Biology subjects

Guzowski, J.

Publications and source records attributed to Guzowski, J..

5 recordsLinked to original sources

bioRxiv2026

High-throughput microfluidic encapsulation of cells in extracellular matrix microbeads for microtissue engineering.

Microfluidics-assisted encapsulation of cells in hydrogel microbeads has found diverse applications in tissue engineering, disease modelling, and drug testing. However, upscaled production of submillimeter-sized microbeads, each encapsulating optimally tens to hundreds of living cells, remains a challenge. Excessive viscous stresses or non-homogeneous crosslinking reactions hamper droplet monodispersity, hydrogel integrity, and/or cell viability. Herein, we present a device consisting of (i) tens of parallel step-emulsifiers arranged radially around the hydrogel inlet and (ii) an in-flow UV crosslinking system capable of continuous generation and crosslinking of up to around 45000 microbeads/min, each encapsulating N~50 cells, at cell viability exceeding 80%. We use gelatin methacryloyl (GelMA) as the biomaterial of choice and establish a unique sweet spot in terms of UV wavelength, irradiance, and exposure time- the latter controlled via the net applied flow rate and the length of the UV-exposed microchannel- at which we achieve an optimal balance between long-term microbead stability and cell viability. In particular, we observe a strong correlation between reduced cell viability and poor crosslinking efficiency, the latter attributed to the presence of a spontaneous core-shell structure. We further study the impact of added methacrylated hyaluronic acid (HAMA) and extracellular matrix (ECM) on cell proliferation and microtissue formation in the case of triple-negative breast cancer (TNBC) cells for future application in drug testing. The upscaled production of the encapsulated microtissues demonstrated here provides new perspectives for both drug testing and cell therapies where the precision in microenvironment formulation as well as production throughput remain of primary importance.

bioengineering↗

bioRxiv2025

Engineering of interconnected microcapillary networks at the mesoscale via magnetic assembly of endothelial-cell 'seeds'.

Despite significant developments in endothelial-cell (EC) manipulation techniques, a proper in vitro model of a functional microvasculature with controlled local interconnectivity under well-defined global architecture is still lacking. Here, we report the generation of such controlled multi-scale vascular net-works via manipulation of tens of sprouting EC seeds. We exploit magnetic patterning to assemble EC-coated superparamagnetic microbeads into ordered arrays and establish effective growth rules gov-erning the development of interconnectivity and directionality of the networks depending on the applied seed-seed spacing. The EC-seed-based approach offers a range of advantages over conventional EC-manipulation techniques including: (i) expedited sprouting, (ii) spatial control over interconnections, (iii) reduction in cell consumption by >100x, and (iv) native high-throughput format. We co-develop multiparametric morphometric analysis tool and demonstrate high-content assessment of drug-induced vascular remodeling in 3D tumor microenvironments. Overall, we propose a uniquely precise and stand-ardized vascular-microtissue engineering tool with applications, e.g., in angiogenesis research, high-throughput drug testing including personalized therapies, and with possible extension to organ-on-chip and tissue-regenerative approaches.

bioengineering↗

bioRxiv2024

High-throughput formulation of reproducible 3D cancer microenvironments for drug testing in myelogenous leukemia

Targeting cancer microenvironment is currently one of the major directions in drug development and preclinical studies in leukemia. Despite the variety of available chronic myelogenous leukemia 3D culture models, the reproducible generation of miniaturized leukemia microenvironments, suitable for high-throughput drug testing, has remained a challenge. Here, we use microfluidics to generate over ten thousand highly monodisperse leukemic-bone marrow hydrogel microbeads per minute. We employ gelatin methacrylate (GelMA) as a model extracellular matrix (ECM) and tune the concentration of the biopolymer, as well as other possible components of the ECM (fibrin, hyaluronic acid), cell concentration and the ratio of leukemic cells to bone marrow cells within the microbeads. This allows to achieve optimal cell viability and the propensity of the encapsulated cells to microtissue formation, while also warranting long-term stability of the microbeads in culture. We administer model kinase inhibitor, imatinib, at various concentrations to the microbeads and, via comparing mono-and co-culture conditions (cancer alone vs cancer-stroma), we find that the stroma-leukemia crosstalk systematically protects the encapsulated cells against the drug-induced cytotoxicity, confirming therefore that our system mimics the physiological stroma-dependent protection. We finally discuss applicability of our model to (i) studying the role of direct-or close-contact interactions between leukemia and bone marrow cells embedded in 3D ECM on the stroma-mediated protection, and (ii) high-throughput screening of anti-cancer therapeutics in personalized therapies.

cancer biology↗

bioRxiv2023

Topological evolution of sprouting vascular networks: from day-by-day analysis to general growth rules.

Engineering tissues with an embedded vasculature of well-controlled topology remains one of the basic problems in biofabrication. Still, little is known about the evolution of topological characteristics of vascular networks over time. Here, we perform a high-throughput day-by-day analysis of tens of microvasculatures that sprout from endothelial-cell coated micrometric beads embedded in an external fibrin gel. We use the bead-assays to systematically analyze (i) macroscopic observables such as the overall length and area of the sprouts, (ii) microscopic observables such as the lengths of segments or the branching angles and their distributions, as well as (iii) general measures of network complexity such as the average number of bifurcations per branch. We develop a custom angiogenic image analysis toolkit and track the evolution of the networks for at least 14 days of culture under various conditions, e.g., in the presence of fibroblasts or with added endothelial growth factor (VEGF). We find that the evolution always consists of three stages: (i) an inactive stage in which cells remain bound to the beads, (ii) a sprouting stage in which the sprouts rapidly elongate and bifurcate, and (iii) the maturation stage in which the growth slows down. We show that higher concentrations of VEGF lead to an earlier onset of sprouting and to a higher number of primary branches, yet without significantly affecting the speed of growth of the individual sprouts. We find that the mean branching angle is weakly dependent on VEGF and typically in the range of 60-75 degrees suggesting that, by comparison with the available Laplacian growth models, the sprouts tend to follow local VEGF gradients. Finally, we observe an exponential distribution of segment lengths, which we interpret as a signature of stochastic branching at a constant bifurcation rate (per unit branch length). Our results, due to high statistical relevance, may serve as a benchmark for predictive models and reveal how the external means of control, such as VEGF concentration, could be used to control the morphology of the vascular networks. We provide guidelines for the fabrication of optimized microvasculatures with potential applications in drug testing or regenerative medicine.

bioengineering↗

bioRxiv2020

Robust Population Single Neuronal Calcium Signal Extraction Using SCOUT Allows for Longitudinal Analysis of Behavior-associated Neural Ensemble Dynamics

In vivo calcium imaging enables simultaneous recording of large neuronal ensembles while engaged in operations such as learning and memory. However, such in vivo optical recordings are typically subject to motion artifact and background contamination from neurons and blood vessels. Further, population cell tracking across multiple recordings is complicated by non-rigid transformation induced by cell movements and field shifts. We introduce the novel method SCOUT for Single-Cell SpatiOtemporal LongitUdinal Tracking, consisting of two crucial parts: (1) imposition of spatial constraints on neuronal footprints extracted from individual optical recordings to improve ROI selection and eliminate false discoveries, and (2) application of a predictor-corrector, using spatiotemporal correlation of extracted neurons across sessions, for population cell tracking across multiple sessions. SCOUT empirically outperforms current methods for cell extraction and tracking in long-term multi-session imaging experiments across multiple brain regions. Application of this method allows for robust longitudinal analysis of contextual discrimination associated neural ensemble dynamics in the hippocampus up to 60 days.

neuroscience↗