Noncontact Multiphysics Probe for Spatiotemporal Resolved Single-Cell Manipulation and Analyses.

Heterogeneity and spatial arrangement of individual cells within tissues are critical to the identity of the host multicellular organism. While current single-cell techniques are capable of resolving heterogeneity, they mostly rely on extracting target cells from their physiological environment and hence lose the spatiotemporal resolution required for understanding cellular networks. Here, a multifunctional noncontact scanning probe that can precisely perform multiple manipulation procedures on living single-cells, while within their physiological tissue environment, is demonstrated. The noncontact multiphysics probe (NMP) consists of fluidic apertures and "hump" shaped electrodes that simultaneously confine reagents and electric signals with a single-cell resolution. The NMP's unique electropermealization-based approach in transferring macromolecules through the cell membrane is presented. The technology's adjustable spatial ability is demonstrated by transfecting adjacent single-cells with different DNA plasmid vectors. The NMP technology also opens the door for controllable cytoplasm extraction from living single-cells. This powerful application is demonstrated by executing multiple time point biopsies on adherent cells without affecting the integrity of the extracted macromolecules or the viability of cells. Furthermore, the NMP's function as an electro-thermal based microfluidic whole-cell tweezer is reported. This work offers a multifunctional tool with unprecedented probing features for spatiotemporal single-cell analysis within tissue samples.

Integration of shallow gradients of Shh and Netrin-1 guides commissural axons.

During nervous system development, gradients of Sonic Hedgehog (Shh) and Netrin-1 attract growth cones of commissural axons toward the floor plate of the embryonic spinal cord. Mice defective for either Shh or Netrin-1 signaling have commissural axon guidance defects, suggesting that both Shh and Netrin-1 are required for correct axon guidance. However, how Shh and Netrin-1 collaborate to guide axons is not known. We first quantified the steepness of the Shh gradient in the spinal cord and found that it is mostly very shallow. We then developed an in vitro microfluidic guidance assay to simulate these shallow gradients. We found that axons of dissociated commissural neurons respond to steep but not shallow gradients of Shh or Netrin-1. However, when we presented axons with combined Shh and Netrin-1 gradients, they had heightened sensitivity to the guidance cues, turning in response to shallower gradients that were unable to guide axons when only one cue was present. Furthermore, these shallow gradients polarized growth cone Src-family kinase (SFK) activity only when Shh and Netrin-1 were combined, indicating that SFKs can integrate the two guidance cues. Together, our results indicate that Shh and Netrin-1 synergize to enable growth cones to sense shallow gradients in regions of the spinal cord where the steepness of a single guidance cue is insufficient to guide axons, and we identify a novel type of synergy that occurs when the steepness (and not the concentration) of a guidance cue is limiting.

The relative composition of actin isoforms regulates cell surface biophysical features and cellular behaviors.

BACKGROUND: Cell surface mechanics is able to physically and biomechanically affect cell shape and motility, vesicle trafficking and actin dynamics. The biophysical properties of cell surface are strongly influenced by cytoskeletal elements. In mammals, tissue-specific expression of six actin isoforms is thought to confer differential biomechanical properties. However, the relative contribution of actin isoforms to cell surface properties is not well understood. Here, we sought to investigate whether and how the composition of endogenous actin isoforms directly affects the biomechanical features of cell surface and cellular behavior. METHODS: We used fibroblasts isolated from wild type (WT), heterozygous (HET) and from knockout (KO) mouse embryos where both ß-actin alleles are not functional. We applied a combination of genome-wide analysis and biophysical methods such as RNA-seq and atomic force microscopy. RESULTS: We found that endogenous ß-actin levels are essential in controlling cell surface stiffness and pull-off force, which was not compensated by the up-regulation of other actin isoforms. The variations of surface biophysical features and actin contents were associated with distinct cell behaviors in 2D and 3D WT, HET and KO cell cultures. Since ß-actin in WT cells and smooth muscle α-actin up-regulated in KO cells showed different organization patterns, our data support the differential localization and organization as a mechanism to regulate the biophysical properties of cell surface by actin isoforms. CONCLUSIONS: We propose that variations in actin isoforms composition impact on the biophysical features of cell surface and cause the changes in cell behavior.

Microfluidic direct writer with integrated declogging mechanism for fabricating cell-laden hydrogel constructs.

Cell distribution and nutrient supply in 3D cell-laden hydrogel scaffolds are critical and should mimic the in vivo cellular environment, but been difficult to control with conventional fabrication methods. Here, we present a microfluidic direct writer (MFDW) to construct 3D cell-laden hydrogel structures with openings permitting media exchange. The MFDW comprises a monolithic microfluidic head, which delivers coaxial streams of cell-laden sodium alginate and calcium chloride solutions to form hydrogel fibers. Fiber diameter is controlled by adjusting the ratio of the volumetric flow rates. The MFDW head is mounted on a motorized stage, which is automatically controlled and moves at a speed synchronized with the speed of fiber fabrication. Head geometry, flow rates, and viscosity of the writing solutions were optimized to prevent the occurrence of curling and bulging. For continuous use, a highly reliable process is needed, which was accomplished with the integration of a declogging conduit supplying a solvent to dissolve the clogging gel. The MFDW was used for layer-by-layer fabrication of simple 3D structures with encapsulated cells. Assembly of 3D structures with distinct fibers is demonstrated by alternatively delivering two different alginate gel solutions. The MFDW head can be built rapidly and easily, and will allow 3D constructs for tissue engineering to be fabricated with multiple hydrogels and cell types.

Ventilation-Based Strategy to Manage Intraoperative Aerosol Viral Transmission in the Era of SARS-CoV-2.

In operating theaters, ventilation systems are designed to protect the patient from airborne contamination for minimizing risks of surgical site infections (SSIs). Ventilation systems often produce an airflow pattern that continuously pushes air out of the area surrounding the operating table, and hence reduces the resident time of airborne pathogen-carrying particles at the patient's location. As a result, patient-released airborne particles due to the use of powered tools, such as surgical smoke and insufflated CO2, typically circulate within the room. This circulation exposes the surgical team to airborne infection-especially when operating on a patient with infectious diseases, including COVID-19. This study examined the flow pattern of functional ventilation configurations in view of developing ventilation-based strategies to protect both the patient and the surgical team from aerosolized infections. A favorable design that minimized particle circulation was deduced using experimentally validated numerical models. The parameters adapted to quantify circulation of airborne particles were particles' half-life and elevation. The results show that the footprint of the outlet ducts and resulting flow pattern are important parameters for minimizing particle circulation. Overall, this study presents a modular framework for optimizing the ventilation systems that permits a switch in operation configuration to suit different operating procedures.

OMEF biochip for evaluating red blood cell deformability using dielectrophoresis as a diagnostic tool for type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) is a prevalent and debilitating disease with numerous health risks, including cardiovascular diseases, kidney dysfunction, and nerve damage. One important aspect of T2DM is its association with the abnormal morphology of red blood cells (RBCs), which leads to increased blood viscosity and impaired blood flow. Therefore, evaluating the mechanical properties of RBCs is crucial for understanding the role of T2DM in cellular deformability. This provides valuable insights into disease progression and potential diagnostic applications. In this study, we developed an open micro-electro-fluidic (OMEF) biochip technology based on dielectrophoresis (DEP) to assess the deformability of RBCs in T2DM. The biochip facilitates high-throughput single-cell RBC stretching experiments, enabling quantitative measurements of the cell size, strain, stretch factor, and post-stretching relaxation time. Our results confirm the significant impact of T2DM on the deformability of RBCs. Compared to their healthy counterparts, diabetic RBCs exhibit ∼27% increased size and ∼29% reduced stretch factor, suggesting potential biomarkers for monitoring T2DM. The observed dynamic behaviors emphasize the contrast between the mechanical characteristics, where healthy RBCs demonstrate notable elasticity and diabetic RBCs exhibit plastic behavior. These differences highlight the significance of mechanical characteristics in understanding the implications for RBCs in T2DM. With its ∼90% sensitivity and rapid readout (ultimately within a few minutes), the OMEF biochip holds potential as an effective point-of-care diagnostic tool for evaluating the deformability of RBCs in individuals with T2DM and tracking disease progression.

Next-Generation Microfluidics for Biomedical Research and Healthcare Applications.

Microfluidic systems offer versatile biomedical tools and methods to enhance human convenience and health. Advances in these systems enables next-generation microfluidics that integrates automation, manipulation, and smart readout systems, as well as design and three-dimensional (3D) printing for precise production of microchannels and other microstructures rapidly and with great flexibility. These 3D-printed microfluidic platforms not only control the complex fluid behavior for various biomedical applications, but also serve as microconduits for building 3D tissue constructs-an integral component of advanced drug development, toxicity assessment, and accurate disease modeling. Furthermore, the integration of other emerging technologies, such as advanced microscopy and robotics, enables the spatiotemporal manipulation and high-throughput screening of cell physiology within precisely controlled microenvironments. Notably, the portability and high precision automation capabilities in these integrated systems facilitate rapid experimentation and data acquisition to help deepen our understanding of complex biological systems and their behaviors. While certain challenges, including material compatibility, scaling, and standardization still exist, the integration with artificial intelligence, the Internet of Things, smart materials, and miniaturization holds tremendous promise in reshaping traditional microfluidic approaches. This transformative potential, when integrated with advanced technologies, has the potential to revolutionize biomedical research and healthcare applications, ultimately benefiting human health. This review highlights the advances in the field and emphasizes the critical role of the next generation microfluidic systems in advancing biomedical research, point-of-care diagnostics, and healthcare systems.

Affinity-Based Microfluidics Combined with Atomic Force Microscopy for Isolation and Nanomechanical Characterization of Circulating Tumor Cells.

In this chapter, we present the materials and methods required to isolate and characterize circulating tumor cells (CTCs) from blood samples of cancer patients based on our newly developed microfluidic technologies. In particular, the devices presented herein are designed to be compatible with at\omic force microscopy (AFM) for post-capture nanomechanical investigation of CTCs. Microfluidics is well-established as a technology for isolating CTCs from the whole blood of cancer patients, and AFM is a gold standard for quantitative biophysical analysis of cells. However, CTCs are very scarce in nature, and those captured using standard closed-channel microfluidic chips are typically inaccessible for AFM procedures. As a result, their nanomechanical properties largely remain unexplored. Thus, given limitations associated with current microfluidic designs, significant efforts are put toward bringing innovative designs for real time characterization of CTCs. In light of this constant endeavor, the scope of this chapter is to compile our recent efforts on two microfluidic technologies, namely, the AFM-Chip and the HB-MFP, which proved to be efficient in isolating CTCs through antibody-antigen interactions, and their subsequent characterization using AFM.

Spike- and nucleocapsid-based gold colloid assay toward the development of an adhesive bandage for rapid SARS-CoV-2 immune response detection and screening.

Immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies are important biomarkers used for the diagnosis and screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in both symptomatic and asymptomatic individuals. These antibodies are highly specific to the spike (S) and nucleocapsid (N) proteins of the SARS-CoV-2 virus. This paper outlines the development steps of a novel hybrid (vertical-lateral-vertical) flow assay in the form of a finger-stick point-of-care device, similar to an adhesive bandage, designed for the timely detection and screening of IgM and IgG immune responses to SARS-CoV-2 infections. The assay, comprising a vertically stacked plasma/serum separation membrane, conjugate pad, and detection (readout) zone, utilizes gold nanoparticles (AuNPs) conjugated with SARS-CoV-2 S and N proteins to effectively capture IgM and IgG antibodies from a pinprick (~15 µL) of blood in just one step and provides results of no immune IgM-/IgG-, early immune IgM+/IgG-, active immune IgM+/IgG+ or immune IgM-/IgG+ in a short amount of time (minutes). The adhesive bandage-like construction is an example of the design of rapid, low-cost, disposable, and easy-to-use tests for large-scale detection and screening in households. Furthermore, the bandage can be easily adjusted and optimized to detect different viral infections as they arise by simply selecting appropriate antigens related to pandemics and outbreaks.

Characterizing circulating tumor cells using affinity-based microfluidic capture and AFM-based biomechanics.

Elasticity and bio-adhesiveness of circulating tumor cells (CTCs) are important biomarkers of cancer. CTCs are rare in blood, thus their capture and atomic force microscopy (AFM)-based biomechanical characterization require use of multifunctional microfluidic device. Here, we describe procedures for fabrication of such device, AFM-Chip, and give details on its use in affinity-based CTC capture, and integration with AFM via reversable physical assembly. In the AFM-Chip, CTC capture is efficient, and transition to AFM characterization is seamless with minimal cell loss. For complete details on the use and execution of this protocol, please refer to Deliorman et al. (2020).

3D Generation of Multipurpose Atomic Force Microscopy Tips.

In this work, 3D polymeric atomic force microscopy (AFM) tips, referred to as 3DTIPs, are manufactured with great flexibility in design and function using two-photon polymerization. With the technology holding a great potential in developing next-generation AFM tips, 3DTIPs prove effective in obtaining high-resolution and high-speed AFM images in air and liquid environments, using common AFM modes. In particular, it is shown that the 3DTIPs provide high-resolution imaging due to their extremely low Hamaker constant, high speed scanning rates due to their low quality factor, and high durability due to their soft nature and minimal isotropic tip wear; the three important features for advancing AFM studies. It is also shown that refining the tip end of the 3DTIPs by focused ion beam etching and by carbon nanotube inclusion substantially extends their functionality in high-resolution AFM imaging, reaching angstrom scales. Altogether, the multifunctional capabilities of 3DTIPs can bring next-generation AFM tips to routine and advanced AFM applications, and expand the fields of high speed AFM imaging and biological force measurements.

Stiffness Assessment and Lump Detection in Minimally Invasive Surgery Using In-House Developed Smart Laparoscopic Forceps.

Minimally invasive surgery (MIS) incorporates surgical instruments through small incisions to perform procedures. Despite the potential advantages of MIS, the lack of tactile sensation and haptic feedback due to the indirect contact between the surgeon's hands and the tissues restricts sensing the strength of applied forces or obtaining information about the biomechanical properties of tissues under operation. Accordingly, there is a crucial need for intelligent systems to provide an artificial tactile sensation to MIS surgeons and trainees. This study evaluates the potential of our proposed real-time grasping forces and deformation angles feedback to assist surgeons in detecting tissues' stiffness. A prototype was developed using a standard laparoscopic grasper integrated with a force-sensitive resistor on one grasping jaw and a tunneling magneto-resistor on the handle's joint to measure the grasping force and the jaws' opening angle, respectively. The sensors' data are analyzed using a microcontroller, and the output is displayed on a small screen and saved to a log file. This integrated system was evaluated by running multiple grasp-release tests using both elastomeric and biological tissue samples, in which the average force-to-angle-change ratio precisely resembled the stiffness of grasped samples. Another feature is the detection of hidden lumps by palpation, looking for sudden variations in the measured stiffness. In experiments, the real-time grasping feedback helped enhance the surgeons' sorting accuracy of testing models based on their stiffness. The developed tool demonstrated a great potential for low-cost tactile sensing in MIS procedures, with room for future improvements. Significance: The proposed method can contribute to MIS by assessing stiffness, detecting hidden lumps, preventing excessive forces during operation, and reducing the learning curve for trainees.

Smart Laparoscopic Grasper Utilizing Force and Angle Sensors for Stiffness Assessment in Minimally Invasive Surgery.

As an alternative to open surgery, minimally invasive surgery (MIS) utilizes small skin incisions as ports to insert an endoscope and surgical tools. MIS offers significant advantages, including reduced pain, shorter recovery times, and better cosmetic outcomes than classical surgeries. However, MIS procedures come at the cost of losing the "sense of touch," which surgeons rely on to examine the tissues under operation, palpate organs, and assessing their conditions. This has encouraged researchers to develop smart MIS tools that provide artificial tactile sensation, mostly using electrical- or optical-based tactile sensors. In this work, we introduce a prototype of a smart laparoscopic grasper integrated with force and angle sensing capabilities via off-the-shelf sensors. The specification and design of the smart grasper are presented, as well as a demonstration on stiffness assessment of elastomeric samples and chicken meat. Overall, our prototype exhibits great potential for MIS applications, with room for future improvements.Clinical Relevance- The development of a smart laparoscopic grasper for MIS applications helps in restoring the tactile sensation to surgeons and enables safe grasping and manipulation of human organs.

Cryopreservable arrays of paper-based 3D tumor models for high throughput drug screening.

Three-dimensional (3D) tumor models have gained increased attention in life-science applications as they better represent physiological conditions of in vivo tumor microenvironments, and thus, possess big potential for guiding drug screening studies. Although various techniques proved effective in growing cancer cells in 3D, their procedures are typically complex, time consuming, and expensive. Here, we present a versatile, robust, and cost-effective method that utilizes a paper platform to create cryopreservable high throughput arrays of 3D tumor models. In the approach, we use custom 3D printed masks along with simple chemistry modifications to engineer highly localized hydrophilic 'virtual microwells', or microspots, on paper for 3D cell aggregation, surrounded by hydrophobic barriers that prevent inter-microspot mixing. The method supports the formation and cryopreservation of 3D tumor arrays for extended periods of storage time. Using MCF-7 and MDA-MB-231 breast cancer cell lines, we show that the cryopreservable arrays of paper-based 3D models are effective in studying tumor response to cisplatin drug treatment, while replicating key characteristics of the in vivo tumors that are absent in conventional 2D cultures. This technology offers a low cost, easy, and fast experimental procedure, and allows for 3D tumor arrays to be cryopreserved and thawed for on-demand use. This could potentially provide unparalleled advantages to the fields of tissue engineering and personalized medicine.

Dielectrophoretic characterization of dendritic cell deformability upon maturation.

We have developed a rapid technique for characterizing the biomechanical properties of dendritic cells using dielectrophoretic forces. It is widely recognized that maturing of dendritic cells modulates their stiffness and migration capabilities, which results in T-cell activation triggering the adaptive immune response. Therefore it is important to develop techniques for mechanophenotyping of immature and mature dendritic cells. The technique reported here utilizes nonuniform electric fields to exert a substantial force on the cells to induce cellular elongation for optical measurements. In addition, a large array of interdigitated electrodes allows multiple cells to be stretched simultaneously. Our results indicate a direct correlation between F-actin activity and deformability observed in dendritic cells, determined through mean fluorescence signal intensity of phalloidin.

Tactile Sensing for Minimally Invasive Surgery: Conventional Methods and Potential Emerging Tactile Technologies.

As opposed to open surgery procedures, minimally invasive surgery (MIS) utilizes small skin incisions to insert a camera and surgical instruments. MIS has numerous advantages such as reduced postoperative pain, shorter hospital stay, faster recovery time, and reduced learning curve for surgical trainees. MIS comprises surgical approaches, including laparoscopic surgery, endoscopic surgery, and robotic-assisted surgery. Despite the advantages that MIS provides to patients and surgeons, it remains limited by the lost sense of touch due to the indirect contact with tissues under operation, especially in robotic-assisted surgery. Surgeons, without haptic feedback, could unintentionally apply excessive forces that may cause tissue damage. Therefore, incorporating tactile sensation into MIS tools has become an interesting research topic. Designing, fabricating, and integrating force sensors onto different locations on the surgical tools are currently under development by several companies and research groups. In this context, electrical force sensing modality, including piezoelectric, resistive, and capacitive sensors, is the most conventionally considered approach to measure the grasping force, manipulation force, torque, and tissue compliance. For instance, piezoelectric sensors exhibit high sensitivity and accuracy, but the drawbacks of thermal sensitivity and the inability to detect static loads constrain their adoption in MIS tools. Optical-based tactile sensing is another conventional approach that facilitates electrically passive force sensing compatible with magnetic resonance imaging. Estimations of applied loadings are calculated from the induced changes in the intensity, wavelength, or phase of light transmitted through optical fibers. Nonetheless, new emerging technologies are also evoking a high potential of contributions to the field of smart surgical tools. The recent development of flexible, highly sensitive tactile microfluidic-based sensors has become an emerging field in tactile sensing, which contributed to wearable electronics and smart-skin applications. Another emerging technology is imaging-based tactile sensing that achieved superior multi-axial force measurements by implementing image sensors with high pixel densities and frame rates to track visual changes on a sensing surface. This article aims to review the literature on MIS tactile sensing technologies in terms of working principles, design requirements, and specifications. Moreover, this work highlights and discusses the promising potential of a few emerging technologies towards establishing low-cost, high-performance MIS force sensing.

A Method to Efficiently Cryopreserve Mammalian Cells on Paper Platforms.

This protocol describes a simple method to cryopreserve mammalian cells within filter papers as an alternative to conventional slow-freezing approach. The method involves treating paper fibers with fibronectin, using low concentrations of the cryoprotectant dimethyl sulfoxide (DMSO), and slow freezing cells to -80 °C at a 1 °C min-1 rate. In our method, the biocompatibility, large surface area, 3D porosity and fiber flexibility of the paper, in combination with the fibronectin treatment, yield recovery of cells comparable to conventional approaches, with no additional fine-tuning to freezing and thawing procedures. We expect that the paper-based cryopreservation method will bring several advantages to the field of preserving mammalian cells, including accommodation of a higher number of cells within a unit volume and no cell loss after release. The method requires a minimal storage space, where paper platforms with large areas can be rolled and/or folded and stored in stocks, and allows for efficient transportation/distribution of cells in an on-demand manner. Moreover, an additional feature of this method includes the formation and cryopreservation of cellular spheroids and 3D cell cultures.

Electrically Actuated Concentration of Microparticles through Levitation and Convective Flows in Evaporating Droplets.

We present an electrically actuated approach for creating a well-defined centered microparticle cluster within a sessile droplet on an interdigitated microelectrodes. The method is demonstrated with different aggregation shapes and particle types including biological cells for 3D microtissue development. AC voltage application induces particle levitation and enhanced-convection through accelerated evaporation. Radial long-range fluid convection evolves along the substrate surface toward the droplet's center and suspended microparticles aggregate within the central stagnation zone, in an interesting occurrence that is opposite to the well-known coffee ring effect. This remarkable approach could open new opportunities in immunoassays, rare cell counting, and 3D cell cultures.

Paper-Based Cell Cryopreservation.

The continuous development of simple and practical cell cryopreservation methods is of great importance to a variety of sectors, especially when considering the efficient short- and long-term storage of cells and their transportation. Although the overall success of such methods has been increased in recent years, there is still need for a unified platform that is highly suitable for efficient cryogenic storage of cells in addition to their easy-to-manage retrieval. Here, a paper-based cell cryopreservation method as an alternative to conventional cryopreservation methods is presented. The method is space-saving, cost-effective, simple and easy to manage, and requires no additional fine-tuning to conventional freezing and thawing procedures to yield comparable recovery of viable cells. It is shown that treating papers with fibronectin solution enhances the release of viable cells post thawing as compared to untreated paper platforms. Additionally, upon release, the remaining cells within the paper lead to the formation and growth of spheroid-like structures. Moreover, it is demonstrated that the developed method works with paper-based 3D cultures, where preformed 3D cultures can be efficiently cryopreserved.

AFM-compatible microfluidic platform for affinity-based capture and nanomechanical characterization of circulating tumor cells.

Circulating tumor cells (CTCs) carried by the patient's bloodstream are known to lead to the metastatic spread of cancer. It is becoming increasingly clear that an understanding of the nanomechanical characteristics of CTCs, such as elasticity and adhesiveness, represents advancements in tracking and monitoring cancer progression and metastasis. In the present work, we describe a combined microfluidic-atomic force microscopy (AFM) platform that uses antibody-antigen capture to routinely isolate and nanomechanically characterize CTCs present in blood samples from prostate cancer patients. We introduce the reversible assembly of a microfluidic device and apply refined and robust chemistry to covalently bond antibodies onto its glass substrate with high density and the desired orientation. As a result, we show that the device can efficiently capture CTCs from patients with localized and metastatic prostate cancer through anti-EpCAM, anti-PSA, and anti-PSMA antibodies, and it is suitable for AFM measurements of captured intact CTCs. When nanomechanically characterized, CTCs originating from metastatic cancer demonstrate decreased elasticity and increased deformability compared to those originating from localized cancer. While the average adhesion of CTCs to the AFM tip surface remained the same in both the groups, there were fewer multiple adhesion events in metastatic CTCs than there were in their counterparts. The developed platform is simple, robust, and reliable and can be useful in the diagnosis and prognosis of prostate cancer as well as other forms of cancer.