Cell Manipulation Method Research Articles

Acoustic Cell Patterning for Structured Cell-Laden Hydrogel Fibers/Tubules.

Cell-laden hydrogel fibers/tubules are one of the fundamentals of tissue engineering. They have been proven as a promising method for constructing biomimetic tissues, such as muscle fibers, nerve conduits, tendon and vessels, etc. However, current hydrogel fiber/tubule production methods have limitations in ordered cell arrangements, thus impeding the biomimetic configurations. Acoustic cell patterning is a cell manipulation method that has good biocompatibility, wide tunability, and is contact-free. However, there are few studies on acoustic cell patterning for fiber production, especially on the radial figure cell arrangements, which mimic many native tissue-like cell arrangements. Here, an acoustic cell patterning system that can be used to produce hydrogel fibers/tubules with tunable cell patterns is shown. Cells can be pre-patterned in the liquid hydrogel before being extruded as cross-linked hydrogel fibers/tubules. The radial patterns can be tuned with different complexities based on the acoustic resonances. Cell viability assays after 72h confirm good cell viability and proliferation. Considering the biocompatibility and reliability, the present method can be further used for a variety of biomimetic fabrications.

Multi-functional single cell manipulation method based on a multichannel micropipette

Cell manipulation techniques that provide versatility in handling various cell types and performing diverse manipulations are essential for advancing biological research. This study presents a novel and adaptable approach utilizing a multifunctional micropipette for precise single-cell operations, including three-dimensional (3D) manipulation, trapping, lysis, and aspiration. The multifunctional micropipette comprises two liquid metal-based channels with high electrical conductivity, along with a negative pressure-based aspiration channel. By utilizing the liquid metal electrodes, the system enables the application of electrical stimulation, inducing both dielectrophoretic force and electroporation on cells. Through the implementation of dielectrophoretic force and integration with a 3D-movable stage, single cells can be patterned, manipulated in 3D, accurately trapped, and separated with a minimal adjacent distance of 10 µm. By utilizing the electroporation phenomenon, rapid lysis of suspension cells can be achieved within 50 ms, while adherent cells can be lysed within 200 ms, utilizing a square-wave electrical signal with a frequency of 1 kHz and an amplitude of 40 Vpp. Furthermore, the study explores the impact of electrode size on the lysis of adherent cell groups. Additionally, the utilization of negative pressure for lysate aspiration is also demonstrated. In summary, the reported technique is versatile for single cell manipulation.

A correlation of conductivity medium and bioparticle viability on dielectrophoresis-based biomedical applications.

Dielectrophoresis (DEP) bioparticle research has progressed from micro to nano levels. It has proven to be a promising and powerful cell manipulation method with an accurate, quick, inexpensive, and label-free technique for therapeutic purposes. DEP, an electrokinetic phenomenon, induces particle movement as a result of polarization effects in a nonuniform electrical field. This review focuses on current research in the biomedical field that demonstrates a practical approach to DEP in terms of cell separation, trapping, discrimination, and enrichment under the influence of the conductive medium in correlation with bioparticle viability. The current review aims to provide readers with an in-depth knowledge of the fundamental theory and principles of the DEP technique, which is influenced by conductive medium and to identify and demonstrate the biomedical application areas. The high conductivity of physiological fluids presents obstacles and opportunities, followed by bioparticle viability in an electric field elaborated in detail. Finally, the drawbacks of DEP-based systems and the outlook for the future are addressed. This article will aid in advancing technology by bridging the gap between bioscience and engineering. We hope the insights presented in this review will improve cell suspension medium and promote DEP-viable bioparticle manipulation for health-care diagnostics and therapeutics.

Acoustophoretic Motion of Leishmania spp. Parasites.

The analysis of cell motion in an acoustic field is of interest as it can lead to new methods of cell separation, isolation and manipulation for diagnosis and treatment of diseases. Studies of the motion of different species of Leishmania parasites during exposure to ultrasonic standing waves in a microfluidic device allowed identification of acoustic responses of these parasites in their promastigote and amastigote forms. Both forms exhibited a positive acoustic contrast factor and were driven toward the pressure node established in the center of the channel by the acoustically induced radiation force (FR). Promastigotes experience calculated FR amplitudes one order of magnitude larger than those experienced by amastigotes because of the measured differences in volume. The aggregates formed at the pressure node have distinct shapes and stability conditions, for both promastigotes and amastigotes.

Dielectrophoresis as a tool for electrophysiological characterization of stem cells.

Dielectrophoresis (DEP), a nonlinear electrokinetic technique caused by Maxwell-Wagner interfacial polarization of neutral particles in an electrolyte solution, is a powerful cell manipulation method used widely for various applications such as enrichment, trapping, and sorting of heterogeneous cell populations. While conventional cell characterization and sorting methods require tagging or labeling of cells, DEP has the potential to manipulate cells in a label-free way. Due to its unique ability to characterize and sort cells without the need of labeling, there is renewed interest in using DEP for stem cell research and regenerative medicine. Stem cells have the potential to differentiate into various lineages, but achieving homogeneous cell phenotypes from an initially heterogeneous cell population is a challenge. Using DEP to efficiently and affordably identify, sort, and enrich either undifferentiated or differentiated stem cell populations in a label-free way would advance their potential uses for applications in tissue engineering and regenerative medicine. This review summarizes recent, significant research findings regarding the electrophysiological characterization of stem cells, with a focus on cellular dielectric properties, i.e., permittivity and conductivity, and on studies that have obtained these measurements using techniques that preserve cell viability, such as crossover frequency. Potential applications for DEP in regenerative medicine are also discussed. Overall, DEP is a promising technique and, when used to characterize, sort, and enrich stem cells, will advance stem cell-based regenerative therapies.

Development of High Throughput Single-Cell Analysis System for Circulating Tumor Cells Based on Digital Micromirror Device

Circulating tumor cells (CTCs) are widely known as useful biomarkers in the liquid biopsies of cancer patients. Recently, in addition to counting the number of CTCs, genetic analysis of CTCs provides critical insights into cancer metastases and add detailed clinical information that enhances patient care. It is widely known that CTCs are genetically heterogeneous, the molecular characterization of CTCs should be performed at the single-cell level. In fact, several studies have identified inter- and intra-patient heterogeneity in the mutational status of CTCs in metastatic breast and lung cancers. These studies also reported that CTCs related to drug resistance are novel therapeutic targets. Thus, genetic analysis of single CTCs has the potential to be widely applicable in personalized medicine and drug discovery. However, CTCs are extremely rare cells that only 1-100 cells are contained in 1 ml of blood, so the recovery of such extremely rare CTCs from the peripheral blood is technically challenging. So far, we have developed a CTC recovery system using a microcavity array (MCA), which demonstrates highly efficient recovery of cancer cells based on differences in cell size and deformability. Furthermore, we have proposed a novel cell manipulation method through the visualization of single cells through hydrogel encapsulation for subsequent genetic analysis of single CTCs. Subsequently, recovered CTCs can be subjected to a hydrogel-encapsulation for the following genetic analysis of single cells. Clinical studies indicated that the genetic analysis of CTCs from lung, pancreatic and gastric cancer patients was successfully achieved. However, this hydrogel encapsulation has limitations in throughput. In this study, we demonstrate a high-throughput single-cell analysis for CTCs using a multiple single-cell encapsulation system with a digital micromirror device (DMD). The novel multiple single-cell encapsulation systemwas composed of two optical systems: the wide-field fluorescence imaging system and the photopolymerization system equipped with DMD. The wide-field fluorescence imaging system using a CMOS sensor was designed to visualize 2D fluorescence imaging of the entire MCA (6.0 × 6.0 mm2). We evaluated the sensitivity of fluorescence detection of the wide-field fluorescence imaging system using the calibration slide. The limit of detection of the wide-field fluorescence imaging system was 2.1 × 102 molecules-Cy3/μm2, which is comparable to conventional fluorescence microscopy (1.0 × 102 molecules-Cy3/μm2). Generally, the expression of cellular marker protein, such as cytokeratin, was estimated to be 1.3 × 103 molecules/μm2. Therefore, our proposed system had enough sensitivity for the detection of antibody-stained CTCs. Furthermore, this wide-field fluorescence imaging system for CTC detection enables the rapid visualization of all stained cells within the field of the MCA via one-shot imaging. This rapid detection method also allows for the rapid completion of single-CTC manipulation. The photopolymerization system for single-cell encapsulation was controlled by the DMD. The curing light (λmax = 365 nm) modulated by the DMD was projected onto an MCA for hydrogel generation, and single-cell isolation by hydrogel-photopolymerization was examined at the optimized conditions. After recovery of cancer cells from whole blood onto MCA and staining by the fluorescent-labeled antibodies, cancer cells were identified by the wide-field imaging system. Then light irradiation (3 sec) was performed by the photopolymerization system. To minimize the area of the hydrogel reserved for a single cell, we newly designed the convex-type hydrogel. In the spike-in experiment, single-cell isolation rates were 97.6% (41/42) for NCI-H1975 cells (non-small cell lung cancer cell) and 94.4% (51/54) for NCI-N87 (gastric cancer cell) that were comparable with our previous study using fluorescence microscopy. With this proposed system, more than 50 single cells were encapsulated simultaneously within 1 min and isolated with an efficiency. Furthermore, single cells were successfully isolated from adjacent cells on the same microcavity without any contamination. Single cancer cell was partially encapsulated on the hydrogel, and the partial encapsulation allows us to subject them to whole genome amplification (WGA). Light irradiation and photopolymerizaion did not affect the quality of the amplified WGA products for single-cell genomic analyses. We are currently investigating single cell genetic analysis of metastatic cancer patients, and this developed multiple single-cell encapsulation system will improve the high throughput single-cell genetic analysis of CTCs in clinical samples. Our system provides an attractive application for other targets such as adherent cells, tissue samples, and microorganisms, in addition to widespread use in the isolation of CTCs for liquid biopsy.

Hurdles to uptake of mesenchymal stem cells and their progenitors in therapeutic products.

Twenty-five years have passed since the first clinical trial utilising mesenchymal stomal/stem cells (MSCs) in 1995. In this time academic research has grown our understanding of MSC biochemistry and our ability to manipulate these cells in vitro using chemical, biomaterial, and mechanical methods. Research has been emboldened by the promise that MSCs can treat illness and repair damaged tissues through their capacity for immunomodulation and differentiation. Since 1995, 31 therapeutic products containing MSCs and/or progenitors have reached the market with the level of in vitro manipulation varying significantly. In this review, we summarise existing therapeutic products containing MSCs or mesenchymal progenitor cells and examine the challenges faced when developing new therapeutic products. Successful progression to clinical trial, and ultimately market, requires a thorough understanding of these hurdles at the earliest stages of in vitro pre-clinical development. It is beneficial to understand the health economic benefit for a new product and the reimbursement potential within various healthcare systems. Pre-clinical studies should be selected to demonstrate efficacy and safety for the specific clinical indication in humans, to avoid duplication of effort and minimise animal usage. Early consideration should also be given to manufacturing: how cell manipulation methods will integrate into highly controlled workflows and how they will be scaled up to produce clinically relevant quantities of cells. Finally, we summarise the main regulatory pathways for these clinical products, which can help shape early therapeutic design and testing.

Optogenetyka i inne metody kontroli aktywności komórek nerwowych

Sterowanie czynnością elektryczną wybranych, specyficznych grup komórek nerwowych lub nawet pojedynczej komórki w mózgu pozwala na zrozumienie tego jak sieci neuronowe przetwarzają i kodują informacje. Możliwość manipulacji aktywnością neuronów jest niezbędna w badaniu obwodów neuronalnych, ich funkcji i roli w zdrowym i chorym mózgu. Obecnie istnieje wiele metod regulacji aktywności komórek nerwowych wykorzystujących różne podejścia i narzędzia. Techniki te różnią się bardzo pod względem specyficzności komórkowej, kinetyki i kierunku zmiany aktywności komórkowej (aktywacja/hamowanie). W niniejszej pracy wymieniamy różne metody kontrolowania aktywności neuronów poczynając od lat 60? ubiegłego wieku, a kończąc na technikach XXI wieku. Najwięcej miejsca przeznaczamy na opis opsyn i rozwoju metod zastosowania optogenetyki. Oddzielny rozdział poświęcamy charakterystyce metod powstałych w ostatnich latach, które wykorzystują nanocząsteczki i optyczną manipulację transkrypcją DNA.

Acoustic Holographic Cell Patterning in a Biocompatible Hydrogel.

Acoustophoresis is promising as a rapid, biocompatible, noncontact cell manipulation method, where cells are arranged along the nodes or antinodes of the acoustic field. Typically, the acoustic field is formed in a resonator, which results in highly symmetric regular patterns. However, arbitrary, nonsymmetrically shaped cell assemblies are necessary to obtain the irregular cellular arrangements found in biological tissues. It is shown that arbitrarily shaped cell patterns can be obtained from the complex acoustic field distribution defined by an acoustic hologram. Attenuation of the sound field induces localized acoustic streaming and the resultant convection flow gently delivers the suspended cells to the image plane where they form the designed pattern. It is shown that the process can be implemented in a biocompatible collagen solution, which can then undergo gelation to immobilize the cell pattern inside the viscoelastic matrix. The patterned cells exhibit F-actin-based protrusions, which indicate that the cells grow and thrive within the matrix. Cell viability assays and brightfield imaging after one week confirm cell survival and that the patterns persist. Acoustophoretic cell manipulation by holographic fields thus holds promise for noncontact, long-range, long-term cellular pattern formation, with a wide variety of potential applications in tissue engineering and mechanobiology.

Capturing magnetic bead-based arrays using perpendicular magnetic anisotropy

Designing and implementing means of locally trapping magnetic beads and understanding the factors underlying the bead capture force are important steps toward advancing the capture-release process of magnetic particles for biological applications. In particular, capturing magnetically labeled cells using magnetic microstructures with perpendicular magnetic anisotropy (PMA) will enable an approach to cell manipulation for emerging lab-on-a-chip devices. Here, a Co (0.2 nm)/Ni (0.4 nm) multilayered structure was designed to exhibit strong PMA and large saturation magnetization (M s ). Finite element simulations were performed to assess the dependence of the capture force on the value of M s . The simulated force profile indicated the largest force at the perimeter of the disks. Arrays of Co/Ni disk structures of (4-7) μm diameter were fabricated and tested in a microchannel with suspended fluorescent magnetic beads. The magnetic beads were captured and localized to the edge of the disks as predicted by the simulations. This approach has been demonstrated to enable uniform assembly of magnetic beads without external fields and may provide a pathway toward precise cell manipulation methods.

Gel-based cell manipulation method for isolation and genotyping of single-adherent cells

Genetic analysis of single-cells is widely recognized as a powerful tool for understanding cellular heterogeneity and obtaining genetic information from rare populations. Recently, many kinds of single-cell isolation systems have been developed to facilitate single-cell genetic analysis. However, these systems mainly target non-adherent cells or cells in a cell suspension. Thus, it is still challenging to isolate single-adherent cells of interest from a culture dish using a microscope. We had previously developed a single-cell isolation technique termed "gel-based cell manipulation" (GCM). In GCM, single-cells could be visualized by photopolymerizable-hydrogel encapsulation that made it easier to isolate the single-cells. In this study, GCM-based isolation of single-adherent cancer cells from a culture dish was demonstrated. Single-adherent cells were encapsulated in a photopolymerizable hydrogel using a microscope and isolated with high efficiency. Furthermore, whole genome amplification and sequencing for the isolated single-adherent cell could be achieved. We propose that the GCM-based approach demonstrated in this study has the potential for efficient analysis of single-adherent cells at the genetic level.

Magnetic Force-Based Microfluidic Techniques for Cellular and Tissue Bioengineering.

Live cell manipulation is an important biotechnological tool for cellular and tissue level bioengineering applications due to its capacity for guiding cells for separation, isolation, concentration, and patterning. Magnetic force-based cell manipulation methods offer several advantages, such as low adverse effects on cell viability and low interference with the cellular environment. Furthermore, magnetic-based operations can be readily combined with microfluidic principles by precisely allowing control over the spatiotemporal distribution of physical and chemical factors for cell manipulation. In this review, we present recent applications of magnetic force-based cell manipulation in cellular and tissue bioengineering with an emphasis on applications with microfluidic components. Following an introduction of the theoretical background of magnetic manipulation, components of magnetic force-based cell manipulation systems are described. Thereafter, different applications, including separation of certain cell fractions, enrichment of rare cells, and guidance of cells into specific macro- or micro-arrangements to mimic natural cell organization and function, are explained. Finally, we discuss the current challenges and limitations of magnetic cell manipulation technologies in microfluidic devices with an outlook on future developments in the field.

Graphene-Augmented Nanofiber Scaffolds Trigger Gene Expression Switching of Four Cancer Cell Types.

Three-dimensional (3D) customized scaffolds are anticipated to provide new frontiers in cell manipulation and advanced therapy methods. Here, we demonstrate the application of hybrid 3D porous scaffolds, representing networks of highly aligned self-assembled ceramic nanofibers, for culturing four types of cancer cells. Ultrahigh aspect ratio (∼107) of graphene augmented fibers of tailored nanotopology is shown as an alternative tool to substantially affect cancerous gene expression, eventually due to differences in local biomechanical features of the cell–matrix interactions. Here, we report a clear selective up- and down-regulation of groups of markers for breast cancer (MDA-MB231), colorectal cancer (CaCO2), melanoma (WM239A), and neuroblastoma (Kelly) depending on only fiber orientation and morphology without application of any other stimulus. Changes in gene expression are also revealed for Mitomycin C treatment of MDA-MB231, making the scaffold a suitable platform for testing of anticancer agents. This allows an opportunity for selective “clean” guidance to a deep understanding of mechanisms of cancer cells progressive growth and tumor formation without possible side effects by manipulation with the specific markers.

Parallel trapping of single motile cells based on vibration-induced flow

We propose an on-chip cell manipulation method for trapping single motile cells in parallel. The proposed method traps large ( $$\gtrsim \,50\,\upmu \hbox {m}$$ ) motile cells in parallel, which is difficult to achieve by conventional cell manipulation methods based on optical, acoustic, electric, or magnetic forces. The trapping method exploits the flow induced by applying a vibration to a microfluidic chip with microstructures on its surface. By applying a rectilinear vibration to a chip with pairs of micropillars, we can trap single motile cells within the local flow generated between the micropillars. Using the proposed method, we trapped single Euglena gracilis cells (of size 50–100 $$\upmu \hbox {m}$$ ) in parallel. Moreover, we evaluate the trapping performance for various micropillar array design parameters and the controllability of the trapping-flow velocity by varying the amplitude of the vibration. The proposed method was then demonstrated in a motility evaluation of motile cells. The demonstration confirms the potential of the proposed method in realizing high-throughput motility evaluations of single motile cells.

Mesenchymal Stem Cells Engineering: Microcapsules-Assisted Gene Transfection and Magnetic Cell Separation.

Stem cell engineering-the manipulation and functionalization of stem cells involving genetic modification-can significantly expand their applicability for cell therapy in humans. Toward this aim, reliable, standardized, and cost-effective methods for cell manipulation are required. Here we explore the potential of magnetic multilayer capsules to serve as a universal platform for nonviral gene transfer, stem cell magnetization, and magnetic cell separation to improve gene transfer efficiency. In particular, the following experiments were performed: (i) a study of the process of internalization of magnetic capsules into stem cells, including capsule co-localization with established markers of endo-lysosomal pathway; (ii) characterization and quantification of capsule uptake with confocal microscopy, electron microscopy, and flow cytometry; (iii) intracellular delivery of messenger RNA and separation of gene-modified cells by magnetic cell sorting (MACS); and (iv) analysis of the influence of capsules on cell proliferation potential. Importantly, based on the internalization of magnetic capsules, transfected cells became susceptible to external magnetic fields, which made it easy to enrich gene-modified cells using MACS (purity ∼95%), and also to influence their migration behavior. In summary, our results underline the high potential of magnetic capsules in stem cell functionalization, namely (i) to increase gene-transfer efficiency and (ii) to facilitate enrichment and targeting of transfected cells. Finally, we did not observe a negative impact of the capsules used on the proliferative capacity of stem cells, proving their high biocompatibility.

Effective cell trapping using PDMS microspheres in an acoustofluidic chip

We present a facile particle-based cell manipulation method using acoustic radiation forces. In this work, we selected several representative particles including poly(lactic-co-glycolic acid) (PLGA) microspheres, silica-coated magnetic microbeads, polydimethylsiloxane (PDMS) microspheres and investigated the responses of these particle systems to ultrasonic standing waves (USWs) in a microfluidic chip. We show that depending on the nature (positive or negative acoustic contrast factors) of the particles, these particle systems display different alignment behaviors along the microfluidic channel under USWs. Specifically, PLGA microspheres and silica-coated magnetic microbeads are able to be aligned in the middle of the microfluidic channel, while PDMS microspheres are translocated to the side walls of the channel, which is beneficial for cell trapping and manipulation. Further results demonstrate that the functional PDMS microspheres with a negative acoustic contrast factor can be used to trap cells to the pressure antinodes in the acoustofluidic chip. Cell viability tests reveal that the ultrasonic manipulation does not exert any harmful effect to the cells. This acoustic-based particle and cell manipulation technique may hold a great promise for the development of rapid, noninvasive, continuous assays for detecting of cells and separation of biological samples.

Functional Salivary Gland Regenerative Therapy for Oral Health

Salivary glands maintain oral health and homeostasis via functional interactions with many organs, including the teeth and the tongue. Dysfunction of salivary glands causes many problems, such as dental caries, bacterial infection, and swallowing dysfunction. Current regenerative therapy for salivary gland tissue repair and whole salivary gland replacement is currently a novel therapeutic concept that may result in full recovery of salivary gland function. The salivary glands arise from reciprocal epithelial and mesenchymal interactions. We developed a novel three-dimensional cell manipulation method that can reproduce organogenesis via the epithelial-mesenchymal interaction. The bioengineered salivary glands develop correct structure and successfully secrete saliva into the oral cavity via reestablishment of the afferent-efferent neural network. The bioengineered salivary glands also improve dry mouth symptoms, such as bacterial infections and swallowing dysfunction. This review summarizes recent findings and technological advances in salivary gland regenerative therapy.

Microfluidic technology for cell hydrodynamic manipulation

In the recent years microfluidic technology has affirmed itself as a powerful tool in medical and biological research. Among the different applications, cell manipulation has been widely investigated. Micro-flowcytometers, micromixers, cell sorters and analyzers are only few examples of the developed devices. Various methods for cell manipulation have been proposed, such as hydrodynamic, magnetic, optical, mechanical, and electrical, in this way categorized according to the manipulating force employed. In particular, when cells are manipulated by hydrodynamic effects, there is no needing of applying external forces. This brings to a simplification in the design and fabrication phase, and at the same time undesired effects on the biological sample are limited. In this paper, we will discuss the physics of the relevant hydrodynamic effects in microfluidics, and how they are exploited for cell manipulation.

Yeast Nanobiotechnology

Yeast nanobiotechnology is a recent field where nanotechniques are used to manipulate and analyse yeast cells and cell constituents at the nanoscale. The aim of this review is to give an overview and discuss nanobiotechnological analysis and manipulation techniques that have been particularly applied to yeast cells. These techniques have mostly been applied to the model yeasts Saccharomyces cerevisiae and Schizosaccaromyces pombe, and the pathogenic model yeast Candida albicans. Nanoscale imaging techniques, such as Atomic Force Microscopy (AFM), super-resolution fluorescence microscopy, and electron microscopy (scanning electron microscopy (SEM), transmission electron microscopy (TEM), including electron tomography) are reviewed and discussed. Other nano-analysis methods include single-molecule and single-cell force spectroscopy and the AFM-cantilever-based nanomotion analysis of living cells. Next, an overview is given on nano/microtechniques to pattern and manipulate yeast cells. Finally, direct contact cell manipulation methods, such as AFM-based single cell manipulation and micropipette manipulation of yeast cells, as well as non-contact cell manipulation techniques, such as optical, electrical, and magnetic cells manipulation methods are reviewed.

Massively-multicellular alignment with the self-aggregate of air bubbles.

This study proposes a cell manipulation method with aggregated air bubbles on cell culture medium. This method requires no additional regents nor devices, except for normal cell-culture materials such as cell culture dishes and pipettes. Bubbles generated by pipetting were spontaneously aggregated with regularity on the whole surface and used as a mask for avoiding cell adhesion after cell-seeding. The diameter of bubbles was able to be controlled by the size of micro-pipette tips. Seeded cells spread to the whole area along the bubble gap. This technique is a surface-tension-driven self-assembly-based method. Using this technique, millions of living cells were successfully aligned into a hexagonal pattern within 300 μm in pattern width on the whole surface of dish for less than 2 h.