Jana Hlinkova

• Esposito, Alessio; Fortunato, Gabriele Maria; Stasi, Mauro Di; Fradinho, Ana C.; Pacetta, Guglielmo & Soriano, Maria Grazia [Show all 16 contributors for this article] (2026). A Biomimetic Eye In Vitro Model to Investigate the Blood-Retinal Barrier Pathophysiology. ACS Biomaterials Science and Engineering. doi: 10.1021/acsbiomaterials.6c00764. Full text in Research Archive Show summary

  Abstract Existing in vivo models for retinal pathologies are costly, time-consuming, and subject to regulatory challenges, promoting interest in advanced in vitro models that replicate the blood-retinal barrier. Challenges related to vascularization and structural multiscale interactions hinder the development of in vitro models, which are useful to study nutrient and drug exchange between the retina and systemic circulation. To address these issues, we developed a blood-retinal barrier in vitro model for studying retinal pathophysiology. It features an electrospun scaffold (Bruch’s membrane) placed between a culture chamber (vitreous humor) and a bioinspired microfluidic network (choroidal vasculature). The network, modelled from indocyanine green angiography of the human retina, is laser-engraved into a 15 mm PDMS disc with channels 70–800 μm wide. PLGA and gelatin electrospun scaffold were investigated to mimic Bruch’s membrane. The scaffold is fixed to the microfluidic network, assembled into a culture chamber, and integrated within a bioreactor. A peristaltic pump ensures a consistent flow in the channels. Biological validation included ARPE-19 cells cultured on Transwell, PLGA, or gelatin membranes, HUVECs within membrane-coupled microchannels, and dual-layer constructs. Cells were examined separately and together, allowing evaluation of epithelial, endothelial, and construct responses. All configurations showed progressive increases in metabolic activity and limited LDH release over 28 days. Cell-seeded constructs reduced FITC–dextran transport relative to acellular controls, with stable values between days 21 and 28. Blank-corrected TEER likewise increased during ARPE-19 culture and plateaued by day 21. PLGA constructs showed the greatest electrical and solute-transport resistance, although acellular controls confirmed a substantial material contribution. Confocal microscopy demonstrated HUVEC attachment and F-actin organization along the microchannels, while ARPE-19 monolayers exhibited continuous junction-associated ZO-1 staining that remained stable between days 21 and 28. Overall, the platform supported long-term epithelial–endothelial culture and provided a stable, perfusable system for studying oBRB transport and cellular interactions.

• Agger, Anne Eriksson; Hlinkova, Jana; Reseland, Janne Elin & Samara, Athina (2026). 3D models for oral inflammation using gingival fibroblasts, lipopolysaccharides and hypoxia | Scientific Reports. Scientific Reports. doi: 10.1038/s41598-026-47648-7. Full text in Research Archive Show summary

  Three-dimensional (3D) in vitro models that more faithfully recapitulate human tissue architecture are essential to advance mechanistic insight into inflammatory diseases. We developed and characterized a 3D human gingival fibroblast spheroid-system to dissect the combined effects of hypoxia and inflammation, two central features of host–pathogen interactions in periodontal disease and other chronic inflammatory conditions. Using controlled oxygen environments and stimulation with E. coli lipopolysaccharide (LPS) and IL-1β, we show that hypoxia alone markedly alters spheroid morphology, producing smaller aggregates with thinner rims, while the combination of hypoxia and inflammation yields the most prominent structural disruption. Cross-section analyses reveal condition-dependent remodeling of extracellular matrix and cytoskeletal organization, with coordinated reductions in fibronectin, collagen I/III, integrin β1, and filamentous actin. Gene expression profiling confirms suppression of extracellular matrix (ECM)-related and adhesion genes under hypoxia and combined stress, whereas secretome analysis demonstrates robust upregulation of pro-inflammatory cytokines and chemokines (IL-6, IL-8, MCP-1, TNFα) and angiogenic VEGFα. Despite these marked structural and immunological shifts, cell viability remained largely preserved. This 3D system captures key features of fibroblast responses to inflammatory and hypoxic cues, providing a human-relevant platform for studies of tissue remodeling, cytokine signaling, and microenvironmental stress relevant to host–pathogen–driven disease.

• Hlinkova, Jana; Dziemidowicz, Karolina; Ullrich, Mathilde M.; Agger, Anne Eriksson; Lian, Aina-Mari & Reseland, Janne Elin [Show all 7 contributors for this article] (2025). Neural growth patterns: how random and aligned fibers guide 3D cell organization and pseudospheroid formation. Frontiers in Bioengineering and Biotechnology. 13. doi: 10.3389/fbioe.2025.1659965. Full text in Research Archive Show summary

  Background and purpose Electrospun biomaterials replicate the structural complexity of the extracellular matrix (ECM), providing mechanical support and promoting neural cell survival and organization. Fiber orientation is a key determinant of neural cell behavior, influencing adhesion, migration, and differentiation. This study investigates how high seeding density combined with fiber directionality shapes SH-SY5Y culture morphology, gene expression, and early network formation; all critical factors for the design of next-generation scaffolds for neural tissue engineering. Methods Polycaprolactone (PCL) scaffolds with either random or aligned fiber orientation were fabricated via monoaxial electrospinning. Human SH-SY5Y neuroblastoma cells were seeded at high density and cultured for 7 days, and cell viability was assessed by lactate dehydrogenase (LDH) activity. Neural, ECM, and differentiation markers were analyzed using quantitative PCR, Luminex cytokine profiling, and confocal immunofluorescence. Results Hydrophobic PCL fibers supported cell adhesion, migration, and proliferation when cells were seeded in small clusters. After 7 days, cell coverage of the fiber-mat was significantly higher on random fibers compared to aligned ones (27.7% vs. 15.8%). Fiber orientation influenced both culture morphology and gene expression. Pseudospheroids formed on both substrates, that differed in perimeter (348.5 µm on random vs. 450.5 µm on aligned fibers, p < 0.05), with no significant difference in thickness (38.4 ± 7.7 µm vs. 43.2 ± 5.5 µm). mRNA expression of connexin 43 and β3-tubulin increased significantly from day 1 to day 7 on random fibers. On aligned fibers, mRNA patterns resembled cells cultured on glass (control), with elevated connexin 31 and doublecortin over time. Immunofluorescence showed early enrichment of nestin on aligned fibers (day 1), and greater expression of β3-tubulin, acetylated tubulin, and connexin 31 on aligned substrates, whereas fibronectin 1 was more prominent on random fibers. Conclusion Fiber orientation significantly affected SH-SY5Y cell behaviour, including adhesion, formation of pseudospheroids, and differentiation marker expression under high-density conditions. Random and aligned fibers elicited distinct structural patterns and molecular responses, highlighting the importance of scaffold architecture in the rational design of neuroregenerative platforms. To our knowledge, this is the first study to describe scaffold-anchored neural pseudospheroids as a distinct model from conventional suspension spheroids.

• Ullrich, Mathilde M.; Pulipaka, Bhavana; Yin, Jing; Hlinkova, Jana; Zhang, Fangyuan & Chan, Michael W [Show all 9 contributors for this article] (2025). Neuroprotective Riluzole-Releasing Electrospun Implants for Spinal Cord Injury. Molecular Pharmaceutics. ISSN 1543-8384. doi: 10.1021/acs.molpharmaceut.4c01270. Full text in Research Archive
• Saghir, Saloua; Hlinkova, Jana; Samara, Athina & Schiavone, Giuseppe (2025). Protocol for fabricating a reusable plate-well insert with a 3D-printed mounter and hydrogel scaffolds for 3D cell culture and functional assays. STAR Protocols. 6(3). doi: 10.1016/j.xpro.2025.104014. Full text in Research Archive Show summary

  We present a protocol for the fabrication of reusable inserts for standard 24-well plates, comprising a 3D-printed mounter and a hydrogel scaffold for 3D cell culture studies. We cover the design and production of the mounter, hydrogel scaffold synthesis, lyophilization for storage, sterilization, and hydrophilization before cell culture. Downstream applications include cytotoxicity assessment, imaging, and qPCR. Streamlining hydrogel preparation and mounting minimizes batch-to-batch variation, reduces costs, and supports synthetic biology studies.

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• Saghir, Saloua; Samara, Athina; Hlinkova, Jana & Schiavone, Giuseppe (2026). REUSABLE PLATE-WELL INSERTS WITH HYDROGEL SCAFFOLDS FOR 3D CELL CULTURE. Full text in Research Archive

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