In: ChemEngineering 2019, 3(2), 48

Abstract: We investigate the interaction of two immiscible fluids in a head-on device geometry, where both fluids are streaming opposite to each other. The simulations are based on the two-dimensional (2D) lattice Boltzmann method (LBM) using the Rothman and Keller (RK) model. We validate the LBM code with several benchmarks such as the bubble test, static contact angle, and layered flow. For the first time, we simulate a head-on device by forcing periodicity and a volume force to induce the flow. From low to high flow rates, three main flow patterns are observed in the head-on device, which are dripping-squeezing, jetting-shearing, and threading. In the squeezing regime, the flow is steady and the droplets are equal. The jetting-shearing flow is not as stable as dripping-squeezing. Moreover, the formation of droplets is shifted downstream into the main channel. The last flow form is threading, in which the immiscible fluids flow parallel downstream to the outlet. In contrast to other studies, we select larger microfluidic channels with 1-mm channel width to achieve relatively high volumetric fluxes as used in chemical synthesis reactors. Consequently, the capillary number of the flow regimes is smaller than 10−5. In conclusion, the simulation compares well to experimental data.

Volker Paul Schulz1, Nima Abbaspour1, Tobias Baumeister2 and Thorsten Röder2

1
Department of Mechanical Engineering, Cooperative State University Mannheim, Coblitzallee 1-9, 68163 Mannheim, Germany
2 Institute of Chemical Process Engineering, Mannheim University of Applied Sciences, Paul-Wittsack-Str. 10, 68163 Mannheim, Germany

 

In: BMC Cancer (2019) 19:402

Abstract
Background: Different 3D-cell culture approaches with varying degrees of complexity have been developed to serve as melanoma models for drug testing or mechanistic studies. While these 3D-culture initiatives are already often superior to classical 2D approaches, they are either composed of only melanoma cells or they are so complex that the behavior of individual cell types is hard to understand, and often they are difficult to establish and expensive.
Methods: This study used low-attachment based generation of spheroids composed of up to three cell types. Characterization of cells and spheroids involved cryosectioning, immunofluorescence, FACS, and quantitative analyses. Statistical evaluation used one-way ANOVA with post-hoc Tukey test or Student’s t-test.
Results: The tri-culture model allowed to track cellular behavior in a cell-type specific manner and recapitulated different characteristics of early melanoma stages. Cells arranged into a collagen-IV rich fibroblast core, a ring of keratinocytes, and groups of highly proliferating melanoma cells on the outside. Regularly, some melanoma cells were also found to invade the fibroblast core. In the absence of melanoma cells, the keratinocyte ring stratified into central basal-like and peripheral, more differentiated cells. Conversely, keratinocyte differentiation was clearly reduced upon addition of melanoma cells. Treatment with the cytostatic drug, docetaxel, restored keratinocyte differentiation and induced apoptosis of external melanoma cells. Remaining intact external melanoma cells showed a significantly increased amount of ABCB5-immunoreactivity.
Conclusions: In the present work, a novel, simple spheroid-based melanoma tri-culture model composed of fibroblasts, keratinocytes, and melanoma cells was described. This model mimicked features observed in early melanoma stages, including loss of keratinocyte differentiation, melanoma cell invasion, and drug-induced increase of ABCB5 expression in external melanoma cells.

Julia Klicks1,2, Christoph Maßlo3, Andreas Kluth4, Rüdiger Rudolf1,2 and Mathias Hafner1,2

1Institute of Molecular and Cell Biology, Mannheim University of Applied Sciences, Paul-Wittsack-Straße 10, 68163 Mannheim, Germany.
2Institute of Medical Technology, Mannheim University of Applied Sciences and Medical Faculty Mannheim of Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany.
3RHEACELL GmbH & Co. KG, Im Neuenheimer Feld 517, 69120 Heidelberg,Germany.
4TICEBA GmbH, Im Neuenheimer Feld 517, 69120 Heidelberg, Germany.

 

In: Front. Cell. Neurosci. 2019, 13, 49

Abstract: Neuromuscular junctions (NMJs) mediate skeletal muscle contractions and play an important role in several neuromuscular disorders when their morphology and function are compromised. However, due to their small size and sparse distribution throughout the comparatively large, inherently opaque muscle tissue the analysis of NMJ morphology has been limited to teased fiber preparations, longitudinal muscle sections, and flat muscles. Consequently, whole mount analyses of NMJ morphology, numbers, their distribution, and assignment to a given muscle fiber have also been impossible to determine in muscle types that are frequently used in experimental paradigms. This impossibility is exacerbated by the lack of optical tissue clearing techniques that are compatible with clear and persistent NMJ stains. Here, we present MYOCLEAR, a novel and highly reproducible muscle tissue clearing protocol. Based on hydrogel-based tissue clearing methods, this protocol permits the labeling and detection of all NMJs in adult hindleg extensor digitorum longus muscles from wildtype and diseased mice. The method is also applicable to adult mouse diaphragm muscles and can be used for different staining agents, including toxins, lectins, antibodies, and nuclear dyes. It will be useful in understanding the distribution, morphological features, and muscle tissue context of NMJs in hindleg muscle whole mounts for biomedical and basic research.

Marion Patrick Ivey Williams1, Matteo Rigon1, Tatjana Straka1,2, Sarah Janice Hörner1,2, Manfred Thiel3, Norbert Gretz4,5, Mathias Hafner1,5, Markus Reischl6 and Rüdiger Rudolf1,2,5,7

1Institute of Molecular and Cell Biology, Mannheim University of Applied Sciences, Mannheim, Germany
2Interdisciplinary Center for Neurosciences, Heidelberg University, Heidelberg, Germany
3Department of Anesthesiology and Surgical Intensive Care Medicine, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
4Medical Faculty Mannheim, Medical Research Center, Heidelberg University, Mannheim, Germany
5Medical Faculty Mannheim, Institute of Medical Technology, Mannheim University of Applied Sciences, Mannheim, Germany
6Institute for Automation and Applied Informatics, Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen, Germany
7Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen, Germany

In: Chemie Ingenieur Technik, 2019, 91, No. 5, 1–7

Abstract: A millistructured reactor and spectroscopic setup for contactless kinetic measurements in oscillating droplets is presented in this work. The polymerization of acrylic acid serves as a model reaction. Design and construction of the reactor focus on the optical access for Raman measurement, the nearly isothermal behavior, and the preservation of droplets during long‐term reactions with a high increase of viscosity. Another key aspect is the possibility of full automation at a later stage.

Torsten Klement, Norbert Kockmann, Thorsten Röder

In: Front. Oncol. 2019, 9, 36.

Abstract: Normally, bone sialoprotein (BSP) is an important contributor to bone micro-calcification. However, it is also highly expressed in bone-metastatic malignancies, including prostate, lung, and breast cancer. In these disorders, BSP correlates with poor prognosis. Its expression in triple-negative breast cancer cells is enhanced by the transcription factor RUNX2, and both, BSP and RUNX2 are under control of IGF-1 and TGFβ1. Knockdown of BSP or its inactivation by specific antibodies were found to reduce the metastatic potential of MDA-MB-231 triple-negative breast cancer cells in xenografts. While the role of BSP in bone metastasis was studied using such in vivo models, valid in vitro test systems to investigate BSP biology have been lacking since this protein is expressed at very low levels in classical 2D cell cultures and the frequently used breast cancer cell line MDA-MB-231 is difficult to grow in 3D. Here, we have developed a long-term 3D spheroid culture model using MDA-MB-231 cells in a sandwich approach using cell embedding between a non-adherent surface and basement membrane extracts. This allowed consistent growth of spheroids for more than 21 days. Also, co-culturing of MDA-MB-231 with CCD-1137Sk fibroblasts yielded stably growing spheroids, suggesting the importance of extracellular matrix (ECM) in this process. In addition, we have set up a novel and simple open source analysis tool to characterize protein expression in 2D cultures and spheroids by immunofluorescence. Using this approach in combination with Western blot analysis, the expression profile of BSP was analyzed. BSP was enriched at the rims of spheroids, both in mono- and co-cultures and its abundance in general correlated with that of TGFβ1 under different conditions, including spheroid maturation, cytostatic treatment, and fibroblast co-culture. Conversely, correlation of IGF-1 and BSP was limited to mono-culture time course profiles. In conclusion, we present novel tools to study the regulation of gene expression in combination with cell proliferation and apoptosis in a long-term 3D model of breast cancer and find dynamic abundance profiles of the metastasis-relevant protein BSP and its regulators.

Valeh Rustamov1,2, Florian Keller1,2, Julia Klicks1,2, Mathias Hafner1,2 and Rüdiger Rudolf1,2

1Institute of Molecular and Cell Biology, Mannheim University of Applied Sciences, Mannheim, Germany
2Institute of Medical Technology of Heidelberg University and Mannheim University of Applied Sciences, Mannheim, Germany

In: Reaction Chemistry & Engineering, 2019, 4, 523

Abstract:
We investigated solvent-free synthesis of the ionic liquid 1-butyl-3-methylimidazolium bromide in a microreactor setup. Time–conversion curves were used to derive a model for the liquid–liquid two-phase system taking both heat transfer and mass transfer into account. Simulations conducted with parameter variations were used to evaluate the general influence of mass and heat transfer coefficients on the conversion and temperature profiles in a flow reactor. A scale-up of the microreactor experiments to production in a millistructured plate reactor can be limited by insufficient heat removal, which results in parametric sensitivity. Thus, a general stability criterion was applied to the reactor model to derive stability diagrams from the calculated temperature profiles. These diagrams provide parameter ranges that ensure stable reactor operation, and can be used to predict the effectiveness of different scale-up concepts. Several concepts were combined in the design of a reaction-specific optimized multi-injection flow reactor with a channel geometry that could be adapted to the predicted local rate of heat generation.

Sebastian Schwolow, Benedikt Mutsch, Norbert Kockmann, Thorsten Röder

 

In: Chemie Ingenieur Technik, 109(7), 1151. ISSN 0009286X

Abstract: This work shows the possibility to measure scattered light from particles, which are exposed to miniscule laser intensities, with the help of a very sensitive single‐photon measurement technique. The advantages for subsequently designed sensors can be found in explosive prevention, avoiding product damage and improved user safety. Measurement systems to examine suspensions and aerosols are described. Laser output is less than 5 µW. The measurements start at 200 nm particle diameter and are carried out in a 90° arrangement as a worst‐case scenario for scattering intensities.

Tobias Teumer, Thomas Hufnagel, Thomas Schäfer, Robert Schlarp‐Horvath, Heike P. Karbstein, Frank‐Jürgen Methner, Matthias Rädle

In: Proteomics Clin. Appl. 2018, 1800029

Abstract: PURPOSE: To facilitate the transition of MALDI-MS Imaging (MALDI-MSI) from basic science to clinical application, it is necessary to analyze formalin-fixed paraffin-embedded (FFPE) tissues. The aim is to improve in situ tryptic digestion for MALDI-MSI of FFPE samples and determine if similar results would be reproducible if obtained from different sites.
EXPERIMENTAL DESIGN: FFPE tissues (mouse intestine, human ovarian teratoma, tissue microarray of tumor entities sampled from three different sites) are prepared for MALDI-MSI. Samples are coated with trypsin using an automated sprayer then incubated using deliquescence to maintain a stable humid environment. After digestion, samples are sprayed with CHCA using the same spraying device and analyzed with a rapifleX MALDI Tissuetyper at 50 µm spatial resolution. Data are analyzed using flexImaging, SCiLS, and R.
RESULTS: Trypsin application and digestion are identified as sources of variation and loss of spatial resolution in the MALDI-MSI of FFPE samples. Using the described workflow, it is possible to discriminate discrete histological features in different tissues and enabled different sites to generate images of similar quality when assessed by spatial segmentation and PCA.
CONCLUSIONS AND CLINICAL RELEVANCE: Spatial resolution and site-to-site reproducibility can be maintained by adhering to a standardized MALDI-MSI workflow.

Ly A1, Longuespée R2, Casadonte R3, Wandernoth P3, Schwamborn K4, Bollwein C4, Marsching C5, Kriegsmann K6, Hopf C5, Weichert W4, Kriegsmann J3, Schirmacher P2, Kriegsmann M2, Deininger SO1.

1Bruker Daltonik GmbH, Bremen, Germany.
2Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany.
3Proteopath GmbH, Trier, Germany.
4Institute of Pathology, Technical University of Munich, Munich, Germany.
5Center for Biomedical Mass Spectrometry and Optical Spectroscopy (CeMOS), Mannheim University of Applied Sciences, Mannheim, Germany.
6Department of Hematology, Oncology and Rheumatology, University Hospital Heidelberg, Heidelberg, Germany.

In: Journal of Sensors and Sensor Systems, 7, 543-549, 2018

Abstract: This paper presents an explosion-proof two-channel Raman photometer designed for chemical process monitoring in hazardous explosive atmospheres. Due to its design, alignment of components is simplified and economic in comparison to spectrometer systems. Raman spectrometers have the potential of becoming an increasingly important tool in process analysis technologies as part of molecular-specific concentration monitoring. However, in addition to the required laser power, which restricts use in potentially explosive atmospheres, the financial hurdle is also high. Within the scope of a proof of concept, it is shown that photometric measurements of Raman scattering are possible. The use of highly sensitive detectors allows the required excitation power to be reduced to levels compliant for operation in potentially explosive atmospheres. The addition of an embedded platform enables stable use as a self-sufficient sensor, since it carries out all calculations internally.

Multi-pixel photon counters (MPPCs) with large detection areas of 1350 µm2 are implemented as detectors. As a result, the sensitivity of the sensor is strongly increased. This gain in sensitivity is primarily achieved through two characteristics: first, the operating principle “avalanche breakdown” to detect single photons is used; second, the size of the image projected onto the MPPC is much bigger than the pixel area in competing Raman-Spectrometers resulting in higher photon flux. This combination enables reduction of the required excitation power to levels compliant for operation in potentially explosive atmospheres. All presented experiments are performed with strongly attenuated laser power of 35 mW. These include the monitoring of the analytes ethanol and hydrogen peroxide as well as the reversible binding of CO2 to amine. Accordingly, the described embedded sensor is ideally suited as a process analytical technology (PAT) tool for applications in environments with limitations on power input.

Marcel Nachtmann1, Shaun Paul Keck1, Frank Braun1, Hanns Simon Eckhardt2, Christoph Mattolat2, Norbert Gretz3, Stephan Scholl4, and Matthias Rädle1

1Institute for Process Control and Innovative Energy Conversion, Mannheim University of Applied Sciences, Mannheim, 68163, Germany
2tec5 AG, Oberursel/Ts, 61440, Germany
3Medical Research Center, Medical Faculty Mannheim, Heidelberg University, Mannheim, 68167, Germany
4Institute for Chemical and Thermal Process Engineering, Technical University Braunschweig, Braunschweig, 38106, Germany

Prof. Dr. Rüdiger Rudolf on the development of "Organoids" based on complex 3D cell cultures for the screening of new bioactive agents for skin care products and biomedicine.

Blickwinkel Structures Oktober 2018
Organoids - drug screening drives the development of new technologies

So far, drug and drug screening has been mostly based on either two-dimensional (2D) cell cultures or animal studies. However, both approaches have significant disadvantages. While 2D cell cultures are inexpensive to make and carry out the experiments, are capable of high throughput rates, and allow the use of cells of human origin, flat petri dishes, because of their rigidity and restriction of contact formation on one side of the cell surface In addition, penetration of the cells with drugs as well as the distribution of waste products, oxygen and nutrients in such 2D cultures is quite different than in the three-dimensional arrays of true human tissue.

Please read on here.

In: Scientific Reports, volume 8, Article number: 11260 (2018)

 

Abstract: Recent advances in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry have enabled whole cell-MALDI mass spectrometry biotyping of drug-treated cultured cells for rapid monitoring of known abundant pharmacodynamic protein markers such as polyacetylated histones. In contrast, generic and automated analytical workflows for discovery of such pharmacodynamic markers, in particular lipid markers, and their use in cellular tests of drug-like compounds are still lacking. Here, we introduce such a workflow and demonstrate its utility for cellular drug-response monitoring of BCR-ABL tyrosine kinase inhibitors in K562 leukemia cells: First, low-molecular mass features indicating drug responses are computationally extracted from groups of MALDI-TOF mass spectra. Then, the lipids/metabolites corresponding to these features are identified by MALDI-Fourier transformation mass spectrometry. To demonstrate utility of the method, we identify the potassium adduct of phosphatidylcholine PC(36:1) as well as heme B, a marker for erythroid differentiation, as markers for a label-free MALDI MS-based test of cellular responses to BCR-ABL inhibitors. Taken together, these results suggest that MALDI-TOF mass spectrometry of lipids and other low molecular mass metabolites could support cell-based drug profiling.

David Weigt, Denis A. Sammour, Timon Ulrich, Bogdan Munteanu & Carsten Hopf

In: International Journal of Molecular Sciences, Vol. 19, Issue 7

Abstract: Vertebrate neuromuscular junctions (NMJs) have been conceived as tripartite synapses composed of motor neuron, Schwann cell, and muscle fiber. Recent work has shown the presence of sympathetic neurons in the immediate vicinity of NMJs and experimental and clinical findings suggest that this plays an eminent role in adult NMJ biology. The present study examined the postnatal development and distribution of sympathetic innervation in different muscles using immunofluorescence, confocal microscopy, and Western blot. This demonstrates the proximity of sympathetic neurons in diaphragm, extensor digitorum longus, tibialis anterior, soleus, and levator auris longus muscles. In extensor digitorum longus muscle, sympathetic innervation of NMJs was quantified from perinatal to adult stage and found to increase up to two months of age. In diaphragm muscle, an extensive network of sympathetic neurons was prominent along the characteristic central synapse band. In summary, these data demonstrate that an elaborate sympathetic innervation is present in several mouse skeletal muscles and that this is often next to NMJs. Although the presence of sympathetic neurons at the perisynaptic region of NMJs increased during postnatal development, many synapses were already close to sympathetic neurons at birth. Potential implications of these findings for treatment of neuromuscular diseases are discussed.

Tatjana Straka1,2,3, Veronica Vita4, Kaltrina Prokshi1,2, Sarah Janice Hörner1,2, Muzamil Majid Khan1,2,3, Marco Pirazzini4, Marion Patrick Ivey Williams1, Mathias Hafner1, Tania Zaglia4,5,6 and Rüdiger Rudolf1,2,3
1 Institute of Molecular and Cell Biology, Mannheim University of Applied Sciences, 68163 Mannheim, Germany
2 Interdisciplinary Center for Neurosciences, Heidelberg University, 69120 Heidelberg, Germany
3 Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, 76344 Eggenstein-Leopoldshafen, Germany
4 Venetian Institute of Molecular Medicine, 35129 Padua, Italy
5 Department of Biomedical Sciences, University of Padua, 35131 Padua, Italy
6 Department of Cardiac Thoracic and Vascular Sciences, University of Padua, 35128 Padua, Italy

In: Frontiers in Molecular Neuroscience - Front. Mol. Neurosci. 2018, 11, 206

Abstract: Alzheimer's disease (AD) is characterized by progressive neurodegeneration and a concomitant loss of synapses and cognitive abilities. Recently, we have proposed that an alteration of neuronal membrane lipid microdomains increases neuronal resistance toward amyloid-β stress in cultured neurons and protects from neurodegeneration in a mouse model of AD. Lipid microdomains are highly enriched in a specific subclass of glycosphingolipids, termed gangliosides. The enzyme glucosylceramide synthase (GCS) catalyzes the rate-limiting step in the biosynthesis of these gangliosides. The present work now demonstrates that genetic GCS deletion in subsets of adult forebrain neurons significantly improves the spatial memory and counteracts the loss of dendritic spines in the hippocampal dentate gyrus of 5x familial AD mice (5xFAD//Ugcgf/f//Thy1-CreERT2//EYFP mice), when compared to 5xFAD//Ugcgf/f littermates (5xFAD mice). Aberrantly activated glial cells and their expression of pro-inflammatory cytokines have emerged as the major culprits for synaptic loss in AD. Typically, astrocytic activation is accompanied by a thickening of astrocytic processes, which impairs astrocytic support for neuronal synapses. In contrast to 5xFAD mice, 5xFAD//Ugcgf/f//Thy1-CreERT2//EYFP display a less pronounced thickening of astrocytic processes and a lower expression of tumor necrosis factor-α and interleukin 1-α in the hippocampus. Thus, this work further emphasizes that GCS inhibition may constitute a potential therapeutic target against AD.

Herzer S1,2, Hagan C1,3, von Gerichten J1,4, Dieterle V1,2, Munteanu B5, Sandhoff R1,4, Hopf C5, Nordström V1,2.

1Division of Cellular and Molecular Pathology, German Cancer Research Center, Heidelberg, Germany.
2Interdisciplinary Center for Neurosciences, Heidelberg University, Heidelberg, Germany.
3Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO, United States.
4Lipid Pathobiochemistry Group, German Cancer Research Center, Heidelberg, Germany.
5Center for Mass Spectrometry (CeMOS), University of Heidelberg and Mannheim University of Applied Sciences, Mannheim, Germany.

In: Bioconjugate Chem., 2018, 29 (5), pp 1525–1533

Abstract: Gold nanoparticles (AuNPs) have widely been used for 70 years in cancer treatment, but only in the last 15 years has the focus been on specific AuNPs with homogeneous size and shape for various areas in science. They constitute a perfect platform for multifunctionalization and therefore enable the enhancement of target affinity. Here we report on the development of tumor specific AuNPs as diagnostic tools intended for the detection of prostate cancer via fluorescence imaging and positron emission tomography (PET). The AuNPs were further evaluated in vitro and in vivo and exhibited favorable diagnostic properties concerning tumor cell uptake, biodistribution, clearance, and tumor retention.

Marc Pretze*† , Andreas Hien‡, Matthias Rädle‡, Ralf Schirrmacher§ , Carmen Wängler∥, and Björn Wängler*†
†Molecular Imaging & Radiochemistry, Department of Clinical Radiology and Nuclear Medicine and ∥Biomedical Chemistry, Medical Faculty Mannheim of Heidelberg University, Mannheim 68167, Germany
‡ Institute of Process Control and Innovative Energy Conversion, Mannheim University of Applied Sciences, Mannheim 68163, Germany
§ Oncologic Imaging, Department of Oncology, University of Alberta, Edmonton 6820, Alberta, Canada

In: Scientific Reports 8, Article number: 313 (2018)

Abstract: Multimodal imaging combines complementary platforms for spatially resolved tissue analysis that are poised for application in life science and personalized medicine. Unlike established clinical in vivo multimodality imaging, automated workflows for in-depth multimodal molecular ex vivo tissue analysis that combine the speed and ease of spectroscopic imaging with molecular details provided by mass spectrometry imaging (MSI) are lagging behind. Here, we present an integrated approach that utilizes non-destructive Fourier transform infrared (FTIR) microscopy and matrix assisted laser desorption/ionization (MALDI) MSI for analysing single-slide tissue specimen. We show that FTIR microscopy can automatically guide high-resolution MSI data acquisition and interpretation without requiring prior histopathological tissue annotation, thus circumventing potential human-annotation-bias while achieving >90% reductions of data load and acquisition time. We apply FTIR imaging as an upstream modality to improve accuracy of tissue-morphology detection and to retrieve diagnostic molecular signatures in an automated, unbiased and spatially aware manner. We show the general applicability of multimodal FTIR-guided MALDI-MSI by demonstrating precise tumor localization in mouse brain bearing glioma xenografts and in human primary gastrointestinal stromal tumors. Finally, the presented multimodal tissue analysis method allows for morphology-sensitive lipid signature retrieval from brains of mice suffering from lipidosis caused by Niemann-Pick type C disease.

Jan-Hinrich Rabe1,2, Denis A. Sammour1,2, Sandra Schulz1,2, Bogdan Munteanu1,2, Martina
Ott3, Katharina Ochs3,5, Peter Hohenberger4, Alexander Marx4, Michael Platten3,4,
Christiane A. Opitz5,6, Daniel S. Ory7 & Carsten Hopf1,2

1Center for Applied Research in Applied Biomedical Mass Spectrometry (ABIMAS), Mannheim University of Applied Sciences, Mannheim, Germany
2Institute of Medical Technology, Heidelberg University and Mannheim University of Applied Sciences, Mannheim, Germany
3German Cancer Consortium (DKTK) CCU Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany
4University Medical Center Mannheim of Heidelberg University, Mannheim, Germany
5Brain Cancer Metabolism Group, German Cancer Research Center (DKFZ), Heidelberg, Germany
6Department of Neurology and National Center of Tumor Diseases, University Hospital Heidelberg, Heidelberg, Germany
7Diabetic Cardiovascular Disease Center and Department of Medicine, Washington University School of Medicine

In: Journal of Cellular Biotechnology 3 (2017) 41–50

Abstract: In recent years, many different methods were introduced for generation of 3D cell culture. However, many currently available three-dimensional techniques are not suitable for certain cell lines and sometimes showed a lack of reproducibility. Therefore, specific protocols for cell lines are needed. In this work, we demonstrate different methods of generating 3D cell culture for SCC4 tongue cancer cell line and discuss their applicability. Using three different methods, tumor spheroids were generated from SCC4 cells and cultured for 20 days. To investigate the influence of initial seeding density on spheroid morphology and size during long term culture, the same set of different cell numbers was used for each method. Using phase-contrast microscopy, spheroids were monitored until day 20 and their sizes were determined. We observed that spheroids were formed within 24 hours regardless of the method and initial cell density. Further, in all groups the spheroid size was maximal at day 2, followed by a decline until day 20. Spheroids remained stable until day 20 independent of initial seeding concentration in all groups. We have generated long-term culture spheroids of SCC4 cells. The size of the spheroids can be influenced by varying the initial cell seeding density until day 20. This may be useful if different sizes of spheroids are required, e.g. in hypoxia research.

Valeh Rustamova,b, Rüdiger Rudolfa,b, Vugar Yagubluc, Hella-Monika Kuhna, Mario Vitacolonnaa,1 and Mathias Hafnera,b,1

aInstitute of Molecular and Cell Biology, Faculty of Biotechnology, Mannheim University
of Applied Sciences, Mannheim, Germany
bInstitute of Medical Technology, Heidelberg University, Heidelberg, Germany
cDepartment of General, Visceral, Vascular, and Thoracic Surgery, Frankfurt Hoechst Hospital,
Frankfurt, Germany

In: Journal of Cellular Biotechnology 3 (2017) 61–80

Abstract: Major pathomechanisms underlying neurodegenerative diseases, such as Parkinson’s Disease, are still not well understood. Induced human pluripotent and rodent embryonic stem cells provide powerful disease models to address neurodegeneration-inducing pathomechanisms on a molecular and cellular level. Our aim is to establish a refined protocol to generate healthy and patient donor stem cell-derived dopaminergic neurons to investigate neurodegenerative events in vitro.METHODSHuman healthy donor- and patient-derived induced pluripotent stem cells were differentiated into stable dopaminergic progenitor cell lines and further differentiated into dopaminergic neurons. Induced pluripotent stem cells, neuronal progenitors and terminally differentiated neurons were characterized by confocal laser microscopy-based immunofluorescence analysis, live cell imaging demonstrating dopamine transporter-specific uptake of a fluorescent substrate and transcriptome analysis. Based on our immunofluorescence analysis, dopaminergic differentiation approaches predominantly yield dopaminergic neurons and GFAP-expressing glial cells. We detected a small partition of GABAergic neurons, yet neither serotonergic nor glutamatergic neurons. Dopaminergic neurons were successfully stained for pre- and postsynaptic and mitochondrial markers. Live cell imaging experiments verified dopamine transporter-dependent uptake of the fluorescent monoamine transporter substrate ASP+. Human stem cell-derived dopaminergic neurons are a suitable cellular system for fluorescence-based experimental approaches to address neurodegenerative events in vitro.

Yasmina Martí a,b,1, Elina Nürnberga,c,1, Sandra Horschitzb, Mathias Hafnerc, Patrick Schlossb, Andreas Meyer-Lindenbergb and Thorsten Laua

aCentral Institute of Mental Health, Hector Institute for Translational Brain Research, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
bCentral Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Department Psychiatry and Psychotherapy, Mannheim, Germany
cInstitute of Molecular and Cell Biology, Mannheim University of Applied Sciences, Mannheim, Germany

In: Journal of Cellular Biotechnology 3 (2017) 21–39

Abstract: Skin fulfils a plethora of eminent physiological functions ranging from physical barrier over immunity shield to the interface mediating social interaction. Prone to several acquired and inherited diseases, skin is therefore a major target of pharmaceutical and cosmetic research. The lack of similarity between human and animal skin and rising ethical concerns in the use of animal models have driven the search for novel realistic three-dimensional skin models. This review provides a survey of contemporary skin models and compares them in terms of applicability, reliability, cost and complexity.

Julia Klicksa, Elena von Molitora, Torsten Ertongur-Fauthb, Rüdiger Rudolfc and Mathias Hafnerc

aMannheim University of Applied Sciences, Institute of Molecular and Cell Biology, Mannheim, Germany
bBRAIN AG, Zwingenberg, Germany
cInstitute of Medical Technology, Heidelberg University, Heidelberg, Germany