Robotic Platform for Parallelized Cultivation and Monitoring of Microbial Growth Parameters in Microwell Plates

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.The enormous variation possibilities of bioprocesses challenge process development to fix a commercial process with respect to costs and time. Although some cultivation systems and some devices for unit operations combine the latest technology on miniaturization, parallelization, and sensing, the degree of automation in upstream and downstream bioprocess development is still limited to single steps. We aim to face this challenge by an interdisciplinary approach to significantly shorten development times and costs. As a first step, we scaled down analytical assays to the microliter scale and created automated procedures for starting the cultivation and monitoring the optical density (OD), pH, concentrations of glucose and acetate in the culture medium, and product formation in fed-batch cultures in the 96-well format. Then, the separate measurements of pH, OD, and concentrations of acetate and glucose were combined to one method. This method enables automated process monitoring at dedicated intervals (e.g., also during the night). By this approach, we managed to increase the information content of cultivations in 96-microwell plates, thus turning them into a suitable tool for high-throughput bioprocess development. Here, we present the flowcharts as well as cultivation data of our automation approach

Automated Cell Treatment for Competence and Transformation of Escherichia coli in a High-Throughput Quasi-Turbidostat Using Microtiter Plates

Metabolic engineering and genome editing strategies often lead to large strain libraries of a bacterial host. Nevertheless, the generation of competent cells is the basis for transformation and subsequent screening of these strains. While preparation of competent cells is a standard procedure in flask cultivations, parallelization becomes a challenging task when working with larger libraries and liquid handling stations as transformation efficiency depends on a distinct physiological state of the cells. We present a robust method for the preparation of competent cells and their transformation. The strength of the method is that all cells on the plate can be maintained at a high growth rate until all cultures have reached a defined cell density regardless of growth rate and lag phase variabilities. This allows sufficient transformation in automated high throughput facilities and solves important scheduling issues in wet-lab library screenings. We address the problem of different growth rates, lag phases, and initial cell densities inspired by the characteristics of continuous cultures. The method functions on a fully automated liquid handling platform including all steps from the inoculation of the liquid cultures to plating and incubation on agar plates. The key advantage of the developed method is that it enables cell harvest in 96 well plates at a predefined time by keeping fast growing cells in the exponential phase as in turbidostat cultivations. This is done by a periodic monitoring of cell growth and a controlled dilution specific for each well. With the described methodology, we were able to transform different strains in parallel. The transformants produced can be picked and used in further automated screening experiments. This method offers the possibility to transform any combination of strain- and plasmid library in an automated high-throughput system, overcoming an important bottleneck in the high-throughput screening and the overall chain of bioprocess development.BMBF, 031L0018A, ERASysApp2 - Verbundprojekt: LEANPROT - Entwicklung einer Systembiologie-Plattform für die Entwicklung von lean-proteome-Escherichia coli-Stämmen - Deutsches Teilprojekt

Toward microbioreactor arrays : a slow-responding xxygen sensor for monitoring of microbial cultures in standard 96-well plates

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.In this study, a slow-responding chemo-optical sensor for dissolved oxygen (DO) integrated into a 96-well plate was developed. The slow response time ensures that the measured oxygen value does not change much during plate transport to the microplate reader. The sensor therefore permits at-line DO measurement of microbial cultures. Moreover, it eliminates the necessity of individual optical measurement systems for each culture plate, as many plates can be measured successively. Combined with the 96-well format, this increases the experimental throughput enormously. The novel sensor plate (Slow OxoPlate) consists of fluorophores suspended in a polymer matrix that were placed into u-bottom 96-well plates. Response time was measured using sodium sulfite, and a t90 value of 9.7 min was recorded. For application, DO values were then measured in Escherichia coli and Saccharomyces cerevisiae cultures grown under fed-batch–like conditions. Depending on the DO sensor’s response time, different information on the oxygenation state of the culture plate was obtained: a fast sensor variant detects disturbance through sampling, whereas the slow sensor indicates oxygen limitation during incubation. A combination of the commercially available OxoPlate and the Slow OxoPlate enables operators of screening facilities to validate their cultivation procedures with regard to oxygen availability.BMBF, 02PJ1150, Plattformtechnologien für automatisierte Bioprozessentwicklung (AutoBio

Online optimal experimental re-design in robotic parallel fed-batch cultivation facilities

We present an integrated framework for the online optimal experimental re-design applied to parallel nonlinear dynamic processes that aims to precisely estimate the parameter set of macro kinetic growth models with minimal experimental effort. This provides a systematic solution for rapid validation of a specific model to new strains, mutants, or products. In biosciences, this is especially important as model identification is a long and laborious process which is continuing to limit the use of mathematical modeling in this field. The strength of this approach is demonstrated by fitting a macro-kinetic differential equation model for Escherichia coli fed-batch processes after 6 h of cultivation. The system includes two fully-automated liquid handling robots; one containing eight mini-bioreactors and another used for automated at-line analyses, which allows for the immediate use of the available data in the modeling environment. As a result, the experiment can be continually re-designed while the cultivations are running using the information generated by periodical parameter estimations. The advantages of an online re-computation of the optimal experiment are proven by a 50-fold lower average coefficient of variation on the parameter estimates compared to the sequential method (4.83% instead of 235.86%). The success obtained in such a complex system is a further step towards a more efficient computer aided bioprocess development. Biotechnol. Bioeng. 2017;114: 610–619. © 2016 Wiley Periodicals, Inc.BMBF, 02PJ1150, Verbundprojekt: Plattformtechnologien für automatisierte Bioprozessentwicklung (AutoBio); Teilprojekt: Automatisierte Bioprozessentwicklung am Beispiel von neuen Nukleosidphosphorylase

Reduced Oct4 expression directs a robust pluripotent state with distinct signaling activity and increased enhancer occupancy by Oct4 and Nanog

10.1016/j.stem.2013.04.023Cell Stem Cell125531-54

Plattformtechnologien für die automatisierte Bioprozessentwicklung

The development of production processes in biotechnology is a time and resource intensive task due to the vast design space to be screened. Therefore, processes are commonly performed at a local optimum. Within the past two decades, miniaturization, parallelization and automation of laboratory work have improved significantly and researchers are now able to carry out thousands of experiments per week in automated facilities. In many cases there is a disagreement between the results of small-scale experiments and data from the production scale due to the different conditions, in which the cells are cultivated. Laboratory scale experiments are often performed as batch cultures in complex media without any instrumentation, while the production processes run under fed-batch conditions in instrumented bioreactors. This discrepancy may result in multiple rounds of experiments, until a feasible strain for scale-up is identified. Therefore, cultivation conditions should be kept consistent throughout the developmental line. In this work, platform technologies for consistent bioprocess development are presented in the form of three publications. First, a novel dissolved oxygen sensor for screening facilities was developed, which can determine the aeration level of cultures grown in 96-well plates that are transported from the incubator to the plate reader. The long response time (t90) of 9.7 min allows an estimation of the oxygenation status during incubation. The sensor detected oxygen limitation in fed-batch cultures of E. coli and S. cerevisiae. In the second part, a workflow for rapid cell lysis buffer optimization using design of experiments (DoE) is presented. Experiments were planned with a DoE software and written as worklists for the liquid handling robot into a laboratory information management system (infoteam iLab-Bio). In three experimental runs, a lysis buffer composition for efficient release of beta-galactosidase from E. coli was determined. The product formation profile of yeast strains was evaluated using parallel fed-batch cultures at the millilitre-scale in the third part. For comparison, A-stat cultivations in a 1.5 L bioreactor were performed, which showed comparable product formation rates. Using these platform technologies, a framework for streamlined experimental planning, execution and data management can be established.Die Entwicklung von Produktionsprozessen in der Biotechnologie ist zeit- und kostenintensiv, insbesondere bei der Produktion von therapeutischen Proteinen und gentechnisch verbesserten Enzymen. Neben allgemeinen Prozessgrößen wie Temperatur, pH und Medienzusammensetzung beeinflussen Produktionsstamm, Expressionssystem und Fusionspartner sowie Prozessführung und die Aufarbeitung des Produktes den Ertrag und damit die Wirtschaftlichkeit eines biotechnologischen Herstellungsprozesses. Die Anzahl an nötigen Laborexperimenten zur Optimierung ist meist zu groß, um manuell durchgeführt zu werden, sodass häufig nur bei einem Quasi-Optimum gearbeitet werden kann. In den letzten zwei Jahrzehnten ist die Miniaturisierung, Parallelisierung und Automatisierung von Experimenten stark vorangeschritten, sodass heute tausende von Versuchen pro Woche durchgeführt werden können. Ob die Daten aus den kleinen Systemen prädiktiv für den Produktionsmaßstab eingesetzt werden können, ist jedoch nicht ohne Zweifel. Daher müssen für die Maßstabsvergrößerung in der Regel zusätzliche Versuchsreihen in Labor- und Pilotbioreaktoren durchgeführt werden. Die Unterschiede zwischen Labor- und Produktionsbedingungen betreffen häufig die Kulturführung und die eingesetzten Medien. Meist wird ein Batch-Prozess mit Komplexmedium im Kleinmaßstab und ein Fed-Batch Prozess mit Mineralsalzmedium in der Produktion eingesetzt. Außerdem erlauben die Screening-Systeme nur Endpunktmessungen und es wird häufig erfahrungsbasiert oder nach Versuch und Irrtum gearbeitet. Die Datenaufzeichnung und -Auswertung geschieht meist manuell. Um die Aussagekraft von Laborexperimenten im Kleinmaßstab zu verbessern, sollten Versuchsbedingungen gewählt werden, die dem Produktionsmaßstab entsprechen. Diese Prozessentwicklungsstrategie wird auch konsistente Bioprozessentwicklung genannt. Im Rahmen dieser Dissertation wurden Plattformtechnologien für konsistente Bioprozessentwicklung erarbeitet und in Form von drei Fallstudien auf unterschiedliche Fragestellungen angewendet. Ein neuartiger Sensor zur Messung von Gelöstsauerstoff in Mikrowellplatten wurde in der ersten Publikation entwickelt. Der Sensor besitzt eine Ansprechzeit von 9,7 Minuten, was die verlässliche Bestimmung von Gelöstsauerstoffwerten in Anlagen ermöglicht, in denen Verzögerungen durch Transportzeiten vom Inkubator zum Photometer vorhanden sind. Mithilfe von Escherichia coli und Saccharomyces cerevisiae Kulturen konnte demonstriert werden, dass der Sensor Sauerstofflimitationen im Inkubator detektieren kann. In Kombination mit einem schnell ansprechenden Sensor ist eine umfassende Charakterisierung des automatisierten Kultivierungssystems möglich. In der zweiten Publikation wurde ein Arbeitsablauf zur schnellen Optimierung des chemisch-enzymatischen Zellaufschlusses entwickelt. Mit Hilfe einer Software für experimentelles Design wurden Versuchspläne erstellt, die in einem Datenbanksystem für den Pipettierroboter abrufbar gespeichert wurden. In drei Experimenten wurde die optimale Mischung von EDTA, Lysozym, Triton X-100 und Polymyxin B bestimmt. Im Vergleich zu kommerziell erhältlichen Produkten wies der Puffer eine vergleichbare Performance auf. Im Rahmen der dritten Publikation wurde das Produktbildungsprofil von Hefekulturen in parallelen miniaturisierten Fed-Batch Kulturen untersucht und eine Abhängigkeit der spezifischen Produktbildungsrate von der spezifischen Wachstumsrate festgestellt. Das Produktbildungsprofil wurde mit einer kontinuierlichen Kultivierung verglichen. Es konnte festgestellt werden, dass die aus den miniaturisierten Kulturen gewonnenen Daten mit dem 1,5 L Bioreaktor vergleichbar sind. Eine Charakterisierung von Hefestämmen in Mikrowellplatten bietet sich demnach an, um zeit- und kostenintensive Experimente in Bioreaktoren zu minimieren. Die entwickelten Plattformtechnologien bieten eine Grundstruktur zur schnelleren Versuchsplanung, -durchführung und -auswertung. Durch die Beibehaltung der Rahmenbedingungen des Produktionsmaßstabes während der Produkt- und Prozessentwicklung können Entwicklungszeit und -kosten von biotechnologischen Produktionsprozessen verringert werden, was schlussendlich zu einer weiteren Verbreitung von nachhaltigen Produktionsmethoden führen wird.BMBF, 02PJ1150, Autobi

Accelerated Bioprocess Development of Endopolygalacturonase-Production with Saccharomyces cerevisiae Using Multivariate Prediction in a 48 Mini-Bioreactor Automated Platform

Mini-bioreactor systems enabling automatized operation of numerous parallel cultivations are a promising alternative to accelerate and optimize bioprocess development allowing for sophisticated cultivation experiments in high throughput. These include fed-batch and continuous cultivations with multiple options of process control and sample analysis which deliver valuable screening tools for industrial production. However, the model-based methods needed to operate these robotic facilities efficiently considering the complexity of biological processes are missing. We present an automated experiment facility that integrates online data handling, visualization and treatment using multivariate analysis approaches to design and operate dynamical experimental campaigns in up to 48 mini-bioreactors (8–12 mL) in parallel. In this study, the characterization of Saccharomyces cerevisiae AH22 secreting recombinant endopolygalacturonase is performed, running and comparing 16 experimental conditions in triplicate. Data-driven multivariate methods were developed to allow for fast, automated decision making as well as online predictive data analysis regarding endopolygalacturonase production. Using dynamic process information, a cultivation with abnormal behavior could be detected by principal component analysis as well as two clusters of similarly behaving cultivations, later classified according to the feeding rate. By decision tree analysis, cultivation conditions leading to an optimal recombinant product formation could be identified automatically. The developed method is easily adaptable to different strains and cultivation strategies, and suitable for automatized process development reducing the experimental times and costs

Aging alters the epigenetic asymmetry of HSC division.

Hematopoietic stem cells (HSCs) balance self-renewal and differentiation to maintain homeostasis. With aging, the frequency of polar HSCs decreases. Cell polarity in HSCs is controlled by the activity of the small RhoGTPase cell division control protein 42 (Cdc42). Here we demonstrate-using a comprehensive set of paired daughter cell analyses that include single-cell 3D confocal imaging, single-cell transplants, single-cell RNA-seq, and single-cell transposase-accessible chromatin sequencing (ATAC-seq)-that the outcome of HSC divisions is strongly linked to the polarity status before mitosis, which is in turn determined by the level of the activity Cdc42 in stem cells. Aged apolar HSCs undergo preferentially self-renewing symmetric divisions, resulting in daughter stem cells with reduced regenerative capacity and lymphoid potential, while young polar HSCs undergo preferentially asymmetric divisions. Mathematical modeling in combination with experimental data implies a mechanistic role of the asymmetric sorting of Cdc42 in determining the potential of daughter cells via epigenetic mechanisms. Therefore, molecules that control HSC polarity might serve as modulators of the mode of stem cell division regulating the potential of daughter cells