MATERIALS AND METHODS
Tissue Models
EpiDermFT™ 3D full thickness in vitro human skin models (MatTek Corp, Ashland, MA) were cultured with EpiDermFT Assay Media (MatTek Corp). Tissues were irradiated with 200mJ/cm2 ultraviolet (UV) light with UV-B filter lamp (Honle, Germany) followed by application of 15 μL of TTC or dH2O (control) and incubated at 37°C and 5% CO2 for 24 hours.
Ex vivo studies were performed using Hyposkin® human skin explants (Genoskin, Salem, MA). After equilibrating tissues with Hyposkin® Medium (Genoskin), 15μL of TTC or dH2O was applied and tissues were incubated at 37°C and 5% CO2. The topical composition was re-applied, and media changed every 24 hours for an additional two days.
Quantitative Real-Time PCR
After incubation, tissues were collected into Invitrogen™ RNAlater® solution (ThermoFisher Scientific, Waltham, MA). mRNA was extracted using the Maxwell® RSC simplyRNA Tissue Kit (Promega, Madison, WI) followed by cDNA synthesis using the Applied Biosystems™ High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific). Gene expression was evaluated using Applied Biosystems™ Fast Advanced Master Mix and pre-designed TaqMan Gene Expression Assays on the QuantStudio7 Flex instrument (ThermoFisher Scientific). Relative expression was calculated compared to UV irradiated control treated samples (in vitro), or to control treated skin (ex vivo), and was normalized to GAPDH.
EpiDermFT™ 3D full thickness in vitro human skin models (MatTek Corp, Ashland, MA) were cultured with EpiDermFT Assay Media (MatTek Corp). Tissues were irradiated with 200mJ/cm2 ultraviolet (UV) light with UV-B filter lamp (Honle, Germany) followed by application of 15 μL of TTC or dH2O (control) and incubated at 37°C and 5% CO2 for 24 hours.
Ex vivo studies were performed using Hyposkin® human skin explants (Genoskin, Salem, MA). After equilibrating tissues with Hyposkin® Medium (Genoskin), 15μL of TTC or dH2O was applied and tissues were incubated at 37°C and 5% CO2. The topical composition was re-applied, and media changed every 24 hours for an additional two days.
Quantitative Real-Time PCR
After incubation, tissues were collected into Invitrogen™ RNAlater® solution (ThermoFisher Scientific, Waltham, MA). mRNA was extracted using the Maxwell® RSC simplyRNA Tissue Kit (Promega, Madison, WI) followed by cDNA synthesis using the Applied Biosystems™ High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific). Gene expression was evaluated using Applied Biosystems™ Fast Advanced Master Mix and pre-designed TaqMan Gene Expression Assays on the QuantStudio7 Flex instrument (ThermoFisher Scientific). Relative expression was calculated compared to UV irradiated control treated samples (in vitro), or to control treated skin (ex vivo), and was normalized to GAPDH.
RESULTS
Key ingredients and mechanisms for TTC are presented in Table 1. The utility of TTC was first tested using in vitro UVdamaged 3D-reconstructed human skin, which contains both dermis and epidermis. Application of TTC resulted in significant upregulation of all ECM-associated genes tested including collagens, elastic fiber proteins and TGFβ1 (Figure 1A), Treatment with TTC selectively upregulated pro-lymphangiogenic VEGFC (Figure 1B), and significant induction of anti-inflammatory IL-10 was observed compared to pro-inflammatory IL-6 (Figure 1C). In addition, expression of key genes involved in major cellular 
