**Acknowledgements**

and the two modifications to protocol (6-well plate and 12-well plate modifications). Indeed, filaggrin and loricrin were detected in healthy skin substitutes, whereas their absence was observed in psoriatic skin substitutes. This therefore confirms that the characteristics associ-

Keratins are intermediate filaments highly involved in epidermal structure and different types are expressed in the varying differentiation stages [48]. K5 and K14 are normally found in the basal layer of the epidermis, and they are progressively replaced by K1 and K10 in suprabasal layers [49]. However, *in vivo* psoriatic skin shows K14 in all layers of the epidermis, including the *stratum corneum*. This therefore suggests that the degradation mechanism of this keratin is altered in psoriasis [48, 50]. Moreover, in such hyperproliferative diseases, a new pair of keratins, K6 and K16, is appearing, causing a decrease in the expression of K1 and K10 [48, 50–52]. In the present work, healthy and psoriatic skin substitutes reconstructed according to the original method and the 6-well plate modification to protocol have demonstrated the same K1 and K14 expression than *in vivo*. Thus, taking together, K1, K14, filaggrin, and loricrin results validated the conservation of psoriatic skin differentiation in the new 6-well plate modification. This suggests that the 6-well method would be a great alternative to the original method. In healthy substitutes produced following the 12-well plate modification, an abnormal presence of K14 and K1 was observed in the *stratum corneum*, showing a less efficient differentiation of keratinocytes. This can probably be explained by the higher number of cells seeded in the culture area. These observations demonstrated that the 6-well plate modification is more effective for the production

Some studies suggested that alterations in the basal membrane of psoriatic skin play an important role in the abnormal proliferation and differentiation of psoriatic keratinocytes [53–55]. Indeed, the expression of proteins such as laminin, which is one of the main proteins that forms the basal membrane, is decreased and disrupted in psoriatic skin unlike in healthy skin. In this last, laminin forms a linear and continuous structure [54]. Laminin expression in our skin substitutes produced with healthy cells regardless of the method (original method and two new modifications) was intense, continuous and more restricted to the basal lamina, demonstrating a good structure of the basal membrane. For psoriatic skin substitutes (original method and 6-well plate modification), the expression of laminin was more distributed through the dermis compared to healthy substitutes, showing disorganization in the basal membrane. For the psoriatic skin substitutes reconstructed according to the 12-well plate modification, laminin staining was more compact and similar to healthy substitute expression. Interestingly, this observation is showing that the basal membrane was more organized, thus less similar to the psoriatic phenotype. These results showed that the 6-well plate modification is more representative of the *in vivo* psoriatic skin

These new modifications to protocol provide several advantages in the production of skin substitutes. Indeed, the 6-well and 12-well plate modifications require almost 3 times fewer

ated with the psoriatic phenotype are preserved with these new methods.

of healthy substitutes than the 12-well plate modification.

than the 12-well plate modification.

**2.3. Conclusion**

206 Cell Culture

The authors acknowledge financial support from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canadian Institutes of Health Research (CIHR) through their joint Collaborative Health Research Projects (CHRP) program. The "Fonds d'Enseignement et de Recherche (FER)" of the Faculty of Pharmacy, Université Laval, Québec, QC, Canada (Isabelle Gendreau scholarship), the "Fonds de Recherche du Québec—Santé (FRQS)" (Alexe Grenier scholarship) and the support of the "Réseau ThéCell du Québec" are also acknowledged. Moreover, Dr. Pouliot is a FRQS career award scholar.
