20 research outputs found
Simplified Bioprinting-Based 3D Cell Culture Infection Models for Virus Detection
Studies of virus–host interactions in vitro may be hindered by biological characteristics of conventional monolayer cell cultures that differ from in vivo infection. Three-dimensional (3D) cell cultures show more in vivo-like characteristics and may represent a promising alternative for characterisation of infections. In this study, we established easy-to-handle cell culture platforms based on bioprinted 3D matrices for virus detection and characterisation. Different cell types were cultivated on these matrices and characterised for tissue-like growth characteristics regarding cell morphology and polarisation. Cells developed an in vivo-like morphology and long-term cultivation was possible on the matrices. Cell cultures were infected with viruses which differed in host range, tissue tropism, cytopathogenicity, and genomic organisation and virus morphology. Infections were characterised on molecular and imaging level. The transparent matrix substance allowed easy optical monitoring of cells and infection even via live-cell microscopy. In conclusion, we established an enhanced, standardised, easy-to-handle bioprinted 3D-cell culture system. The infection models are suitable for sensitive monitoring and characterisation of virus–host interactions and replication of different viruses under physiologically relevant conditions. Individual cell culture models can further be combined to a multicellular array. This generates a potent diagnostic tool for propagation and characterisation of viruses from diagnostic samples.Peer Reviewe
Enhanced Proliferation of Monolayer Cultures of Embryonic Stem (ES) Cell-Derived Cardiomyocytes Following Acute Loss of Retinoblastoma
Background: Cardiomyocyte (CM) cell cycle analysis has been impeded because of a reliance on primary neonatal cultures of poorly proliferating cells or chronic transgenic animal models with innate compensatory mechanisms. Methodology/Principal Findings: We describe an in vitro model consisting of monolayer cultures of highly proliferative embryonic stem (ES) cell-derived CM. Following induction with ascorbate and selection with puromycin, early CM cultures are.98 % pure, and at least 85 % of the cells actively proliferate. During the proliferative stage, cells express high levels of E2F3a, B-Myb and phosphorylated forms of retinoblastoma (Rb), but with continued cultivation, cells stop dividing and mature functionally. This developmental transition is characterized by a switch from slow skeletal to cardiac TnI, an increase in binucleation, cardiac calsequestrin and hypophosphorylated Rb, a decrease in E2F3, B-Myb and atrial natriuretic factor, and the establishment of a more negative resting membrane potential. Although previous publications suggested that Rb was not necessary for cell cycle control in heart, we find following acute knockdown of Rb that this factor actively regulates progression through the G1 checkpoint and that its loss promotes proliferation at the expense of CM maturation. Conclusions/Significance: We have established a unique model system for studying cardiac cell cycle progression, and show in contrast to previous reports that Rb actively regulates both cell cycle progression through the G1 checkpoint an
Non-lethal heat shock protects gnotobiotic Artemia franciscana larvae against virulent Vibrios
Establishment and characterization of a three-dimensional cell culture model for Cowpox infections
Zellkultivierungen stellen ein elementares Werkzeug für die Erforschung vielfältiger biologischer Phänomene dar. In der Virologie werden Infektionen in vitro fast ausschließlich in Monolayer-Kulturen charakterisiert und erforscht, in denen Zellen flach ausgebreitet auf starren Oberflächen kultiviert werden und im Vergleich zum in vivo Status der gleichen Zellen deutliche Unterschiede bezüglich Morphologie und Physiologie aufweisen. Im Gegensatz dazu werden in einigen Teilen von Forschungsfeldern wie der Onkologie und der Stammzellforschung, aber auch für spezielle virale Erreger, deren Bedürfnisse keine andere Art der Kultivierung zulassen, bereits seit Jahrzehnten alternative Kultivierungsmethoden verwendet. Bei diesen Alternativen handelt es sich um dreidimensionale (3D) Gewebekulturen, in denen Zellen in vivo-ähnliche Charakteristika aufweisen und somit ein sehr gutes Abbild des jeweiligen Gewebes und untersuchten biologischen Phänomens liefern. Für Infektionen mit Orthopockenviren (OPV), deren Analyse aufgrund des zoonotischen Potentials einiger Vertreter wie den Kuhpockenviren (CPXV) einerseits und der Bedrohung eines Missbrauchs als bioterroristisches Agens des humanpathogenen Variola Virus andererseits, von hoher Relevanz für die öffentliche Gesundheit ist, wurden bis dato nur wenige Untersuchungen in 3D-Kulturmodellen publiziert. Diese beschränkten sich hauptsächlich auf morphologische Charakterisierungen und die Analyse der Wirksamkeit verschiedener antiviraler Substanzen. Das Ziel dieser Arbeit war daher die Etablierung eines 3D-Zellkulturmodells, das einerseits zur weiterführenden Erforschung von OPV Infektionen beitragen sollte und andererseits einfacher zu handhaben und an ein breiteres Spektrum möglicher Wirtszellen adaptierbar sein sollte als bisher etablierte 3D-Ansätze. Zu diesem Zweck wurde als Fundament für die 3D-Kulturen eine dezellularisierte biologische extrazelluläre Matrix (EZM) verwendet, die mit verschiedenen Zelltypen besiedelt werden konnte und somit für die Untersuchung unterschiedlicher Viren geeignet war. Uninfizierte epitheliale 3D-Zellkulturen wiesen im Vergleich zu korrespondierenden Monolayer-Kulturen distinkte Unterschiede bezüglich Zellmorphologie, Differenzierungsstatus sowie Polarisation auf und zeigten auf Transkriptomebene ein ähnliches Expressionsmuster wie ihr in vivo Gegenstück – die menschliche Epidermis. Um detailliertere Erkenntnisse über die Virusreplikation von OPV und deren Interaktionen mit den Wirtszellen innerhalb eines komplexen biologischen Systems zu erhalten, wurden zwei epitheliale 3D-Infektionsmodelle mit dem CPXV-Stamm Brighton Red (BR) als Modellvirus etabliert. Durch die 3D-Kultivierung der Zelllinie Vero E6 konnte bei einer Infektion mit CPXV eine im Vergleich zur Monolayer-Kultur deutlich verlängerte Zellviabilität bei annähernd gleichbleibenden Virustitern erzielt werden. Weiterhin war in diesem Modell, wie auch bei der Weiterentwicklung der 3D-Kulturen mit primären humanen Keratinozyten, eine effektivere Virusreifung in Zelllysaten und Überständen zu erreichen. Während die Virusreplikation in 3D- und Monolayer-Kultur ansonsten sehr ähnlich ablief, gab es in primären 3D-Kulturen erhebliche Unterschiede im Wirtszelltranskriptom, die sich signifikant auf die Regulation spezifischer biologischer Prozesse auswirkten. Zudem konnte durch die Inhibition des epidermalen Wachstumsfaktorrezeptors, der sich anhand von Vorergebnissen und früherer Arbeiten unserer Arbeitsgruppe als vielversprechendes Ziel für eine antivirale Therapie mit Gefitinib anbot, erstmals gezeigt werden, dass eine Virusinfektion in Abhängigkeit der Kultivierungsart in 3D-kultivierten Zellen wesentlich effizienter gehemmt werden kann als in korrespondierenden Monolayer-Kulturen. Das in dieser Arbeit etablierte Kultivierungsmodell stellt ein vielfältig einsetzbares, reproduzierbares und im Vergleich zu komplexeren 3D-Ansätzen einfacher zu handhabendes 3D-Zellkulturmodell für Zelllinien und Primärzellen dar. Durch das gewebeähnliche Verhalten der Zellen können in vitro prädiktivere Daten für das Geschehen in vivo generiert und mögliche Zielmoleküle für antivirale Substanzen ermittelt werden, die in konventionellen Monolayer-Kulturen aufgrund des artifiziellen Wachstums der Zellen unter Umständen nicht von Relevanz sind. Andersherum trägt die 3D-Kultivierung auch dazu bei, die mitunter langwierige Erforschung von Zielmolekülen zu vermeiden, die durch Versuche in Monolayer-Kulturen identifiziert werden und unter Umständen lediglich kultivierungsabhängige Artefakte darstellen. Aufgrund der besseren Vorhersage der Wirkungsweise von Substanzen in vivo können so eventuell notwendige Tierversuche präziser geplant und auf ein Minimum reduziert werden und somit nur noch die aussichtsreichsten Kandidaten in weiterführende, kostenintensive klinische Studien überführt werden.Cell cultivations reflect a fundamental tool for research on diverse biological phenomena. Research on viruses in vitro is mainly carried out in conventional monolayer cultures where cells grow outstretched and flattened on rigid surfaces. Compared to in vivo conditions monolayer cultured cells show distinct differences in morphology and physiology. Unlike in virology, three-dimensional cell cultivation methods are used in different fields of research such as oncology or stem cell research since several decades because of their in vivo like characteristics and therefore high similarity to the respective organs or investigated biological phenomena. But also a few viruses whose biological needs are linked to more complex and differentiated tissues are studied in 3D cultures since more than twenty years. So far, infections with orthopoxviruses (OPV) which are highly relevant for public health because the zoonotic potential of some species such as cowpox virus (CPXV) as well as the threat of Variola virus being misused as a biological weapon were rarely studied in 3D culture models. The few existing studies were limited to morphological characterizations and some efficacy studies of antiviral substances. Therefore, the aim of the study was on the one hand the establishment of an easy-to-handle 3D cell culture model for comprehensive investigations of OPV infections. Further, the 3D culture system should allow adaption to a broad spectrum of cells for the study of different viruses. For this reason, a decellularized biological extracellular matrix (ECM) was used to generate 3D cultures with several cell types. Uninfected epithelial 3D cultures showed distinct differences regarding cell morphology, differentiation, and polarization compared to the corresponding monolayer cultures. Their gene expression patterns exhibited strong similarities to their in vivo counterparts – the human epidermis. For more detailed analysis of OPV replication and virus-host cell interactions within a complex biological system two epithelial 3D infection models with CPXV Brighton Red (BR) as model virus were established. Cell viability in CPXV-infected 3D cultures established with the Vero E6 cell line was distinctly prolonged compared to the corresponding monolayer cultures producing nearly stable viral titers. In this infection model as well as in an advanced 3D culture system with primary human keratinocytes efficiency of virus maturation in cell lysates and in supernatants was strongly elevated. However, while overall virus replication in 3D and monolayer cultures was comparable, considerable differences in the host cell transcriptome were observed which resulted in a significant differential regulation of specific biological processes. Based on the data from the 3D culture and on data from previous studies of our working group, the epidermal growth factor receptor emerged as a promising target for a host-directed antiviral therapy that could be specifically inhibited by gefitinib. With this approach it was shown for the first time that virus inhibition was cell culture model dependent with a strongly enhanced efficiency in 3D cultures. In summary, this versatile and reproducible 3D culture system for cell lines and primary human cells is far easier to handle than many previously established 3D approaches. Due to the tissue-like behavior of the cells, data generated in vitro will be more predictive for the in vivo situation. Therefore, 3D cultivation allows the discovery of novel target molecules for antiviral treatment otherwise missed due the artificial nature of conventional monolayer cultivation. Vice versa, 3D cultivation may help reducing potential lengthy and expensive research on target molecules only identified as cultivation artefacts in monolayer cultures. Due to an improved prediction of the in vivo mechanisms of substances based on 3D cultivation, animal experiments can be planned more precisely and therefore ultimately could be reduced to a minimum. In consequence, only the most promising candidate substances would finally reach the cost-intensive clinical trials
Differential Efficacy of Novel Antiviral Substances in 3D and Monolayer Cell Culture
Repurposing of approved drugs that target host functions also important for virus replication promises to overcome the shortage of antiviral therapeutics. Mostly, virus biology including initial screening of antivirals is studied in conventional monolayer cells. The biology of these cells differs considerably from infected tissues. 3D culture models with characteristics of human tissues may reflect more realistically the in vivo events during infection. We screened first, second, and third generation epidermal growth factor receptor (EGFR)-inhibitors with different modes of action and the EGFR-blocking monoclonal antibody cetuximab in a 3D cell culture infection model with primary human keratinocytes and cowpox virus (CPXV) for antiviral activity. Antiviral activity of erlotinib and osimertinib was nearly unaffected by the cultivation method similar to the virus-directed antivirals tecovirimat and cidofovir. In contrast, the host-directed inhibitors afatinib and cetuximab were approx. 100-fold more efficient against CPXV in the 3D infection model, similar to previous results with gefitinib. In summary, inhibition of EGFR-signaling downregulates virus replication comparable to established virus-directed antivirals. However, in contrast to virus-directed inhibitors, in vitro efficacy of host-directed antivirals might be seriously affected by cell cultivation. Results obtained for afatinib and cetuximab suggest that screening of such drugs in standard monolayer culture might underestimate their potential as antivirals.Peer Reviewe
»Ich habe mich nie wirklich für diese beiden Labels interessiert« Forschende Unternehmer*innen und unternehmerische Forscher*innen im Design
Design allein ist nicht genug – deshalb bietet der HKB-MA Design die Vertiefungen ›Entrepreneurship‹ und ›Research‹ an. Um Projekte zu entwickeln, die über traditionelle Dienstleistungen im Design hinausgehen, eignen sich die Studierenden darin auch unternehmerische und forschende Fähigkeiten an.Design alone is not enough – which is why the HKB-MA Design offers the specialisations ‘Entrepreneurship’ and ‘Research’. In order to develop projects that go beyond traditional design services, students also acquire entrepreneurial and research skills in these areas
Rotational object discrimination by pigeons
Four experiments examined the discrimination of directional object motion by pigeons. Four pigeons were tested in a go/no-go procedure with video stimuli of geons rotating right or left around their central y-axis. This directional discrimination was learned in 7 to 12 sessions and was not affected by changes in object starting orientation, but did require the coherent ordering of the videos' successive frames. Subsequent experiments found no or little transfer of this motion discrimination to novel objects. Experiments varying the speed of rotation and degrees of apparent motion per frame revealed that both factors strongly affected the discrimination. Finally, tests with partial occlusion of different portions of a rotating object suggested that the majority of the object was likely involved in determining rotational direction. These experiments indicate that pigeons can exclusively use motion cues to judge relative object motion. They also suggest the pigeons may have used a specific representation of the motion sequences of each object to make the discrimination
Twenty-four hours, or five days, of continuous sleep deprivation or experimental sleep fragmentation do not alter thirst or motivation for water reward in rats
The Functional Anatomy of the Deep Facial Fat Compartments: A Detailed Imaging-Based Investigation
BACKGROUND
Injection of soft-tissue fillers into the facial fat compartments is frequently performed to ameliorate the signs of facial aging. This study was designed to investigate the functional anatomy of the deep facial fat compartments and to provide information on the effects of injected material in relation to age and gender differences.
METHODS
Forty fresh frozen cephalic specimens of 17 male and 23 female Caucasian body donors (mean age, 76.9 ± 13.1 years; mean body mass index, 23.6 ± 5.3 kg/m(2)) were investigated. Computed tomographic and magnetic resonance imaging procedures were carried out using colored contrast-enhanced materials with rheologic properties similar to commercially available soft-tissue fillers. Anatomical dissections were performed to guide conclusions.
RESULTS
No statistically significant influences of age or gender were detected in the investigated sample. Increased amounts of injected contrast agent did not correlate with inferior displacement of the material in any of the investigated compartments: deep pyriform, deep medial cheek, deep lateral cheek, deep nasolabial (located within the premaxillary space), and the medial and lateral sub-orbicularis oculi fat.
CONCLUSIONS
Increasing volume in the deep midfacial fat compartments did not cause inferior displacement of the injected material. This underscores the role of deep soft-tissue filler injections (i.e., in contact with the bone) in providing support for overlying structures and resulting in anterior projection
Preliminary investigation of the antibacterial efficacy of a handheld Plasma Gun source for endodontic procedures
The investigation of the efficiency of a handheld Plasma Gun (PG) in the decontamination of tooth root canals is here reported. The antibacterial efficacy of the PG was first assessed on Enterococcus faecalis contaminated agar plates to determine optimal operating conditions that were then quantitatively evaluated treating contaminated liquid suspensions. Moreover, two different procedures for the inactivation of bacteria in realistic tooth models, resembling procedures conventionally adopted in endodontic practice, were investigated: (A) irrigation of contaminated tooth models with plasma activated water (indirect treatment) produced using the PG and (B) direct exposure of the contaminated tooth models to the plasma plume produced by the PG (direct treatment). The experiments were performed with the root canal model both in wet (root canal filled with bacteria suspension) and in dry (root canal contaminated and dried) conditions. From the obtained results, the direct treatment under dry conditions turned out to be the most effective, leading to a bacterial load mean reduction of 4.1