Electroporation of cells is a widely-used tool to transport molecules such as proteins or nucleic acids into cells or to extract cellular material. However, bulk methods for electroporation do not offer the possibility to selectively porate subpopulations or single cells in heterogeneous cell samples. To achieve this, either presorting or complex single-cell technologies are required currently. In this work, we present a microfluidic flow protocol for selective electroporation of predefined target cells identified in real-time by high-quality microscopic image analysis of fluorescence and transmitted light. While traveling through the microchannel, the cells are focused by dielectrophoretic forces into the microscopic detection area, where they are classified based on image analysis techniques. Finally, the cells are forwarded to a poration electrode and only the target cells are pulsed. By processing a heterogenically stained cell sample, we were able to selectively porate only target cells (green-fluorescent) while non-target cells (blue-fluorescent) remained unaffected. We achieved highly selective poration with >90% specificity at average poration rates of >50% and throughputs of up to 7200 cells per hour.

Continuous flow cell sorting based on image analysis is a powerful concept that exploits spatially-resolved features in cells, such as subcellular protein localisation or cell and organelle morphology, to isolate highly specialised cell types that were previously inaccessible to biomedical research, biotechnology, and medicine. Recently, sorting protocols have been proposed that achieve impressive throughput by combining ultra-high flow rates with sophisticated imaging and data processing protocols. However, moderate image quality and high complex experimental setups still prevent the full potential of image-activated cell sorting from being a general-purpose tool. Here, we present a new low-complexity microfluidic approach based on high numerical aperture wide-field microscopy and precise dielectrophoretic cell handling. It provides high-quality images with unprecedented resolution in image-activated cell sorting (i.e., 216 nm). In addition, it also allows long image processing times of several hundred milliseconds for thorough image analysis, while ensuring reliable and low-loss cell processing. Using our approach, we sorted live T cells based on subcellular localisation of fluorescence signals and demonstrated that purities above 80% are possible while targeting maximum yields and sample volume throughputs in the range of μl min-1. We were able to recover 85% of the target cells analysed. Finally, we ensure and quantify the full vitality of the sorted cells cultivating the cells for a period of time and through colorimetric viability tests.

We present a method for label-free imaging and sorting of cancer cells in blood, which is based on a dielectrophoretic microfluidic chip and label-free interferometric phase microscopy. The chip used for imaging has been embedded with dielectrophoretic electrodes, and therefore it can be used to sort the cells based on the decisions obtained during the cell flow by the label-free quantitative imaging method. Hence, we obtained a real-time, automatic, label-free imaging flow cytometry with the ability to sort the cells during flow. To validate our model, we combined into the label-free imaging interferometer a fluorescence imaging channel that indicated the correctness of the label-free sorting. We have achieved above 98% classification success and 69% sorting accuracy at flow rates of 4 to 7 mu L hr(-1). In the future, this method is expected to help in label-free sorting of circulating tumor cells in blood following an initial state-of-the-art cell enrichment.

In vitro cultured neuronal networks with defined connectivity are required to improve neuronal cell culture models. However, most protocols for their formation do not provide sufficient control of the direction and timing of neurite outgrowth with simultaneous access for analytical tools such as immunocytochemistry or patch-clamp recordings. Here, we present a proof-of-concept for the dynamic (i.e., time-gated) control of neurite outgrowth on a cell culture substrate based on 2D-micropatterned coatings of thermoresponsive polymers (TRP). The pattern consists of uncoated microstructures where neurons can readily adhere and neurites can extend along defined pathways. The surrounding regions are coated with TRP that does not facilitate cell or neurite growth at 33 °C. Increasing the ambient temperature to 37 °C renders the TRP coating cell adhesive and enables the crossing of gaps coated with TRP by neurites to contact neighboring cells. Here, we demonstrate the realization of this approach employing human neuronal SH-SY5Y cells and human induced neuronal cells. Our results suggest that this approach may help to establish a spatiotemporal control over the connectivity of multinodal neuronal networks.

With the advent of single-cell technologies comes the necessity for efficient protocols to process single cells. We combine dielectrophoresis with open source computer vision programming to automatically control the trajectories of single cells inside a microfluidic device. Using real-time image analysis, individual cells are automatically selected, isolated and spatially arranged.

Event Abstract Back to Event Switchable cell adhesive microstructures to grow defined neuronal networks Philipp Wysotzki1*, Jessica Schröder1, Laura V. Behm2, 3, Susanna Gerike2, 4, Felix Pfisterer2, Claus Duschl2, Michael Kirschbaum2, Jan Gimsa1 and Werner Baumann1 1 University of Rostock, Department of Biophysics, Germany 2 Fraunhofer-Institut für Zelltherapie und Immunologie (IZI), Branch Bioanalytics and Bioprocesses, Germany 3 Berlin-Brandenburgisches Zentrum für Regenerative Therapien, Charité Universitätsmedizin, Germany 4 Freie Universität Berlin, Germany The molecular structure of TRPs (thermo-responsive polymers) alters below and above a certain temperature. In experiments with neuronal cell lines (SH-SY5Y), we used TRP, which repelled cells below 33 °C and favored cell adhesion above 37 °C. For a more detailed characterization of the TRP properties, we determined the initial cell adhesion forces with single cell force spectroscopy. For the temporal and spatial control of the formation of neuronal circuits in vitro, micro-patterned structures with thermo-responsive surfaces were developed. Glass chips were produced with heating micro-structures covered by a Si3N4 passivation layer and coated with gold (Figure 1, gold). Experiments with primary neuronal mouse cells, which did not favor direct adhesion to TRP surfaces required micro-structuration of the TRP surfaces. Thiol-gold chemistry was used to coat the chip surfaces with TRP. The gold surface featured micro-structured round spots with protruding trenches, which prevented TRP adsorption but permitted the adhesion of single cells directly at the Si3N4 passivation layer (Figure 1, blue). The trenches, which connected the cell adhesion spots served to guide the axons of the cells to their neighbors (Figure 1, green). Cell adhesion to the feeder areas, trenches and cell adhesion spots could be enhanced by their specific surface modification with (3-aminopropyl)-triethoxysilane. The cell spots were connected to feeder areas on both sides of the chips (Figure 1, mint green). A gap in the trench to each neighboring cell prevented the direct connection of the axons to the neighbor cells (Figure 1, red). Heating micro-structures were located underneath the gaps. They permitting to switch between the cell adhesive and non-adhesive states of the TRP cover layer. Our final goal is the cultivation of primary neurons in defined network structures and the control of the connection in cellular arrays. Figure 1: Micro-structured chip for an array of four cells with two heating structures, four cell adhesion spot, and guiding trenches (blue: single cell adhesion spot, green: axon guide, red: thermo-switchable gap, gold: TRP-coated gold, mint green: feeder area). Scale bar: 100 µm. Figure 1 Acknowledgements The authors are grateful to the DFG (German Research Council) for funding the Projekt "NeuroTRP" (#290023374) Keywords: Thermo-responsive polymers, neuronal circuits, primary neuronal cells, Directed growth, microsystems Conference: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018. Presentation Type: Poster Presentation Topic: Neural Networks Citation: Wysotzki P, Schröder J, Behm LV, Gerike S, Pfisterer F, Duschl C, Kirschbaum M, Gimsa J and Baumann W (2019). Switchable cell adhesive microstructures to grow defined neuronal networks. Conference Abstract: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays. doi: 10.3389/conf.fncel.2018.38.00102 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 15 Mar 2018; Published Online: 17 Jan 2019. * Correspondence: Mr. Philipp Wysotzki, University of Rostock, Department of Biophysics, Rostock, Germany, 18057, Germany, Philipp.wysotzki@uni-rostock.de Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Philipp Wysotzki Jessica Schröder Laura V Behm Susanna Gerike Felix Pfisterer Claus Duschl Michael Kirschbaum Jan Gimsa Werner Baumann Google Philipp Wysotzki Jessica Schröder Laura V Behm Susanna Gerike Felix Pfisterer Claus Duschl Michael Kirschbaum Jan Gimsa Werner Baumann Google Scholar Philipp Wysotzki Jessica Schröder Laura V Behm Susanna Gerike Felix Pfisterer Claus Duschl Michael Kirschbaum Jan Gimsa Werner Baumann PubMed Philipp Wysotzki Jessica Schröder Laura V Behm Susanna Gerike Felix Pfisterer Claus Duschl Michael Kirschbaum Jan Gimsa Werner Baumann Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

The precise measurement of surface temperatures on the microscale is of major importance for lab-on-a-chip applications that deal with temperature-dependent processes. For that, thermometric methods that combine high temperature resolution with feasibility, flexibility and applicability in aqueous environments are strongly required. Here, we present and characterize an easy approach for surface temperature measurements based on thin films of commercially available sol-gel that contain the temperature sensitive fluorophore Rhodamine B. These films can be applied onto various surfaces in an easy two-step process and allow for temperature mapping on the microscale under both dry and liquid conditions. To demonstrate the potential of the approach we measured the temperature distribution at the surface of resistive microheaters. We analyzed minute surface temperature gradients and conducted time-dependent measurements. This demonstrated the high resolution regarding temperature (< 1 degrees C), space (lower mu m-range in one dimension), and time (sub-seconds-range) that can be achieved with the described approach under dry as well as under liquid conditions. Good correlation of our experimental results to those from finite element-based simulations confirmed the reliability of the approach. (C) 2017 Published by Elsevier B.V.

A major challenge in the field of optical imaging of live cells is achieving rapid, 3D, and noninvasive imaging of isolated cells without labeling. If successful, many clinical procedures involving analysis and sorting of cells drawn from body fluids, including blood, can be significantly improved. A new label‐free tomographic interferometry approach is presented. This approach provides rapid capturing of the 3D refractive‐index distribution of single cells in suspension. The cells flow in a microfluidic channel, are trapped, and then rapidly rotated by dielectrophoretic forces in a noninvasive and precise manner. Interferometric projections of the rotated cell are acquired and processed into the cellular 3D refractive‐index map. Uniquely, this approach provides full (360°) coverage of the rotation angular range around any axis, and knowledge on the viewing angle. The experimental demonstrations presented include 3D, label‐free imaging of cancer cells and three types of white blood cells. This approach is expected to be useful for label‐free cell sorting, as well as for detection and monitoring of pathological conditions resulting in cellular morphology changes or occurrence of specific cell types in blood or other body fluids.

Understanding the dynamics of signal transduction processes that are induced by cell-cell or cell-surface interactions requires the physical stimulation of the cells of interest on a single-cell level and without any ill-defined contacting of their cell membrane. However, standard cell culture techniques are inapplicable for this task as they do not provide cell and particle handling at sufficiently high spatial and temporal resolution and are limited to ensemble measurements. Here, we present a novel process line for the individual stimulation of single cells with bioactive surfaces, like other cells or particles, and the simultaneous analysis of the induced cytosolic calcium signals. The method is based on a microfluidic lab-on-a-chip environment that allows for contactless cell and particle handling by dielectrophoretic forces.

Wound repair is a quiescent mechanism to restore barriers in multicellular organisms upon injury. In chronic wounds, however, this program prematurely stalls. It is known that patterns of extracellular signals within the wound fluid are crucial to healing. Extracellular pH (pHe) is precisely regulated and potentially important in signaling within wounds due to its diverse cellular effects. Additionally, sufficient oxygenation is a prerequisite for cell proliferation and protein synthesis during tissue repair. It was, however, impossible to study these parameters in vivo due to the lack of imaging tools. Here, we present luminescent biocompatible sensor foils for dual imaging of pHe and oxygenation in vivo. To visualize pHe and oxygen, we used time-domain dual lifetime referencing (tdDLR) and luminescence lifetime imaging (LLI), respectively. With these dual sensors, we discovered centripetally increasing pHe-gradients on human chronic wound surfaces. In a therapeutic approach, we identify pHe-gradients as pivotal governors of cell proliferation and migration, and show that these pHe-gradients disrupt epidermal barrier repair, thus wound closure. Parallel oxygen imaging also revealed marked hypoxia, albeit with no correlating oxygen partial pressure (pO2)-gradient. This highlights the distinct role of pHe-gradients in perturbed healing. We also found that pHe-gradients on chronic wounds of humans are predominantly generated via centrifugally increasing pHe-regulatory Na+/H+-exchanger-1 (NHE1)-expression. We show that the modification of pHe on chronic wound surfaces poses a promising strategy to improve healing. The study has broad implications for cell science where spatial pHe-variations play key roles, e.g. in tumor growth. Furthermore, the novel dual sensors presented herein can be used to visualize pHe and oxygenation in various biomedical fields.

The prospect of novel therapeutic approaches has renewed the current interest in the fusion of rare cells, like stem cells or primary immune cells. While conventional techniques are only capable of mass fusion, lab-on-a-chip systems often still lack an acceptable method for making the cells available after processing. Here, we present a microfluidic approach for electrofusion on the single-cell level that offers high control over the cells both before and after fusion. For cell pairing and fusion, we employed dielectrophoresis and AC voltage pulses, respectively. Each cell has been characterized and selected before they were paired, fused and released from the fluidic system for subsequent analysis and cultivation. The successful experimental evaluation of our system was further corroborated by numerical simulations. We obtained fusion efficiencies of more than 30% for individual pairs of mouse myeloma and B cell blasts and showed the proliferating ability of the hybrid cells 3 d after fusion. Since aggregates of more than two cells can be fused, the technique could also be developed further for generating giant cells for low-noise electrophysiology in the context of semi-automated pharmaceutical screening procedures.

In order to elucidate the dynamics of cellular processes that are induced in context with intercellular communication, defined events along the signal transduction cascade and subsequent activation steps have to be analyzed on the level of individual cells and correlated with each other. Here we present an approach that allows the initiation of cell-cell or cell-particle interactions and the analysis of cellular reactions within various regimes while the identity of each individual cell is preserved. It utilizes dielectrophoresis (DEP) and microfluidics in a lab-on-chip system. With high spatial and temporal precision we contacted single T cells with functionalized microbeads and monitored their immediate cytosolic Ca(2+) response. After this, the cells were released from the chip system and cultivated further. Expression of the activation marker molecule CD69 was analyzed the next day and correlated with the previously recorded Ca(2+) signal for each individual cell. We found a significant difference in the patterns of Ca(2+) traces between activated and non-activated cells, which shows that Ca(2+) signals in T cells can provide early information about a later reaction of the cell. Although T cells are non-excitable cells, we also observed irregular Ca(2+) transients upon exposure to the DEP field only. These Ca(2+) signals depended on exposure time, electric field strength and field frequency. By minimizing their occurrence rate, we could identify experimental conditions that caused the least interference with the physiology of the cell.

The gentle and careful in vitro processing of live cells is essential in order to make them available to future therapeutic applications. We present a protocol for the activation of single-T cells based on the contact formation with individual anti-CD3/anti-CD28 presenting microbeads in a lab-on-chip environment. The chips consist of microfluidic channels and microelectrodes for performing dielectrophoretic manipulation employing a.c. electric fields. The dielectrophoretic guiding elements allow the assembly of cell–bead pairs while avoiding ill-defined physical contacts with their environment. After overnight cultivation of the manipulated cells, 77% of the bead-associated T cells expressed the activation marker molecule CD69. Physiological stress on the cells was shown to be mainly due to the single-cell cultivation and not to the manipulation in the chips. The same approach could be useful for the in vitro regulation of stem cell differentiation.

We present a simple lab-on-chip device for handling small samples of delicate cells, e.g. stem cells. It uses a combination of sedimentation and dielectrophoresis. The transport of cells is driven by gravitation. Dielectrophoresis uses radio-frequency electric fields for generating particle-selective forces dependent on size and polarisability. Electrodes along the channels hold particles and/or cells in a defined position and deflect them towards different outlets. The absence of external pumping and the integration of injection and sampling ports allow the processing of tiny sample volumes. Various functions are demonstrated, such as contact-free cell trapping and cell/particle sorting. Pairs of human cells and antibody-coated beads, as they are formed for T cell activation, are separated from unbound beads. The cells experience only low stress levels compared with the stress levels in dielectrophoresis systems, where transport depends on external pumping. Our device is a versatile yet simple tool that finds applications in cellular biotechnology, in particular when an economic solution is required.