Background: Recently, investigators have been trying to apply the by-products as well as stem cells themselves to various fields such as pharmaceuticals, medical devices, quasi-drug, cosmetis, etc. We aimed to comfirm the antisenescence effect of 3D cultured adipose tissue-derived mesenchymal stem cell-conditioned medium (3D cultured ADMSCs-CM) and develop them as cosmetic raw materials for anti-aging purposes. Methods: We investigated the effect of 3D cultured ADMSCs-CM on collagen production and performed efficacy tests to evaluate the effect of a cream-based cosmetic product containing the medium using various methods, such as dermal density, skin moisture retention, and so on. Results: Analysis of the effect of ADMSCs-CM on skin regeneration and production of collagen showed 1.5-fold (2D cultured ADMSCs-CM) and 2.5-fold (3D cultured ADMSCs-CM) increase in expressions of procollagen and 4-fold (2D cultured ADMSCs-CM) and 5-fold (3D cultured ADMSCs-CM) increase in the expression of collagen compared with control. In addition, related gene expression was also increased. We conducted a human skin test using a cream-based product containing 3D cultured ADMSCs-CM. In skin texture assessment, skin roughness decreased by 11.94% at the application site and 3.74% at the non-application site after 3 weeks of use. Compared with before cream use, after 2 and 4 weeks of substance use, the skin elasticity analysis showed an increase in the elasticity value by 5.97% and 9.34%, respectively, and the improvement of small wrinkles was 5.01% and 6.23%, respectively. After 2 and 4 weeks of test substance use, dermal density analysis showed 6.97% and 12.53% increase, respectively. Skin moisture retention analysis showed skin moisture maintained at 543.60% and 452.38%, respectively, immediately after one-time use and after 20 min of cool breeze exposure compared with before application of the test substance. Conclusions: As raw material for cosmetic products, 3D cultured ADMSCs-CM prevented skin aging by promoting collagen production, restoring damaged skin, and increasing dermal density. Therefore, 3D cultured ADMSCs-CM can be widely applied to maintain and improve skin condition.

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