254 research outputs found
Entwicklung und Automatisierung 3D-gedruckter mikrofluidischer Systeme zur Integration und Kultivierung adhärenter Zellkulturen
Mikrofluidische Systeme werden zur Manipulation von Flüssigkeiten auf Mikroebene eingesetzt. Von ihnen profitieren insbesondere Biowissenschaften durch die Reduktion von Reagenzien und die Automatisierung ganzer Arbeitsabläufe. Die Mikrostrukturierung erlaubt zudem die Entwicklung neuartiger mikrofluidischer Zellkultursysteme wie den organ-on-a-chip Systemen. Diese Systeme zeichnen sich durch eine höhere physiologische Relevanz gegenüber klassischen in vitro Systemen aus und können zur Rekonstruktion einzelner Organfunktionen genutzt werden.
Aufgrund ihrer komplizierten Fertigung wird jedoch der Zugang zu diesen Systemen für Biowissenschaftler:innen er-schwert, sodass ihr Potential noch kaum in kommerziellen Produkten realisiert werden konnte. Eine Lösung bietet die additive Fertigung (3D-Druck) mikrofluidischer Systeme, durch die die unkomplizierte Herstellung eigener Prototypen an Ort und Stelle ermöglicht wird. Um den 3D-Druck jedoch auch für die Herstellung mikrofluidischer Zellkultursysteme nutzen zu können, benötigt es deutlich mehr Biokompatibilitätsstudien zu neuen 3D-Druckmaterialien.
In diesem Sinne wurde in dem ersten Teil dieser Arbeit die in vitro Biokompatibilität eines aus Polyacrylat bestehenden, hitzebeständigen 3D-Druckmaterials sowie dessen Eignung für die Heißdampfsterilisation untersucht. Dabei konnte eine Biokompatibilität gegenüber adhärenten Mausfibroblasten und Hefezellen nachgewiesen werden. Diese Ergebnis-se ermöglichen somit den Einsatz des Materials für die Zellkultur. Die Biokompatibilität blieb auch nach Heißdampfsteri-lisation unbeeinträchtigt, sodass mit diesem Material gedruckte Zellkultursysteme unkompliziert sterilisiert werden können. Im Gegensatz dazu erwies sich das Material für menschliche embryonale Nierenzellen in Suspension als schädlich, was die Bedeutung einer auf den Organismus und die Anwendung zugeschnittenen Biokompatibilitätsprü-fung verdeutlicht.
Im zweiten Teil dieser Arbeit wurde das evaluierte 3D-Druckmaterial zur Herstellung eines vollautomatischen mikroflui-dischen Ventilsystems eingesetzt, dessen Nutzen anschließend durch die Automatisierung eines Zellkulturassays als Machbarkeitsstudie demonstriert wurde. Alle mikrofluidischen Komponenten inklusive Anschlüsse, Mikromischer, Mikroventile und Auslässe wurden dabei in einem Stück gefertigt. Die kostengünstige und leicht zu steuernde Aktuation der 3D-gedruckten Ventilmembranen durch Servomotoren ist ein komplett neuer Ansatz. Die Automatisierung des Sys-tems erfolgte durch einen Raspberry Pi Computer sowie selbst entwickelter Python Skripte. Durch den kompakten Com-puter wird die portable und ferngesteuerte Verwendung des Ventilsystems ermöglicht. Nachdem eine zuverlässige Mischgenauigkeit sowie die hohe Robustheit der Ventile gezeigt werden konnte, wurde das mikrofluidische Ventilsys-tem zur Automatisierung eines Zytotoxizitätsassays als Machbarkeitsstudie verwendet. Das von der Konzentration des Toxins abhängige Zellwachstum wurde dabei mittels Lebendzellmikroskopie und Bildverarbeitung automatisiert ausge-wertet. Die Ergebnisse wurden anschließend mit denen eines pipettierten Assays verglichen. Beide Assays zeigten ein fast identisches Wachstumsverhalten, das die Eignung des Systems für die Zellkultur beweist.
Letztendlich konnte durch den 3D-Druck in Kombination mit der Biokompatibilitätsbestimmung eines 3D-Druckmaterials die Automatisierung von Zellkulturassays durch ein neu entwickeltes, 3D-gedrucktes mikrofluidisches Ventilsystem ermöglicht werden. Mit der Veröffentlichung der 3D-Modelle und Skripte ist es Wissenschaftler:innen nun möglich, das System an ihre eigenen Anwendungen anzupassen.Deutsche Forschungsgemeinschaft/Emmy Noether/346772917/E
3D-Printed Microfluidic Perfusion System for Parallel Monitoring of Hydrogel-Embedded Cell Cultures
The use of three-dimensional (3D) cell cultures has become increasingly popular in the contexts of drug discovery, disease modelling, and tissue engineering, as they aim to replicate in vivo-like conditions. To achieve this, new hydrogels are being developed to mimic the extracellular matrix. Testing the ability of these hydrogels is crucial, and the presented 3D-printed microfluidic perfusion system offers a novel solution for the parallel cultivation and evaluation of four separate 3D cell cultures. This system enables easy microscopic monitoring of the hydrogel-embedded cells and significantly reduces the required volumes of hydrogel and cell suspension. This cultivation device is comprised of two 3D-printed parts, which provide four cell-containing hydrogel chambers and the associated perfusion medium chambers. An interfacing porous membrane ensures a defined hydrogel thickness and prevents flow-induced hydrogel detachment. Integrated microfluidic channels connect the perfusion chambers to the overall perfusion system, which can be operated in a standard CO2-incubator. A 3D-printed adapter ensures the compatibility of the cultivation device with standard imaging systems. Cultivation and cell staining experiments with hydrogel-embedded murine fibroblasts confirmed that cell morphology, viability, and growth inside this cultivation device are comparable with those observed within standard 96-well plates. Due to the high degree of customization offered by additive manufacturing, this system has great potential to be used as a customizable platform for 3D cell culture applications
Automation of cell culture assays using a 3D-printed servomotor-controlled microfluidic valve system
Microfluidic valve systems show great potential to automate mixing, dilution, and time-resolved reagent supply within biochemical assays and novel on-chip cell culture systems. However, most of these systems require a complex and cost-intensive fabrication in clean room facilities, and the valve control element itself also requires vacuum or pressure sources (including external valves, tubing, ports and pneumatic control channels). Addressing these bottlenecks, the herein presented biocompatible and heat steam sterilizable microfluidic valve system was fabricated via high-resolution 3D printing in a one-step process - including inlets, micromixer, microvalves, and outlets. The 3D-printed valve membrane is deflected via miniature on-chip servomotors that are controlled using a Raspberry Pi and a customized Python script (resulting in a device that is comparatively low-cost, portable, and fully automated). While a high mixing accuracy and long-term robustness is established, as described herein the system is further applied in a proof-of-concept assay for automated IC50 determination of camptothecin with mouse fibroblasts (L929) monitored by a live-cell-imaging system. Measurements of cell growth and IC50 values revealed no difference in performance between the microfluidic valve system and traditional pipetting. This novel design and the accompanying automatization scripts provide the scientific community with direct access to customizable full-time reagent control of 2D cell culture, or even novel organ-on-a-chip systems
3D-printed microfluidic perfusion system for parallel monitoring of hydrogel-embedded cell cultures
The use of three-dimensional (3D) cell cultures has become increasingly popular in the contexts of drug discovery, disease modelling, and tissue engineering, as they aim to replicate in vivo-like conditions. To achieve this, new hydrogels are being developed to mimic the extracellular matrix. Testing the ability of these hydrogels is crucial, and the presented 3D-printed microfluidic perfusion system offers a novel solution for the parallel cultivation and evaluation of four separate 3D cell cultures. This system enables easy microscopic monitoring of the hydrogel-embedded cells and significantly reduces the required volumes of hydrogel and cell suspension. This cultivation device is comprised of two 3D-printed parts, which provide four cell-containing hydrogel chambers and the associated perfusion medium chambers. An interfacing porous membrane ensures a defined hydrogel thickness and prevents flow-induced hydrogel detachment. Integrated microfluidic channels connect the perfusion chambers to the overall perfusion system, which can be operated in a standard CO2-incubator. A 3D-printed adapter ensures the compatibility of the cultivation device with standard imaging systems. Cultivation and cell staining experiments with hydrogel-embedded murine fibroblasts confirmed that cell morphology, viability, and growth inside this cultivation device are comparable with those observed within standard 96-well plates. Due to the high degree of customization offered by additive manufacturing, this system has great potential to be used as a customizable platform for 3D cell culture applications
Towards green 3D-microfabrication of Bio-MEMS devices using ADEX dry film photoresists
Current trends in miniaturized diagnostics indicate an increasing demand for large quantities of mobile devices for health monitoring and point-of-care diagnostics. This comes along with a need for rapid but preferably also green microfabrication. Dry film photoresists (DFPs) promise low-cost and greener microfabrication and can partly or fully replace conventional silicon-technologies being associated with high-energy demands and the intense use of toxic and climate-active chemicals. Due to their mechanical stability and superior film thickness homogeneity, DFPs outperform conventional spin-on photoresists, such as SU-8, especially when three-dimensional architectures are required for micro-analytical devices (e.g. microfluidics). In this study, we utilize the commercial epoxy-based DFP ADEX to demonstrate various application scenarios ranging from the direct modification of microcantilever beams via the assembly of microfluidic channels to lamination-free patterning of DFPs, which employs the DFP directly as a substrate material. Finally, kinked, bottom-up grown silicon nanowires were integrated in this manner as prospective ion-sensitive field-effect transistors in a bio-probe architecture directly on ADEX substrates. Hence, we have developed the required set of microfabrication protocols for such an assembly comprising metal thin film deposition, direct burn-in of lithography alignment markers, and polymer patterning on top of the DFP
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Towards Green 3D-Microfabrication of Bio-MEMS Devices Using ADEX Dry Film Photoresists
Current trends in miniaturized diagnostics indicate an increasing demand for large quantities of mobile devices for health monitoring and point-of-care diagnostics. This comes along with a need for rapid but preferably also green microfabrication. Dry film photoresists (DFPs) promise low-cost and greener microfabrication and can partly or fully replace conventional silicon-technologies being associated with high-energy demands and the intense use of toxic and climate-active chemicals. Due to their mechanical stability and superior film thickness homogeneity, DFPs outperform conventional spin-on photoresists, such as SU-8, especially when three-dimensional architectures are required for micro-analytical devices (e.g. microfluidics). In this study, we utilize the commercial epoxy-based DFP ADEX to demonstrate various application scenarios ranging from the direct modification of microcantilever beams via the assembly of microfluidic channels to lamination-free patterning of DFPs, which employs the DFP directly as a substrate material. Finally, kinked, bottom-up grown silicon nanowires were integrated in this manner as prospective ion-sensitive field-effect transistors in a bio-probe architecture directly on ADEX substrates. Hence, we have developed the required set of microfabrication protocols for such an assembly comprising metal thin film deposition, direct burn-in of lithography alignment markers, and polymer patterning on top of the DFP
An Efficient Way to Screen Inhibitors of Energy-Coupling Factor (ECF) Transporters in a Bacterial Uptake Assay
Herein, we report a novel whole-cell screening assay using Lactobacillus casei as a model microorganism to identify inhibitors of energy-coupling factor (ECF) transporters. This promising and underexplored target may have important pharmacological potential through modulation of vitamin homeostasis in bacteria and, importantly, it is absent in humans. The assay represents an alternative, cost-effective and fast solution to demonstrate the direct involvement of these membrane transporters in a native biological environment rather than using a low-throughput in vitro assay employing reconstituted proteins in a membrane bilayer system. Based on this new whole-cell screening approach, we demonstrated the optimization of a weak hit compound (2) into a small molecule (3) with improved in vitro and whole-cell activities. This study opens the possibility to quickly identify novel inhibitors of ECF transporters and optimize them based on structure–activity relationships
In vitro biocompatibility evaluation of a heat-resistant 3D printing material for use in customized cell culture devices
Additive manufacturing (3D printing) enables the fabrication of highly customized and complex devices and is therefore increasingly used in the field of life sciences and biotechnology. However, the application of 3D-printed parts in these fields requires not only their biocompatibility but also their sterility. The most common method for sterilizing 3D-printed parts is heat steam sterilization—but most commercially available 3D printing materials cannot withstand high temperatures. In this study, a novel heat-resistant polyacrylate material for high-resolution 3D Multijet printing was evaluated for the first time for its resistance to heat steam sterilization and in vitro biocompatibility with mouse fibroblasts (L929), human embryonic kidney cells (HEK 293E), and yeast (Saccharomyces cerevisiae (S. cerevisiae)). Analysis of the growth and viability of L929 cells and the growth of S. cerevisiae confirmed that the extraction media obtained from 3D-printed parts had no negative effect on the aforementioned cell types, while, in contrast, viability and growth of HEK 293E cells were affected. No different effects of the material on the cells were found when comparing heat steam sterilization and disinfection with ethanol (70%, v/v). In principle, the investigated material shows great potential for high-resolution 3D printing of novel cell culture systems that are highly complex in design, customized and easily sterilizable—however, the biocompatibility of the material for other cell types needs to be re-evaluated
Groundwater flow dynamics in the complex aquifer system of Gidabo River Basin (Ethiopian Rift): a multi-proxy approach
An Efficient Way to Screen Inhibitors of Energy-Coupling Factor (ECF) Transporters in a Bacterial Uptake Assay
Herein, we report a novel whole-cell screening assay using Lactobacillus casei as a model
microorganism to identify inhibitors of energy-coupling factor (ECF) transporters. This promising
and underexplored target may have important pharmacological potential through modulation of
vitamin homeostasis in bacteria and, importantly, it is absent in humans. The assay represents an
alternative, cost-effective and fast solution to demonstrate the direct involvement of these membrane
transporters in a native biological environment rather than using a low-throughput in vitro assay
employing reconstituted proteins in a membrane bilayer system. Based on this new whole-cell
screening approach, we demonstrated the optimization of a weak hit compound (2) into a small
molecule (3) with improved in vitro and whole-cell activities. This study opens the possibility to
quickly identify novel inhibitors of ECF transporters and optimize them based on structure–activity
relationships
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