Effect of Retinoic Acid on HaCaT and NIH-3T3 cells in an in vitro 3D Collagen Cell Culture Skin Model

Abstract

Human skin aging is characterized by epidermal and dermal thinning, loss of elasticity, and wrinkles. Keratinocytes, the most common type of skin cell and fibroblasts, present in the cellular stroma beneath the skin’s surface, each play a role in aging. Using these cell types in in vitro research can reveal a deeper understanding of the dermatological function and cellular changes in aged skin. 3D cell culture techniques provide an opportunity to use these cell types in a model that can more accurately mimic human skin. Treatment of aging skin is of interest to both medical and consumer communities. Retinoic acid (RA) is a metabolite of vitamin A and retinol that assists in cell proliferation, differentiation, and immune functions. Over the counter (OTC) and prescription retinoids are common topical products used for anti-aging and acne treatments. This study seeks to determine the impact of topical retinoid creams on keratinocyte proliferation and morphology in 3D cell culture models of aged and unaged human skin. NIH-3T3 fibroblasts were embedded in a 3D collagen matrix of varying thickness, and HaCaT keratinocytes were seeded on top of the matrix at varying seeding densities to mimic aging and youthful skin. 0.1 µM 0.025% tretinoin and 0.1 µM 0.1% adapalene topical creams were prepared in culture medium and used to treat cells daily, on alternate days, or just once during a week-long period. Alamar Blue assays and microscopy showed that tretinoin treatment was cytotoxic at this concentration, with a single treatment reducing cell viability by ~43% compared to the untreated control. Adapalene treatment, while showing significantly greater cell proliferation than tretinoin, did not exceed the proliferation of the untreated control. It is understood that retinol increases cell turnover by killing cells rapidly, so it is proposed that in our model, the rate of proliferation does not overcome the rate of cell death. Cell viability trends remain similar between young and old skin model treatments. Future studies should focus on creating a 3D model more accurately representing the aging in vivo skin environment where keratinocytes can be readily differentiated from epidermal stem cells. KEYWORDS: HaCaTs; NIH-3T3; Retinoids; Retinoic Acid; Tretinoin; Adapalene; 3D Collagen Gel; Proliferation