potential within various bioassay applications such as medical disease diagnosis, pathogen detection, and medical testing. The aim of this paper is to present the design and development of a simple traveling wave ferro-microfluidic device and system rig purposed for the potential manipulation and magnetophoretic separation of cells in water-based ferrofluids. This paper details in full: (1) a method for tailoring cobalt ferrite nanoparticles for specific diameter size ranges (10-20 nm), (2) the development of a ferro-microfluidic device for potentially separating cells and magnetic nanoparticles, (3) the development of a water-based ferrofluid with magnetic nanoparticles and non-magnetic microparticles, and (4) the design and development of a system rig for producing the electric field within the ferro-microfluidic channel device for magnetizing and manipulating nonmagnetic particles in the ferro-microfluidic channel. The results reported in this work demonstrate a proof of concept for magnetophoretic manipulation and separation of magnetic and non-magnetic particles in a simple ferro-microfluidic device. This work is a design and proof-of-concept study. The design reported in this model is an improvement over existing magnetic excitation microfluidic system designs in that heat is efficiently removed from the circuit board to allow a range of input currents and frequencies to manipulate non-magnetic particles. Although this work did not analyze the separation of cells from magnetic particles, the results demonstrate that non-magnetic (surrogates for cellular materials) and magnetic entities can be separated and, in some cases, continuously pushed through the channel based on amperage, size, frequency, and electrode spacing. The results reported in this work establish that the developed ferro-microfluidic device may potentially be used as an effective platform for microparticle and cellular manipulation and sorting.

Microring resonator based disposable ultrasound-sensing chronic cranial window

Chronic cranial window (CCW) has been widely used to provide optical access to the brain cortex for longitudinal imaging, while preserving the physiological environments of the brain. Various optical imaging modalities have been demonstrated with CCW for longitudinal brain imaging, including single-photon and multi-photon fluorescence microscopy, optical coherence tomography, and photoacoustic microscopy (PAM). However, sophisticated surgeries and windows designs are still required for chronic imaging and direct cellular recording or manipulation because the surgical implantation of CCW precludes direct physical access to the brain other than optical access. Here, we report an active CCW with integrated ultrasound sensor based on a transparent microring resonator (MRR). The MRR is fabricated on a quartz substrate by using low-cost soft nanoimprinting lithography (sNIL) and then integrated on a glass window with optical fibers for disposable ultrasound-sensing CCW (usCCW). Encapsulating MRR inside an acoustic impedance matched protection layer further improves its reliability for in vivo applications over the observed period of a month. The functions of the active usCCW are experimentally validated through longitudinal (PAM) imaging of cortical vasculature in live mice over a 28-days period.

Microfluidic Platforms for Single Cell Analysis: Applications in Cellular Manipulation and Optical Biosensing

Cellular heterogeneity of any tissue or organ makes it challenging to identify and study the impact and the treatment of any disease. In this context, analysis of cells at an individual level becomes highly relevant for throwing light on the heterogeneous nature of cells. Single cell analysis can be used to gain insights into an overall view of any disease, thereby holding great applications in health diagnosis, disease identification, drug screening, and targeted delivery. Various conventional methods, such as flow cytometry, are used to isolate and study single cells. Still, these methods are narrower in scope due to certain limitations, including the associated processing/run times, the economy of reagents, and sample preparation. Microfluidics, an emerging technology, overcomes such limitations and is now being widely applied to develop tools for the isolation, analysis, and parallel manipulation of single cells. This review systematically compiles various microfluidic tools and techniques involved in single cell investigation. The review begins by highlighting the applications of microfluidics in single cell sorting and manipulation, followed by emphasizing microfluidic platforms for single cell analysis, with a specific focus on optical sensing-based detection in a high-throughput fashion, and ends with applications in cancer cell studies.

Fabrication of three-lobed magnetic microrobots for cell transportation.

Mobile microrobots have the potential to transform medical treatments based on therapeutic delivery. Specifically, microrobots are promising candidates for cell transportation in cell-based therapies. Despite recent progress in cellular manipulation by microrobots, there is a significant need to design and fabricate microrobots to advance the field further. In this work, we present a facile approach to manufacturing three-lobed microrobots by a bench-top procedure. The microrobots are actuated by a harmless magnetic field which makes them biofriendly. Chemically, these microrobots are made of organosilica. The microrobots showed equally good control in both the open-loop and closed-loop settings. The three-lobed microrobots have two modes of motion during the open-loop control experiments. We employed these two modes for single-cell transportation. Our results show that the three-lobed microbots are very promising for cell transportation in a fluid.