Biology subjects

Jacak, J.

Publications and source records attributed to Jacak, J..

8 recordsLinked to original sources

bioRxiv2025

Hybrid MPL Scaffolds with Nanoscale Mechanobiology for Bone-on-Chip

Engineering physiologically relevant 3D microenvironments is critical for studying cell behavior and advancing regenerative medicine. We present a new hybrid scaffold, fabricated via MultiPhoton Lithography (MPL), that integrates synthetic polymers (BisSR/CEA) with methacrylated collagen type I (Coll-MA) for single-cell enclosure and long-term culture. This is the first demonstration of a 3D MPL-printed biodegradable scaffold that mimics bone-like stiffness and allows spatially controlled, biodegradation-driven remodeling. The nanoscale feature size and mechanical properties are validated using Atomic Force Microscopy (AFM), while the nanoscale bioactivity of the scaffold is confirmed through Single-Molecule Localization Microscopy (SMLM). We track vinculin, a focal adhesion protein, with single-molecule resolution during mesenchymal stem cell (MSC) expansion and osteogenic differentiation. A new finding is time-dependent axial migration of vinculin clusters, independent of scaffold composition. Despite similar mechanosensing profiles, hybrid scaffolds significantly enhance osteogenic marker expression (collagen I, osteocalcin), revealing that scaffold bioactivity and geometry, not stiffness alone, direct stem cell fate. Cell expansion is highly dependent on scaffold composition, showing a biodegradation-driven remodeling over time. This platform offers a new tool to study cell-matrix interactions at the single-cell and single-molecule level and holds promise for Organ-on-Chip systems (e.g. bone-cartilage interface models), and personalized regenerative therapies.

bioengineering↗

bioRxiv2025

Bactericidal Membrane Attack Complex formation initiates at the new pole of E. coli

Human immune protection against bacteria critically depends on activation of the complement system. The direct bacteriolytic activity of complement molecules against Gram-negative bacteria acts via the formation of Membrane Attack Complex (MAC) pores. Bactericidal MAC pores damage the bacterial outer membrane, leading to destabilization of the inner membrane. Although it is well-established that inner membrane damage is crucial for bacterial cell death, the critical event causing MAC-mediated inner membrane damage remains elusive. Here we questioned whether the bacterial cell envelope possesses vulnerable spots for MAC pores to insert. We followed the localization of MAC pores on E. coli over time using fluorescence microscopy and elucidate that MAC deposition initiates at the new bacterial pole, which induces inner membrane damage and halts bacterial division. MAC components C8 and C9 preferentially localize at new bacterial poles, while C3b localizes randomly on the bacterial surface. This suggests that preferential MAC localization is determined by one of the initial steps of MAC formation. These findings provide valuable information about the interplay between immune components and the Gram-negative cell envelope.

microbiology↗

bioRxiv2025

3D Multiphoton Nanolithography with Bioresorbable Amino Acid-Based Resins

We demonstrate that the newly designed amino acid phosphordiamidate-resins (APdA), containing vinyl reactive groups for polymerization, can be utilized to fabricate sub - 100 nm features through 3D multiphoton lithography. We have quantitatively analyzed the feature size, Youngs modulus, and functionalization of the nanostructures using atomic force and single-molecule fluorescence microscopy. Our results indicate that the polymer backbone, composed of either valine or alanine, imparts hydrophobic properties to the monomer, restricting the swelling of the polymeric nanostructure to 8% in aqueous environments. Despite minimal swelling, experiments revealed an up to 10-fold change of Youngs modulus for dry versus wet conditions. To enhance the versatility of the APdA-based structures, we incorporated biotin functionalization and utilized it for the immobilization of extracellular vesicles. Hence, these findings highlight the potential of APdA-based nanolithography photoresists for biomedicine and nanotechnology applications.

biophysics↗

bioRxiv2024

Immortalization of mesenchymal stromal cells by hTERT does not affect the functional properties of secreted extracellular vesicles

Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) have emerged as promising and safe therapeutic agents, however, donor heterogeneities, limited replicative life span and changes in the cellular phenotype throughout in vitro cultivation remain major hurdles for scalable EV production. For these reasons, this study aims to investigate the use of hTERT immortalized ( telomerized) MSCs as a potential source for efficient, standardized, reliable MSC-EVs production by comparing parental primary to their telomerized MSC counterparts. We observed that hTERT expression does not affect cell morphology or cellular doubling time, while ensuring unlimited, stable in vitro propagation. In addition, telomerized WJ-MSCs maintained the canonical expression profile of surface markers and the tri-lineage differentiation potential of their primary counterparts. In terms of EV characteristics, the immortalization by hTERT expression did not affect size, number, cargo composition or biological activity regarding anti-inflammatory, anti-fibrotic and wound healing properties in vitro. In summary, the use of hTERT to immortalize MSCs leads to the creation of cell lines that continuously produce MSC-EVs without altering any key functionalities of the cells or resulting EVs. This suggests that telomerization of human cells from single donors is a promising strategy for generating cell factories that can produce EVs in standardized conditions and at scale and with standardization.

cell biology↗

bioRxiv2024

Snorkel-tag Based Affinity Chromatography for Recombinant Extracellular Vesicle Purification

Extracellular vesicles (EVs) are lipid nanoparticles and play an important role in cell-cell communications, making them potential therapeutic agents and allowing to engineer for targeted drug delivery. The expanding applications of EVs in next generation medicine are still limited by existing tools for scaling standardized EV production, single EV tracing and analytics, and thus provide only a snapshot of tissue-specific EV cargo information. Here, we present CD81, an EV surface marker protein, genetically fused to series of tags with additional transmembrane domain to be displayed on the EV surface, which we term Snorkel-tag. This system enables to affinity purify EVs from complex matrices in a non-destructive form. In future applications, this strategy will allow generating transgenic animals to enable tracing and analyzing EVs, and their cargo in physiological and pathophysiological set-ups, and facilitate the development of EV based diagnostic tools in murine models which can be translated to humans.

bioengineering↗

bioRxiv2023

Explanation of saltatory conduction in myelinated axons and of micro-saltatory conduction in C fibers of pain sensation by a wave-type ion plasmonic mechanism of stimulus kinetics

Ion plasmon-polariton model of stimulus in myelinated axons and in C fibers of pain sensation is developed. This solves a long standing problem in neuroscience of by 2 - 3 orders of magnitude discrepancy between the observed fast speed of the saltatory conduction in myelinated axons or in C fibers with the upper limit of diffusive ion current velocity in these axons. The latter, described in the framework of so-called cable model, is too low in axons because of poor conductivity of neuron inner cytosol. The compliance with observations has been achieved upon plasmonic model of ionic local oscillations synchronized in periodically corrugated axons and propagating with high speed in the form of wave-type plasmon-polariton without any net diffusion current, thus not limited by resistivity. The new model of stimulus in myelinated axons reveals the different controlling role of myelin than previously thought from cable model. The control mechanism in non-myelinated C fibers is also proposed in agreement with observations. Recognition of plasmon model of neural signaling may be important for identifying a new targets for the future treatment at demyelination diseases and for fighting pain.

neuroscience↗

bioRxiv2020

Comprehensive label-free characterization of extracellular vesicles and their surface proteins

Interest in mesenchymal stem cell derived extracellular vesicles (MSC-EVs) as therapeutic agents has dramatically increased over the last decade. Preclinical studies show that MSC-EVs have anti-apoptotic and neuroprotective effects, boost wound healing, and improve the integration of allogeneic grafts through immunomodulation. Current approaches to the characterization and quality control of EV-based therapeutics include particle tracking techniques, Western blotting, and advanced cytometry, but standardized methods are lacking. In this study, we established and verified quartz crystal microbalance (QCM) as highly sensitive label-free immunosensing technique for characterizing clinically approved umbilical cord MSC-EVs enriched by tangential flow filtration and ultracentrifugation. Using QCM in conjunction with common characterization methods, we were able to specifically detect EVs via EV (CD9, CD63, CD81) and MSC (CD44, CD49e, CD73) markers and gauge their prevalence. Additionally, we characterized the topography and elasticity of these EVs by atomic force microscopy (AFM), enabling us to distinguish between EVs and non-vesicular particles (NVPs) in a therapeutic formulation. This measurement modality makes it possible to identify EV sub-fractions, discriminate between EVs and NVPs, and to characterize EV surface proteins, all with minimal sample preparation and using label-free measurement devices with low barriers of entry for labs looking to widen their spectrum of characterization techniques. Our combination of QCM with impedance measurement (QCM-I) and AFM measurements provides a robust multi-marker approach to the characterization of clinically approved EV formulations and opens the door to improved quality control.

molecular biology↗

bioRxiv2019

SVF-derived extracellular vesicles carry characteristic miRNAs in lipedema

Lipedema is a chronic, progressive disease of adipose tissue with lack of consistent diagnostic criteria. The aim of this study was a thorough comparative characterization of extracellular microRNAs from the stromal vascular fraction (SVF) of healthy and lipedema adipose tissue. For this, we analyzed 187 extracellular microRNAs in concentrated conditioned media (cCM) and specifically in small extracellular vesicles (sEVs) enriched thereof by size exclusion chromatography. No significant difference in median particle size and concentration was observed between sEV fractions in healthy and lipedema. We found the majority of miRNAs located predominantly in cCM compared to sEV enriched fraction. Surprisingly, hierarchical clustering of the most variant miRNAs showed that only sEV miRNA profiles - but not cCM miRNAs - were impacted by lipedema. Seven sEV miRNAs (miR-16-5p, miR-29a-3p, miR-24-3p, miR-454-p, miR-144-5p, miR-130a-3p, let-7c-5p) were differently regulated in lipedema and healthy, whereas only one cCM miRNA (miR-188-5p) was significantly downregulated in lipedema. Comparing SVF from healthy and lipedema patients, we identified sEVs as the lipedema relevant miRNA fraction. This study contributes to identify the potential role of SVF secreted miRNAs in lipedema.

molecular biology↗