Recently, various types of in vitro-reconstructed 3D skin models have been developed for drug testing and disease modeling. Herein, we structurally and functionally validated a self-assembled reconstructed skin equivalent (RSE) and developed an IL-17a-induced in vitro psoriasis-like model using a self-assembled RSE. The tissue engineering approach was used to construct the self-assembled RSE. The dermal layer was generated using fibroblasts secreting their own ECM, and the epidermal layer was reconstructed by seeding keratinocytes on the dermal layer. To generate the psoriatic model, IL-17A was added to the culture medium during the air–liquid interface culture period. Self-assembled RSE resulted in a fully differentiated epidermal layer, a well-established basement membrane, and dermal collagen deposition. In addition, self-assembled RSE was tested for 20 reference chemicals according to the Performance Standard of OECD TG439 and showed overall sensitivity, specificity, and accuracy of 100%, 90%, and 95%, respectively. The IL-17a-treated psoriatic RSE model exhibited psoriatic epidermal characteristics, such as epidermal hyperproliferation, parakeratosis, and increased expression of KRT6, KRT17, hBD2, and S100A9. Thus, our results suggest that a self-assembled RSE that structurally and functionally mimics the human skin has a great potential for testing various drugs or cosmetic ingredients and modeling inflammatory skin diseases.

Hair follicle stem cells, located in the bulge region of the outer root sheath, are multipotent epithelial stem cells capable of differentiating into epidermal, sebaceous gland, and hair shaft cells. Efficient culturing of these cells is crucial for advancements in dermatology, regenerative medicine, and skin model development. This investigation aimed to develop a protocol for isolating enriched bulge-derived epithelial cells from scalp specimens to produce tissue-engineered substitutes. The epithelium, including hair follicles, was separated from the dermis using thermolysin, followed by microdissection of the bulge region. Epithelial stem cells were isolated using enzymatic dissociation to create a single-cell suspension and compared with the direct explant culture and a benchmark method which isolates cells from the epidermis and pilosebaceous units. After 8 days of culture, the enzymatic digestion of microdissected bulges yielded 5.3 times more epithelial cells compared to explant cultures and proliferated faster than the benchmark method. Cells cultured from all methods exhibited comparable morphology and growth rates. The fully stratified epidermis of tissue-engineered skin was similar, indicating comparable differentiation potential. This enzymatic digestion method improved early-stage cell recovery and expansion while maintaining keratinocyte functionality, offering an efficient hair bulge cell-extraction technique for tissue engineering and regenerative medicine applications.

Hypertrophic scarring is a common complication in severely burned patients who undergo autologous skin grafting. Meshed skin grafts tend to contract during wound healing, increasing the risk of pathological scarring. Although various technologies have been used to study cellular contraction, current methods for measuring contractile forces at the tissue level are limited and do not replicate the complexity of native tissues. Self-assembled skin substitutes (SASSs) were developed at the "Centre de recherche en organogénèse expérimentale de l'Université Laval/LOEX" (LOEX) and are used as permanent full-thickness skin grafts. The autologous skin substitutes are produced using the self-assembly method, allowing the cultured cells to produce their own extracellular matrix (ECM) leading to a tissue-engineered substitute resembling the native skin. The level of contraction of the SASSs during the fabrication process is patient-dependent. Thus, because of its architecture and composition, SASS is an interesting model to study skin contraction in vitro. Unfortunately, standard measurement methods are unsuited for SASS contraction assessment, mainly due to incompatibilities between the SASS manufacturing process and the current contraction force measurement methods. Here, we present an innovative contraction measurement method specifically designed to quantify the contractile behavior of tissue-engineered substitutes, without disrupting the protocol of production. The method uses C-shape anchoring frames that closes at different speed and magnitude according to the tissue contractile behavior. A finite element analysis model is then used to associate the frame deformation to a contractile force amplitude. This paper shows that the method can be used to measure the contraction force of tissues produced with cells displaying different contractile properties, such as primary skin fibroblasts and myofibroblasts. It can also be used to study the effects of cell culture conditions on tissue contraction, such as serum concentration. This protocol can be easily and affordably applied and tuned to many regenerative medicine applications or contraction-related pathological studies.

Stratum Corneum (SC) formation in the human epidermis requires lipid processing. Lipoxygenases (LOXs) such as 12R-Lipoxygenase (12R-LOX) and Epidermis-type lipoxygenase 3 (eLOX-3) contribute to this process. Mutations in their genes cause Autosomal Recessive Congenital Ichthyosis (ARCI) in patients. On the other hand, 12S-lipoxygenase (12S-LOX) is expressed in the human epidermis, but its role still remains to be clarified. The involvement of eLOX-3, 12R, and 12S-LOX in conditions or processes such as skin photodamage, wound healing, psoriasis, and atopic dermatitis is suggested but still remains unclear. In order to eventually gain a better understanding of the role of these LOXs in such processes, models of Tissue-Engineered Skins (TESs) with an impaired expression for the native form of either eLOX-3, 12R-LOX, or 12S-LOX were produced using CRISPR-Cas9(D10A) technology. All three models showed impaired keratinocyte differentiation and changes in the prevalence or the size of lipid droplets within the most superficial layers, thus reproducing features observed in ARCI and supporting a role for 12S-LOX in SC formation. Since eLOX-3 and 12R-LOX depleted TES’s reproduced features observed in ARCI, such models can be considered as reliable tools for the functional studies of these LOXs in the human epidermis.