Modular air-liquid interface aerosol exposure system (MALIES) to study toxicity of nanoparticle aerosols in 3D-cultured A549 cells in vitro. - In: Archives of toxicology, ISSN 1432-0738, Bd. 98 (2024), 4, S. 1061-1080
We present a novel lung aerosol exposure system named MALIES (modular air-liquid interface exposure system), which allows three-dimensional cultivation of lung epithelial cells in alveolar-like scaffolds (MatriGrids®) and exposure to nanoparticle aerosols. MALIES consists of multiple modular units for aerosol generation, and can be rapidly assembled and commissioned. The MALIES system was proven for its ability to reliably produce a dose-dependent toxicity in A549 cells using CuSO4 aerosol. Cytotoxic effects of BaSO4- and TiO2-nanoparticles were investigated using MALIES with the human lung tumor cell line A549 cultured at the air-liquid interface. Experiments with concentrations of up to 5.93 × 10^5 (BaSO4) and 1.49 × 10^6 (TiO2) particles/cm^3, resulting in deposited masses of up to 26.6 and 74.0 µg/cm^2 were performed using two identical aerosol exposure systems in two different laboratories. LDH, resazurin reduction and total glutathione were measured. A549 cells grown on MatriGrids® form a ZO-1- and E-Cadherin-positive epithelial barrier and produce mucin and surfactant protein. BaSO4-NP in a deposited mass of up to 26.6 µg/cm^2 resulted in mild, reversible damage (˜ 10% decrease in viability) to lung epithelium 24 h after exposure. TiO2-NP in a deposited mass of up to 74.0 µg/cm^2 did not induce any cytotoxicity in A549 cells 24 h and 72 h after exposure, with the exception of a 1.7 fold increase in the low exposure group in laboratory 1. These results are consistent with previous studies showing no significant damage to lung epithelium by short-term treatment with low concentrations of nanoscale BaSO4 and TiO2 in in vitro experiments.
https://doi.org/10.1007/s00204-023-03673-3
Comparison of a 3D co-culture and a mini organ culture by testing barium sulphate and titanium dioxide nanoparticle aerosols. - In: Naunyn-Schmiedeberg's archives of pharmacology, ISSN 1432-1912, Bd. 396 (2023), 1, P055, S. S37
https://doi.org/10.1007/s00210-023-02397-6
Advanced 3D cell culturing and monitoring system. - Ilmenau : Universitätsbibliothek, 2023. - 1 Online-Ressource (213 Seiten)
Technische Universität Ilmenau, Dissertation 2023
Die vorgelegte Doktorarbeit stellt ein 3D-Zellkultursystem mit einem vollautomatisierten analytischen, biochemischen Assay und vollautomatisierter Kultivierung mit Mediumwechsel vor. Dieses integrierte Kultivierungs- und Analysesystem wurde in dieser Arbeit mit 3D-Hepatozytenkulturen in Polycarbonat-MatriGrid®-Gerüsten für das 3D-Wachstum von Zellkulturen prototypisiert. Das System perfundiert MatriGrid- Kulturen kontinuierlich mit Wirkstoff-ergänztem Medium und führt bei Bedarf eine Bewertung der Wirkstofftoxizität durch Beobachtung und Messung der Konzentration eines Indikators, des Biomarkers Albumin, durch. Das System kann die MatriGrid-Kultur mit unterschiedlichen Flussraten perfundieren, automatisierte Medienwechsel durchführen und mit dem mitgelieferten ELISA-Modul Proben des zu analysierenden Kulturmediums nach Bedarf untersuchen. Das System unterstützt die parallele Kultivierung von Zellen in mehreren Bioreaktoren. Das Fluidnetzwerk wurde aus Materialien konstruiert, die wenig Proteine und kleine Moleküle binden, absorbieren oder adsorbieren, um seine Anwendung für niedrige Biomarkerkonzentrationen und Langzeitexperimente zu erweitern. Die Doktorarbeit beschreibt das Systemdesign, den Aufbau, das Testen und die Verifikation unter Verwendung von 3D-gewachsenen HepaRG-Zellkulturen. Die zeitabhängige Wirkung von APAP auf die Albuminsekretion wurde über 96 h untersucht, wobei sowohl mit dem neu entwickelten System als auch konventionell in Mikrotiterplatten, gemessen wurde. Es zeigte sich, dass die Ergebnisse vergleichbar sind. Dieses Resultat belegt die Verwendung des Systems als eigenständiges Gerät, das in Echtzeit arbeitet und in der Lage ist, gleichzeitig Zellkultur- und Mediumanalyse in mehreren Bioreaktoren durchzuführen, mit erhöhter Zuverlässigkeit der 3D-Kultivierung, in einfacher Handhabung und Messung. Auf diese Weise soll das neu entwickelte 3D-Zellkultivierungs- und Analysesystem 3D-Zellkultivierungstechniken und -experimente für weitere Forschungsgruppen bekannt machen.
https://doi.org/10.22032/dbt.59056
Visible-light-assisted donor-acceptor-Stenhouse-adduct-based reversible photoswitching on a laser-structurable OrmoComp substrate. - In: ACS applied polymer materials, ISSN 2637-6105, Bd. 5 (2023), 10, S. 8631-8640
Laser-assisted nanolithography of commercially available photoresists is offering a limitless designing opportunity in the micro- and nanostructuring of 3D organotypic cell culture scaffolds. Among them, chemically functionalized OrmoComp has shown promising improvement in cell adhesion that paves the way to assemble cellular entities on a desirable geometry. Establishing a photoswitchable chemistry on the OrmoComp surface may offer an additional degree of freedom to manipulate the surface chemistry locally and selectively. We have established the methods for functionalization of the photopolymerized OrmoComp surface with visible-light-switchable donor-acceptor Stenhouse adducts. Unlike other polymers, a photopolymerized OrmoComp surface appears to be optimal for reversible photothermal switching, offering the possibility to influence surface properties like absorption and hydrophilicity tremendously. Light-assisted chemical modulation between colored triene-2-ol and colorless cyclopentenone can be achieved to a size region as narrow as 20 μm. Thermal reversion to the original triene-2-ol state can be analyzed spectroscopically and observed with the naked eye.
https://doi.org/10.1021/acsapm.3c01766
Meldrum’s acid furfural conjugate MAFC: a new entry as chromogenic sensor for specific amine identification. - In: Molecules, ISSN 1420-3049, Bd. 28 (2023), 18, 6627, S. 1-17
Bioactive amines are highly relevant for clinical and industrial application to ensure the metabolic status of a biological process. Apart from this, generally, amine identification is a key step in various bioorganic processes ranging from protein chemistry to biomaterial fabrication. However, many amines have a negative impact on the environment and the excess intake of amines can have tremendous adverse health effects. Thus, easy, fast, sensitive, and reliable sensing methods for amine identification are strongly searched for. In the past few years, Meldrum’s acid furfural conjugate (MAFC) has been extensively explored as a starting material for the synthesis of photoswitchable donor-acceptor Stenhouse adducts (DASA). DASA formation hereby results from the rapid reaction of MAFC with primary and secondary amines, which has so far been demonstrated through numerous publications for different applications. The linear form of the MAFC-based DASA exhibits intense pink coloration due to its linear conjugated triene-2-ol conformation, which has inspired researchers to use this easy synthesizable molecule as an optical sensor for primary, secondary, and biogenic amines. Due to its new entry into amine identification, a collection of the literature exclusively on MAFC is demanded. In this mini review, we intend to present the state-of-the-art of MAFC as an optical molecular sensor in hopes to motivate researchers to find even more applications of MAFC-based sensors and methods that pave the way to their usage in medicinal applications.
https://doi.org/10.3390/molecules28186627
In vitro-Amplifikation humaner hämatopoetischer Stammzellen im 3D-System. - In: Biospektrum, ISSN 1868-6249, Bd. 28 (2022), 5, S. 489-492
A promising strategy to increase the numbers of hematopoietic stem cells (HSCs) for clinical applications, like stem cell transplantation, is offered by advanced in vitro culture systems. We developed artificial 3D bone marrow-like scaffolds made of polydimethylsiloxane (PDMS) mimicking the natural HSC niche in vitro. These 3D PDMS scaffolds in combination with an optimized culture medium allow the amplification of high numbers of undifferentiated HSCs by activating specific molecular signaling pathways.
https://doi.org/10.1007/s12268-022-1798-2
From microtiter plates to droplets - there and back again. - In: Micromachines, ISSN 2072-666X, Bd. 13 (2022), 7, 1022, S. 1-13
Droplet-based microfluidic screening techniques can benefit from interfacing established microtiter plate-based screening and sample management workflows. Interfacing tools are required both for loading preconfigured microtiter-plate (MTP)-based sample collections into droplets and for dispensing the used droplets samples back into MTPs for subsequent storage or further processing. Here, we present a collection of Digital Microfluidic Pipetting Tips (DMPTs) with integrated facilities for droplet generation and manipulation together with a robotic system for its operation. This combination serves as a bidirectional sampling interface for sample transfer from wells into droplets (w2d) and vice versa droplets into wells (d2w). The DMPT were designed to fit into 96-deep-well MTPs and prepared from glass by means of microsystems technology. The aspirated samples are converted into the channel-confined droplets’ sequences separated by an immiscible carrier medium. To comply with the demands of dose-response assays, up to three additional assay compound solutions can be added to the sample droplets. To enable different procedural assay protocols, four different DMPT variants were made. In this way, droplet series with gradually changing composition can be generated for, e.g., 2D screening purposes. The developed DMPT and their common fluidic connector are described here. To handle the opposite transfer d2w, a robotic transfer system was set up and is described briefly.
https://doi.org/10.3390/mi13071022
MatriGrid® based biological morphologies: tools for 3D cell culturing. - In: Bioengineering, ISSN 2306-5354, Bd. 9 (2022), 5, 220, S. 1-41
Recent trends in 3D cell culturing has placed organotypic tissue models at another level. Now, not only is the microenvironment at the cynosure of this research, but rather, microscopic geometrical parameters are also decisive for mimicking a tissue model. Over the years, technologies such as micromachining, 3D printing, and hydrogels are making the foundation of this field. However, mimicking the topography of a particular tissue-relevant substrate can be achieved relatively simply with so-called template or morphology transfer techniques. Over the last 15 years, in one such research venture, we have been investigating a micro thermoforming technique as a facile tool for generating bioinspired topographies. We call them MatriGrid®s. In this research account, we summarize our learning outcome from this technique in terms of the influence of 3D micro morphologies on different cell cultures that we have tested in our laboratory. An integral part of this research is the evolution of unavoidable aspects such as possible label-free sensing and fluidic automatization. The development in the research field is also documented in this account.
https://doi.org/10.3390/bioengineering9050220
Automated analysis of acetaminophen toxicity on 3D HepaRG cell culture in microbioreactor. - In: Bioengineering, ISSN 2306-5354, Bd. 9 (2022), 5, 196, S. 1-16
Real-time monitoring of bioanalytes in organotypic cell cultivation devices is a major research challenge in establishing stand-alone diagnostic systems. Presently, no general technical facility is available that offers a plug-in system for bioanalytics in diversely available organotypic culture models. Therefore, each analytical device has to be tuned according to the microfluidic and interface environment of the 3D in vitro system. Herein, we report the design and function of a 3D automated culture and analysis device (3D-ACAD) which actively perfuses a custom-made 3D microbioreactor, samples the culture medium and simultaneously performs capillary-based flow ELISA. A microstructured MatriGrid® has been explored as a 3D scaffold for culturing HepaRG cells, with albumin investigated as a bioanalytical marker using flow ELISA. We investigated the effect of acetaminophen (APAP) on the albumin secretion of HepaRG cells over 96 h and compared this with the albumin secretion of 2D monolayer HepaRG cultures. Automated on-line monitoring of albumin secretion in the 3D in vitro mode revealed that the application of hepatotoxic drug-like APAP results in decreased albumin secretion. Furthermore, a higher sensitivity of the HepaRG cell culture in the automated 3D-ACAD system to APAP was observed compared to HepaRG cells cultivated as a monolayer. The results support the use of the 3D-ACAD model as a stand-alone device, working in real time and capable of analyzing the condition of the cell culture by measuring a functional analyte. Information obtained from our system is compared with conventional cell culture and plate ELISA, the results of which are presented herein.
https://doi.org/10.3390/bioengineering9050196
Bio-inspired 3D micro structuring of a liver lobule via direct laser writing: a comparative study with SU-8 and SUEX. - In: Journal of laser applications, ISSN 1938-1387, Bd. 34 (2022), 1, 012007, S. 012007-1-012007-12
Real biological tissues show a great variety of different geometric morphologies with special features on different geometric scales. An interesting example is the liver lobule that is the basic subunit of a liver. The lobule is a quasihexagonal macroscopic structure with periodic like so-called sinusoidal elements with structural features on the micro- and macroscale made of proteins, cells, and fluids. Various tools from micromachining and nanotechnology have demonstrated their capabilities to construct micromorphologies precisely, but even the reconstruction of such a system in technical polymers is challenging. In this work, the rapidly evolving technique of multiphoton polymerization has been explored for the construction of a scaffold that mimics the micromorphology of the liver with high resolution and detail up to the millimeter scale. At the end, a highly complex fluidically perfusable structure was achieved and simulations showed that the occurring shear stress, fluid velocity, and stream lines are comparable to the native liver lobule. Hereby, the photoresists SU-8 and SUEX TDFS were compared in terms of their processability, achievable resolution, and suitability for the intended application. Our results have shown that SUEX needs lower writing velocities but is easier to process and achieves a considerable higher resolution than SU-8. The scaffold could provide a base frame with a geometrically defined morphology for hepatic cells to adhere to, which could act as a starting point for cells to build new liver tissue for further integration in more complex systems.
https://doi.org/10.2351/7.0000433