Publications of InIT at TU IlmenauPublications of InIT at TU Ilmenau
Results: 92
Created on: Sat, 02 Dec 2023 23:12:00 +0100 in 0.0956 sec

Zeußel, Lisa; Schober, Andreas; Ullmann, Fabian; Krischok, Stefan; Heinrich, Doris; Singh, Sukhdeep
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.
Zeußel, Lisa; Singh, Sukhdeep
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.
Marx-Blümel, Lisa; Marx, Christian; Schober, Andreas; Beck, James F.
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.
Henkel, Thomas; Mayer, Günter; Hampl, Jörg; Cao-Riehmer, Jialan; Ehrhardt, Linda; Schober, Andreas; Groß, Gregor Alexander
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.
Mai, Patrick; Hampl, Jörg; Bača, Martin; Brauer, Dana; Singh, Sukhdeep; Weise, Frank; Borowiec, Justyna; Schmidt, André; Küstner, Johanna Merle; Klett, Maren; Gebinoga, Michael; Schroeder, Insa S.; Markert, Udo R.; Glahn, Felix; Schumann, Berit; Eckstein, Diana; Schober, Andreas
MatriGrid® based biological morphologies : tools for 3D cell culturing. - In: Bioengineering, ISSN 2306-5354, Bd. 9 (2022), 5, 220, S. 1-41, insges. 41 S.

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.
Bača, Martin; Brauer, Dana; Klett, Maren; Fernekorn, Uta; Singh, Sukhdeep; Hampl, Jörg; Groß, Gregor Alexander; Mai, Patrick; Friedel, Karin; Schober, Andreas
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.
Zeußel, Lisa; Hampl, Jörg; Weise, Frank; Singh, Sukhdeep; Schober, Andreas
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.
Borowiec, Justyna;
Fabrication and characterization of microstructured scaffolds for complex 3D cell cultures. - Ilmenau : Universitätsbibliothek, 2021. - 1 Online-Ressource (XII, 140 Seiten)
Technische Universität Ilmenau, Dissertation 2021

In einem natürlichen Gewebe wird das zelluläre Verhalten durch Stimuli der Mikroumgebung reguliert. Verschiedene chemische, mechanische und physikalische Reize befinden sich in einem lokalen Milieu und versorgen die Zellen mit einem biologischen Kontext. Im Vergleich zur in vivo Situation, zeigen Standard 2D in vitro Zellkulturmodelle viele Unterschiede in der zellulären Mikroumgebung und können infolgedessen eine Veränderung der Zellantwort verursachen. Die Schaffung einer physiologisch realistischeren Umgebung auf künstlichem Substrat ist ein Schlüsselfaktor für die Entwicklung zuverlässiger Plattformen, die es den kultivierten Zellen ermöglichen, sich natürlicher zu verhalten. Daher sind neuartige Substrate auf Biomaterialbasis mit maßgeschneiderten Eigenschaften sehr gefragt. Die Mikrotechnik ist ein leistungsstarkes Werkzeug, das bei der Herstellung der Funktionsgerüste hilft, um verschiedene Eigenschaften der in vivo Umgebung zu reproduzieren und auf in vitro Bedingungen zu übertragen. Die Gerüstkonstruktionsparameter können manipuliert werden, um die für das jeweilige Gewebe spezifischen Anforderungen zu erfüllen. Eine der grundlegenden Einschränkungen bei aktuellen Herstellungsverfahren ist jedoch die Unfähigkeit, mehrere Gerüsteigenschaften auf vorgefertigte Weise in eine einzelne Gerüststruktur zu integrieren. Diese Dissertation befasst sich mit Gerüstmikrofabrikations- und Oberflächenmodifikations-techniken, welche die Mikrostrukturierungstechnologie verwenden und die gleichzeitige Kontrolle über verschiedene Gerüsteigenschaften ermöglichen. Diese Ansätze bei der Mikrofabrikation von Polymergerüsten werden verwendet, um physikalische und chemische Eigenschaften bereitzustellen, die für die Leberzellkultur optimiert sind. Die physikochemischen Aspekte, die die zelluläre Mikroumgebung von Lebergewebe in vivo ausmachen, werden diskutiert und anschließend werden relevante Technologien vorgestellt, mit denen einige dieser Aspekte in vitro reguliert werden können. Im ersten Teil dieser Arbeit wird ein neuartiges zweistufiges Verfahren zur Herstellung von Polymergerüsten mit mikroporöser Struktur und definierter Topographie gezeigt. Um 3D-Matrizen mit integrierter Porosität zu erhalten, wurde nach der Herstellung mikroporöser Folien ein Mikrostrukturierungsprozess unter Verwendung der Mehrschicht Polymer-Thermoformtechnologie durchgeführt. Diese Methoden wurden verwendet, um Substrate für die organotypische 3D-Hepatozytenkultivierung herzustellen. Poröse Gerüste mit Mikrokavitäten wurden aus lösungsmittelgegossenen und phasengetrennten Polymilchsäure (PLA) Folien gebildet. Die Proben wurden auf grundlegende mechanische und Oberflächenspezifische Eigenschaften sowie auf die Zellleistung untersucht. Um einen Bezugspunkt für die Bewertung der hergestellten Matrices bereitzustellen, wurden PLA-Gerüste mit zuvor beschriebenen Substraten auf Polycarbonat (PC)-Basis mit ähnlicher Geometrie verglichen. HepG2-Zellen, die in PLA-Gerüsten kultiviert wurden, zeigten eine gewebeartige 3D-Aggregation und eine erhöhte Sekretionsrate von Albumin im Vergleich zu PC-Gerüsten. Anschließend wurde dieses zweistufige Herstellungsverfahren verwendet, um schnell abbaubare Gerüste für die gerüstfreie Zellblatttechnik herzustellen. Gerüste mit kontrollierter Porosität und Topographie, die die Schlüsselmerkmale von Lebersinusoiden nachahmen, wurden aus Poly(milch-co-glykolsäure) (PLGA)-Copolymer hergestellt und für den in vitro Abbau in Zellkultur charakterisiert. Um die Beziehung zwischen dem Abbau des Gerüsts und der Organisation der Zellen in der PLGA-Matrix aufzudecken, wurde die Lebensfähigkeit und Morphologie der kultivierten Zellen zusammen mit der Morphologie des Gerüsts untersucht. Im zweiten Teil dieser Arbeit wurden verschiedene technische Lösungen für die gerichtete Strukturierung mikroporöser Polymergerüste bewertet und ihre Eignung zur Erzeugung einer benutzerdefinierten lebenswichtigen oligozellulären Morphologie auf künstlichem Substrat vorgestellt. Besonderes Augenmerk wurde auf das 3D-Mikrokontaktdruckverfahren (3DµCP) gelegt, das die Vorteile des Mikrothermoformens und des Mikrokontaktdrucks kombiniert und eine räumlich-zeitliche Kontrolle über morphologische und chemische Merkmale in einem einzigen Schritt ermöglicht. Um das Potenzial dieser Technik aufzuzeigen, wurden Gerüste mit bestimmten Mikrostrukturen wie Kanäle mit verschiedenen Tiefen und Breiten sowie komplexere Muster hergestellt und verschiedene ECM-Moleküle gleichzeitig in die vordefinierten Geometrien übertragen. Die Gültigkeit des 3DµCP-Prozesses wurde durch mikroskopische Messungen, Fluoreszenzfärbung und Testen der Substrate auf Zelladhäsionsantwort gezeigt. Schließlich wird in dieser Arbeit die Herstellungsmethode zur Erzeugung komplexer Gerüste für die 3D- und gesteuerte Co-Kultivierung von Leberzellen vorgestellt. Polymermatrizen, die die grundlegende Leberarchitektur replizieren und somit eine gut organisierte Leberzellzusammensetzung ermöglichen, wurden erfolgreich unter Verwendung der 3DµCP-Methode hergestellt. Auf der Polycarbonatoberfläche wurden gleichzeitig chemische und topografische Leitfäden in Form sinusförmiger Strukturen strukturiert. Um die 3D-Gewebemikrostruktur zu replizieren, wurden EA.hy926- und HepG2-Zellen auf beiden Seiten des strukturierten porösen Gerüsts Co-kultiviert und anschließend einander gegenüber gestapelt, wodurch zugehörige Kanäle zur Bildung einer Kapillare führen. Das Potenzial unseres 3DµCP-strukturierten Gerüsts für die gerichtete Co-Kultivierung von Zellen wurde unter statischen Zellkulturbedingungen demonstriert. Am Ende wurden Gerüste für die weiteren Anwendungen im perfundierten Bioreaktorsystem angepasst.
Zeußel, Lisa; Aziz, Carlos; Schober, Andreas; Singh, Sukhdeep
pH-dependent selective colorimetric detection of proline and hydroxyproline with Meldrum's acid-furfural conjugate. - In: Chemosensors, ISSN 2227-9040, Bd. 9 (2021), 12, 343, S. 1-13

Activated 2-furfural gives intense color formation when reacted with amines, due to a ring opening reaction cascade that furnishes a conjugated molecular system. Unique colorimetric characteristic of this reaction makes it an interesting candidate for developing chemosensors operating in visible range. Among many activated 2-furfural derivatives, Meldrum's acid furfural conjugate (MAFC) recently gained significant interest as colorimetric chemosensor. MAFC has been explored as selective chemosensor for detecting amines in solution, secondary amines on polymer surfaces and even nitrogen rich amino acids (AA) in aqueous solution. In this work, the pH dependency of MAFC-AA reaction is explored. It was found that proline gives an exceptionally fast colored reaction at pH 11, whereas at other pHs, no naked eye color product formation was observed. The reaction sequence including ring opening reaction upon nucleophilic addition of cyclic amine of proline resulting in a conjugated triene was confirmed by NMR titrations. The highly pH dependent reaction can e.g., potentially be used to detect proline presence in biological samples. An even more intense color formation takes place in the reaction of natural proline derivative 4-hydroxyproline. The detection limit of proline and 4-hydroxyproline with MAFC solution was found to be 11 [my]M and 6 [my)M respectively.
Marx-Blümel, Lisa; Marx, Christian; Sonnemann, Jürgen; Weise, Frank; Hampl, Jörg; Frey, Jessica; Rothenburger, Linda; Cirri, Emilio; Rahnis, Norman; Koch, Philipp; Groth, Marco; Schober, Andreas; Wang, Zhao-Qi; Beck, James F.
Molecular characterization of hematopoietic stem cells after in vitro amplification on biomimetic 3D PDMS cell culture scaffolds. - In: Scientific reports, ISSN 2045-2322, Bd. 11 (2021), 21163, S. 1-14

Hematopoietic stem cell (HSC) transplantation is successfully applied since the late 1950s. However, its efficacy can be impaired by insufficient numbers of donor HSCs. A promising strategy to overcome this hurdle is the use of an advanced ex vivo culture system that supports the proliferation and, at the same time, maintains the pluripotency of HSCs. Therefore, we have developed artificial 3D bone marrow-like scaffolds made of polydimethylsiloxane (PDMS) that model the natural HSC niche in vitro. These 3D PDMS scaffolds in combination with an optimized HSC culture medium allow the amplification of high numbers of undifferentiated HSCs. After 14 days in vitro cell culture, we performed transcriptome and proteome analysis. Ingenuity pathway analysis indicated that the 3D PDMS cell culture scaffolds altered PI3K/AKT/mTOR pathways and activated SREBP, HIF1α and FOXO signaling, leading to metabolic adaptations, as judged by ELISA, Western blot and metabolic flux analysis. These molecular signaling pathways can promote the expansion of HSCs and are involved in the maintenance of their pluripotency. Thus, we have shown that the 3D PDMS scaffolds activate key molecular signaling pathways to amplify the numbers of undifferentiated HSCs ex vivo effectively.