The development of anin vitrohuman hair follicle organoid with a complexity similar to thatin vivo.

In vitrohair follicle (HF) models are currently limited toex vivoHF organ cultures (HFOCs) or 2D models that are of low availability and do not reproduce the architecture or behavior of the hair, leading to poor screening systems. To resolve this issue, we developed a technology for the construction of a humanin vitrohair construct based on the assemblage of different types of cells present in the hair organ. First, we demonstrated that epithelial cells, when isolatedin vitro, have similar genetic signatures regardless of their dissection site, and their trichogenic potential is dependent on the culture conditions. Then, using cell aggregation techniques, 3D spheres of dermal papilla (DP) were constructed, and subsequently, epithelial cells were added, enabling the production and organization of keratins in hair, similar to what is seenin vivo. These reconstructed tissues resulted in the following hair compartments: K71 (inner root-sheath), K85 (matrix region), K75 (companion layer), and vimentin (DP). Furthermore, the new hair model was able to elongate similarly toex vivoHFOC, resulting in a shaft-like shape several hundred micrometers in length. As expected, when the model was exposed to hair growth enhancers, such as ginseng extract, or inhibitors, such as TGF-B-1, significant effects similar to thosein vivowere observed. Moreover, when transplanted into skin biopsies, the new constructs showed signs of integration and hair bud generation. Owing to its simplicity and scalability, this model fully enables high throughput screening of molecules, which allows understanding of the mechanism by which new actives treat hair loss, finding optimal concentrations, and determining the synergy and antagonism among different raw materials. Therefore, this model could be a starting point for applying regenerative medicine approaches to treat hair loss.

Impact of microstructure on cell behavior and tissue mechanics in collagen and dermal decellularized extra-cellular matrices.

Skin models are used for many applications such as research and development or grafting. Unfortunately, most lack a proper microenvironment producing poor mechanical properties and inaccurate extra-cellular matrix composition and organization. In this report we focused on mechanical properties, extra-cellular matrix organization and cell interactions in human skin samples reconstructed with pure collagen or dermal decellularized extra-cellular matrices (S-dECM) and compared them to native human skin. We found that Full-thickness S-dECM samples presented stiffness two times higher than collagen gel and similar to ex vivo human skin, and proved for the first time that keratinocytes also impact dermal mechanical properties. This was correlated with larger fibers in S-dECM matrices compared to collagen samples and with a differential expression of F-actin, vinculin and tenascin C between S-dECM and collagen samples. This is clear proof of the microenvironment's impact on cell behaviors and mechanical properties. STATEMENT OF SIGNIFICANCE: In vitro skin models have been used for a long time for clinical applications or in vitro knowledge and evaluation studies. However, most lack a proper microenvironment producing a poor combination of mechanical properties and appropriate biological outcomes, partly due to inaccurate extra-cellular matrix (ECM) composition and organization. This can lead to limited predictivity and weakness of skin substitutes after grafting. This study shows, for the first time, the importance of a complex and rich microenvironment on cell behaviors, matrix macro- and micro-organization and mechanical properties. The increased composition and organization complexity of dermal skin decellularized extra-cellular matrix populated with differentiated cells produces in vitro skin models closer to native human skin physiology.

A perfusable vascularized full-thickness skin model for potential topical and systemic applications.

Vascularization of reconstructed tissues is one of the remaining hurdles to be considered to improve both the functionality and viability of skin grafts and the relevance ofin vitroapplications. Our study, therefore, sought to develop a perfusable vascularized full-thickness skin equivalent that comprises a more complex blood vasculature compared to existing models. We combined molding, auto-assembly and microfluidics techniques in order to create a vascularized skin equivalent representing (a) a differentiated epidermis with a physiological organization and correctly expressing K14, K10, Involucrin, TGM1 and Filaggrin, (b) three perfusable vascular channels with angiogenic sprouts stained with VE-Caderin and Collagen IV, (c) an adjacent microvascular network created via vasculogenesis and connected to the sprouting macrovessels. Histological analysis and immunostaining of CD31, Collagen IV, Perlecan and Laminin proved the integrity of vascular constructs. In order to validate the vascularized skin potential of topical and systemic applications, caffeine and minoxidil, two compounds with different chemical properties, were topically applied to measure skin permeability and benzo[a]pyrene pollutant was systemically applied to evaluate systemic delivery. Our results demonstrated that perfusion of skin reconstructs and the presence of a complex vascular plexus resulted in a more predictive and reliable model to assess respectively topical and systemic applications. This model is therefore aimed at furthering drug discovery and improving clinical translation in dermatology.

Xeno-free production of human embryonic stem cells in stirred microcarrier systems using a novel animal/human-component-free medium.

Currently, stem cell research faces a major bottleneck related to the low efficiency of methods to produce large quantities of human embryonic stem cells (ESC) for use in clinical trials. Most culture media currently employed for human ESC cultivation contain animal compounds, and cells are grown in static flasks. Besides the immediate contamination with nonhuman compounds, cell expansion in flasks tends to be laborious and nonefficient. Here we cultured human ESC in stirred microcarrier (MC) systems using an animal/human-component-free medium, to overcome both issues. The method developed to culture cells on suspended beads combined the use of polymeric MCs in stirred vessels with an optimized culture medium free of supplements of animal and human origin. This approach generated approximately 160 million cells within 6 days, which were shown to remain pluripotent. The process developed herein provides a step forward toward therapy due to the economic advantages in the production of human ESC and to their consequent low immunogenic potential.