Lotz, C. | bioRxiv Science

bioRxiv2026

Validation of optical coherence tomography as a tool to identify differentiation key drivers in 3D in vitro conjunctival models

Conjunctival in vitro models present a valuable system to investigate conjunctival tissue homeostasis and pathologies. Combinations of collagen and fibroblasts as a stroma equivalent and the supplementation with serum have been reported to promote the differentiation of epithelial cells. However, how the individual factors affect differentiation of ocular surface cells is insufficiently understood. In this study, we analyzed the effect of serum concentration, a collagen matrix, and fibroblasts on conjunctival differentiation in a 3D in vitro model. For this purpose, we developed a computational analysis pipeline for the quantification of optical coherence tomography (OCT) data sets, allowing a time resolved, non-invasive assessment of conjunctiva epithelium differentiation, including goblet cell density. High-resolution dynamic full-field OCT (D-FFOCT) was employed to verify the identity of goblet cells. Conjunctival markers were further analyzed via histology, real-time quantitative PCR, and ELISA to confirm the data of the OCT analysis pipeline. We found that serum is required to induce epithelial differentiation while higher concentrations of 5 - 10% impaired epithelial development. The culture on a collagen matrix increased conjunctival markers upon stimulation with serum, while the co-culture with fibroblasts increased epithelial stratification. Increased serum concentration resulted in the increased occurrence of goblet cells of up to 20 cells/mm{superscript 2}. Altogether, the complementary analyses confirmed the quantified OCT data. Summarized, we identified the combination of serum (3%), collagen, and fibroblasts as a condition resulting in the highest physiological resemblance. Altogether, our study emphasizes the need for fine-tuning of culture conditions for 3D in vitro models. SIGNIFICANCE STATEMENTPhysiologically relevant in vitro conjunctiva models are essential for studying ocular surface homeostasis and disease. Our study refines 3D conjunctival culture conditions to more closely resemble native tissue by systematically identifying serum concentration, collagen scaffolds and fibroblasts as key drivers of epithelial differentiation. By applying non-invasive optical coherence tomography analysis, we enable longitudinal assessment of tissue maturation, addressing the need for non-destructive, repeatable tissue analysis. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=164 HEIGHT=200 SRC="FIGDIR/small/698626v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@13efafaorg.highwire.dtl.DTLVardef@1cf4529org.highwire.dtl.DTLVardef@c5c63forg.highwire.dtl.DTLVardef@5a85c8_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

The role of type IV pilus in the interaction of Neisseria gonorrhoeae with a corneal epithelium tissue model

Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea, can infect intact corneal epithelium, leading to inflammation, cornea perforation, and blindness. Models for studying this type of gonococcal infection are few and limited. We have tested cornea models based on an immortalized hTCEpi corneal epithelium cell line for infection research using four derivatives of the N. gonorrhoeae MS11 strain that differ in pilus expression. We show that bacterial adherence depends on the functional type IV pilus in the early stages of infection, and that the formation of bacterial microcolonies and biofilms on the model surface leads to tissue destruction. The infection induces a specific cytokine response characterized by an increase in the secretion of IL-8 and TNF-, but not of IL-6. The testing of trifluoperazine, a drug that induces pilus retraction, on infected corneal tissue models showed that the drug strongly diminished the number of adherent gonococci only when applied simultaneously with bacteria, and not when bacteria were allowed to form microcolonies on the tissue surface. Our work describes, for the first time, hTCEpi-based corneal epithelium tissue models as a useful tool for investigating N. gonorrhoeae infection, with potential for application in high-throughput studies and drug screening.

microbiology↗

bioRxiv2020

Structural basis for the allosteric regulation of Human Topoisomerase 2α

The human type IIA topoisomerases (Top2) are essential enzymes that regulate DNA topology and chromosome organization. The Top2 isoform is a prime target for antineoplastic compounds used in cancer therapy that form ternary cleavage complexes with the DNA. Despite extensive studies, structural information on this large dimeric assembly is limited to the catalytic domains, hindering the exploration of allosteric mechanism governing the enzyme activities and the contribution of its non-conserved C-terminal domain (CTD). Herein we present cryo-EM structures of the entire human Top2 nucleoprotein complex in different conformations solved at subnanometer resolutions. Our data unveils the molecular determinants that fine tune the allosteric connections between the ATPase domain and the DNA binding/cleavage domain. Strikingly, the reconstruction of the DNA-binding/cleavage domain uncovers a linker leading to the CTD, which plays a critical role in modulating the enzymes activities and opens perspective for the analysis of post-translational modifications.

biochemistry↗