INTRODUCTION: The historical concentration of beauty marketing to the young and beautiful has thankfully given way to a broader fairer attention to women of all ages, and even men, with global cosmetics increasingly becoming a mainstream accepted globally. However, cosmetics for all ages is a challenge not to be underestimated. Young skin has significantly different characteristics to older skin. Thinning characteristics of older skin with changes in matrix protein glycosaminoglycan combinations, lower collagen and hyaluronic acid, damaged elastin and increased water loss from the barrier all lead to differences in how cosmetics react on older skin. The more sinusoidal nature of older skin in general and tendance towards wrinkles has important implications for how cosmetic ingredients are adsorbed to the skin. In addition, many of the OECD recommended tests are based on out of date manually constructed epidermal models, which more often than not contain pooled keratinocytes from many donors, with no regard for immunology at all.
For this reason, we have advanced our 3D bioprinted full thickness skin models towards creating a variety of age-related models, with same-donor cells (rare for in vitro skin models) to reproduce the level of thickness and sinusoidal behavior seen in real life with the aim to help advance efficacy testing.
METHODS: Human keratinocytes and fibroblasts were expanded from donations of human male and female skin donated following elective surgery and full ethical consent from donors ranging from 1 years old to 67 years old, both male and female. Cells at passage 2 or 3 were selected for optimal growth and mixed with a bioink (CELLINK, Sweden), into which epidermal adhesion proteins were added and cartridged into a CELLINK pneumatic 3D bioprinting system, after modelization with Sketchup and SLIC3R slicing software (described at IFSCC2020, Henry Maso award 2022). Full thickness epidermal-dermal skin same-donor models were printed at different thicknesses and with additional or subtracted laminin and collagen substitution. Delayed epidermal maturation was also investigated by promotion of the dermal-epidermal junction before airlifting the model. Subsequent models made with younger and older donors were then harvested for histological analysis to evaluate structure and function of the models.
RESULTS: Even before modulating conditions of the models, it was clear that older donor samples have a tendency to grow in a slightly slower manner with more capability to form sinusoidal junctions between the epidermis and dermis. Younger donors tended to mature faster and with a flatter less wrinkled surface. By using a sacrificial bioprinting ink it was also possible to delay the formation of the epidermal-dermal junction and allow the formation of deeper wrinkle-like grooves seen in older skin, which was extremely important for kinetic adsorption measurements with not only ingredients but also finished products, something which is not always considered in efficacy testing.
DISCUSSION: The overall structure of the models allowed for a more ‘real world’ situation and given that both female and male models were effectively created from same donor cell sources, the immunology of these models can be considered more realistic in terms of safety testing, since the minor histocompatibility antigen system was not interfering in the same donor model. Given that final real-human donor testing can show irritation in finished products which previously passed in vitro testing, it is perhaps time to re-evaluate the safety testing parameters of lab models to be more immunologically sound and help the development of safe cosmetics.
CONCLUSION: It is now possible to select age-groups in efficacy testing which will allow for targeted product development in vitro to relate to in vivo final donor testing of cosmetics with better consideration of the human immunology involved.