Biology subjects

Winkler, T. E.

Publications and source records attributed to Winkler, T. E..

7 recordsLinked to original sources

bioRxiv2026

Overcoming oxygen impermeability in PDMS-free organ-on-a-chip microfluidics with nanoporous thermoplastic

Oxygen availability is a critical yet all-too-often overlooked variable in organ-on-a-chip (OoC) systems. PDMS-based microfluidics remain the most common approach to facilitating oxygen equilibration with the incubator environment, but the materials tendency to ad- and absorb small hydrophobic molecules can pose significant concerns for pharmacological and toxicological studies. Yet there remains a lack of alternative gas-exchange materials feasible for OoC integration, even as the use of thermoplastic microfluidics in particular has otherwise proliferated. Here, we present commercially available track-etched nanoporous polycarbonate (50 nm pores, 1.18% porosity, [~]0.1 {euro}/cm2) as a practical alternative to polydimethylsiloxane (PDMS) for gas exchange in OoC. We show that nanoporous polycarbonate provides a thermoplastic material with an oxygen permeability of 3290 {+/-} 240 fs mol / kg, over an order of magnitude higher than PDMS. We demonstrate integration into existing lamination-based thermoplastic microfluidic fabrication workflows with sustained leak-free operation well above physiologically relevant pressures. We find that nanoporous polycarbonate does not compromise cell viability, but that high water vapor permeance necessitates a high-humidity environment around the device - though thickness-normalized water vapor permeability is notably similar to PDMS. We validate the OoC application with Caco-2 intestinal epithelial cells by monitoring oxygen levels during the critical cell attachment phase, with nanoporous polycarbonate allowing for maintenance of stable oxygen tension, in stark contrast to severe hypoxia in nonporous controls within 30 minutes. We further show that this uncontrolled hypoxia correlates with a time-delayed increase in cellular hypoxia inducible factor-1 reporter expression. Overall, our findings position nanoporous polycarbonate as a low-cost, mechanically robust, and fabrication-friendly alternative that can bring controlled oxygen availability to PDMS-free microfluidics and OoC.

bioengineering↗

bioRxiv2024

Single-cell proteomics of Arabidopsis leaf mesophyll identifies drought stress-related proteins

The application of single-cell omics tools to biological systems can provide unique insights into diverse cellular populations and their heterogeneous responses to internal and external perturbations. Thus far, most single-cell studies in plant systems have been limited to RNA-sequencing approaches, which only provide indirect readouts of cellular functions. Here, we present a single-cell proteomics workflow for plant cells that integrates tape-sandwich protoplasting, piezoelectric cell sorting, nanoPOTS sample preparation, and FAIMS-based MS data acquisition method for label-free single-cell proteomics analysis of Arabidopsis leaf mesophyll cells. From a single leaf protoplast, over 3,000 proteins were quantified with high precision. The workflow is demonstrated to identify stress associated changes in protein abundance by analyzing >80 protoplasts from well-watered and water-deficit stressed plants. Additionally, we describe a new approach for constructing covarying protein networks at the single-cell level and demonstrate how single-cell protein covariation analysis can reveal previously unrecognized protein functions while also capturing stress-induced changes in protein-protein dynamics. HighlightsO_LIThis study describes the first application of scProteomics to study abiotic stress-induced proteome regulation in leaf mesophyll cells C_LIO_LIFrom a single Arabidopsis mesophyll protoplast, [~]2800 proteins on average were quantified with high precision using label free scProteomics C_LIO_LIscProteomics of water-deficit Arabidopsis leaves revealed known and novel proteins involved in drought response C_LIO_LISingle-protoplast proteomics revealed water deficit-induced changes in protein covariation networks independent of changes in abundance C_LI

plant biology↗

bioRxiv2022

Astrocyte 3D Culture and Bioprinting using Peptide Functionalized Hyaluronan Hydrogels

The often-forgotten astrocytes play an important role in the central nervous system, contributing to the development of and maintenance of synapses, recycling of neurotransmitters, and the pathophysiology of various neurodegenerative diseases. Hydrogels can provide improved support and attachment for the culture of astrocytes in 3D models, which could further be used to advance clinical in vivo like tissue models of numerous diseases. For full applicability, these gels must be of scalable and defined origin and with stable attachment elements, such as peptides. In this study, the generation of a functional 3D astrocyte model is presented using a hyaluronan-based hydrogel system conjugated with the peptide sequences cyclic RGD (cRGD) and IKVAV, known promoters of cell attachment. Encapsulation of the neuroblastoma cell line SH-SY5Y and glioblastoma cell line U87 is successfully demonstrated over a 6-day culture period. The presence of the peptides cRGD and IKVAV does not change the cells viability. Human fetal primary astrocytes (FPA) are further tested for the 3D culture in these materials, similarly, showing that the peptides have no effect on the viability over a 6-day culture period. mRNA expression analysis reveals no biologically significant changes in the 3D cultures FPA or the U87 cells. Morphological analysis, on the other hand, revealed that FPA have a higher degree of interactions with the hyaluronan-based gels compared to the cell lines. This interaction is enhanced by peptide conjugation, in particular cRGD. Finally, we demonstrated that the peptide conjugated hydrogels could be used for bioprinting of FPA, opening up for defined neural astrocytic co-culture.

bioengineering↗

bioRxiv2021

Bioorthogonally Cross-Linked Hyaluronan-Laminin Hydrogels for 3D Neuronal Cell Culture and Biofabrication

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 towards 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 we show a modular hydrogel system for neural 3D cell culture, based on hyaluronan (HA) and poly(ethylene glycol) (PEG), that is cross-linked and functionalized with human recombinant LN 521 using bioorthogonal copper-free click chemistry. Encapsulated human neuroblastoma cells demonstrate high viability and grow into spheroids. 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 syringe extruded lt-NES, which can facilitate the development of advanced neuronal tissue and disease models and translation of stem cell-based therapies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/461549v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1ef2167org.highwire.dtl.DTLVardef@79f22eorg.highwire.dtl.DTLVardef@ae8d3org.highwire.dtl.DTLVardef@3ba3da_HPS_FORMAT_FIGEXP M_FIG The authors present an extracellular matrix mimicking hydrogel for 3D culture of neural cell models. Based on hyaluronic acid and poly(ethylene glycol), the hydrogel immobilizes recombinant laminin 521, associated with neuronal development. The study demonstrates support of neuroblastoma cell viability, spontaneous human neuroepithelial stem cell differentiation, and the protective effect of the hydrogels during bioprinting and syringe needle ejection. C_FIG

bioengineering↗

bioRxiv2021

Sorption of neuropsychopharmaca in microfluidic materials for in-vitro studies

Sorption (i.e., ad- & ab-sorption) of small-molecule compounds to polydimethylsiloxane (PDMS) is widely acknowledged. However, studies to date have largely been conducted under atypical conditions for microfluidic applications (lack of perfusion, lack of biological fluids); especially considering the biological studies such as Organs-on-Chips where small-molecule sorption poses the largest concern. Here, we present the first study of small-molecule sorption under relevant conditions for microphysiological systems, focusing on a standard geometry for biological barrier studies that find application in pharmacokinetics. We specifically assess the sorption of a compound panel including 15 neuropsychopharmaca at in-vivo concentration levels. We consider devices constructed from PDMS as well as two material alternatives (off-stoichiometry thiol-ene-epoxy, or tape/polycarbonate laminates). Moreover, we study the much-neglected impact of peristaltic pump tubing, an essential component of the recirculating systems required to achieve in-vivo-like perfusion shear stresses. We find that choice of device material does not significantly impact sorption behavior in our barrier-on-chip-type system. Our PDMS observations in particular suggest that excessive compound sorption observed in prior studies is not sufficiently described by compound hydrophobicity or other suggested predictors. Critically, we show that sorption by peristaltic tubing, including the commonly-utilized PharMed BPT, dominates over device sorption even on an area-normalized basis, let alone at the typically much larger tubing surface areas. Our findings highlight the importance of validating compound dosages in Organ-on-Chip studies, as well as the need for considering tubing materials with equal or higher care than device materials.

bioengineering↗

bioRxiv2021

Continuous monitoring reveals protective effects of N-acetylcysteine amide on an isogenic microphysiological model of the neurovascular unit

Microphysiological systems mimic the in-vivo cellular ensemble and microenvironment with the goal of providing more human-like models for biopharmaceutical research. We report 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-minute temporal resolution. We showcase its capabilities 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 us to capture formation and breakdown of our blood-brain barrier model, consisting of co-cultured hiPSC-derived endothelial-like and astrocyte-like cells. We observe clear cellular disruption when exposing the BBB-on-chip to the nitrosative stressor linsidomine, and report on the barrier permeability and barrier-protective effects of the antioxidant N-acetylcysteine amide. Using metabolomic network analysis, we further find drug-induced changes consistent with prior literature regarding, e.g., cysteine and glutathione involvement. A model like ours 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.

bioengineering↗

bioRxiv2020

Low-cost microphysiological systems: Feasibility study of a tape-based barrier-on-chip system for small intestine modeling

We see affordability as a key challenge in making organs-on-chips accessible to a wider range of users, particularly outside the highest-resource environments. Here, we present an approach to barrier-on-a-chip fabrication based on double-sided pressure-sensitive adhesive tape and off-the-shelf polycarbonate. Besides a low materials cost, common also to PDMS or thermoplastics, it requires minimal ({euro} 100) investment in laboratory equipment, yet at the same time is suitable for upscaling to industrial roll-to-roll manufacture. We evaluate our microhpysiological system with an epithelial (C2BBe1) barrier model of the small intestine, studying the biological effects of permeable support pore size, as well as stimulation with a common food compound (chili pepper-derived capsaicinoids). The cells form tight and continuous barrier layers inside our systems, with comparable permeability but superior epithelial polarization compared to Transwell culture, in line with other perfused microphysiological models. Permeable support pore size is shown to weakly impact barrier layer integrity as well as the metabolic cell profile. Capsaicinoid response proves distinct between culture systems, but we show that impacted metabolic pathways are partly conserved, and that cytoskeletal changes align with previous studies. Overall, our tape-based microphysiolgical system proves to be a robust and reproducible approach to studying physiological barriers, in spite of its low cost.

bioengineering↗