Publikationsliste Julian Riba

• Originalarbeiten in wissenschaftlichen Fachzeitschriften
• Buchbeiträge
• Kurzbeiträge
• Konferenzbeiträge

Originalarbeiten in wissenschaftlichen Fachzeitschriften

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2020

• 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

• J. F. Cahill, J. Riba, V. Kertesz
  Rapid, Untargeted Chemical Profiling of Single Cells in Their Native Environment
  2019 Anal Chem, Band: 91, Seiten: 6118 - 6126
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  Kurzfassung

  We report a method that enables untargeted, high throughput, and quantitative mass spectrometric analysis of single cells from cell suspension without needing additional sample preparation procedures (e.g., molecular tagging) through the combination of single-cell printer technology and liquid vortex capture−mass spectrometry (SCP-LVC-MS). The operating principle behind the SCP-LVC-MS technology is single cell isolation via small droplet piezoelectric ejection followed by capture of the droplet into an LVC-MS sampling probe. Once exposed to an appropriate solvent, the cell is lysed, extracted, and analyzed by MS. The SCP-LVC-MS approach was validated by measuring the lipid composition of microalgae, Chlamydomonas reinhardtii (ChRe) and Euglena gracilis (EuGr), and HeLa cells in their native growth media. Numerous diacylglyceryltrimethylhomo-Ser (DGTS), phosphatidylcholine (PC), monogalactosyldiacylglycerol (MGDG), and digalactosyldiacylglycerol (DGDG) lipids were observed in single cells. Continuous solvent flow ensures that cells are analyzed rapidly, and no signal carryover between cells is observed. ChRe and EuGr microalgae mixed together in the same solution were differentiated cell-by-cell in real-time based on differences between levels of diacylglyceryltrimethylhomo-Ser (DGTS) and phosphatidylcholine (PC) lipids measured in each cell. Several DGTS lipids present in ChRe were quantified with single-cell resolution by normalizing to a DGTS(32:0) internal standard added to the LVC probe solvent during analysis. Quantitative peak areas were validated by comparing to bulk lipid extracts. Lastly, peak area distributions comprised of hundreds of cells were compared for ChRe after 5 days of nitrogen-limited and normal growth conditions, which show clear differences and the ability to resolve cellular population differences with single-cell resolution.

2018

• 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.

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.

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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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.
• 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.
• 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

• 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, T. Gleichmann, R. Zengerle, P. Koltay
  Label-free sorting and deposition of single bacterial cells using the Single-Cell Printer technology
  2015 6th International conference on analysis of microbial cells at the single cell level, Retz/Austria, 19. - 22. Juli 2015
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  Kurzfassung

  We present a modified Single-Cell Printer (SCP) for sorting and deposition of individual bacterial cells by: •Label-free optical cell detection •Confinement of single cells in 35 picoliter droplets •Non-contact drop-on-demand deposition onto a variety of substrates We show that Escherichia coli cells can be deposited with a single cell printing efficiency of 78 %. Further, we deposited individual cells from a heterogeneous sample directly onto agar plates for subsequent clonal culturing.
• 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.

2014

• J. Riba, L. Gutzweiler, L. Riegger, P. Koltay, R. Zengerle, A. Gross
  A picoliter dispenser with disposable cartridges for precise and contact-free injection of DNA into open microfluidic structures
  2014 2nd MFHS Conference 2014, Freiburg, 08 - 10.Okt. 2014 , Seiten: 84 - 87
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  Kurzfassung

  We present a new disposable liquid handling system addressing the lower picoliter volume range and allowing for precise and contact-free injection of biopolymer samples into open microfluidic structures. Droplet placement precision of single stranded DNA (ssDNA) solutions up to 50 μM and a DNA reference ladder is investigated by high resolution optical monitoring. We present three different sample injection methods that account for evaporation protection of picoliter samples. Finally, we show the applicability of the dispenser by a successful electrophoretic separation via injecting ~70 pL of a DNA solution into a 200-300 μm wide gel line.