The combination of three different photoresists into a single direct laser written 3D microscaffold permits functionalization with two bioactive full-length proteins. The cell-instructive microscaffolds consist of a passivating framework equipped with light activatable constituents featuring distinct protein-binding properties. This allows directed cell attachment of epithelial or fibroblast cells in 3D.

Mimicking the properties of the extracellular matrix is crucial for developing in vitro models of the physiological microenvironment of living cells. Among other techniques, 3D direct laser writing (DLW) has emerged as a promising technology for realizing tailored 3D scaffolds for cell biology studies. Here, results based on DLW addressing basic biological issues, e.g., cell‐force measurements and selective 3D cell spreading on functionalized structures are reviewed. Continuous future progress in DLW materials engineering and innovative approaches for scaffold fabrication will enable further applications of DLW in applied biomedical research and tissue engineering.

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Diseases caused by protozoan parasites are responsible for considerable morbidity and mortality, especially in developing countries. The most prevalent parasitic disease is malaria, but leishmaniasis is also considered to be a genuine emerging disease, afflicting worldwide over 12 million people in 88 countries with an annual incidence of about 2 million (2). Lately, leishmaniasis has become better known to the industrialized countries after eight Americans were infected during Operation Desert Storm (11) and especially because of the highly problematic coincidence of visceral leishmaniasis and AIDS in southern Europe (1).The advancement of antileishmanial chemotherapy has been widely neglected in the past decades, leaving pentavalent antimonials, sodium stibogluconate, and meglumine antimonate as the first-line drugs for visceral and cutaneous leishmaniasis despite their variable efficacies and severe side effects (1). There is an obvious need for new drugs with structures and mechanisms of action different from those of drugs in use to date. Nature has been a source for important antiparasitic drugs in the past. Most of these are plant derived (e.g., quinine and artemisinin) (5, 19), but an increasing number have been isolated from microorganisms (amphotericin B and ivermectins) (20).The fungal metabolite aphidicolin (compound 1, Fig. 1 and Table 1) was isolated from Nigrospora sphaerica and was first described as a highly active drug for inhibiting cell division and synchronizing cell cycles in experimental medicine (10,14). Aphidicolin (compound 1) is a tetracyclic diterpene antibiotic with a bridged ring system rarely found among diterpenes. As reported in recent publications, aphidicolin has been tested for antiparasitic potential against Trypanosoma spp. (7, 17), Leishmania spp. (13, 18), and Entamoeba histolytica (12). Nolan (13) reported on selective inhibition of leishmanial and mammalian DNA polymerases. Furthermore, aphidicolin also possesses antineoplastic activity (3, 15). Aphidicolin is cytotoxic for neuroblastoma cells, while not significantly affecting the viability of normal cells (3). Its toxic dose in mice is quite high (60 mg/kg of body weight), indicating a wide pharmacological window.Despite the caveats, the antiparasitic efficacy and in vivo tolerance prompted us to further investigate the antileishmanial potential of aphidicolin and 17 of its semisynthetic derivatives. The parent aphidicolin structure was chemically modified at specific regions to allow a rational structure-activity analysis among this group of tetracyclic diterpenes derived from microbiological sources. MATERIALS AND METHODSCompounds. All compounds (Fig. 1) were produced by AnalytiCon AG, Potsdam, Germany. Purity was determined by high-performance liquid chromatography and nuclear magnetic resonance spectroscopy. Amphotericin B and miltefosin (Sigma, Munich, Germany) were used as standard drugs for positive controls. All compounds were first dissolved in dimethyl sulfoxide at 20 mg/ml and stored frozen before being dilu...

Patients suffering from acute myeloid leukemia (AML) show highly heterogeneous clinical outcomes. Next to variabilities in patient-specific parameters influencing treatment decisions and outcome, this is due to differences in AML biology. In fact, different genetic drivers may transform variable cells of origin and co-exist with additional genetic lesions (e.g., as observed in clonal hematopoiesis) in a variety of leukemic (sub)clones. Moreover, AML cells are hierarchically organized and contain subpopulations of more immature cells called leukemic stem cells (LSC), which on the cellular level constitute the driver of the disease and may evolve during therapy. This genetic and hierarchical complexity results in a pronounced phenotypic variability, which is observed among AML cells of different patients as well as among the leukemic blasts of individual patients, at diagnosis and during the course of the disease. Here, we review the current knowledge on the heterogeneous landscape of AML surface markers with particular focus on those identifying LSC, and discuss why identification and targeting of this important cellular subpopulation in AML remains challenging.

As mechanical properties of cell culture substrates matter, methods for mechanical characterization of scaffolds on a relevant length scale are required. We used multiple particle tracking microrheology to close the gap between elasticity determined from bulk measurements and elastic properties sensed by cells. Structure and elasticity of macroporous, three-dimensional cryogel scaffolds from mixtures of hyaluronic acid (HA) and collagen (Coll) were characterized. Both one-component gels formed homogeneous networks, whereas hybrid gels were heterogeneous in terms of elasticity. Most strikingly, local elastic moduli were significantly lower than bulk moduli presumably due to non-equilibrium chain conformations between crosslinks. This was more pronounced in Coll and hybrid gels than in pure HA gels. Local elastic moduli were similar for all gels, irrespective of their different swelling ratio and bulk moduli. Fibroblast cell culture proved the biocompatibility of all investigated compositions. Coll containing gels enabled cell migration, adhesion and proliferation inside the gels.

Mechanotransduction via yes-associated protein (YAP) is a central mechanism for decision-making in mouse embryonic stem cells (mESCs). Nuclear localization of YAP is tightly connected to pluripotency and increases the cell division rate (CDR). How the geometry of the extracellular environment influences mechanotransduction, thereby YAP localization, and decision-making of single isolated mESCs is largely unknown. To investigate this relation, we produced well-defined 2D and 2.5D microenvironments and monitored CDR and subcellular YAP localization in single mESCs hence excluding cell–cell interactions. By systematically varying size and shape of the 2D and 2.5D substrates we observed that the geometry of the growth environment affects the CDR. Whereas CDR increases with increasing adhesive area in 2D, CDR is highest in small 2.5D micro-wells. Here, mESCs attach to all four walls and exhibit a cross-shaped cell and nuclear morphology. This observation indicates that changes in cell shape are linked to a high CDR. Inhibition of actomyosin activity abrogate these effects. Correspondingly, nuclear YAP localization decreases in inhibitor treated cells, suggesting a relation between cell shape, intracellular forces, and cell division rate. The simplicity of our system guarantees high standardization and reproducibility for monitoring stem cell reactions and allows addressing a variety of fundamental biological questions on a single cell level.

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