In Vitro Models for Evaluating Anti-Aging Formulations: Biomolecular Efficacy, Quality, and Safety Assessment
Keywords:
Anti-aging formulations, in vitro models, 2D skin models, 3D skin models, organ-on-chipAbstract
Introduction: The advancement of anti-aging formulations, both topical and injectable, requires robust preclinical strategies to ensure their biological effectiveness, safety, and quality. In vitro models have become pivotal in this process, offering ethically sound and mechanistically insightful platforms that reduce reliance on animal testing.
Objectives This review critically examines the spectrum of in vitro systems currently employed for the preclinical evaluation of anti-aging formulations.
Methods: We analyzed the range of in vitro systems currently used, from conventional 2D cultures to sophisticated 3D skin equivalents and organ-on-chip technologies, to evaluate their contribution to preclinical testing.
Results: These models contribute to understanding biomolecular pathways, formulation stability, and toxicological profiles.
Conclusions: Regulatory trends and translational challenges are discussed, highlighting the integration of in vitro methods with omics and computational tools as a promising frontier in aesthetic medicine.
References
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21. Cruz AM, Gonçalves MC, Marques MS, Veiga F, Paiva-Santos AC, Pires PC. In vitro models for anti-aging efficacy assessment: A critical update in dermocosmetic research. Cosmetics. 2023;10(2):66. doi:10.3390/cosmetics10020066.
22. Lombardi F, Augello FR, Ciafarone A, Ciummo V, Altamura S, Cinque B, Palumbo P. 3D models currently proposed to investigate human skin aging and explore preventive and reparative approaches: A descriptive review. Biomolecules. 2024;14(9):1066. doi:10.3390/biom14091066.
23. Kaplan BLF, Hoberman AM, Slikker W Jr, et al. Protecting human and animal health: The road from animal models to new approach methods. Pharmacol Rev. 2024;76(2):251-266. doi:10.1124/pharmrev.123.000967.
24. Henkler F, Tralau T, Tentschert J, et al. Risk assessment of nanomaterials in cosmetics: A European Union perspective. Arch Toxicol. 2012;86(11):1641-1646.
25. Letsiou S. Tracing skin aging process: A mini-review of in vitro approaches. Biogerontology. 2021;22(3):261-272. doi:10.1007/s10522-021-09916-z.
26. Quílez C, Bebiano LB, Jones E, et al. Targeting the complexity of in vitro skin models: A review of cutting-edge developments. J Invest Dermatol. 2024;144(12):2650-2670.
27. Russell WM. The development of the three Rs concept. Altern Lab Anim. 1995;23(3):298-304.
28. Hubrecht RC, Carter E. The 3Rs and humane experimental technique: Implementing change. Animals (Basel). 2019;9(10):754. doi:10.3390/ani9100754.
29. Deniz FSS, Orhan IE, Filipek PA, et al. Evaluation of the anti-aging properties of ethanolic extracts from selected plant species and propolis by enzyme inhibition assays and 2D/3D cell culture methods. Pharmaceuticals (Basel). 2025;18(3):439. doi:10.3390/ph18030439.
30. Hartmann C, Herling L, Hartmann A, et al. Systematic estimation of biological age of in vitro cell culture systems by an age-associated marker panel. Front Aging. 2023;4:1129107. doi:10.3389/fragi.2023.1129107.
31. Asselineau D, Farage M, Miller K, Maibach H. Reconstructed skin to create in vitro flexible models of skin aging: New results and prospects. In: Farage M, Miller K, Maibach H, eds. Textbook of Aging Skin. Berlin, Heidelberg: Springer; 2015. doi:10.1007/978-3-642-27814-3_48-2.
32. Jensen C, Teng Y. Is it time to start transitioning from 2D to 3D cell culture? Front Mol Biosci. 2020;7:33. doi:10.3389/fmolb.2020.00033.
33. Caloni F, De Angelis I, Hartung T. Replacement of animal testing by integrated approaches to testing and assessment (IATA): A call for in vivitrosi. Arch Toxicol. 2022;96(7):1935-1950. doi:10.1007/s00204-022-03299-x.
34. Wistner SC, Rashad L, Slaughter G. Advances in tissue engineering and biofabrication for in vitro skin modeling. Bioprinting. 2023;35:e00306. doi:10.1016/j.bprint.2023.e00306.
35. Duval K, Grover H, Han LH, et al. Modeling physiological events in 2D vs. 3D cell culture. Physiology (Bethesda). 2017;32(4):266-277. doi:10.1152/physiol.00036.2016.
36. Urzì O, Gasparro R, Costanzo E, et al. Three-dimensional cell cultures: The bridge between in vitro and in vivo models. Int J Mol Sci. 2023;24(15):12046. doi:10.3390/ijms241512046.
37. Flaten GE, Palac Z, Engesland A, et al. In vitro skin models as a tool in optimization of drug formulation. Eur J Pharm Sci. 2015;75:10-24. doi:10.1016/j.ejps.2015.02.018.
38. Lombardi F, Palumbo P, Augello FR, et al. Type I collagen suspension induces neocollagenesis and myodifferentiation in fibroblasts in vitro. Biomed Res Int. 2020;2020:6093974. doi:10.1155/2020/6093974.
39. Lombardi F, Augello FR, Artone S, et al. Efficacy of probiotic Streptococcus thermophilus in counteracting TGF-β1-induced fibrotic response in normal human dermal fibroblasts. J Inflamm (Lond). 2022;19(1):27. doi:10.1186/s12950-022-00324-9.
40. Augello FR, Lombardi F, Artone S, et al. Evaluation of the effectiveness of an innovative polycomponent formulation on adult and aged human dermal fibroblasts. Biomedicines. 2023;11(9):2410. doi:10.3390/biomedicines11092410.
41. Lee J, Bae IH, Choi J, et al. Development of a full-thickness human skin equivalent using human hair keratinocytes and fibroblasts. J Dermatol Sci. 2021;101(3):174-181. doi:10.1016/j.jdermsci.2021.01.004.
42. Ataç B, Wagner I, Horland R, et al. Skin and hair on-a-chip: Hair follicle formation and drug delivery in a vascularized microfluidic device. Lab Chip. 2013;13(18):3555-3561. doi:10.1039/c3lc50227a.
43. Wufuer M, Lee G, Hur W, et al. Skin-on-a-chip model simulating inflammation, edema and drug-based treatment. Sci Rep. 2016;6:37471. doi:10.1038/srep37471.
44. Mori N, Morimoto Y, Takeuchi S. Skin integrated with perfusable vascular channels on a chip. Biomaterials. 2017;116:48-56. doi:10.1016/j.biomaterials.2016.11.040.
45. Abaci HE, Guo Z, Doucet Y, Jacków J, Christiano AM. Next generation human skin constructs as advanced tools for drug development. Exp Biol Med (Maywood). 2017;242(17):1657-1668. doi:10.1177/1535370217719785.
46. Lee SH, Lee J, Lee J, et al. Construction of a human skin equivalent model using a collagen hydrogel containing fibroblasts and keratinocytes. J Vis Exp. 2020;(159):e61185. doi:10.3791/61185.
47. Kim BS, Lee J, Lee J, et al. Development of a vascularized skin equivalent model using 3D bioprinting and perfusion culture system. Biotechnol Bioeng. 2020;117(6):1853-1863. doi:10.1002/bit.27201.
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51. Lee J, Bae IH, Choi J, et al. Development of a full-thickness human skin equivalent using human hair keratinocytes and fibroblasts. J Dermatol Sci. 2021;101(3):174-181. doi:10.1016/j.jdermsci.2021.01.004.
52. Ataç B, Wagner I, Horland R, et al. Skin and hair on-a-chip: Hair follicle formation and drug delivery in a vascularized microfluidic device. Lab Chip. 2013;13(18):3555-3561. doi:10.1039/c3lc50227a.
53. Wufuer M, Lee G, Hur W, et al. Skin-on-a-chip model simulating inflammation, edema and drug-based treatment. Sci Rep. 2016;6:37471. doi:10.1038/srep37471.
54. Mori N, Morimoto Y, Takeuchi S. Skin integrated with perfusable vascular channels on a chip. Biomaterials. 2017;116:48-56. doi:10.1016/j.biomaterials.2016.11.040.
55. Abaci HE, Guo Z, Doucet Y, Jacków J, Christiano AM. Next generation human skin constructs as advanced tools for drug development. Exp Biol Med (Maywood). 2017;242(17):1657-1668. doi:10.1177/1535370217719785.
56. Lee SH, Lee J, Lee J, et al. Construction of a human skin equivalent model using a collagen hydrogel containing fibroblasts and keratinocytes. J Vis Exp. 2020;(159):e61185. doi:10.3791/61185.
57. Kim BS, Lee J, Lee J, et al. Development of a vascularized skin equivalent model using 3D bioprinting and perfusion culture system. Biotechnol Bioeng. 2020;117(6):1853-1863. doi:10.1002/bit.27201.
58. Mori N, Morimoto Y, Takeuchi S. Skin-on-a-chip model with perfusable vasculature for studying inflammation and drug response. Lab Chip. 2018;18(14):2323-2330. doi:10.1039/c8lc00438a.
59. Abaci HE, Guo Z, Coffman A, et al. Human skin constructs with spatially controlled vasculature using primary and iPSC-derived endothelial cells. Adv Healthc Mater. 2016;5(14):1800-1807. doi:10.1002/adhm.201600167.
60. Kim BS, Park Y, Park S. Generation of 3D skin model using 3D bioprinting technology. J Dermatol Sci. 2022;105(2):92-94. doi:10.1016/j.jdermsci.2022.01.005.
61. Lee J, Bae IH, Choi J, et al. Development of a full-thickness human skin equivalent using human hair keratinocytes and fibroblasts. J Dermatol Sci. 2021;101(3):174-181. doi:10.1016/j.jdermsci.2021.01.004.
62. Ataç B, Wagner I, Horland R, et al. Skin and hair on-a-chip: Hair follicle formation and drug delivery in a vascularized microfluidic device. Lab Chip. 2013;13(18):3555-3561. doi:10.1039/c3lc50227a.
63. Wufuer M, Lee G, Hur W, et al. Skin-on-a-chip model simulating inflammation, edema and drug-based treatment. Sci Rep. 2016;6:37471. doi:10.1038/srep37471.
64. Mori N, Morimoto Y, Takeuchi S. Skin integrated with perfusable vascular channels on a chip. Biomaterials. 2017;116:48-56. doi:10.1016/j.biomaterials.2016.11.040.
65. Abaci HE, Guo Z, Doucet Y, Jacków J, Christiano AM. Next generation human skin constructs as advanced tools for drug development. Exp Biol Med (Maywood). 2017;242(17):1657-1668. doi:10.1177/1535370217719785.
66. Lee SH, Lee J, Lee J, et al. Construction of a human skin equivalent model using a collagen hydrogel containing fibroblasts and keratinocytes. J Vis Exp. 2020;(159):e61185. doi:10.3791/61185.
67. Kim BS, Lee J, Lee J, et al. Development of a vascularized skin equivalent model using 3D bioprinting and perfusion culture system. Biotechnol Bioeng. 2020;117(6):1853-1863. doi:10.1002/bit.27201.
68. Mori N, Morimoto Y, Takeuchi S. Skin-on-a-chip model with perfusable vasculature for studying inflammation and drug response. Lab Chip. 2018;18(14):2323-2330. doi:10.1039/c8lc00438a.
69. Abaci HE, Guo Z, Coffman A, et al. Human skin constructs with spatially controlled vasculature using primary and iPSC-derived endothelial cells. Adv Healthc Mater. 2016;5(14):1800-1807. doi:10.1002/adhm.201600167.
70. Kim BS, Park Y, Park S. Generation of 3D skin model using 3D bioprinting technology. J Dermatol Sci. 2022;105(2):92-94. doi:10.1016/j.jdermsci.2022.01.005.
71. Lee J, Bae IH, Choi J, et al. Development of a full-thickness human skin equivalent using human hair keratinocytes and fibroblasts. J Dermatol Sci. 2021;101(3):174-181. doi:10.1016/j.jdermsci.2021.01.004.
72. Ataç B, Wagner I, Horland R, et al. Skin and hair on-a-chip: Hair follicle formation and drug delivery in a vascularized microfluidic device. Lab Chip. 2013;13(18):3555-3561. doi:10.1039/c3lc50227a.
73. Wufuer M, Lee G, Hur W, et al. Skin-on-a-chip model simulating inflammation, edema and drug-based treatment. Sci Rep. 2016;6:37471. doi:10.1038/srep37471.
74. Mori N, Morimoto Y, Takeuchi S. Skin integrated with perfusable vascular channels on a chip. Biomaterials. 2017;116:48-56. doi:10.1016/j.biomaterials.2016.11.040.
75. Abaci HE, Guo Z, Doucet Y, Jacków J, Christiano AM. Next generation human skin constructs as advanced tools for drug development. Exp Biol Med (Maywood). 2017;242(17):1657-1668. doi:10.1177/1535370217719785.
76. Lee SH, Lee J, Lee J, et al. Construction of a human skin equivalent model using a collagen hydrogel containing fibroblasts and keratinocytes. J Vis Exp. 2020;(159):e61185. doi:10.3791/61185.
77. Kim BS, Lee J, Lee J, et al. Development of a vascularized skin equivalent model using 3D bioprinting and perfusion culture system. Biotechnol Bioeng. 2020;117(6):1853-1863. doi:10.1002/bit.27201.
78. Mori N, Morimoto Y, Takeuchi S. Skin-on-a-chip model with perfusable vasculature for studying inflammation and drug response. Lab Chip. 2018;18(14):2323-2330. doi:10.1039/c8lc00438a.
79. Abaci HE, Guo Z, Coffman A, et al. Human skin constructs with spatially controlled vasculature using primary and iPSC-derived endothelial cells. Adv Healthc Mater. 2016;5(14):1800-1807. doi:10.1002/adhm.201600167.
80. Kim BS, Park Y, Park S. Generation of 3D skin model using 3D bioprinting technology. J Dermatol Sci. 2022;105(2):92-94. doi:10.1016/j.jdermsci.2022.01.005.
81. Lee J, Bae IH, Choi J, et al. Development of a full-thickness human skin equivalent using human hair keratinocytes and fibroblasts. J Dermatol Sci. 2021;101(3):174-181. doi:10.1016/j.jdermsci.2021.01.004.
82. Ataç B, Wagner I, Horland R, et al. Skin and hair on-a-chip: Hair follicle formation and drug delivery in a vascularized microfluidic device. Lab Chip. 2013;13(18):3555-3561. doi:10.1039/c3lc50227a.
83. Wufuer M, Lee G, Hur W, et al. Skin-on-a-chip model simulating inflammation, edema and drug-based treatment. Sci Rep. 2016;6:37471. doi:10.1038/srep37471.
84. Mori N, Morimoto Y, Takeuchi S. Skin integrated with perfusable vascular channels on a chip. Biomaterials. 2017;116:48-56. doi:10.1016/j.biomaterials.2016.11.040.
85. Abaci HE, Guo Z, Doucet Y, Jacków J, Christiano AM. Next generation human skin constructs as advanced tools for drug development. Exp Biol Med (Maywood). 2017;242(17):1657-1668. doi:10.1177/1535370217719785.
86. Lee SH, Lee J, Lee J, et al. Construction of a human skin equivalent model using a collagen hydrogel containing fibroblasts and keratinocytes. J Vis Exp. 2020;(159):e61185. doi:10.3791/61185.
87. Kim BS, Lee J, Lee J, et al. Development of a vascularized skin equivalent model using 3D bioprinting and perfusion culture system. Biotechnol Bioeng. 2020;117(6):1853-1863. doi:10.1002/bit.27201.
88. Mori N, Morimoto Y, Takeuchi S. Skin-on-a-chip model with perfusable vasculature for studying inflammation and drug response. Lab Chip. 2018;18(14):2323-2330. doi:10.1039/c8lc00438a.
89. Abaci HE, Guo Z, Coffman A, et al. Human skin constructs with spatially controlled vasculature using primary and iPSC-derived endothelial cells. Adv Healthc Mater. 2016;5(14):1800-1807. doi:10.1002/adhm.201600167.
90. Kim BS, Park Y, Park S. Generation of 3D skin model using 3D bioprinting technology. J Dermatol Sci. 2022;105(2):92-94. doi:10.1016/j.jdermsci.2022.01.005.
91. Abaci HE, Jacków J, Doucet Y, Christiano AM. Human skin constructs with hair follicles for modeling hair disorders and drug testing. Exp Dermatol. 2018;27(5):453-455. doi:10.1111/exd.13591.
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Copyright (c) 2026 Gino Orsini, Francesca Rosaria Augello, Alessia Ciafarone, Maria Grazia Cifone, Maurizio Giuliani
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