Arkadiusz Pierzchalski

Microfluidic impedance‐based flow cytometry

Microfabricated flow cytometers can detect, count, and analyze cells or particles using microfluidics and electronics to give impedance‐based characterization. Such systems are being developed to provide simple, low‐cost, label‐free, and portable solutions for cell analysis. Recent work using microfabricated systems has demonstrated the capability to analyze micro‐organisms, erythrocytes, leukocytes, and animal and human cell lines. Multifrequency impedance measurements can give multiparametric, high‐content data that can be used to distinguish cell types. New combinations of microfluidic sample handling design and microscale flow phenomena have been used to focus and position cells within the channel for improved sensitivity. Robust designs will enable focusing at high flowrates while reducing requirements for control over multiple sample and sheath flows. Although microfluidic impedance‐based flow cytometers have not yet or may never reach the extremely high throughput of conventional flow cytometers, the advantages of portability, simplicity, and ability to analyze single cells in small populations are, nevertheless, where chip‐based cytometry can make a large impact.

Potential of a cytomics top-down strategy for drug discovery.

It takes about 10 to 15 years and roughly 800 mln

Label-free hybridoma cell culture quality control by a chip-based impedance flow cytometer

to bring a new drug to the market. Only 10% of drug molecules entering clinical trials succeed and only 3 out of 10 drugs generate enough profit to pay back for the investment. Drug targets may be searched by hypothesis driven modeling of molecular networks within and between cells by systems biology. However, there is the potential to simplify the search for new drugs and drug targets by an initial top-down cytomics phase. The cytomics approach i) requires no detailed a-priori knowledge on mechanisms of drug activity or complex diseases, ii) is hypothesis driven for the investigated parameters (genome, transcriptome, proteome, metabolome a.o.) and iii) is hypothesis-free for data analysis. Moreover it iv) carries the potential to uncover unknown molecular interrelations as a prerequisite for later new hypothesis driven modeling and research strategies. A set of discriminatory parameter patterns (molecular hotspots) describing the cellular model (mechanism of drug action) can be identified by differential molecular cell phenotyping. Hereby, the immediate modeling of existing complexities by bottom-up oriented systems biology is avoided. The review focuses on the fast technological developments of molecular single cell analysis in recent years. They comprise a multitude of sensitive new molecular markers as well as various new image and flow cytometric high-content screening methods as facilitators of the cytomics concept. New bioinformatic tools enable the extraction of relevant molecular hotspots in description of cellular models, being required for the subsequent molecular reverse engineering phase by systems biology.

Immunological monitoring of extracorporeal photopheresis after heart transplantation

Impedance flow cytometry (IFC) was evaluated as a possible alternative to fluorescence-based methods for on-line quality monitoring of hybridoma cells. Hybridoma cells were cultured at different cell densities and viability was estimated by means of IFC and fluorescence-based flow cytometry (FCM). Cell death was determined by measuring the impedance phase value at high frequency in low conductivity buffer. IFC data correlate well with reference FCM measurements using AnnexinV and 7-AAD staining. Hybridoma cells growing at different densities in cell culture revealed a density-dependent subpopulation pattern. Living cells of high density cultures show reduced impedance amplitudes, indicating particular cellular changes. Dead cell subpopulations become evident in cultures with increasing cell densities. In addition, a novel intermediate subpopulation, which most probably represents apoptotic cells, was identified. These results emphasize the extraordinary sensitivity of high frequency impedance measurements and their suitability for hybridoma cell culture quality control.

Cytomics and nanobioengineering.

Extracorporeal photopheresis (ECP) has been used as a prophylactic and therapeutic option to avoid and treat rejection after heart transplantation (HTx). Tolerance‐inducing effects of ECP such as up‐regulation of regulatory T cells (Tregs) are known, but specific effects of ECP on regulatory T cell (Treg) subsets and dendritic cells (DCs) are lacking. We analysed different subsets of Tregs and DCs as well as the immune balance status during ECP treatment after HTx. Blood samples were collected from HTx patients treated with ECP for prophylaxis (n = 9) or from patients with histologically proven acute cellular rejection (ACR) of grade ≥ 1B (n = 9), as well as from control HTx patients without ECP (HTxC; n = 7). Subsets of Tregs and DCs as well as different cytokine levels were analysed. Almost 80% of the HTx patients showed an effect to ECP treatment with an increase of Tregs and plasmacytoid DCs (pDCs). The percentage of pDCs before ECP treatment was significantly higher in patients with no ECP effect (26·3% ± 5·6%) compared to patients who showed an effect to ECP (9·8% ± 10·2%; P = 0·011). Analysis of functional subsets of CD4+CD25highCD127low Tregs showed that CD62L‐, CD120b‐ and CD147‐positive Tregs did not differ between the groups. CD39‐positive Tregs increased during ECP treatment compared to HTxC. ECP‐treated patients showed higher levels for T helper type 1 (Th1), Th2 and Th17 cytokines. Cytokine levels were higher in HTx patients with rejection before ECP treatment compared to patients with prophylactic ECP treatment. We recommend a monitoring strategy that includes the quantification and analysis of Tregs, pDCs and the immune balance status before and up to 12 months after starting ECP.

An Innovative Cascade System for Simultaneous Separation of Multiple Cell Types

The finding that an individuals genome differs as much as by many million variants from that of the human reference assembly diminished the great enthusiasm that every disease could be predicted based on nucleotide polymorphisms. Even individual cells of an organ may be specifically equipped to perform specific tasks and that the information of individual cells in a cell system is key information to understand function or dysfunction. Therefore, cytomics received great attention during the last years as it allows to quantitatively and qualitatively analyzing great number of individual cells, cell constituents, and of their intracellular and functional interactions in a cellular system and also giving the concept of analysis of these data.

Cytocidal effect of interleukin 1 (IL-1) on HeLa cells is mediated by both soluble and transmembrane tumor necrosis factor (TNF)

Isolation of different cell types from one sample by fluorescence activated cell sorting is standard but expensive and time consuming. Magnetic separation is more cost effective and faster by but requires substantial effort. An innovative pluriBead-cascade cell isolation system (pluriSelect GmbH, Leipzig, Germany) simultaneously separates two or more different cell types. It is based on antibody-mediated binding of cells to beads of different size and their isolation with sieves of different mesh-size. For the first time, we validated the pluriSelect system for simultaneous separation of CD4+- and CD8+-cells from human EDTA-blood samples. Results were compared with those obtained by magnetic activated cell sorting (MACS; two steps -first isolation of CD4+, then restaining of the residual cell suspension with anti-human CD8+ MACS antibody followed by the second isolation). pluriSelect separation was done in whole blood, MACS separation on density gradient isolated mononuclear cells. Isolated and residual cells were immunophenotyped by 7-color 9-marker panel (CD3; CD16/56; CD4; CD8; CD14; CD19; CD45; HLA-DR) using flow cytometry. Cell count, purity, yield and viability (7-AAD exclusion) were determined. There were no significant differences between both systems regarding purity (MACS (median[range]: 92.4% [91.5-94.9] vs. pluriSelect 95% [94.9-96.8])) of CD4+ cells, however CD8+ isolation showed lower purity by MACS (74.8% [67.6-77.9], pluriSelect 89.9% [89.0-95.7]). Yield was not significantly different for CD4 (MACS 58.5% [54.1-67.5], pluriSelect 67.9% [56.8-69.8]) and for CD8 (MACS 57.2% [41.3-72.0], pluriSelect 67.2% [60.0-78.5]). Viability was slightly higher with MACS for CD4+ (98.4% [97.8-99.0], pluriSelect 94.1% [92.1-95.2]) and for CD8+-cells (98.8% [98.3-99.1], pluriSelect 86.7% [84.2-89.9]). pluriSelect separation was substantially faster than MACS (1h vs. 2.5h) and no pre-enrichment steps were necessary. In conclusion, pluriSelect is a fast, simple and gentle system for efficient simultaneous separation of two and more cell subpopulation directly from whole blood and provides a simple alternative to magnetic separation.

Prenatal phthalate exposure associates with low regulatory T-cell numbers and atopic dermatitis in early childhood: Results from the LINA mother-child study

Interleukin 1 (IL-1) is a pleiotropic cytokine able to induce cytocidal effect. The aim of the presented work was to analyze the mechanism of IL-1-induced cytocidal effect in HeLa cells in the presence of cycloheximide (CHX). We found that the pattern of IL-1-induced cell death shares significant similarities with the effect of tumor necrosis factor (TNF) in these cells. Subsequently, we identified IL-1 cytotoxicity as an indirect effect. The supernatant collected from the cells treated with IL-1 and CHX showed toxic activity towards IL-1-resistant while TNF-sensitive A9 cells. Furthermore, antibodies neutralizing TNF blocked HeLa cell death induced by IL-1/CHX. TNF was then detected in HeLa cells by means of flow cytometry, fluorescence microscopy and ELISA of detergent-soluble cell extracts. In the presence of an inhibitor of TNF sheddase (TACE), the cytotoxic effect of IL-1/CHX and the amount of TNF protein in detergent-soluble cell extracts were enhanced. These results suggest that in response to interleukin 1/CHX, the amount of transmembrane TNF is increased. Taken together, we demonstrated that the mechanism of IL-1 cytotoxic activity in HeLa cells in the presence of CHX depends on the function of soluble and transmembrane TNF.

Observing the invisible: molecular and cellular imaging by multimodal virtual anatomy.

Citation for published version (APA): Herberth, G., Pierzchalski, A., Feltens, R., Bauer, M., Röder, S., Olek, S., ... Lehmann, I. (2017). Prenatal phthalate exposure associates with low regulatory T-cell numbers and atopic dermatitis in early childhood: Results from the LINA mother-child study. Journal of Allergy and Clinical Immunology, 139(4), 1376-1379.e8. https://doi.org/10.1016/j.jaci.2016.09.034

Flow cytometric assay for analysis of cytotoxic effects of potential drugs on human peripheral blood leukocytes

COMMENTARY Molecular imaging with both new instrumentation and data analysis has become an exciting tool for in vivo whole animal imaging. As shown in the impressive review by Wessels et al. (1) scientists now can very precisely distinguish details of a small animal’s anatomy with high resolution. The authors demonstrate that with these new instrumentations one could perform molecular imaging at the cellular and molecular level. Resolution of state of the art instruments can go below that of single cells and even micro capillaries can be made visible with noninvasive technologies. That is indeed ‘‘virtual anatomy.’’ But is this cytometry? Even with access to such highly sophisticated technologies, can we stoichiometrically analyze cells in vivo in a way that is familiar to cytometrists? The modern imaging technologies like miniaturized nuclear magnetic resonance imaging (lMRI) as well as flat-panel volumetric computed tomography (fpVCT) and micro-computed tomography (lCT) are able to visualize morphology (‘‘virtual histology’’) but in most cases lack single cell based information (1–3). Using fluorescent tags such as quantum dots (4), a plethora of fluorescent proteins (5), and switchable molecular colors (PS-CFP, PA-GFP) (6) gives the substantial advantage of imaging selectively labelled cells in vivo (within the body) with a very good signal-to-noise ratio. But this is a doubleedged sword. Even though deep tissue penetration with near IR light (NIR; 650–900 nm) is possible and with polychromatic analysis different cells and their function could be simultaneously recorded, the anatomical resolution is very poor (7). Presently, there are two different imaging approaches for small animals: (a) imaging techniques with real 3D recordings (lMRI and fpVCT, lCT) for excellent imaging of tissues, blood vessels, cartilage, and bones and (b) methods to detect specific fluorescence dyes with high specificity and sensitivity. A combination of both modalities—real 3D reconstruction of anatomical structures with a high resolution detection of fluorescence—is not satisfactory with today’s techniques. Nevertheless, tissue penetration by fluorescence limits the application to a depth of hundredths of micrometres (two-photon imaging) (8) up to not more than a few centimetres (e.g. by spectral imaging) (9). Combined or merged with additional techniques which provide deep penetration and real 3D imaging not only virtual anatomy is feasible but also acquisition of fluorescence information at a single cell level in vivo. A combination of both approaches would thereby allow for the exact positioning of the detected fluorescence in the animal. Merging of different modalities has been realized already in some set-ups allowing for functional (molecular) and structural (anatomical) information from the same machine (10). Yet there is no one modality that is ideal for all experimental studies (3). A similar approach was proposed by Weissleder and coworkers who introduced fluorescence molecular tomography in vivo (as the combination of 3D tissue analysis with fluorescence detection) (11).