Establishment of a Human Blood-Brain Barrier Co-culture Model Mimicking the Neurovascular Unit Using Induced Pluri- and Multipotent Stem Cells

Summary In vitro models of the human blood-brain barrier (BBB) are highly desirable for drug development. This study aims to analyze a set of ten different BBB culture models based on primary cells, human induced pluripotent stem cells (hiPSCs), and multipotent fetal neural stem cells (fNSCs). We systematically investigated the impact of astrocytes, pericytes, and NSCs on hiPSC-derived BBB endothelial cell function and gene expression. The quadruple culture models, based on these four cell types, achieved BBB characteristics including transendothelial electrical resistance (TEER) up to 2,500 Ω cm2 and distinct upregulation of typical BBB genes. A complex in vivo-like tight junction (TJ) network was detected by freeze-fracture and transmission electron microscopy. Treatment with claudin-specific TJ modulators caused TEER decrease, confirming the relevant role of claudin subtypes for paracellular tightness. Drug permeability tests with reference substances were performed and confirmed the suitability of the models for drug transport studies

The blood-brain barrier is dysregulated in COVID-19 and serves as a CNS entry route for SARS-CoV-2.

Neurological complications are common in COVID-19. Although SARS-CoV-2 has been detected in patients' brain tissues, its entry routes and resulting consequences are not well understood. Here, we show a pronounced upregulation of interferon signaling pathways of the neurovascular unit in fatal COVID-19. By investigating the susceptibility of human induced pluripotent stem cell (hiPSC)-derived brain capillary endothelial-like cells (BCECs) to SARS-CoV-2 infection, we found that BCECs were infected and recapitulated transcriptional changes detected in vivo. While BCECs were not compromised in their paracellular tightness, we found SARS-CoV-2 in the basolateral compartment in transwell assays after apical infection, suggesting active replication and transcellular transport of virus across the blood-brain barrier (BBB) in vitro. Moreover, entry of SARS-CoV-2 into BCECs could be reduced by anti-spike-, anti-angiotensin-converting enzyme 2 (ACE2)-, and anti-neuropilin-1 (NRP1)-specific antibodies or the transmembrane protease serine subtype 2 (TMPRSS2) inhibitor nafamostat. Together, our data provide strong support for SARS-CoV-2 brain entry across the BBB resulting in increased interferon signaling

Poly(2-ethyl-2-oxazoline-co-N-propylethylene imine)s by controlled partial reduction of poly(2-ethyl-2-oxazoline) : synthesis, characterization and cytotoxicity

The partial reduction of poly(2-ethyl-2-oxazoline) was investigated. A series of poly(2-ethyl-2-oxazoline-co-N-propylethylene imine)s were synthesized by direct reduction using lithium aluminum hydride or borane/dimethylsulfide (BH3/DMS), respectively. It is shown that the degree of reduction can be readily controlled either by the reaction time when using an excess of LiAlH4 or by the stoichiometry of BH3/DMS, as was demonstrated by H-1-NMR spectroscopy. Differential scanning calorimetry revealed that the glass transition temperature of the products decreased with increasing degree of reduction up to 25% of reduction, above which no glass transition could be detected. Moreover, acid-base titration showed a very pronounced, reduction degree dependent buffering capacity of these polymers between pH 4 and 8, which is of great interest, e.g. in the context of endosomal escape. This control over the reduction allows to tailor the synthesis of partially cationic polymers on the basis of poly(2-oxazoline)s, which, in combination over the hydrophilic/lipophilic balance through the side chain length allows a tight control over materials properties. Such materials may be interesting, inter alia, for biomaterials or organic electronics.Peer reviewe

Screening applications to test cellular fitness in transwell® models after nanoparticle treatment

Nanoparticles (NPs) in biotechnology hold great promise for revolutionizing medical treatments and therapies. In order to bring NPs into clinical application there is a number of preclinical in vitro and in vivo tests, which have to be applied before. The initial in vitro evaluation includes a detailed physicochemical characterization as well as biocompatibility tests, among others. For determination of biocompatibility at the cellular level, the correct choice of the in vitro assay as well as NP pretreatment is absolutely essential. There are a variety of assay technologies available that use standard plate readers to measure metabolic markers to estimate the number of viable cells in culture. Each cell viability assay has its own set of advantages and disadvantages. Regardless of the assay method chosen, the major factors critical for reproducibility and success include: (1) choosing the right assay after comparing optical NP properties with the read-out method of the assay, (2) verifying colloidal stability of NPs in cell culture media, (3) preparing a sterile and stable NP dispersion in cell culture media used in the assay, (4) using a tightly controlled and consistent cell model allowing appropriate characterization of NPs. This chapter will briefly summarize these different critical points, which can occur during biocompatibility screening applications of NPs

Establishment of a human blood-brain barrier co-culture model mimicking the neurovascular unit using induced pluri- and multipotent stem cells

In vitro models of the human blood-brain barrier (BBB) are highly desirable for drug development. This study aims to analyze a set of ten different BBB culture models based on primary cells, human induced pluripotent stem cells (hiPSCs), and multipotent fetal neural stem cells (fNSCs). We systematically investigated the impact of astrocytes, pericytes, and NSCs on hiPSC-derived BBB endothelial cell function and gene expression. The quadruple culture models, based on these four cell types, achieved BBB characteristics including transendothelial electrical resistance (TEER) up to 2,500 Ω cm2 and distinct upregulation of typical BBB genes. A complex in vivo-like tight junction (TJ) network was detected by freeze-fracture and transmission electron microscopy. Treatment with claudin-specific TJ modulators caused TEER decrease, confirming the relevant role of claudin subtypes for paracellular tightness. Drug permeability tests with reference substances were performed and confirmed the suitability of the models for drug transport studies. In this article, Metzger and colleagues present the establishment of physiologically relevant human blood-brain barrier quadruple culture models based on induced pluripotent and multipotent stem cells. The novel model can be used as a powerful tool in pharmaceutical drug research and ADMET studies, which may allow a more reliable translation of promising drug candidates into clinical application

In Vitro Blood-Brain Barrier Permeability and Cytotoxicity of an Atorvastatin-Loaded Nanoformulation Against Glioblastoma in 2D and 3D Models

Inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase of the family of statins have been suggested as therapeutic options in various tumors. Atorvastatin is a statin with the potential to cross the blood-brain barrier; however, the concentrations necessary for a cytotoxic effect against cancer cells exceed the concentrations achievable via oral administration, which made the development of a novel atorvastatin formulation necessary. We characterized the drug loading and basic physicochemical characteristics of micellar atorvastatin formulations and tested their cytotoxicity against a panel of different glioblastoma cell lines. In addition, activity against tumor spheroids formed from mouse glioma and mouse cancer stem cells, respectively, was evaluated. Our results show good activity of atorvastatin against all tested cell lines. Interestingly, in the three-dimensional (3D) models, growth inhibition was more pronounced for the micellar formulation compared to free atorvastatin. Finally, atorvastatin penetration across a blood-brain barrier model obtained from human induced-pluripotent stem cells was evaluated. Our results suggest that the presented micelles may enable much higher serum concentrations than possible by oral administration; however, if transport across the blood-brain barrier is sufficient to reach the therapeutic atorvastatin concentration for the treatment of glioblastoma via intravenous administration remains unclear.Peer reviewe

Calcium fluoride based multifunctional nanoparticles for multimodal imaging

New multifunctional nanoparticles (NPs) that can be used as contrast agents (CA) in different imaging techniques, such as photoluminescence (PL) microscopy and magnetic resonance imaging (MRI), open new possibilities for medical imaging, e.g., in the fields of diagnostics or tissue characterization in regenerative medicine. The focus of this study is on the synthesis and characterization of CaF2:(Tb3+,Gd3+) NPs. Fabricated in a wet-chemical procedure, the spherical NPs with a diameter of 5–10 nm show a crystalline structure. Simultaneous doping of the NPs with different lanthanide ions, leading to paramagnetism and fluorescence, makes them suitable for MR and PL imaging. Owing to the Gd3+ ions on the surface, the NPs reduce the MR T1 relaxation time constant as a function of their concentration. Thus, the NPs can be used as a MRI CA with a mean relaxivity of about r = 0.471 mL·mg−1·s−1. Repeated MRI examinations of four different batches prove the reproducibility of the NP synthesis and determine the long-term stability of the CAs. No cytotoxicity of NP concentrations between 0.5 and 1 mg·mL−1 was observed after exposure to human dermal fibroblasts over 24 h. Overall this study shows, that the CaF2:(Tb3+,Gd3+) NPs are suitable for medical imaging

In Vitro Blood-Brain-Barrier Permeability and Cytotoxicity of Atorvastatin-Loaded Nanoformulation Against Glioblastoma in 2D and 3D Models

Inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase of the family of statins have been suggested as therapeutic options in various tumors. Atorvastatin is a statin with potential to cross the blood-brain-barrier, however, the concentrations necessary for a cytotoxic effect against cancer cells exceeds the concentration achievable via oral administration, which made the development of a novel atorvastatin formulation necessary. We characterized the drug loading and basic physicochemical characteristics of micellar atorvastatin formulations and tested their cytotoxicity against a panel of different glioblastoma cell lines. In addition, activity against tumor spheroids formed from mouse glioma and mouse cancer stem cells, respectively, was evaluated. Our results show good activity of atorvastatin against all tested cell lines. Interestingly, in the 3D models, growth inhibition was more pronounced for the micellar formulation compared to free atorvastatin. Finally, atorvastatin penetration across a blood-brain-barrier model obtained from human induced-pluripotent stem cells was evaluated. Our results suggest that the presented micelles may enable much higher serum concentrations than possible by oral administration, however, if transport across the blood-brain-barrier is sufficient to reach therapeutic atorvastatin concentration for the treatment of glioblastoma via intravenous administration remains unclear.<br /