Isolation efficiency of collagenase and EDTA for the culture of corneal endothelial cells.

Purpose: Tissue engineering of the corneal endothelium, as well as cell therapy, has been proposed as an alternative approach for the treatment of corneal endotheliopathies. These approaches require in vitro amplification of functional corneal endothelial cells (CECs). The goal of this study was to compare two common isolation methods, collagenase A and EDTA (EDTA), and determine whether they influence cell viability, morphology, and barrier function. Methods: Human eye bank research-grade corneas were used to isolate and cultivate CECs. All donors were more than 40 years old. Two Descemet membranes from the same donor were used separately to compare the collagenase A and EDTA cell isolation methods. The number of isolated cells, cell viability, morphology, and barrier functionality were compared. Results: A higher isolation efficiency of viable CECs and a higher circularity index (endothelial morphology) were obtained using collagenase A. Passage 3 cells presented similar barrier functionalities regardless of the isolation method. Conclusions: This study showed that isolation of CECs using collagenase A yields higher isolation efficiency than EDTA, delaying the loss of endothelial morphology for early passage cells.

The Vision Health Research Network and its commitment to the scholarly development of its trainees.

The Vision Health Research Network (VHRN) is a provincial scientific organization that aims to improve the ocular health of patients across Quebec by supporting local research endeavors in vision health. The VHRN Student Committee, composed of 288 trainees with diverse backgrounds, has demonstrated its commitment to the scholarly development of its members by providing leadership opportunities, creating networking events, increasing visibility of researchers-in-training and encouraging professional advancement through educational workshops and funding programs. In this article, we review the contributions of the VHRN Student Committee and discuss its future projects.

Lung CD200 Receptor Activation Abrogates Airway Hyperresponsiveness in Experimental Asthma.

In allergic asthma, homeostatic pathways are dysregulated, which leads to an immune response toward normally innocuous antigens. The CD200-CD200 receptor pathway is a central regulator of inflammation, and CD200 expression was recently found to be down-regulated in circulating leukocytes of patients with asthma. Given the antiinflammatory properties of CD200, we investigated whether local delivery of recombinant CD200 (rCD200) could reinstate lung homeostasis in an experimental model of asthma. Brown Norway rats were sensitized with ovalbumin (OVA) and alum. rCD200 was intratracheally administered 24 hours before OVA challenge, and airway responsiveness to methacholine was measured 24 hours after the allergen challenge. Inflammation was also assessed by measuring cell recruitment and cytokine levels in bronchoalveolar lavages, as well as lung and draining lymph node accumulation of dendritic cells (DCs) and T cells. In sensitized rats, rCD200 abolished airway hyperresponsiveness, whereas the sham treatment had no effect. In addition, rCD200 strongly reduced OVA-induced lung accumulation of myeloid DCs, CD4(+) T cells, and T helper type 2 cells. This was associated with a strong reduction of OVA-induced IL-13 level and with an increase of IL-10 in supernatants of bronchoalveolar lavages. Lung eosinophilia and draining lymph node accumulation of myeloid DCs and T cells were not affected by rCD200. Overall, these data reveal that rCD200 can inhibit airway hyperresponsiveness in a model of asthma by a multistep mechanism associated with local alterations of the T cell response and the cytokine milieu.

Live virus neutralizing antibodies against pre and post Omicron strains in food and retail workers in Québec, Canada.

Background: Measuring the ability of SARS-CoV-2 antibodies to neutralize live viruses remains an effective approach to quantify the level of protection of individuals. We assessed the neutralization activity against the ancestral SARS-CoV-2, Delta, Omicron BA.1, BA.2, BA.2.12.1, BA.4 and BA.5 strains, in 280 vaccinated restaurant/bar, grocery and hardware store workers in Québec, Canada. Methods: Participants were recruited during the emergence of Omicron BA.1 variant. The neutralizing activity of participant sera was assessed by microneutralization assay. Results: Serum neutralizing antibody (NtAb) titers of all participants against the ancestral SARS-CoV-2 strain were comparable with those against Delta variant (ranges of titers 10-2032 and 10-2560, respectively), however, their response was significantly reduced against Omicron BA.1, BA2, BA.2.12.1, BA.4 and BA.5 (10-1016, 10-1016, 10-320, 10-80 and 10-254, respectively). Individuals who received 2 doses of vaccine had significantly reduced NtAb titers against all SARS-CoV-2 strains compared to those infected and then vaccinated (≥1 dose), vaccinated (≥2 doses) and then infected, or those who received 3 doses of vaccine. Participants vaccinated with 2 or 3 doses of vaccine and then infected had the highest NtAb titers against all SARS-CoV-2 strains tested. Conclusion: We assessed for the first time the NtAb response in food and retail workers. We found that vaccination prior to the emergence of Omicron BA.1 was associated with higher neutralizing activity against pre-Omicron variants, suggesting the importance of updating vaccines to increase antibody response against new SARS-CoV-2 variants. Vaccination followed by infection was associated with higher neutralizing activity against all SARS-CoV-2 strains tested.

TGF-ß-Mediated Modulation of Cell-Cell Interactions in Postconfluent Maturing Corneal Endothelial Cells.

Purpose: Transforming growth factor-beta (TGF-ß) is known to influence many cell functions. In the corneal endothelium, TGF-ß1 exerts contextual effects, promoting endothelial-mesenchymal transition in proliferating cells and enhancing barrier integrity in early confluent maturing cells. Herein, we studied how TGF-ß isoforms participate in the formation of corneal endothelial intercellular junctions. Methods: Corneal endothelial cells (CECs) were cultured using a two-phase media approach. When CECs reached confluence, the proliferation medium was replaced with maturation medium, which was supplemented or not with TGF-ß isoforms. The cell morphology (circularity index), intercellular junction protein expression, trans-endothelial electrical resistance (TEER), and permeability of 7-day postconfluent CECs were assessed. Gene transcription and signaling pathways that were activated following maturation in the presence of TGF-ß2 were also studied. The beneficial effect of TGF-ß2 on CEC maturation was evaluated using ex vivo corneas mounted on a corneal bioreactor. Results: The results showed increases in circularity index, membrane localization of junction-related proteins, and TEER when TGF-ß isoforms were individually added during the maturation phase, and TGF-ß2 was the most effective isoform. Gene profiling revealed an increase in extracellular matrix-related gene expression. In ex vivo cell adhesion experiments, CECs that were matured in the presence of TGF-ß2 had a higher circularity index and cell density and exhibited cell membrane-localized junction-related protein expression at earlier time points. Conclusions: These results suggest that TGF-ß2 can strengthen cell-cell and cell-substrate adhesion, which accelerates barrier integrity establishment and thus enhances CEC functionality.

In Vitro Expansion of Corneal Endothelial Cells for Transplantation.

The corneal endothelium forms a leaky barrier between the corneal stroma and the aqueous humor of the anterior chamber. This cell monolayer maintains the corneal stroma in a state of relative dehydration, a process called deturgescence, which is required in order to obtain corneal stromal transparency. Endothelial dysfunctions lead to visual impairment that ultimately can only be treated surgically via the corneal transplantation of a functional endothelium. Shortages of corneas suitable for transplantation has motivated research toward new alternatives involving in vitro corneal endothelial cell (CEC) expansion.This chapter describes current methods that allow isolate and culture CECs. In brief, Descemet membrane is peeled out of the cornea and digested in order to obtain CECs. Cells are then seeded and cultured.

TGF-ß1 promotes cell barrier function upon maturation of corneal endothelial cells.

Human corneal endothelial cells (HCECs) easily become fibroblastic-like when cultured, rendering them unsuitable for tissue engineering of the cornea. Transforming growth factor ß (TGF-ß) could be a key factor in this phenomenon; however, TGF-ß is also known to maintain the endothelium in a quiescent state in vivo. This work aimed to compare the effects of TGF-ß1 on the phenotype of HCECs during the proliferation and maturation phases. Our results show that addition of TGF-ß1 during the active proliferation phase produced fibroblastic HCECs and loss of the cell junction markers ZO-1 and n-cadherin, independent from the presence of epidermal growth factor (EGF). By contrast, addition of TGF-ß1 in maturation media containing few mitogens led to an endothelial phenotype and functional cell junctions as HCECs developed a high trans-endothelial resistance. Furthermore, addition of AG-1478, an epithelial growth factor receptor inhibitor, enhanced the gain of the endothelial phenotype and cell barrier function. Overall, these results show that TGF-ß1 can be used to promote the formation of a typical leaky endothelial barrier during the maturation phase of cultured HCECs. A two-phase culture of HCECs using distinct proliferation and maturation media could also be key for developing ideal HCEC culture conditions.