Publikationsliste Dr. Stefan Zimmermann

• Originalarbeiten in wissenschaftlichen Fachzeitschriften
• Reviews/Übersichtsartikel in wissenschaftlichen Fachzeitschriften
• Buchbeiträge
• Kurzbeiträge
• Konferenzbeiträge

Originalarbeiten in wissenschaftlichen Fachzeitschriften

 zurück zur Übersicht aller Publikationen

2024

• V. Zieger, E. Woehr, S. Zimmermann, D. Frejek, P. Koltay, R. Zengerle, S. Kartmann
  Automated Nanodroplet Dispensing for Large-Scale Spheroid Generation via Hanging Drop and Parallelized Lossless Spheroid Harvesting
  2024 Micromachines, Band: 15, Seite: 231

2023

• J. Weygant, F. Koch, K. Adam, K. Tröndle, R. Zengerle, G. Finkenzeller, S. Kartmann, P. Koltay, S. Zimmermann
  A Drop-on-Demand Bioprinting Approach to Spatial Arrangement of Multiple Cell Types and Monitoring Their Cell–Cell Interactions towards Vascularization Based on Endothelial Cells and Mesenchymal Stem Cells
  2023 Cells, Band: 12, Nummer: 4, Seite: 646
• D. Grijalva Garces, S. Strauß, S. Gretzinger, B. Schmieg, T. Jüngst, J. Groll, L. Meinel, I. Schmidt, H. Hartmann, K. Schenke-Layland, N. Brandt, M. Selzer, S. Zimmermann, P. Koltay, A. Southan, G. E. M. Tovar, S. Schmidt, A. Weber, T. Ahlfeld, M. Gelinsky, T. Scheibel, R. Detsch, A. R. Boccaccini, T. Naolou, C. Lee-Thedieck, C. Willems, T. Groth, S. Allgeier, B. Köhler, T. Friedrich, H. Briesen, J. Buchholz, D. Paulus, A. von Gladiss, J. Hubbuch
  On the reproducibility of extrusion-based bioprinting: round robin study on standardization in the field
  2023 Biofabrication, Band: 16, Seite: 015002
• V. Zieger, D. Frejek, S. Zimmermann, G. A. A. Miotto, P. Koltay, R. Zengerle, S. Kartmann
  Towards Label-Free Standardization in 3d Cell Culture: Automated, Selective and Gentle High-Throughput Handling of Spheroids and Organoids via Novel Pick-Flow-Drop Principle
  2023 Adv Healthc Mater, Seite: e2303350

2022

• K. Tröndle, G. Miotto, L. Rizzo, R. Pichler, F. Koch, P. Koltay, R. Zengerle, S. S. Lienkamp, S. Kartmann, S. Zimmermann
  Deep Learning-Assisted Nephrotoxicity Testing with Bioprinted Renal Spheroids
  2022 Int. J. Bioprint, Band: 8, Nummer: 2, Seite: 528
• V. Burchak, F. Koch, L. Siebler, S. Haase, V. K. Horner, X. Kempter, G. B. Stark, U. Schepers, A. Grimm, S. Zimmermann, P. Koltay, S. Strassburg, G. Finkenzeller, F. Simunovic, F. Lampert
  Evaluation of a Novel Thiol–Norbornene-Functionalized Gelatin Hydrogel for Bioprinting of Mesenchymal Stem Cells
  2022 Int. J. Mol. Sci., Band: 23, Seite: 7939
• F. Koch, O. Thaden, S. Conrad, K. Tröndle, G. Finkenzeller, R. Zengerle, S. Kartmann, S. Zimmermann, P. Koltay
  Mechanical properties of polycaprolactone (PCL) scaffolds for hybrid 3D-bioprinting with alginate-gelatin hydrogel
  2022 J. Mech. Behav. Biomed. Mat., Band: 130, Seite: 105219

2021

• T. Gross, C. Jeney, D. Halm, G. Finkenzeller, G. B. Stark, R. Zengerle, P. Koltay, S. Zimmermann
  Characterization of CRISPR/Cas9 RANKL knockout mesenchymal stem cell clones based on single-cell printing technology and Emulsion Coupling assay as a low-cellularity workflow for single-cell cloning
  2021 Plos One, Band: 16, Nummer: 3, Seite: e0238330
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  Kurzfassung

  The homogeneity of the genetically modified single-cells is a necessity for many applications such as cell line development, gene therapy, and tissue engineering and in particular for regenerative medical applications. The lack of tools to effectively isolate and characterize CRISPR/Cas9 engineered cells is considered as a significant bottleneck in these applications. Especially the incompatibility of protein detection technologies to confirm protein expression changes without a preconditional large-scale clonal expansion creates a gridlock in many applications. To ameliorate the characterization of engineered cells, we propose an improved workflow, including single-cell printing/isolation technology based on fluorescent properties with high yield, a genomic edit screen (Surveyor assay), mRNA RT-PCR assessing altered gene expression, and a versatile protein detection tool called emulsion-coupling to deliver a high-content, unified single-cell workflow. The workflow was exemplified by engineering and functionally validating RANKL knockout immortalized mesenchymal stem cells showing bone formation capacity of these cells. The resulting workflow is economical, without the requirement of large-scale clonal expansions of the cells with overall cloning efficiency above 30% of CRISPR/Cas9 edited cells. Nevertheless, as the single-cell clones are comprehensively characterized at an early, highly parallel phase of the development of cells including DNA, RNA, and protein levels, the workflow delivers a higher number of successfully edited cells for further characterization, lowering the chance of late failures in the development process.
• F. Koch, O. Thaden, K. Tröndle, R. Zengerle, S. Zimmermann, P. Koltay
  Open-source hybrid 3D-bioprinter for simultaneous printing of thermoplastics and hydrogels
  2021 HardwareX, Band: 10, Seite: e00230

2020

• F. Koch, K. Tröndle, G. Finkenzeller, R. Zengerle, S. Zimmermann, P. Koltay
  Generic method of printing window adjustment for extrusion-based 3D-bioprinting to maintain high viability of mesenchymal stem cells in an alginate-gelatin hydrogel
  2020 Bioprinting, Seite: e00094
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  Kurzfassung

  Over the last decade, bioprinting of artificial tissues has been developed into a significant field of research. With an increasing number of printing technologies and bioinks used in bioprinting, its complexity increases as both the printing technology and the properties of the bioink influence the cell biological functionality and printing accuracy of the printed tissue. Therefore, optimization of bioprinting processes often remains a challenge, which could be solved by a smart fine-tuning of the process parameters. We present a novel method to adjust the printing window for extrusion-based bioprinting on the basis of a two-step assessment to determine process parameters such as nozzle size, extrusion flow rate, and printing temperature. First, a suitable printing temperature is deduced from the bioink properties and second nozzle size and extrusion flow rate is selected in a way that the immediate cell damage after printing is reduced. For both steps only basic rheological properties of the bioinks need to be known as well as detailed knowledge of the cell survival in the bioink for different shear stresses. This method is applied to an exemplary alginate-gelatin hydrogel to show how the printing temperature affects the achievable printing accuracy. For this bioink, viability of immortalized mesenchymal stem cells (iMSC) decreases with about 4% per thousand Pascal increase in maximum shear stress. For different combinations of flow rate, nozzle size and nozzle shape it is shown, that only the maximum shear stress experienced by the iMSCs influences average cell viability. Factors like flow rate, nozzle size and shape only play an indirect role by influencing the maximum shear stress and individually have no significant influence on cell viability. The experimental results allow a direct adjustment of printing parameters for the presented combination of hydrogel and cell type but are not limited to it. For other bioinks, the described generic method can be easily used to systematically adjust the printing parameters. For this purpose, only the basic rheological properties and the influence of shear stress on cell survival need to be known and process parameters can be set concerning the respective application.
• P. Rukavina, F. Koch, M. Wehrle, K. Tröndle, G. B. Stark, P. Koltay, S. Zimmermann, R. Zengerle, F. Lampert, S. Strassburg, G. Finkenzeller, F. Simunovic
  In vivo evaluation of bioprinted prevascularized bone tissue
  2020 Biotechnol Bioeng, Seiten: 1 - 10
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  Kurzfassung

  Bioprinting can be considered as a progression of the classical tissue engineering approach, in which cells are randomly seeded into scaffolds. Bioprinting offers the advantage that cells can be placed with high spatial fidelity within three‐dimensional tissue constructs. A decisive factor to be addressed for bioprinting approaches of artificial tissues is that almost all tissues of the human body depend on a functioning vascular system for the supply of oxygen and nutrients. In this study, we have generated cuboid prevascularized bone tissue constructs by bioprinting human adipose‐derived mesenchymal stem cells (ASCs) and human umbilical vein endothelial cells (HUVECs) by extrusion‐based bioprinting and drop‐on‐demand (DoD) bioprinting, respectively. The computer‐generated print design could be verified in vitro after printing. After subcutaneous implantation of bioprinted constructs in immunodeficient mice, blood vessel formation with human microvessels of different calibers could be detected arising from bioprinted HUVECs and stabilization of human blood vessels by mouse pericytes was observed. In addition, bioprinted ASCs were able to synthesize a calcified bone matrix as an indicator of ectopic bone formation. These results indicate that the combined bioprinting of ASCs and HUVECs represents a promising strategy to produce prevascularized artificial bone tissue for prospective applications in the treatment of critical‐sized bone defects.
• M. Trotter, D. Juric, Z. Bagherian, N. Borst, K. Gläser, T. Meissner, F. von Stetten, A. Zimmermann
  Inkjet-Printing of Nanoparticle Gold and Silver Ink on Cyclic Olefin Copolymer for DNA-Sensing Applications
  2020 Sensors, Band: 20, Nummer: 5, Seite: 1333
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  Kurzfassung

  Inkjet technology as a maskless, direct-writing technology offers the potential for structured deposition of functional materials for the realization of electrodes for, e.g., sensing applications. In this work, electrodes were realized by inkjet-printing of commercial nanoparticle gold ink on planar substrates and, for the first time, onto the 2.5D surfaces of a 0.5 mm-deep microfluidic chamber produced in cyclic olefin copolymer (COC). The challenges of a poor wetting behavior and a low process temperature of the COC used were solved by a pretreatment with oxygen plasma and the combination of thermal (130 °C for 1 h) and photonic (955 mJ/cm²) steps for sintering. By performing the photonic curing, the resistance could be reduced by about 50% to 22.7 μΩ cm. The printed gold structures were mechanically stable (optimal cross-cut value) and porous (roughness factors between 8.6 and 24.4 for 3 and 9 inkjet-printed layers, respectively). Thiolated DNA probes were immobilized throughout the porous structure without the necessity of a surface activation step. Hybridization of labeled DNA probes resulted in specific signals comparable to signals on commercial screen-printed electrodes and could be reproduced after regeneration. The process described may facilitate the integration of electrodes in 2.5D lab-on-a-chip systems.
• J. Riba, J. Schoendube, S. Zimmermann, P. Koltay, R. Zengerle
  Single-cell dispensing and ‘realtime’cell classification using convolutional neural networks for higher efficiency in single-cell cloning
  2020 nature scientific reports, Band: 10, Seite: 1193
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  Kurzfassung

  Single-cell dispensing for automated cell isolation of individual cells has gained increased attention in the biopharmaceutical industry, mainly for production of clonal cell lines. Here, machine learning for classification of cell images is applied for ‘real-time’ cell viability sorting on a single-cell printer. We show that an extremely shallow convolutional neural network (CNN) for classification of lowcomplexity cell images outperforms more complex architectures. Datasets with hundreds of cell images from four different samples were used for training and validation of the CNNs. The clone recovery, i.e. the fraction of single-cells that grow to clonal colonies, is predicted to increase for all the samples investigated. Finally, a trained CNN was deployed on a c.sight single-cell printer for ‘real-time’ sorting of a CHO-K1 cells. On a sample with artificially damaged cells the clone recovery could be increased from 27% to 73%, thereby resulting in a significantly faster and more efficient cloning. Depending on the classification threshold, the frequency at which viable cells are dispensed could be increased by up to 65%. This technology for image-based cell sorting is highly versatile and can be expected to enable cell sorting by computer vision with respect to different criteria in the future.

2019

• K. Tröndle, F. Koch, G. Finkenzeller, G. B. Stark, R. Zengerle, P. Koltay, S. Zimmermann
  Bioprinting of high cell density constructs leads to controlled lumen formation with self‐assembly of endothelial cells
  2019 Journal of Tissue Engineering and Regenerative Medicine, Band: 13, Nummer: 10, Seiten: 1883 - 1895
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  Kurzfassung

  Active nutrient supply and waste product removal are key requirements for the fabrication of long term viable and functional tissue constructs of considerable size. This work aims to contribute to the fabrication of artificial perfusable networks with a bioprinting process, based on drop‐on‐demand (DoD) printing of primary endothelial cell (EC) suspension bioink (25 · 106 ± 3 · 106 cells/ml). The process results in prescribed lumen between two hydrogel layers, allowing its integration in common layering based bioprinting processes. Low volume bioink droplets (appr. 10 nl) as building blocks, were deposited between two fibrin or collagen I layers to realize shapeable, cell‐rich aggregates. Unattainable with manual positioning, DoD printing allowed precise fabrication of various designs, such as spheroidal‐, line‐shaped and Y‐branch cellular structures, with a mean lateral extension of 285 ± 81 μm. For basic characterization, the cell suspension building blocks were systematically compared to preformed spheroids of the same cell type, passage and number. Post printing investigations of initially loose cell arrangements showed self‐assembly and formation of central lumen with a mean cross‐sectional area of Ølumen = 6400 μm2 at day 3, lined by a single layer of CD31 positive ECs, as evaluated by confocal microscopy. Originating from this main lumen smaller, undirected side‐branches (Øbranches = 740 μm2) were formed by sprouting cells, inducing a first step towards a simplistic hierarchically organized network. These lumen could prospectively help for tissue construct perfusion in vitro or, potentially, as niche for angiogenesis of host vascularization in implants.
• M. Wehrle, F. Koch, S. Zimmermann, P. Koltay, R. Zengerle, G. B. Stark, S. Strassburg, G. Finkenzeller
  Examination of Hydrogels and Mesenchymal Stem Cell Sources for Bioprinting of Artificial Osteogenic Tissues
  2019 Cellular and Molecular Bioengineering, Seiten: 1 - 15
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  Kurzfassung

  Mesenchymal stem cells (MSCs) represent a very important cell source in the field of regenerative medicine and for bone and cartilage tissue engineering applications. Three-dimensional (3D) bioprinting has the potential to improve the classical tissue engineering concept as this technique allows the printing of cells with high spatial control of cell allocation within a 3D construct. In this study, we systematically compared different hydrogel blends for 3D bioprinting of MSCs by testing their cytocompatibility, ability to support osteogenic differentiation and their mechanical properties. In addition, we compared four different MSC populations isolated from different human tissues for their osteogenic differentiation capacity in combination with different hydrogels. The aim of this study was to identify the best MSC source and the most suitable hydrogel blend for extrusion-based bioprinting of 3D large-scaled osteogenic constructs.

2018

• C.H. Tsai, X. Wu, D.H. Kuan, S. Zimmermann, R. Zengerle, P. Koltay
  Digital hydraulic drive for microfluidics and miniaturized cell culture devices based on shape memory alloy actuators
  2018 J Micromech Microeng, Band: 28, Seite: 084001
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  Kurzfassung

  In order to culture and analyze individual living cells, microfluidic cell culture and manipulation of cells becomes an increasingly important topic. Such microfluidic systems allow for exploring the phenotypic differences between thousands of genetically identical cells or pharmacological tests in parallel, which is impossible to achieve by traditional macroscopic cell culture methods. Therefore, plenty of microfluidic devices have been developed for cell biological studies like cell culture, cell sorting, and cell lysis in the past. However, these devices are still limited by the external pressure sources which most of the time are large in size and have to be connected by fluidic tubing leading to complex and delicate systems.
 In order to provide a miniaturized, more robust actuation system a novel, compact and low power consumption Digital Hydraulic Drive (DHD) has been developed that is intended for use in portable and automated systems for microfluidic applications. The DHD consists of a shape memory alloy (SMA) actuator and a pneumatic cylinder. The switching time of the digital modes (pressure ON vs. OFF) can be adjusted from 1 second to minutes. Thus, the DHDs might have many applications for driving microfluidic devices. In this work different implementations of DHDs are presented and their performance is characterized by experiments. In particular, it will be shown that DHDs can be used for microfluidic large-scale integration (mLSI) valve control (256 valves in parallel) as well as potentially for droplet-based microfluidic systems. As further application example high-throughput mixing of cell cultures (96 wells in parallel) are demonstrated employing the DHD and a so called "functional lid" (FL) approach, to enable a miniaturized microbioreactor in a regular 96-well micro well plate.
• J. M. Stosch, A. Heumüller, C. Niemöller, S. Bleul, M. Rothenberg-Thurley, J. Riba, N. Renz, K. Szarc vel Szic, D. Pfeifer, M. Follo, H. L. Pahl, S. Zimmermann, J. Duyster, J. Wehrle, M. Lübbert, K. H. Metzeler, R. Claus, H. Becker
  Gene mutations and clonal architecture in myelodysplastic syndromes and changes upon progression to acute myeloid leukaemia and under treatment
  2018 Brit J Haematol, Band: 182, Seiten: 830 - 842
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  Kurzfassung

  Knowledge of the molecular and clonal characteristics in the myelodysplastic syndromes (MDS) and during progression to acute myeloid leukaemia (AML) is essential to understand the disease dynamics and optimize treatment. Sequencing serial bone marrow samples of eight patients, we observed that MDS featured a median of 3 mutations. Mutations in genes involved in RNA-splicing or epigenetic regulation were most frequent, and exclusively present in the major clone. Minor subclones were distinguishable in three patients. As the MDS progressed, a median of one mutation was gained, leading to clonal outgrowth. No AML developed genetically independent of a pre-existing clone. The gained mutation mostly affected genes encoding signalling proteins. Additional acquisition of genomic aberrations frequently occurred. Upon treatment, emergence of new clones could be observed. As confirmed by single-cell sequencing, multiple mutations in identical genes in different clones were present within individual patients. DNA-methylation profiling in patients without identification of novel mutations in AML revealed methylation changes in individual genes. In conclusion, our data complement previous observations on the mutational and clonal characteristics in MDS and at progression. Moreover, DNA-methylation changes may be associated with progression in single patients. Redundancy of mutated genes in different clones suggests fertile grounds promoting clonal selection or acquisition.

2017

• L. Benning, L. Gutzweiler, K. Tröndle, J. Riba, R. Zengerle, P. Koltay, S. Zimmermann, G. B. Stark, G. Finkenzeller
  Assessment of hydrogels for bioprinting of endothelial cells
  2017 J Biomed Mater Res A, Seiten: 935 - 947
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  Kurzfassung

  In tissue engineering applications, vascularization can be accomplished by co-implantation of tissue forming cells and endothelial cells (ECs), whereby the latter are able to form functional blood vessels. The use of three-dimensional (3D) bioprinting technologies has the potential to improve the classical tissue engineering approach because these will allow the generation of scaffolds with high spatial control of endothelial cell allocation. This study focuses on a side by side comparisons of popular commercially available bioprinting hydrogels (matrigel, fibrin, collagen, gelatin, agarose, Pluronic F-127, alginate and alginate/gelatin) in the context of their physicochemical parameters, their swelling/degradation characteristics, their biological effects on vasculogenesis-related EC parameters and their printability. The aim of this study was to identify the most suitable hydrogel or hydrogel combination for inkjet printing of ECs to build pre-vascularized tissue constructs. Most tested hydrogels displayed physicochemical characteristics suitable for inkjet printing. However, Pluronic F-127 and the alginate/gelatin blend were rapidly degraded when incubated in cell culture medium. Agarose, Pluronic F-127, alginate and alginate/gelatin hydrogels turned out to be unsuitable for bioprinting of ECs because of their non-adherent properties and/or their incapability to support EC proliferation. Gelatin was able to support EC proliferation and viability but was unable to support endothelial cell sprouting. Our experiments revealed fibrin and collagen to be most suitable for bioprinting of ECs, because these hydrogels showed acceptable swelling/degradation characteristics, supported vasculogenesis-related EC parameters and showed good printability. Moreover, ECs in constructs of preformed spheroids survived the printing process and formed capillary-like cords.
• L. Benning, L. Gutzweiler, K. Tröndle, J. Riba, R. Zengerle, P. Koltay, S. Zimmermann, G.B. Stark, G. Finkenzeller
  Cytocompatibility testing of hydrogels toward bioprinting of mesenchymal stem cells
  2017 J Biomed Mater Res A, Band: 105, Seiten: 3231 - 3241
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  Kurzfassung

  Mesenchymal stem cells (MSCs) represent a very attractive cell source for tissue engineering applications aiming at the generation of artificial bone substitutes. The use of three-dimensional bioprinting technologies has the potential to improve the classical tissue engineering approach because bioprinting will allow the generation of hydrogel scaffolds with high spatial control of MSC allocation within the bioprinted construct. In this study, we have performed direct comparisons between commercially available hydrogels in the context of their cytocompatibility toward MSCs and their physicochemical parameters with the aim to identify the most suitable hydrogel for drop-on-demand (DoD) printing of MSCs. In this context, we examined matrigel, fibrin, collagen, gelatin, and gelatin/alginate at various hydrogel concentrations. Matrigel, fibrin, collagen, and gelatin were able to support cell viability, but the latter showed a limited potential to promote MSC proliferation. We concentrated our study on fibrin and collagen hydrogels and investigated the effect of hydroxyapatite (HA) inclusion. The inclusion of HA enhanced proliferation and osteogenic differentiation of MSCs and prevented degradation of fibrin in vitro. According to viscosity and storage moduli measurements, HA-blends displayed physicochemical characteristics suitable for DoD printing. In bioprinting experiments, we confirmed that fibrin and collagen and their respective HA-blends represent excellent hydrogels for DoD-based printing as evidenced by high survival rates of printed MSCs.
• L. Gutzweiler, S. Kartmann, K. Troendle, L. Benning, G. Finkenzeller, R. Zengerle, P. Koltay, B. Stark, S. Zimmermann
  Large scale production and controlled deposition of single HUVEC spheroids for bioprinting applications
  2017 Biofabrication, Band: 9 (2), Seite: 02502
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  Kurzfassung

  We present 1.) a fast and automated method for large scale production of HUVEC spheroids based on the hanging drop method and 2.) a novel method for well-controlled lateral deposition of single spheroids by drop-on-demand printing. Large scale spheroid production is achieved via printing 1536 droplets of HUVEC cell suspension having a volume of 1 µl each within 3 minutes at a pitch of 2.3 mm within an array of 48 x 32 droplets onto a flat substrate. Printing efficiencies between 97.9% and 100% and plating efficiencies between 87.3% and 100% were achieved. Harvested spheroids (consisting of approx. 250 HUVECs each) appear uniform in size and shape. After incubation and harvesting, the spheroids are deposited individually in user-defined patterns onto hydrogels using an automated drop-on-demand dispenser setup. Controlled by an image detection algorithm focusing the dispenser nozzle, droplets containing exactly one spheroid are printed onto a substrate, while all other droplets are discarded. Using this approach an array of 6 x 3 HUVEC spheroids with intermediate distances of 500 µm embedded in fibrin was generated. Successful progress of spheroid sprouting and merging of neighboring sprouts was observed during the first 72 hours of incubation indicating a good viability of the deposited spheroids.

2016

• J. Riba, T. Gleichmann, S. Zimmermann, R. Zengerle, P. Koltay
  Label-free isolation and deposition of single bacterial cells from heterogeneous samples for clonal culturing
  2016 Scientific Reports, Band: 6, Seite: 32837
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  Kurzfassung

  The isolation and analysis of single prokaryotic cells down to 1 μm and less in size poses a special challenge and requires micro-engineered devices to handle volumes in the picoliter to nanoliter range. Here, an advanced Single-Cell Printer (SCP) was applied for automated and label-free isolation and deposition of bacterial cells encapsulated in 35 pl droplets by inkjet-like printing. To achieve this, dispenser chips to generate micro droplets have been fabricated with nozzles 20 μm in size. Further, the magnification of the optical system used for cell detection was increased. Redesign of the optical path allows for collision-free addressing of any flat substrate since no compartment protrudes below the nozzle of the dispenser chip anymore. The improved system allows for deterministic isolation of individual bacterial cells. A single-cell printing efficiency of 93% was obtained as shown by printing fluorescent labeled E. coli. A 96-well plate filled with growth medium is inoculated with single bacteria cells on average within about 8 min. Finally, individual bacterial cells from a heterogeneous sample of E. coli and E. faecalis were isolated for clonal culturing directly on agar plates in user-defined array geometry.
• J. Riba, N. Renz, C. Niemöller, S. Bleul, D. Pfeifer, J. M. Stosch, K. H. Metzeler, B. Hackanson, M. Lübbert, J. Duyster, P. Koltay, R. Zengerle, R. Claus, S. Zimmermann, H. Becker
  Molecular Genetic Characterization of Individual Cancer Cells Isolated via Single-Cell Printing
  2016 Plos One, Band: 6, Seite: 32837
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  Kurzfassung

  Intratumoral genetic heterogeneity may impact disease outcome. Gold standard for dissecting clonal heterogeneity are single-cell analyses. Here, we present an efficient workflow based on an advanced Single-Cell Printer (SCP) device for the study of gene variants in single cancer cells. To allow for precise cell deposition into microwells the SCP was equipped with an automatic dispenser offset compensation, and the 384-microwell plates were electrostatically neutralized. The ejection efficiency was 99.7% for fluorescent beads (n = 2304) and 98.7% for human cells (U-2 OS or Kasumi-1 cancer cell line, acute myeloid leukemia [AML] patient; n = 150). Per fluorescence microscopy, 98.8% of beads were correctly delivered into the wells. A subset of single cells (n = 81) was subjected to whole genome amplification (WGA), which was successful in all cells. On empty droplets, a PCR on LINE1 retrotransposons yielded no product after WGA, verifying the absence of free-floating DNA in SCP-generated droplets. Representative gene variants identified in bulk specimens were sequenced in single-cell WGA DNA. In U-2 OS, 22 of 25 cells yielded results for both an SLC34A2 and TET2 mutation site, including cells harboring the SLC34A2 but not the TET2 mutation. In one cell, the TET2 mutation analysis was inconclusive due to allelic dropout, as assessed via polymorphisms located close to the mutation. Of Kasumi-1, 23 of 33 cells with data on both the KIT and TP53 mutation site harbored both mutations. In the AML patient, 21 of 23 cells were informative for a TP53 polymorphism; the identified alleles matched the loss of chromosome arm 17p. The advanced SCP allows efficient, precise and gentle isolation of individual cells for subsequent WGA and routine PCR/sequencing-based analyses of gene variants. This makes single-cell information readily accessible to a wide range of applications and can provide insights into clonal heterogeneity that were indeterminable solely by analyses of bulk specimens.

Reviews/Übersichtsartikel in wissenschaftlichen Fachzeitschriften

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2015

• A. Gross, J. Schoendube, S. Zimmermann, M. Steeb, R. Zengerle, P. Koltay
  Technologies for Single-Cell Isolation
  2015 Int J Mol Sci, Band: 16, Seiten: 16897 - 16919
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  Kurzfassung

  The handling of single cells is of great importance in applications such as cell line development or single-cell analysis, e.g., for cancer research or for emerging diagnostic methods. This review provides an overview of technologies that are currently used or in development to isolate single cells for subsequent single-cell analysis. Data from a dedicated online market survey conducted to identify the most relevant technologies, presented here for the first time, shows that FACS (fluorescence activated cell sorting) respectively Flow cytometry (33% usage), laser microdissection (17%), manual cell picking (17%), random seeding/dilution (15%), and microfluidics/lab-on-a-chip devices (12%) are currently the most frequently used technologies. These most prominent technologies are described in detail and key performance factors are discussed. The survey data indicates a further increasing interest in single-cell isolation tools for the coming years. Additionally, a worldwide patent search was performed to screen for emerging technologies that might become relevant in the future. In total 179 patents were found, out of which 25 were evaluated by screening the title and abstract to be relevant to the field.

Buchbeiträge

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2018

• J. Riba, S. Zimmermann, P. Koltay
  Technologies for Automated Single Cell Isolation
  In: Handbook of Single Cell Technologies
  2018, Springer Nature, T. S. Santra, F.-G. Tseng, T. S. Santra, F.-G. Tseng, ISBN: 978-981-10-4857-9
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  Kurzfassung

  The isolation of individual cells has gained tremendous importance with the advent of new methods for highly parallel single-cell analysis. A prerequisite for effective clonal cultivation or single-cell analysis is the efficient isolation of individual cells from liquid cell suspensions. This review provides an overview of technologies that are used to automate the isolation of single cells for subsequent cultivation or analysis. First, currently available technologies are classified based on their major technical characteristics. Then, the most prominent technologies such as limiting dilution, FACS, single-cell printing, hydrodynamic trapping, droplet microfluidics, and cell manipulation by external forces are described in detail. Furthermore, the individual features of each technology with focus on throughput, isolation efficiency, level of automation, flexibility in terms of cell types, and their suitability for specific downstream processing and analysis methods are discussed. In contrast to previous works, this review provides a classification approach for single-cell isolation technologies according to performance requirements, makes specific reference to methods for the isolation of microbial cells, and discusses sample input requirements, which is an important aspect in particular for diagnostic purposes.

Kurzbeiträge

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2017

• J. Riba, S. Zimmermann, B. Steimle, A. Gross
  Vereinzelung von prokaryotischen und eukaryotischen Zellen
  2017 BIOspektrum, Band: 23, Seiten: 298 - 300

Konferenzbeiträge

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2023

• V. Zieger, D. Frejek, S. Zimmermann, P. Koltay, R. Zengerle, S. Kartmann
  A novel platform for automated and efficient handling of scaffold-free 3D cell-culture models enables well-controlled large-scale 3D in vitro drug screening
  2023 SLAS 2023 Conference and Exhibition, San Diego, CA, USA, 25.02. - 01.03.2023
• V. Zieger, E. Wöhr, S. Zimmermann, D. Frejek, P . Koltay, R. Zengerle, S. Kartmann
  Automated large scale spheroid generation via hanging drop and efficient transfer into physiological mimicking microenvironment
  2023 Transducers, Kyoto/Japan, May 25-29, 2023
• V. Zieger, D. Frejek, S. Zimmermann, P. Koltay, R. Zengerle, S. Kartmann
  Selective high-throughput deposition of single spheroids towards automated 3D in vitro cell culture
  2023 3D Cell Culture 2023, Freiburg, 17. - 19.04.2023

2022

• L. Riek, F. Koch, D. Frejek, L. Zausch, R. Zengerle, P. Koltay, S. Kartmann, S. Zimmermann
  A Bioprinting Fidelity Imager (BioFI) for a standardized characterization of bioprinting processes
  2022 SLAS Europe 2022 Conference and Exhibition, Dublin, Ireland, May 26, 2022
• V. Zieger, D. Frejek, G. Miotto, L. Riek, S. Zimmermann, P. Koltay, R. Zengerle, S. Kartmann
  Selective and automated high-throughput deposition of spheroids for scalable 3D-bioprinting
  2022 Biofabrication 2022, Pisa, Italy, September, 25-28, 2022
• F. Koch, L. Riek, D. Frejek, L. Zausch, S. Zimmermann, S. Kartmann, R. Zengerle, A. Osorio-Madrazo, P. Koltay
  Towards standardized characterization of droplet-based bioprinting processes
  2022 Biofabrication 2022, Pisa, Italy, September, 25-28, 2022
• T. Lange, T. Groß, Á. Jeney, J. Scherzinger, E. S. C. Niemöller, S. Zimmermann, P. Koltay, F. von Stetten, R. Zengerle, C. Jeney
  Validation of scRNA-seq fold changes by single-cell reverse transcription digital PCR
  2022 Single-Cell Genomics, Gordon Research Conference, Les Diablerets, VD, Switzerland, May 1- 6, 2022

2021

• J. Weygant, F. Koch, K. Troendle, G. Finkenzeller, R. Zengerle, S. Kartmann, S. Zimmermann, P. Koltay
  Drop-on-Demand Bioprinting Approach For Precise Alignment and Interaction Studies of Different Cell Types
  2021 International Conference on Biofabrication, Australia, 27. – 29.09.2021 (online)
• K. Tröndle, A. Itani, F. Koch, R. Zengerle, P. Koltay, S. Zimmermann
  Drop-on-demand bioprinting solutions for the fabrication of 3D cell culture systems
  2021 DECHEMA 3D Cell Culture Freiburg (online), 05.-07.05.2021
• F. Koch, K. Tröndle, G. Finkenzeller, P. Rukavina, R. Zengerle, P. Koltay, S. Zimmermann
  Hybrider 3D-Biodruck zur künstlichen Herstellung von Knochen / Using hybrid processes for 3D-bioprinting of artificial bone tissue
  2021 MST-Kongress, Ludwigsburg, 08.-10.11.2021
• M. Trotter, D. Juric, Z. Bagherian, N. Borst, K. Gläser, F. von Stetten, A. Zimmermann
  Integration of nanoporous gold electrodes in microfluidic chambers by inkjet-printing for biosensing applications
  2021 MST-Kongress, Ludwigsburg, 08.-10.11.2021

2020

• K. Tröndle, A. Itani, F. Koch, R. Zengerle, S. Zimmermann, P. Koltay
  Fabrication and fluidic integration of self-assembled cellular microtubules for nephron-on-chip applications
  2020 MicroTAS 2020, 04.-09.10.2020, virtual

2019

• C. Siber, L. Lautscham, J. Schoendube, P. Reichert, F. Stumpf, S. Zimmermann, R. Zengerle, P. Koltay
  Effective acoustic field generation in diposable dispensing cartridges for acustophoretic particle focusing
  2019 Transducers 2019, Berlin, 23.06. - 27.06.2019
• F. Koch, M. Wehrle, K. Tröndle, P. Koltay, G. Finkenzeller, R. Zengerle, S. Zimmermann
  Rapid assessment of combined drop on demand and extrusion-based bioprinting with controlled shear stress and high shape fidelity
  2019 Transducers 2019 - EUROSENSORS XXXIII 23. -27. Juni 2019 - Berlin, Germany
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  Kurzfassung

  We present a novel combination of drop on demand (DoD) and extrusion-based bioprinting to generate highprecision patterns of cells inside large hydrogel volumes. Extrusion-based bioprinting has the great advantage of enabling a fast deposition of high viscous cell-loaded hydrogel with reasonable precision. Compromises between high shape fidelity and cell viability, as well as short process times often require many iterations of optimizing process parameters and varying compositions of the hydrogel. To limit the multitude of parameters during extrusion-based bioprinting, a method for rapid process assessment was developed. This enables to define limits for printing temperature, flow rate and nozzle size from basic rheological measurements with regard to the biological and mechanical requirements. The combination of extrusion-based bioprinting with DoD bioprinting allows for precise deposition of low viscous cell suspension and adjustable concentrations of crosslinking agent. Together, the technologies were used to print a bone replacement model by using the predefined process parameters. Adiposed-derived stem cells (ASC) prone to osteogenic differentiation were homogenously extruded in a cuboid structure of 10x10x5 mm. Human umbilical vein endothelial cells (HUVEC) were printed as highly dense cell suspension lines inside the extruded hydrogel to allow a potential vascularization of the structure in vivo.
• F. Koch, M. Wehrle, K. Tröndle, P. Koltay, G. Finkenzeller, R. Zengerle, S. Zimmermann
  Rapid assessment of extrusion based bioprinting by controlling shear stresses on cells
  2019 Transducers 2019, Berlin, 23.06. - 27.06.2019

2018

• C.-H. Tsai, D.- H. Kuan, S. Zimmermann, J. Schoendube, A. Gross, R. Zengerle, P. Koltay
  A highliy parallel microbioreactor for cell line development based on a microtiter plate with functional microfluidic lid
  2018 MicroTAS 2018, 11. -15. November 2018, Kaohsiung / Taiwan
• K. Tröndle, S. Kartmann, L. Gutzweiler, R. Zengerle, P. Koltay, S. Zimmermann
  Bioprinting with spheroids: Automated large-scale production and deposition
  2018 3D Cell Culture 2018, 5. - 7. Juni 2018, Freiburg
• C. Siber, F. Stumpf, J. Schöndube, S. Zimmermann, P. Koltay, L. Lautscham
  Velocity field analysis of acoustophoretic focusing in a single-cell printing system
  2018 Acoustofluidics, Lille/France, 29. – 31.08.2018

2017

• S. Zimmermann, J. Riba, R. Zengerle, P. Koltay
  A single-cell printer as a versatile tool for cell line development and single-cell analysis
  2017 LAPASO - Microfluidics for label-free particle sorting, Lund / Sweden, 05. - 06.09.2017
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  Kurzfassung

  Single-cell analysis emerged as a promising approach to decipher the heterogeneity of complex cell populations such as tumors. Furthermore, the proof of monoclonality is a regulatory requirement in cell line development, where a cell population producing a therapeutic protein in a bioreactor has to originate from a single cell. Within the advent of such diverse applications, a number of single-cell isolation technologies have been developed and adapted for different requirements [1]. Limitations of these technologies regarding the uniqueness and the integrity of the cells can be overcome by a single-cell printer (scp), a laboratory device developed by the University of Freiburg and commercialized by the spin-off company cytena.
• C.H. Tsai, S. Zimmermann, R. Zengerle, P. Koltay
  An autonomous microbioreactor based on a functional lid in 96-well microplate format
  2017 MFHS, Twente / The Netherlands, 04. - 06.10.2017
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  Kurzfassung

  We have developed an integrated microbioreactor (MBR) based on a disposable functional lid (FL) and a standard microtiter plate (MTP), which only requires one pressure source for parallel and reciprocal mixing of cells cultured in suspension in the MTP as a prerequisite in pharmaceutical cell line development (CLD). The mixing principle of the FL does not only allows for working with significantly smaller culture volumes (30 μl), but also reduces the requirements of external processing stations.
• J. Riba, H. Becker, P. Koltay, R. Zengerle, S. Zimmermann
  Assay miniaturization for the genetic analysis of individual cells enabled by single-cell printing and nanoliter liquid handling
  2017 Keystone Symposia Conference – Single Cell Omics, Stockholm, Sweden, 26. -30.May 2017
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  Kurzfassung

  Automated single-cell isolation and low volume liquid handling for reagent cost reduction are needed to mature single-cell sequencing assays into routine clinical analysis methods. Here, we combine our single-cell printer (scp) for the isolation and deposition of individual cells with non-contact dispensing for reagent dosage in the nanoliter range. This enables us to downscale the reaction volumes of a single-cell whole-genome-amplification (WGA) assay from 50 µl to 2.5 µl using standard 384-well PCR plates.
• S. Zimmermann, J. Schoendube, A. Gross, B. Steimle, L. Lautscham, K. Pfleghar, T. Christmann, B. Werdelmann, F. Koch, J. Riba, P. Koltay, R. Zengerle, M. Pirsch
  Cell line development by single-cell printing and cell imaging
  2017 Cell Line Development & Engineering, Amsterdam, 23.-25. April 2017
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  Kurzfassung

  In biopharmaceutical production the proof of monoclonality is a regulatory requirement for the development of clonal cell lines. A complementary approach based on a single-cell printer (scp, cytena GmbH) and a NYONE cell imager (SYNENTEC GmbH) is used to produce truly monoclonal cell lines and compared to standard single-cell isolation technologies like limited dilution and FACS. Different CHO cell lines were used on different microtiter plate types in addition to cell-equivalent beads for testing the system.
• C.H. Tsai, X.Y. Wu, S. Zimmermann, R. Zengerle, P. Koltay
  Digital Hydraulic Drive For Microfluidic Large-scale Integration System Based On Shape Memory Alloy Actuators
  2017 Transducers 2017, Kaohsiung/Taiwan, 18.-22.06.2017,
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  Kurzfassung

  We present a small size and low power Digital Hydraulic Drive (DHD) intended for use in miniaturized and portable control systems for microfluidic large-scale integration (mLSI) chips. The main components of a DHD are a pneumatic cylinder and a shape memory alloy (SMA) actuator. Depending on different types of SMA actuators, the DHD is not only able to provide digital hydraulic pulses (switching time smaller 1 s), but can maintain a steady state up to 24 hours. A single DHD enables to control 256 valves in parallel.
• J. Riba, J. C. Niemöller, N. Renz, S. Bleul, J. M. Stosch, P. Koltay, R. Zengerle, R. Claus, H. Becker, S. Zimmermann
  Genetic Analysis of Individual Cancer Cells Isolated via Single-Cell Printing
  2017 Single-Cell Genomics Workshop Paris, Paris/France, 27. - 28.06.2017
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  Kurzfassung

  Single-cell genomics emerged as a promising tool to analyze the heterogeneity of complex cancer cell populations by characterizing the genome of each individual cell. In acute myeloid leukemia (AML), single-cell sequencing in addition to bulk next generation sequencing (NGS) is particularly useful for deciphering complex clonal architectures. Here, we present an efficient workflow based on an advanced Single-Cell Printer (scp) for the study of gene variants in single cancer cells. The ejection efficiency was 99.7% for fluorescent beads (n = 2304) and 98.7% for human cells (U-2OS or Kasumi-1 cancer cell line, or AML patient; n = 150). Per fluorescence microscopy, 98.8% of beads were correctly delivered into the wells. A subset of single cells (n = 81) was subjected to whole genome amplification (WGA), which was successful in all cells. On empty droplets, a PCR on LINE1 retrotransposons yielded no product after WGA, verifying the absence of free-floating DNA in SCP-generated droplets. Representative gene variants identified in bulk specimens were sequenced in single-cell WGA DNA. In U-2 OS, 22 of 25 cells yielded results for both an SLC34A2 and TET2 mutation site, including cells harboring the SLC34A2 but not the TET2 mutation. In one cell, the TET2 mutation analysis was inconclusive due to allelic dropout, as assessed via polymorphisms located close to the mutation. Of Kasumi-1, 23 of 33 cells with data on both the KIT and TP53 mutation site harbored both mutations. In the AML patient, 21 of 23 cells were informative for a TP53 variant; the identified alleles matched the loss of chromosome arm 17p. The advanced SCP allows efficient, precise and gentle isolation of individual cells for subsequent WGA and routine PCR/sequencing-based analyses of gene variants.
• J.M. Stosch, A. Heumüller, S. Bleul, C. Niemöller, M. Rothenberg-Thurley, N. Renz, J. Riba, K. Szarc vel Szic, D. Pfeifer, S. Zimmermann, J. Duyster, M. Lübbert, J. Wehrle, K.H. Metzeler, R. Claus, H. Becker
  MDS progression to AML associates with acquisition of single driver mutations with consequent changes in clonal architecture and occurrence of multiple clones with mutations in identical clones
  2017 DGHO, Stuttgart 29.09. – 03.10. 2017
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  Kurzfassung

  Development of acute myeloid leukemia (AML) from myelodysplastic syndromes (MDS) associates with acquisition of genetic aberrations. Here, we studied serial samples of patients with MDS and subsequent AML in order to decipher changes in the mutation profiles and clonal evolution.
• J. Stosch, A. Heumüller, S. Bleul, C. Niemöller, M. Rothenberg-Thurley, N. Renz, J. Riba, K. Szarc vel Szic, D. Pfeifer, A. Nieters, S. Zimmermann, J. Duyster, M. Lübbert, J. Wehrle, K. Metzeler, R. Claus, H. Becker
  Progression of MDS to AML features gain of single driver mutations with consequent changes in clonal composition and occurence of multiple clones with mutations in identical genes
  2017 European Hematology Association (EHA), Madrid/Spain, 22.-25.06.2017
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  Kurzfassung

  Mutations in MDS are few in number, but enriched in genes involved in RNA-splicing or epigenetic regulation; gain of single driver mutations leads to clonal outgrowth and thus, AML. Subsequent treatment can change the mutational and clonal profile. Mutations in identical genes occur in different clones, as confirmed by single-cell analyses; this suggests a fertile ground (e.g. microenvironment) for such mutations in a patient and may lead to (a therapeutically exploitable) competition of clones.

2016

• S. Zimmermann, J. Schoendube, A. Gross, B. Steimle, T. Christmann, B. Werdelmann, P. Koltay, M. Pirsch
  Establishing clonal cell lines by single-cell printing and cell imaging
  2016 Cell Line Development & Engineering, Wien (AU), 11. - 13.04.2016
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  Kurzfassung

  The proof of monoclonality is a regulatory requirement for the development of clonal cell lines for biopharmaceutical production. Here we describe a complementary approach based on a single-cell printer (SCP, cytena GmbH) and a NyONE cell imager (SynenTec GmbH) to produce truly monoclonal cell lines derived from CHO-K1 cell progenitors with high efficiencies.
• J. Riba, J.C. Niemöller, J. Schoendube, A. Gross, S. Bleul, R. Claus, J. Duyster, H. Becker, P. Koltay, R. Zengerle, S. Zimmermann
  Single-Cell Printing for the genomic analysis of eukaryotic and prokaryotic cells
  2016 Single Cell Biology, Hinxton, UK, 8-10 March 2016
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  Kurzfassung

  Single-cell genomics emerged as a promising tool to analyze the heterogeneity of complex cell populations by characterizing the genome of each individual cell. Prerequisite for precise single-cell genomic analysis is an efficient cell isolation. Conventional isolation workflows or instruments are often not able to isolate both eukaryotic and prokaryotic cells or require complex and cost-intensive devices. We previously demonstrated that the Single-Cell Printer (SCP) allows for isolating and depositing individual mammalian cells with high viability rates for monoclonal culturing. Similar to an inkjet printer, the SCP uses a piezo-driven dispenser chip to generate free-flying micro-droplets. A camera system coupled with computer-assisted image processing enables the detection of cells in the chip nozzle and the printing of droplets containing exactly one cell. Here, we demonstrate an advanced SCP that is capable of isolating individual eukaryotic and prokaryotic cells down to 1 µm in size for subsequent single-cell genomic analysis. Using a commercially available whole-genome amplification (WGA) kit (Qiagen REPLI-gTM) with reduced reaction volumes, the genomes of 25 individually printed mammalian cancer cells (U2OS osteosarcoma cell line) were amplified with 100 % success rate as quantified with a QubitTM assay. Eight representative WGA samples were analyzed by Sanger sequencing revealing the U2OS-specific mutations in the SLC34A2 (c.1538G>T) and TET2 (c.1394C>T) genes. We further amplified the genome of 14 individual bacteria cells (E.coli and E.faecalis) with 93 % success rate. Finally, we sequenced part of the 16S rRNA gene of individual bacteria cells enabling the successful taxonomic classification on a phylum level. In conclusion, we show that the SCP allows for efficient and highly automated single-cell deposition of both eukaryotic and prokaryotic cells. Furthermore, the data demonstrate the applicability of the SCP for the analysis of genetic aberrations in single cancer cells at the base pair level and for 16S rRNA-based taxonomic classification of individual microorganisms.

2015

• J. Riba, T. Gleichmann, P. Koltay, R. Zengerle, S. Zimmermann
  A new tool for label-free isolation and deposition of single bacteria cells
  2015 New Approaches and Concepts in Microbiology, EMBL Heidelberg, Germany, 11.-14. October 2015
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  Kurzfassung

  Increasing interest in single-cell analysis throughout life sciences and industry has aroused demand for technologies to separate and handle individual cells. We previously demonstrated that the Single-Cell Printer (SCP) can be used to sort and deposit single mammalian cells onto various substrates for subsequent monoclonal culturing and single-cell genomics. The SCP exploits drop-on-demand printing and automatic image recognition to print 35-200 pl droplets containing single-cells. Compared to other technologies like FACS systems the SCP can address various substrates with high precision and employs a disposable cartridge to prevent cross-contamination. Here, we present an advanced version of the instrument with high-resolution optical detection that has been developed with the aim to detect and deposit bacterial cells. For the first time, we show label-free deposition of single bacterial cells using the SCP. By printing single-cell arrays of GFP expressing E. coli we found that the single-cell printing efficiency of our first prototype yields 78 %. We further demonstrate that the instrument can be used to separate individual cells from a heterogeneous sample followed by clonal culturing. Therefore, we deposit arrays of 100 individual cells from a mixture of E. faecalis and E. coli directly on agar plates for subsequent incubation overnight. After 10 hours we count on average 66 clearly visible clonal cultures. Using light microscopy we show that the two strains were evenly printed and resulted in clonal colonies. In the future we aim to apply the technology to enable highly automated single-cell genomics for phylogenetic studies of environmental samples and to finger-printing of rare bacterial species from clinical samples via mass spectroscopy.
• J. Riba, J. C. Niemöller, S. Zimmermann, J. Schoendube, S. Bleul, P. Koltay, R. Zengerle, R. Claus, H. Becker, A. Gross
  Single-cell printing for the genetic analysis of cancer cells
  2015 Single cell genomics 2015, Utrecht, The Netherlands, 16. – 18. September 2015
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  Kurzfassung

  We present a workflow for the isolation and genetic analysis of single cancer cells based on a Single-Cell Printer (SCP): • Printing of single cells from the osteosarcoma cell line U2OS in wells of a 384-well microtiter plate (MTP) • Whole genome amplification (WGA) of single-cell DNA at reduced reagent volumes • Multiplex PCR on LINE1 retrotransposons • Analysis of U2OS-specific mutations Following this workflow, a single-cell printing efficiency of 98% and uniform DNA yields after WGA were achieved. LINE1 retrotransposons could be detected in all WGA samples, and mutations in the TET2 and the SLC34A2 gene, respectively.