Development of a Novel Platform Technology to Enhance the Growth of Human Skin Models in vitro for the Use in Biomedical Research

Abstract

The skin is the most exposed organ of the body and endures a wide range of external insults. Consequently, it is often the first to encounter potentially toxic substances and plays a critical role in the testing regimes mandated by regulatory authorities. The OECD has already approved several in vitro skin models for substance testing, significantly reducing the reliance on animal experimentation. Despite these advancements, the application of current skin models remains limited. A key limitation is the short longevity of in vitro models, which challenges their use in extended studies, such as those required for chronic exposure testing. A promising solution lies in the development of dynamic culture systems, which have been shown to improve tissue morphogenesis, functionality, and preservation compared to static cultures.
A novel dynamic culture platform based on a stirring principle was developed, and 3D printing technology was leveraged to manufacture customized cell culture components. This system was applied for the dynamic culture of a bioengineered full thickness skin model, characterized by its de novo synthesis of dermal extracellular matrix proteins by human dermal fibroblasts, facilitated through Transforming Growth Factor-beta 1 (TGF-ß1) supplementation. Additionally, indicative insights into the system’s flow properties were gained by using computational fluid dynamics as a supportive exploratory approach. Exposing dermal equivalents to high stirring speeds in vitro enhanced scaffold infiltration, increased extracellular matrix (ECM) deposition, and amplified myofibroblast differentiation. The observation of an activated dermal tissue state under high flow rates has been reported in literature before and demonstrated the generation of high flow velocities through high stirring speeds, exceeding the physiological ranges typically experienced by dermal fibroblasts. When dynamic flow was applied to accelerate dermal tissue maturation at high stirring speeds, substantial changes at the full thickness stage were observed, including augmented epidermal activation. While accelerated dermal maturation reduced model generation time, tissue activation may impair the establishment of a homeostatic state, potentially compromising the longevity of the in vitro model.
The design of the dynamic stirred system was optimized aiming at flow properties within a physiologically relevant range of dermal interstitial flow. Further, the dynamic protocol was adapted to establish a TGF-ß1 supplementation-free method. This enabled the generation of robust dermal equivalents with a de novo synthesised ECM, significantly reduced myofibroblast differentiation, and a shorter model formation time. These dermal equivalents were successfully used to build full thickness skin models with a reduced activation state and a stratified, fully differentiated epidermis. Models generated with this protocol may facilitate earlier establishment of tissue homeostasis, with the potential to extend in vitro longevity.
Finally, dynamic culture at the air-liquid interface in the optimized stirred system demonstrated superior tissue preservation in full thickness skin models over an extended culture period of five weeks compared to static cultures. This work reports, for the first time, the use of a stirred dynamic platform for the culture of a bioengineered full thickness skin model. The results demonstrate the great potential of this methodology to enhance the generation of skin models while providing a simplified set-up that is easy to handle and adaptable to other in vitro models.