Production of Tissue-Engineered Skin Substitutes for Clinical Applications: Elimination of Serum.

Tissue-engineered skin substitutes (TESs) are used as a treatment for severe burn injuries. Their production requires culturing both keratinocytes and fibroblasts. The methods to grow these cells have evolved over the years, but bovine serum is still commonly used in the culture medium. Because of the drawbacks associated with the use of serum, it would be advantageous to use serum-free media for the production of TESs. In a previous study, we developed a serum-free medium (Surge SFM) for the culture of keratinocytes. Herein, we tested the use of this medium, together with a commercially available serum-free medium for fibroblasts (Prime XV), to produce serum-free TESs. Our results show that serum-free TESs are macroscopically and histologically similar to skin substitutes produced with conventional serum-containing media. TESs produced with either culture media expressed keratin 14, Ki-67, transglutaminase 1, filaggrin, type I and IV collagen, and fibronectin comparably. Mechanical properties, such as contraction and tensile strength, were comparable between TESs cultured with and without serum. Serum-free TESs were also successfully grafted onto athymic mice for a six-month period. In conclusion, Surge SFM and Prime XV serum-free media could be used to produce high quality clinical-grade skin substitutes.

Eicosapentaenoic acid reduces the proportion of IL-17A-producing T cells in a 3D psoriatic skin model.

Psoriasis is a skin disease presenting as erythematous lesions with accentuated proliferation of epidermal keratinocytes, infiltration of leukocytes, and dysregulated lipid metabolism. T cells play essential roles in the disease. n-3 polyunsaturated fatty acids are anti-inflammatory metabolites, which exert an immunosuppressive effect on healthy T cells. However, the precise mechanistic processes of n-3 polyunsaturated fatty acids on T cells in psoriasis are still unrevealed. In this study, we aimed to evaluate the action of eicosapentaenoic acid (EPA) on T cells in a psoriatic skin model produced with T cells. A coculture of psoriatic keratinocytes and polarized T cells was prepared using culture media, which was either supplemented with 10 µM EPA or left unsupplemented. Healthy and psoriatic skin substitutes were produced according to the self-assembly method. In the coculture model, EPA reduced the proportion of IL-17A-positive cells, while increasing that of FOXP3-positive cells, suggesting an increase in the polarization of regulatory T cells. In the 3D psoriatic skin model, EPA normalized the proliferation of psoriatic keratinocytes and diminished the levels of IL-17A. The expression of the proteins of the signal transducer and activator of transcription was influenced following EPA supplementation with downregulation of the phosphorylation levels of signal transducer and activator of transcription 3 in the dermis. Finally, the NFκB signaling pathway was modified in the EPA-supplemented substitutes with an increase in Fas amounts. Ultimately, our results suggest that in this psoriatic model, EPA exerts its anti-inflammatory action by decreasing the proportion of IL-17A-producing T cells.

Evaluation of an interprofessional primary healthcare team as a new model of primary care in Quebec: a protocol for a type 2 effectiveness-implementation hybrid study.

INTRODUCTION: One family medicine group (FMG) in Quebec has commenced a 5-year pilot project, which is herein referred to as the Archimède model, to implement a patient-centred model based on interprofessional care and the optimal use of healthcare providers' practice scopes. A research project will be conducted to: (1) assess this model's effect on the FMG's operational performance, and its users' resource utilisation at the public health system level; (2) investigate its optimisation with respect to professional roles, interprofessional teamwork and patient-centredness and (3) document users' experience with the model. The aim of this article is to describe the protocol that will be used for this research. METHODS AND ANALYSIS: A hybrid implementation approach (type 2 model) will be used. We will collect both quantitative and qualitative data. Regarding the quantitative dimension, and because this is a single-unit intervention study, we will use either or both synthetic control methods and one-sample generalised linear models for analyses at the FMG level. To evaluate the broader impact of Archimède on the public health system, we will use mixed-effects models and propensity score matching methods. Regarding the qualitative research dimension, using an interpretative descriptive approach, we will document users' experience and identify the factors that optimise professional scopes of practice, collaborative practices and patient-centredness. We will conduct individual in-depth semistructured interviews with healthcare providers, administrative staff, stakeholders involved in the Archimède model implementation and patients. ETHICS AND DISSEMINATION: This study was approved by the Ethics Committee of the Sectoral Research in Population Health and Primary Care of the Centre intégré universitaire de santé et de services sociaux de la Capitale-Nationale (#2019-1503). The results of the investigation will be presented to the stakeholders involved in the advisory committees and at several scientific conferences. Manuscripts will be submitted to peer-reviewed journals.

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.

Influence of the Postmortem/Storage Time of Human Corneas on the Properties of Cultured Limbal Epithelial Cells.

Besides being a powerful model to study the mechanisms of corneal wound healing, tissue-engineered human corneas (hTECs) are sparking interest as suitable substitutes for grafting purposes. To ensure the histological and physiological integrity of hTECs, the primary cultures generated from human cornea (identified as human limbal epithelial cells (hLECs) that are used to produce them must be of the highest possible quality. The goal of the present study consisted in evaluating the impact of the postmortem/storage time (PM/ST) on their properties in culture. hLECs were isolated from the entire cornea comprising the limbus and central cornea. When grown as monolayers, short PM/ST hLECs displayed increased daily doublings and generated more colonies per seeded cells than long PM/ST hLECs. Moreover, hLECs with a short PM/ST exhibited a markedly faster wound closure kinetic both in scratch wound assays and hTECs. Collectively, these results suggest that short PM/ST hLECs have a greater number of highly proliferative stem cells, exhibit a faster and more efficient wound healing response in vitro, and produce hTECs of a higher quality, making them the best candidates to produce biomaterial substitutes for clinical studies.

Moyamoya Disease Susceptibility Gene RNF213 Regulates Endothelial Barrier Function.

BACKGROUND: Variants in the ring finger protein 213 (RNF213) gene are known to be associated with increased predisposition to cerebrovascular diseases development. Genomic studies have identified RNF213 as a major risk factor of Moyamoya disease in East Asian descendants. However, little is known about the RNF213 (ring finger protein 213) biological functions or its associated pathogenic mechanisms underlying Moyamoya disease. METHODS: To investigate RNF213 loss-of-function effect in endothelial cell, stable RNF213-deficient human cerebral endothelial cells were generated using the CRISPR-Cas9 genome editing technology. RESULTS: In vitro assays, using RNF213 knockout brain endothelial cells, showed clear morphological changes and increased blood-brain barrier permeability. Downregulation and delocalization of essential interendothelial junction proteins involved in the blood-brain barrier maintenance, such as PECAM-1 (platelet endothelial cell adhesion molecule-1), was also observed. Brain endothelial RNF213-deficient cells also showed an abnormal potential to transmigration of leukocytes and secreted high amounts of proinflammatory cytokines. CONCLUSIONS: Taken together, these results indicate that RNF213 could be a key regulator of cerebral endothelium integrity, whose disruption could be an early pathological mechanism leading to Moyamoya disease. This study also further reinforces the importance of blood-brain barrier integrity in the development of Moyamoya disease and other RNF213-associated diseases.

Irradiated Human Fibroblasts as a Substitute Feeder Layer to Irradiated Mouse 3T3 for the Culture of Human Corneal Epithelial Cells: Impact on the Stability of the Transcription Factors Sp1 and NFI.

Because of the worldwide shortage of graftable corneas, alternatives to restore visual impairments, such as the production of a functional human cornea by tissue engineering, have emerged. Self-renewal of the corneal epithelium through the maintenance of a sub-population of corneal stem cells is required to maintain the functionality of such a reconstructed cornea. We previously reported an association between stem cell differentiation and the level to which they express the transcription factors Sp1 and NFI. In this study, we investigated the impact of replacing irradiated 3T3 (i3T3) murine fibroblast feeder cells by irradiated human corneal fibroblasts (iHFL) on the expression of Sp1 and NFI and evaluated their contribution to the proliferative properties of human corneal epithelial cells (hCECs) in both monolayer cultures and human tissue engineered corneas (hTECs). hCECs co-cultured with iHFL could be maintained for up to two more passages than when they were grown with i3T3. Western Blot and electrophoretic mobility shift assays (EMSAs) revealed no significant difference in the feeder-layer dependent increase in Sp1 at both the protein and DNA binding level, respectively, between HCECs grown with either i3T3 or iHFL. On the other hand, a significant increase in the expression and DNA binding of NFI was observed at each subsequent passage when hCECs were co-cultured along with i3T3. These changes were found to result from an increased expression of the NFIA and NFIB isoforms in hCECs grown with i3T3. Exposure of hCECs to cycloheximide revealed an increased stability of NFIB that likely resulted from post-translational glycosylation of this protein when these cells were co-cultured with i3T3. In addition, iHFL were as efficient as i3T3 at preserving corneal, slow-cycling, epithelial stem cells in the basal epithelium of the reconstructed hTECs. Furthermore, we observed an increased expression of genes whose encoded products promote hCECs differentiation along several passages in hCECs co-cultured with either type of feeder layer. Therefore, the iHFL feeder layer appears to be the most effective at maintaining the proliferative properties of hCECs in culture most likely by preserving high levels of Sp1 and low levels of NFIB, which is known for its gene repressor and cell differentiation properties.

Isolation and Culture of Human Keratinocytes.

Culturing keratinocytes to form coherent epithelial tissue sheets has improved the treatment of extensively burned patients. Keratinocyte culture is also used to investigate various cellular and molecular mechanisms involved in different skin pathologies. To preserve stem cells during epithelial cell culture, reliable methods and conditions are of the utmost importance. Properly cultured keratinocytes will exhibit a consistent cuboid morphology and can proliferate for many passages. This chapter details materials needed and methods for all aspects of efficient keratinocyte culture for clinical applications, namely tissue sampling and transportation, isolation, routine culture, subculture, and cryopreservation.

Qualitatively Monitoring Binding and Expression of the Transcription Factors Sp1 and NFI as a Useful Tool to Evaluate the Quality of Primary Cultured Epithelial Stem Cells in Tissue Reconstruction.

Electrophoretic mobility shift assays and Western blots are simple, efficient, and rapid methods to study DNA-protein interactions and protein expression, respectively. Primary cultures and subcultures of epithelial cells are widely used for the production of tissue-engineered substitutes and are gaining popularity as a model for gene expression studies. The preservation of stem cells through the culture process is essential to produce high quality substitutes. However, the increase in the number of cell passages is associated with a decrease in their ability to proliferate until senescence is reached. This process is likely to be mediated by the altered expression of nuclear-located transcription factors such as Sp1 and NFI, whose expression has been documented to be required for cell adhesion, migration, and differentiation. In some of our recent studies, we observed a correlation between reconstructed tissues exhibiting poor histological and structural characteristics and a low expression of Sp1 in their constituting epithelial cells. Therefore, monitoring both the expression and DNA binding of these transcription factors in human skin and corneal epithelial cells is a useful tool for characterizing the quality of primary cultured epithelial cells.

Specialized Living Wound Dressing Based on the Self-Assembly Approach of Tissue Engineering.

There is a high incidence of failure and recurrence for chronic skin wounds following conventional therapies. To promote healing, the use of skin substitutes containing living cells as wound dressings has been proposed. The aim of this study was to produce a scaffold-free cell-based bilayered tissue-engineered skin substitute (TES) containing living fibroblasts and keratinocytes suitable for use as wound dressing, while considering production time, handling effort during the manufacturing process, and stability of the final product. The self-assembly method, which relies on the ability of mesenchymal cells to secrete and organize connective tissue sheet sustaining keratinocyte growth, was used to produce TESs. Three fibroblast-seeding densities were tested to produce tissue sheets. At day 17, keratinocytes were added onto 1 or 3 (reference method) stacked tissue sheets. Four days later, TESs were subjected either to 4, 10, or 17 days of culture at the air⁻liquid interface (A/L). All resulting TESs were comparable in terms of their histological aspect, protein expression profile and contractile behavior in vitro. However, signs of extracellular matrix (ECM) digestion that progressed over culture time were noted in TESs produced with only one fibroblast-derived tissue sheet. With lower fibroblast density, the ECM of TESs was almost completely digested after 10 days A/L and lost histological integrity after grafting in athymic mice. Increasing the fibroblast seeding density 5 to 10 times solved this problem. We conclude that the proposed method allows for a 25-day production of a living TES, which retains its histological characteristics in vitro for at least two weeks.

Are the Effects of the Cholera Toxin and Isoproterenol on Human Keratinocytes' Proliferative Potential Dependent on Whether They Are Co-Cultured with Human or Murine Fibroblast Feeder Layers?

Human keratinocyte culture has provided the means to treat burns, wounds and skin pathologies. To date, to efficiently culture keratinocytes, cells are cultured on an irradiated feeder layer (iFL), either comprising human (iHFL) or murine (i3T3FL) fibroblasts, and the culture medium is supplemented with a cyclic adenosine monophosphate (cAMP) accumulation inducing agent such as isoproterenol (ISO) or cholera toxin (CT). Previous studies have characterized how the feeder layer type and the cAMP inducer type influence epithelial cells' phenotype independently from one another, but it is still unknown if an optimal combination of feeder layer and cAMP inducer types exists. We used sophisticated statistical models to search for a synergetic effect of feeder layer and cAMP inducer types on human keratinocytes' proliferative potential. Our data suggests that, when culturing human keratinocytes, using iHFL over i3T3FL increases population doublings and colony-forming efficiency through signaling pathways involving Ak mouse strain thymoma (Akt, also known as protein kinase B) isoforms 1 to 3, signal transducer and activator of transcription 5 (STAT5), p53, and adenosine monophosphate activated protein kinase α1 (AMPKα1). Both tested cAMP inducers ISO and CT yielded comparable outcomes. However, no significant synergy between feeder layer and cAMP inducer types was detected. We conclude that, to promote human keratinocyte growth in the early passages of culture, co-culturing them with a human feeder layer is preferable to a murine feeder layer.