Validation of biological recognition elements for signal transduction as first step in the development of whole cell biosensors

Choosing the proper combination of receptor element, cell type and measurable signal requires major consideration for developing cell-based biosensors. In order to use physiologically relevant cellular responses towards (geno)toxic conditions, information on the mechanism of action and of the expected outcome of exposure needs to be considered

Radiation Response of Murine Embryonic Stem Cells

To understand the mechanisms of disturbed differentiation and development by radiation, murine CGR8 embryonic stem cells (mESCs) were exposed to ionizing radiation and differentiated by forming embryoid bodies (EBs). The colony forming ability test was applied for survival and the MTT test for viability determination after X-irradiation. Cell cycle progression was determined by flow cytometry of propidium iodide-stained cells, and DNA double strand break (DSB) induction and repair by γH2AX immunofluorescence. The radiosensitivity of mESCs was slightly higher compared to the murine osteoblast cell line OCT-1. The viability 72 h after X-irradiation decreased dose-dependently and was higher in the presence of leukemia inhibitory factor (LIF). Cells exposed to 2 or 7 Gy underwent a transient G2 arrest. X-irradiation induced γH2AX foci and they disappeared within 72 h. After 72 h of X-ray exposure, RNA was isolated and analyzed using genome-wide microarrays. The gene expression analysis revealed amongst others a regulation of developmental genes (Ada, Baz1a, Calcoco2, Htra1, Nefh, S100a6 and Rassf6), downregulation of genes involved in glycolysis and pyruvate metabolism whereas upregulation of genes related to the p53 signaling pathway. X-irradiated mESCs formed EBs and differentiated toward cardiomyocytes but their beating frequencies were lower compared to EBs from unirradiated cells. These results suggest that X-irradiation of mESCs deregulate genes related to the developmental process. The most significant biological processes found to be altered by X-irradiation in mESCs were the development of cardiovascular, nervous, circulatory and renal system. These results may explain the X-irradiation induced-embryonic lethality and malformations observed in animal studies

Hypergravity attenuates Reactivity in Primary Murine Astrocytes

Neuronal activity is the key modulator of nearly every aspect of behavior, affecting cognition, learning, and memory as well as motion. Hence, disturbances of the transmission of synaptic signals are the main cause of many neurological disorders. Lesions to nervous tissues are associated with phenotypic changes mediated by astrocytes becoming reactive. Reactive astrocytes form the basis of astrogliosis and glial scar formation. Astrocyte reactivity is often targeted to inhibit axon dystrophy and thus promote neuronal regeneration. Here, we aim to understand the impact of gravitational loading induced by hypergravity to potentially modify key features of astrocyte reactivity. We exposed primary murine astrocytes as a model system closely resembling the in vivo reactivity phenotype on custom-built centrifuges for cultivation as well as for live-cell imaging under hypergravity conditions in a physiological range (2g and 10g). We revealed spreading rates, migration velocities, and stellation to be diminished under 2g hypergravity. In contrast, proliferation and apoptosis rates were not affected. In particular, hypergravity attenuated reactivity induction. We observed cytoskeletal remodeling of actin filaments and microtubules under hypergravity. Hence, the reorganization of these key elements of cell structure demonstrates that fundamental mechanisms on shape and mobility of astrocytes are affected due to altered gravity conditions. In future experiments, potential target molecules for pharmacological interventions that attenuate astrocytic reactivity will be investigated. The ultimate goal is to enhance neuronal regeneration for novel therapeutic approache

Streamlining Culture Conditions for the Neuroblastoma Cell Line SH-SY5Y: A Prerequisite for Functional Studies

The neuroblastoma cell line SH-SY5Y has been a well-established and very popular in vitro model in neuroscience for decades, especially focusing on neurodevelopmental disorders, such as Parkinson’s disease. The ability of this cell type to differentiate compared with other models in neurobiology makes it one of the few suitable models without having to rely on a primary culture of neuronal cells. Over the years, various, partly contradictory, methods of cultivation have been reported. This study is intended to provide a comprehensive guide to the in vitro cultivation of undifferentiated SH-SY5Y cells. For this purpose, the morphology of the cell line and the differentiation of the individual subtypes are described, and instructions for cell culture practice and long-term cryoconservation are provided. We describe the key growth characteristics of this cell line, including proliferation and confluency data, optimal initial seeding cell numbers, and a comparison of different culture media and cell viability during cultivation. Furthermore, applying an optimized protocol in a long-term cultivation over 60 days, we show that cumulative population doubling (CPD) is constant over time and does not decrease with incremental passage, enabling stable cultivation, for example, for recurrent differentiation to achieve the highest possible reproducibility in subsequent analyses. Therefore, we provide a solid guidance for future research that employs the neuroblastoma cell line SH-SY5

Hypergravity attenuates Reactivity in Primary Murine Astrocytes

Neuronal activity is the key modulator of nearly every aspect of behavior, affecting cognition, learning and memory as well as motion. Alterations or even disruptions of the transmission of synaptic signals are the main cause of many neurological disorders. Lesions to nervous tissues are associated with phenotypic changes mediated by astrocytes becoming reactive. Reactive astrocytes form the basis of astrogliosis and glial scar formation. Astrocyte reactivity is often targeted to inhibit axon dystrophy and thus promote neuronal regeneration. Here, we use increased gravitational (mechanical) loading induced by hypergravity to identify a potential method to modify key features of astrocyte reactivity. We exposed primary murine astrocytes as a model system closely resembling the reactivity phenotype in vivo on custom-built centrifuges for cultivation as well as for livecell imaging under hypergravity conditions in a physiological range (2g and 10g). This resulted in significant changes to astrocyte morphology, behavior and reactivity phenotypes, with the ultimate goal being to enhance neuronal regeneration for novel therapeutic approaches

The Use of ProteoTuner Technology to Study Nuclear Factor κB Activation by Heavy Ions

Nuclear factor κB (NF-κB) activation might be central to heavy ion-induced detrimental processes such as cancer promotion and progression and sustained inflammatory responses. A sensitive detection system is crucial to better understand its involvement in these processes. Therefore, a DD-tdTomato fluorescent protein-based reporter system was previously constructed with human embryonic kidney (HEK) cells expressing DD-tdTomato as a reporter under the control of a promoter containing NF-κB binding sites (HEK-pNFκB-DD-tdTomato-C8). Using this reporter cell line, NF-κB activation after exposure to different energetic heavy ions (¹⁶O, 95 MeV/n, linear energy transfer—LET 51 keV/µm; ¹²C, 95 MeV/n, LET 73 keV/μm; ³⁶Ar, 95 MeV/n, LET 272 keV/µm) was quantified considering the dose and number of heavy ions hits per cell nucleus that double NF-κB-dependent DD-tdTomato expression. Approximately 44 hits of ¹⁶O ions and ≈45 hits of ¹²C ions per cell nucleus were required to double the NF-κB-dependent DD-tdTomato expression, whereas only ≈3 hits of ³⁶Ar ions were sufficient. In the presence of Shield-1, a synthetic molecule that stabilizes DD-tdTomato, even a single particle hit of ³⁶Ar ions doubled NF-κB-dependent DD-tdTomato expression. In conclusion, stabilization of the reporter protein can increase the sensitivity for NF-κB activation detection by a factor of three, allowing the detection of single particle hits’ effects

Streamlining Culture Conditions for the Neuroblastoma Cell Line SH-SY5Y: A Prerequisite for Functional Studies

The neuroblastoma cell line SH-SY5Y has been a well-established and very popular in vitro model in neuroscience for decades, especially focusing on neurodevelopmental disorders, such as Parkinson’s disease. The ability of this cell type to differentiate compared with other models in neurobiology makes it one of the few suitable models without having to rely on a primary culture of neuronal cells. Over the years, various, partly contradictory, methods of cultivation have been reported. This study is intended to provide a comprehensive guide to the in vitro cultivation of undifferentiated SH-SY5Y cells. For this purpose, the morphology of the cell line and the differentiation of the individual subtypes are described, and instructions for cell culture practice and long-term cryoconservation are provided. We describe the key growth characteristics of this cell line, including proliferation and confluency data, optimal initial seeding cell numbers, and a comparison of different culture media and cell viability during cultivation. Furthermore, applying an optimized protocol in a long-term cultivation over 60 days, we show that cumulative population doubling (CPD) is constant over time and does not decrease with incremental passage, enabling stable cultivation, for example, for recurrent differentiation to achieve the highest possible reproducibility in subsequent analyses. Therefore, we provide a solid guidance for future research that employs the neuroblastoma cell line SH-SY5Y

Die Etablierung der Neuroblastomzelllinie SY5Y als Modellsystem zur Abschätzung strahleninduzierter Schäden neuronaler Zellen bei einer bemannten Marsmission

Die kosmische Strahlung ist der größte Risikofaktor für die bemannte Raumfahrt, insbesondere für Langzeitmissionen wie die Reise zum Mars. Die kosmische Strahlung ist in sich inhomogen und setzt sich aus verschiedensten Komponenten und Strahlenqualitäten zusammen. Eine Vorhersage von Hoch-Dosis-Ereignissen wie Sonnenstürmen ist schwierig bis unmöglich. Eine ausreichende Abschirmung ist derzeit technisch nicht umsetzbar und beim heutigen Stand der Entwicklung sogar kontraproduktiv. Daher muss das Verständnis der Wirkung ionisierender Strahlung auf den Menschen und insbesondere auf das nur wenig erforschte Gehirn vertieft werden. In dieser Arbeit wird die Neuroblastomzelllinie SY5Y als mögliches Modellsystem für die Auswirkungen ionisierender Strahlung auf das menschliche Gehirn untersucht sowie zell- und strahlenbiologisch etabliert

Induktion von neuronaler Regeneration durch Hypergravitation

Einleitung: Eine uneingeschränkte Funktion von neuronalen Zellen ist unerlässlich für jeden Aspekt des menschlichen Verhaltens. Wahrnehmung, Erinnerung, Lernen sowie jegliche Art von Bewegung sind grundsätzlich von der Aktivität unseres Nervensystems abhängig. Eine Ursache oder Konsequenz der meisten neurologischen Erkrankungen sind daher Veränderungen oder Störungen der synaptischen Aktivität. Um wirksame Behandlungen von neurologischen Störungen zu entwickeln, müssen synaptische Signale wieder in das zuvor verletzte neuronale Netzwerk integriert werden. Besonders nach schweren Läsionen, wie z.B. nach Traumata und Rückenmarksverletzungen, sowie durch Beeinflussung von Tumorgewebe oder epileptischen Anfällen wird vermehrt Narbengewebe gebildet, welches die Regeneration von neuronalen Ausläufern und damit synaptischen Signalen in das verletzte Gewebe nahezu vollständig inhibiert. Diese Art von Verletzung kann daher zu irreparablen Schäden der mentalen oder physischen Leistungsfähigkeit von Patienten führen. Fragestellung: Wir streben an, den sehr ineffizienten Vorgang der neuronalen Regeneration nach Verletzungen von Nervengewebe durch die Exposition in Hypergravitation zu induzieren. Unsere Hypothese ist, dass artifiziell erhöhte Schwerkraft eine potentiell sehr innovative und wirkungsvolle Methode darstellen könnte, um Komponenten des neuronalen Zytoskelettes zu stabilisieren, was weiterhin dazu führen sollte, dass die Projektionen der Nervenzellen der Inhibition des neuronalen Narbengewebes entgegenwirken und in gesteigertem Maße wachsen (regenerieren) können. Daher sollten die Ausläufer unter Einfluss von Hypergravitation viel ausgeprägter in der Lage sein auszuwachsen und sich anschließend neu in geschädigtes Gewebe zu integrieren. Weiterhin soll die neuronale Entwicklung und Aktivität bei Exposition mit ionisierender Strahlung untersucht werden, um Strahlung als Risikofaktor für neurodegenerative Erkrankungen z.B. in der bemannten Raumfahrt zu bewerten. Methodik: In der vorliegenden Studie werden primäre murine hippokampale Neuronen eingesetzt, welche ein nah-verwandtes Modell-System für humane Nervenzellen darstellen. Die neuronale Entwicklung unter dem Einfluss von Hypergravitation (2g) und Strahlenexposition wird bei allen Entwicklungsstadien im Vergleich zu Kontrollen (1g, unbestrahlt) untersucht, wie z.B. das Neuritenwachstum, die Polarisation, die Synaptogenese, sowie abschließend die Integration in ein maturiertes, funktionelles neuronales Netzwerk. Ergebnisse: Die Exposition von primären Neuronen an erhöhte Gravitation (2g) induzierte ein gesteigertes Auswachsen initialer Neuriten (ca. 30%), sowie ein erhöhtes Neuriten-Wachstum (Elongation) (ca. 20%) im Vergleich zu Kontrollen bei 1g. In späteren Entwicklungsstadien wurden trotz potentiellen Veränderungen des neuronalen Zytokslettes maturierte synaptische Kontakte ausgebildet. Weiterhin wurden primäre Astrozyten (Glia-Zellen, die neuronales Narbengewebe nach Verletzungen bilden) durch Hypergravitation in ihrem Wachstum und ihrer Ausbreitung (Narbenbildung) gehemmt. Diese Beobachtungen sind in großer Übereinstimmung mit einem stabilisierten Tubulin- und einem destabilisierten Aktin-Zytoskelett. Schlussfolgerungen: Unsere Ergebnisse belegen, dass neuronale Regeneration durch den Einfluss von Hypergravitation in primären Neuronen induziert und das Wachstum von primären Astrozyten (Glia-Zellen) durch die Kultivierung unter Hypergravitationsbedingungen gehemmt wird. Dieser Ansatz kann für weitere Studien angewandt werden, um die grundlegenden Mechanismen aufzuklären und die Effizienz neuronaler Regenerationsprozesse steigern zu können

Cell death bypass mechanisms in DNA damage response of mammalian cells after exposure with heavy ions relevant for Space radiation environment

For humans in space, there are two limiting factors namely the microgravity and the galactic cosmic radiation. For sake of better risk assessment for the astronaut, the cellular response to such ionizing radiation qualities, as predominate in Space, needs to be better understood. One of the key elements to be investigated is the cell cycle control as a central element in DNA damage response being a central switch for cellular decision making between life and death. Thus cell death bypass mechanisms in DNA damage response of mammalian cells will be investigated after heavy ion exposure relevant for the Space radiation environment. The RBE-LET dependency will be determined for different biological endpoints. Of special interest are cellular survival, the activation of transcription factors and the identification of genes expressed in DNA damage response after high LET exposure.平成26年度放射線医学総合研究所重粒子線がん治療装置等共同利用研究報告