In vitro induction of hair follicle signatures using human dermal papilla cells encapsulated in fibrin microgels.

Cellular spheroids have been described as an appropriate culture system to restore human follicle dermal papilla cells (hFDPc) intrinsic properties; however, they show a low and variable efficiency to promote complete hair follicle formation in in vivo experiments. In this work, a conscientious analysis revealed a 25% cell viability in the surface of the dermal papilla spheroid (DPS) for all culture conditions, questioning whether it is an appropriate culture system for hFDPc. To overcome this problem, we propose the use of human blood plasma for the generation of fibrin microgels (FM) with encapsulated hFDPc to restore its inductive signature, either in the presence or in the absence of blood platelets. FM showed a morphology and extracellular matrix composition similar to the native dermal papilla, including Versican and Collagen IV and increasing cell viability up to 85%. While both systems induce epidermal invaginations expressing hair-specific keratins K14, K15, K71, and K75 in in vitro skin cultures, the number of generated structures increases from 17% to 49% when DPS and FM were used, respectively. These data show the potential of our experimental setting for in vitro hair follicle neogenesis with wild adult hFDPc using FM, being a crucial step in the pursuit of human hair follicle regeneration therapies.

Evaluation of different methodologies for primary human dermal fibroblast spheroid formation: automation through 3D bioprinting technology.

Cell spheroids have recently emerged as an effective tool to recapitulate native microenvironments of living organisms in anin vitroscenario, increasing the reliability of the results obtained and broadening their applications in regenerative medicine, cancer research, disease modeling and drug screening. In this study the generation of spheroids containing primary human dermal fibroblasts was approached using the two-widely employed methods: hanging-drop and U-shape low adhesion plate (LA-plate). Moreover, extrusion-based three-dimensional (3D) bioprinting was introduced to achieve a standardized and scalable production of cell spheroids, decreasing considerably the possibilities of human error. This was ensured when U-shape LA-plates were used, showing an 85% formation efficiency, increasing up to a 98% when it was automatized using the 3D bioprinting technologies. However, sedimentation effect within the cartridge led to a reduction of 20% in size of the spheroid during the printing process. Hyaluronic acid (HA) was chosen as viscosity enhancer to supplement the bioink and overcome cell sedimentation within the cartridge due to the high viability values exhibited by the cells-around 80%-at the used conditions. Finally, (ANCOVA) of spheroid size over time for different printing conditions stand out HA 0.4% (w/v) 60 kDa as the viscosity-improved bioink that exhibit the highest cell viability and spheroid formation percentages. Besides, not only did it ensure cell spheroid homogeneity over time, reducing cell sedimentation effects, but also wider spheroid diameters over time with less variability, outperforming significantly manual loading.