| 1. |
- Banis, G. E., et al.
(författare)
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The binding effect of proteins on medications and its impact on electrochemical sensing : Antipsychotic clozapine as a case study
- 2017
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Ingår i: Pharmaceuticals. - : MDPI AG. - 1424-8247. ; 10:3
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Tidskriftsartikel (refereegranskat)abstract
- Clozapine (CLZ), a dibenzodiazepine, is demonstrated as the optimal antipsychotic for patients suffering from treatment-resistant schizophrenia. Like many other drugs, understanding the concentration of CLZ in a patient’s blood is critical for managing the patients’ symptoms, side effects, and overall treatment efficacy. To that end, various electrochemical techniques have been adapted due to their capabilities in concentration-dependent sensing. An open question associated with electrochemical CLZ monitoring is whether drug–protein complexes (i.e., CLZ bound to native blood proteins, such as serum albumin (SA) or alpha-1 acid-glycoprotein (AAG)) contribute to electrochemical redox signals. Here, we investigate CLZ-sensing performance using fundamental electrochemical methods with respect to the impact of protein binding. Specifically, we test the activity of bound and free fractions of a mixture of CLZ and either bovine SA or human AAG. Results suggest that bound complexes do not significantly contribute to the electrochemical signal for mixtures of CLZ with AAG or SA. Moreover, the fraction of CLZ bound to protein is relatively constant at 31% (AAG) and 73% (SA) in isolation with varying concentrations of CLZ. Thus, electrochemical sensing can enable direct monitoring of only the unbound CLZ, previously only accessible via equilibrium dialysis. The methods utilized in this work offer potential as a blueprint in developing electrochemical sensors for application to other redox-active medications with high protein binding more generally. This demonstrates that electrochemical sensing can be a new tool in accessing information not easily available previously, useful toward optimizing treatment regimens.
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| 2. |
- Elhami Nik, Farzad, et al.
(författare)
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Low-Cost PVD Shadow Masks with Submillimeter Resolution from Laser-Cut Paper
- 2020
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Ingår i: Micromachines. - Basel : MDPI. - 2072-666X. ; 11:7
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Tidskriftsartikel (refereegranskat)abstract
- We characterize an affordable method of producing stencils for submillimeter physical vapor deposition (PVD) by using paper and a benchtop laser cutter. Patterning electrodes or similar features on top of organic or biological substrates is generally not possible using standard photolithography. Shadow masks, traditionally made of silicon-based membranes, circumvent the need for aggressive solvents but suffer from high costs. Here, we evaluate shadow masks fabricated by CO2 laser processing from quantitative filter papers. Such papers are stiff and dimensionally stable, resilient in handling, and cut without melting or redeposition. Using two exemplary interdigitated electrode designs, we quantify the line resolution achievable with both high-quality and standard lenses, as well as the positional accuracy across multiple length scales. Additionally, we assess the gap between such laser-cut paper masks and a substrate, and quantify feature reproduction onto polycarbonate membranes. We find that ~100 µm line widths are achievable independent of lens type and that average positional accuracy is better than ±100 µm at 4”-wafer scale. Although this falls well short of the micron-size features achievable with typical shadow masks, resolution in the tenths to tens of millimeters is entirely sufficient for applications from contact pads to electrochemical cells, allowing new functionalities on fragile materials.
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| 3. |
- Jury, Michael, 1984-, et al.
(författare)
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Bioorthogonally Cross‐Linked Hyaluronan–Laminin Hydrogels for 3D Neuronal Cell Culture and Biofabrication
- 2022
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Ingår i: Advanced Healthcare Materials. - : Wiley. - 2192-2640 .- 2192-2659. ; 11:11
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Tidskriftsartikel (refereegranskat)abstract
- Laminins (LNs) are key components in the extracellular matrix of neuronal tissues in the developing brain and neural stem cell niches. LN-presenting hydrogels can provide a biologically relevant matrix for the 3D culture of neurons toward development of advanced tissue models and cell-based therapies for the treatment of neurological disorders. Biologically derived hydrogels are rich in fragmented LN and are poorly defined concerning composition, which hampers clinical translation. Engineered hydrogels require elaborate and often cytotoxic chemistries for cross-linking and LN conjugation and provide limited possibilities to tailor the properties of the materials. Here a modular hydrogel system for neural 3D cell cultures, based on hyaluronan and poly(ethylene glycol), that is cross-linked and functionalized with human recombinant LN-521 using bioorthogonal copper-free click chemistry, is shown. Encapsulated human neuroblastoma cells demonstrate high viability and grow into spheroids. Long-term neuroepithelial stem cells (lt-NES) cultured in the hydrogels can undergo spontaneous differentiation to neural fate and demonstrate significantly higher viability than cells cultured without LN. The hydrogels further support the structural integrity of 3D bioprinted structures and maintain high viability of bioprinted and syringe extruded lt-NES, which can facilitate biofabrication and development of cell-based therapies.
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| 4. |
- Last, Torben, et al.
(författare)
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Self-sealing MEMS spray-nozzles to prevent bacterial contamination of portable inhalers for aqueous drug delivery
- 2022
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Ingår i: Biomedical microdevices (Print). - : Springer Nature. - 1387-2176 .- 1572-8781. ; 24:3
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Tidskriftsartikel (refereegranskat)abstract
- Pulmonary drug delivery by portable inhalers is the gold standard in lung disease therapy. An increasing focus on environmentally friendly inhalation currently spurs the development of propellant-free devices. However, the absence of propellants in the drug creates a need for suitable sealing systems that can ensure the pathogenic safety of devices. Traditionally, liquid drug inhalers incorporate a spray nozzle and a separate check valve. Here we show a fully integrated MEMS -based spray system for aqueous drug solutions and demonstrate its bacterial safety. The device comprises a thin silicon membrane with spray orifices, which self-seal against a compliant parylene valve seat underneath. This sealing system prevents bacterial ingrowth in its default closed state, while actuation lifts the membrane from the valve seat upon pressurization and sprays an inhalable aerosol from the nozzles. To seal against bacterial contamination effectively, we found that a contact force between the valve seat and the membrane (featuring the spray nozzles) is needed. In our testing, both self-sealing and an otherwise identical unvalved version of the spray chip can be bacterially safe in continued use when thoroughly cleaned of excess fluids and subjected to low bacterial loads for brief periods. However, when directly exposed to 10(7) CFU/ml of our test organism Citrobacter rodentium for 24 h, unvalved systems become contaminated in nearly 90% of cases. In contrast, self-sealing spray chips reduced contamination probability by 70%. This development may enable preservative-free drug formulations in portable inhalers that use propellant-free aqueous drug solutions.
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| 5. |
- Matthiesen, Isabelle, et al.
(författare)
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Astrocyte 3D culture and bioprinting using peptide functionalized hyaluronan hydrogels
- 2023
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Ingår i: Science and Technology of Advanced Materials. - : Informa UK Limited. - 1468-6996 .- 1878-5514. ; 24:1
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Tidskriftsartikel (refereegranskat)abstract
- Astrocytes play an important role in the central nervous system, contributing to the development of and maintenance of synapses, recycling of neurotransmitters, and the integrity and function of the blood-brain barrier. Astrocytes are also linked to the pathophysiology of various neurodegenerative diseases. Astrocyte function and organization are tightly regulated by interactions mediated by the extracellular matrix (ECM). Engineered hydrogels can mimic key aspects of the ECM and can allow for systematic studies of ECM-related factors that govern astrocyte behaviour. In this study, we explore the interactions between neuroblastoma (SH-SY5Y) and glioblastoma (U87) cell lines and human fetal primary astrocytes (FPA) with a modular hyaluronan-based hydrogel system. Morphological analysis reveals that FPA have a higher degree of interactions with the hyaluronan-based gels compared to the cell lines. This interaction is enhanced by conjugation of cell-adhesion peptides (cRGD and IKVAV) to the hyaluronan backbone. These effects are retained and pronounced in 3D bioprinted structures. Bioprinted FPA using cRGD functionalized hyaluronan show extensive and defined protrusions and multiple connections between neighboring cells. Possibilities to tailor and optimize astrocyte-compatible ECM-mimicking hydrogels that can be processed by means of additive biofabrication can facilitate the development of advanced tissue and disease models of the central nervous system.
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| 6. |
- Matthiesen, Isabelle, et al.
(författare)
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Continuous Monitoring Reveals Protective Effects of N‐Acetylcysteine Amide on an Isogenic Microphysiological Model of the Neurovascular Unit
- 2021
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Ingår i: Small. - : Wiley. - 1613-6810 .- 1613-6829. ; 17:32, s. 2101785-
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Tidskriftsartikel (refereegranskat)abstract
- Microphysiological systems mimic the in vivo cellular ensemble and microenvironment with the goal of providing more human-like models for biopharmaceutical research. In this study, the first such model of the blood-brain barrier (BBB-on-chip) featuring both isogenic human induced pluripotent stem cell (hiPSC)-derived cells and continuous barrier integrity monitoring with <2 min temporal resolution is reported. Its capabilities are showcased in the first microphysiological study of nitrosative stress and antioxidant prophylaxis. Relying on off-stoichiometry thiol–ene–epoxy (OSTE+) for fabrication greatly facilitates assembly and sensor integration compared to the prevalent polydimethylsiloxane devices. The integrated cell–substrate endothelial resistance monitoring allows for capturing the formation and breakdown of the BBB model, which consists of cocultured hiPSC-derived endothelial-like and astrocyte-like cells. Clear cellular disruption is observed when exposing the BBB-on-chip to the nitrosative stressor linsidomine, and the barrier permeability and barrier-protective effects of the antioxidant N-acetylcysteine amide are reported. Using metabolomic network analysis reveals further drug-induced changes consistent with prior literature regarding, e.g., cysteine and glutathione involvement. A model like this opens new possibilities for drug screening studies and personalized medicine, relying solely on isogenic human-derived cells and providing high-resolution temporal readouts that can help in pharmacodynamic studies.
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| 7. |
- Matthiesen, Isabelle, et al.
(författare)
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Metabolic Assessment of Human Induced Pluripotent Stem Cells-Derived Astrocytes and Fetal Primary Astrocytes : Lactate and Glucose Turnover
- 2022
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Ingår i: Biosensors. - : MDPI AG. - 2079-6374. ; 12:10, s. 839-
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Tidskriftsartikel (refereegranskat)abstract
- Astrocytes represent one of the main cell types in the brain and play a crucial role in brain functions, including supplying the energy demand for neurons. Moreover, they are important regulators of metabolite levels. Glucose uptake and lactate production are some of the main observable metabolic actions of astrocytes. To gain insight into these processes, it is essential to establish scalable and functional sources for in vitro studies of astrocytes. In this study, we compared the metabolic turnover of glucose and lactate in astrocytes derived from human induced pluripotent stem cell (hiPSC)-derived Astrocytes (hiAstrocytes) as a scalable astrocyte source to human fetal astrocytes (HFAs). Using a user-friendly, commercial flow-based biosensor, we could verify that hiAstrocytes are as glycogenic as their fetal counterparts, but their normalized metabolic turnover is lower. Specifically, under identical culture conditions in a defined media, HFAs have 2.3 times higher levels of lactate production compared to hiAstrocytes. In terms of glucose, HFAs have 2.1 times higher consumption levels than hiAstrocytes at 24 h. Still, as we describe their glycogenic phenotype, our study demonstrates the use of hiAstrocytes and flow-based biosensors for metabolic studies of astrocyte function.
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| 8. |
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| 9. |
- Matthiesen, Isabelle
(författare)
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Recreating the microenvironment of the neurovascular unit
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The neurovascular unit (NVU) comprises the blood-brain-barrier (BBB) and its surrounding astrocytes, pericytes and neurons that are embedded in the extracellular matrix (ECM). As the main function of the BBB is to protect the brain from inlet of pathogens and toxins, the specialized endothelial cells that keep the barrier tight will also hinder the passage of pharmaceuticals. Understanding the detailed microenvironment and cellular interactions involved in the development of the neurovascular unit is, therefore, an important step towards designing CNS-targeting pharmaceuticals that can pass into the brain. At the same time, the initial steps of pharmaceutical development often involve the use of animal based in vitro models with poor human translation; thus, there is a great need for novel methods to better mimic the complexity of the human NVU. Apart from conventional cell culture models, the use of micro-engineered devices, microphysiological systems (MPS), have gained popularity. The use of MPS allows for fabrication of tissue-like structures using stem cells and provide more in vivo-like parameters in terms of physical cues and dynamic flow. Various materials have been explored for chip fabrication, and biological and synthetic ECM-mimicking hydrogels have been developed for cell encapsulation. Unfortunately, models developed to date often lack either: i) relevant and reproducible cell sources, ii) materials that allow for easy chip fabrication where sensors can be integrated to understand metabolic effects and barrier integrity, or iii) animal-free defined ECM-mimicking scaffolds that support the culture of sensitive cells. This thesis presents an isogenic model of the BBB using iPSC-derived endothelial cells and astrocytes cultured in a MPS made from the non-absorbing polymer OSTE+ that allows for easy fabrication and integration of interdigitated gold electrodes for continuous barrier integrity monitoring. The model presents barrier-protective effects of the BBB-penetrating drug NACA. To better understand the metabolic attributes of astrocytes, a flow-cell sensor is evaluated for the measurement of glucose and lactate turnover during a ketogenic diet. The results imply that such a sensor is valuable for the measurement of metabolic changes and can, in the future, be integrated into MPSs.Furthermore, a model of early neuronal development is realized by using defined copper-free click chemistry to conjugate laminin to a hyaluronic-based hydrogel system for the differentiation of neuroepithelial stem cells. The use of the hydrogel is validated for bioprinting, and the first-ever printed neuroepithelial stem cells are presented. In another study astrocyte 3D culture and bioprinting is evaluated in peptide conjugated hyaluronic-based hydrogels. Unique attachment and spreading of human fetal astrocytes is observed while the common glioblastoma U87 cells display a rounded up morphology. The results of the hydrogel study imply that the defined chemistry of the hydrogel is suitable for both neuroepithelial stem cells, U87 and fetal primary astrocytes, and can in the future be integrated into MPS to circumvent the use of animal derived matrices. In summary, these results provide solutions to some of the problems to date and lay the ground work for the continuation of the development of human-relevant MPS of the NVU.
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| 10. |
- Winkler, Thomas, Ph.D., et al.
(författare)
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Continuous monitoring of isogenic blood-brain barrier integrity in a pdms-free microphysiological system
- 2020
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Ingår i: MicroTAS 2020 - 24th International Conference on Miniaturized Systems for Chemistry and Life Sciences. - : Chemical and Biological Microsystems Society. ; , s. 963-964
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Konferensbidrag (refereegranskat)abstract
- We present a microphysiological system (MPS) of the human blood-brain barrier (BBB) that uniquely combines three key advantages - continuous monitoring, PDMS-free fabrication, and cellular relevance - over existing MPS, and we demonstrate BBB formation, breakdown, and rescue. Specifically, we model nitrosative stress - strongly implicated in brain-related disorders from stroke to Alzheimer's - and its prevention using a BBB-permeable antioxidant. This kind of MPS paves the way toward patient-specific BBB modeling for time-resolved studies of drug kinetics and/or side effects towards personalized treatment planning.
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