Dermal fibroblast cell‐derived exosomes for atopic dermatitis: In‐vitro test

The exosome, a kind of extracellular vesicle with a diameter in the range of 50–200 nm, has been extensively investigated in treating various skin issues, such as skin aging mitigation and cutaneous wound healing stimulation, and atopic dermatitis (AD). 1–3 Compared to stem cell therapy, advantageous properties, such as a long half-life, small size, favorable penetration, and low immunogenicity, make exosomes more favorable and beneficial. 4 Although exosomes have been extensively investigated for their application in skin damage repair, the only sources shown to be effective are those derived from mesenchymal stem cells (MSCs). 5 However, exosome isolation from MSCs accompanies multiple drawbacks, such as the time and effort required for MSC culture and expansion, the risk of contamination with red blood cells during the isolation process, neovascularization potential, diminished cell viability, and the extreme invasiveness of removing MSCs from bone marrow. 6 Therefore, finding an alternative cell source for exosome isolation has become imperative for the effective application of exosomes inthe repair of dysfunctional skin. Dermal fibroblasts (DFs) are the predominant cell type in the dermis and are responsible for regulating the extracellular matrix (ECM) while playing important roles in the maintenance of the normal structure and function of the skin. According to the previous study, DFs are considered a useful cell line in which to study exosome-related treatments because these cells are easily cultivated. In this article, we suggest DFs for the extraction of exosomes because, in contrast with MSCs, fibroblasts more closely resembletheskinandmaybeobtainedfromtheskinusinglessinvasive techniques. 7 This study thus aimed to determine if DF-derived exosomes are a promising candidate for skin damage repair. To investigate the effects of DF exosomes

panies multiple drawbacks, such as the time and effort required for MSC culture and expansion, the risk of contamination with red blood cells during the isolation process, neovascularization potential, diminished cell viability, and the extreme invasiveness of removing MSCs from bone marrow. 6 Therefore, finding an alternative cell source for exosome isolation has become imperative for the effective application of exosomes in the repair of dysfunctional skin. Dermal fibroblasts (DFs) are the predominant cell type in the dermis and are responsible for regulating the extracellular matrix (ECM) while playing important roles in the maintenance of the normal structure and function of the skin. According to the previous study, DFs are considered a useful cell line in which to study exosome-related treatments because these cells are easily cultivated. In this article, we suggest DFs for the extraction of exosomes because, in contrast with MSCs, fibroblasts more closely resemble the skin and may be obtained from the skin using less invasive techniques. 7 This study thus aimed to determine if DF-derived exosomes are a promising candidate for skin damage repair. To investigate the effects of DF exosomes on skin permeability barrier protection, we evaluated the expression levels of skin permeability barrier maintenance biomarkers in keratinocytes treated with 1-chloro-2,4-dinitrobenzene (DNCB).
We harvested exosomes from conditioned media of fibroblast cells (Gibco, Grand Island, NY, USA) using differential ultracentrifugation.
When the fibroblast cells reached ∼95% confluency in a 150-mm petri dish, they were treated with cycloheximide (5-100 µg/ml) to generate a stress-induced condition. Post-treatment-conditioned media Kwangho Yoo and Nikita Thapa contributed equally to this work.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. were collected and subjected to differential centrifugation. Lastly, pellets containing harvested exosomes were separated and collected from the ultracentrifuged supernatant, resuspended in phosphatebuffered saline, and stored at −80 • C for further examination. Light microscopy revealed that the cultured fibroblasts exhibited a typical spindle/fibroblast-like structure while adhering to the culture vessel surface ( Figure 1A). The characterization of the harvested exosomes from the fibroblast cell culture-conditioned media was done by nanoparticle tracking analysis (NTA). NTA was used to determine the particle number and size of the isolated exosomes. ZetaView (Analytik, Cambridge, UK) was used to determine the vesicle concentration and size distribution. The mean particle sizes of the isolated fibroblast exosomes were 215.4 ± 116.1 nm, with a concentration of 2.33×10 10 ± 2.22×10 9 particles/ml. ( Figure 1B) The cytotoxicity of the isolated exosomes was least at concentrations of 1×10 4 , 1×10 5 , and 1×10 6 particles/ml after 24 and 48 h of treatment. However, when the exosome concentration was 1×10 7 particles/ml, we detected considerable cytotoxicity after 24 h of treatment. Interestingly, the cytotoxic effects on the HaCaT cells were further increased after 48 h of treatment with the harvested exosomes. exosomes derived from autologous DFs were found to more efficiently ameliorate senescence-related tissue damage and promote cutaneous wound healing, which indicates that HDF-exosomes can be potential candidates for protecting and repairing skin damage. Furthermore, exosomes isolated from conditioned media during three-dimensional dermal fibroblast culture were shown to stimulate ECM synthesis and secretion from dermal fibroblasts, as well as cellular migration and proliferation, in part through interleukin-6 signaling. These results imply that DF-derived exosomes participate in numerous stages of dermal ECM formation, thus contributing to skin damage repair, and illustrating their potential mechanism. 10 However, no study has investigated the mechanism used by DF exosomes on skin permeability barrier protection. This article attempts to evaluate the therapeutic significance of DF-derived exosomes on skin epidermal marker proteins. Hence, we evaluated the expression levels of skin permeability barrier maintenance biomarkers in keratinocytes treated with DNCB, a skin irritant, and inducer of dermatitis, including AD.
Though exosome cytotoxicity has been the subject of some investigations, it is now believed that exosomes generated from fibroblasts do not exhibit considerable cytotoxicity. This is probably because exosomes are naturally secreted cell-derived vesicles that are engaged in regular physiological functions. 4 However, it is still essential to emphasize that the safety and efficiency of fibroblast-derived exosomes in clinical settings require further investigation. In our study, the cytotoxicity of the harvested exosomes from DFs using the MTT assay was evaluated before determining their repair efficacy. The optimal concentration was identified from four different exosome concentrations (1×10 4 , 1×10 5 , 1×10 6 , and 1×10 7 particles/ml). Our data showed that DF-derived exosomes at concentrations of 1×10 4  Notably, in this study, the optimal exosome concentration was determined, which showed the maximum efficacy in expressing biomarkers related to the integrity and inflammation of the permeability barrier in HaCaT cells. All three exosome concentrations (1×10 4 , 1×10 5 , and 1×10 7 particles/ml) have shown significant effects on protein expression, but the 1×10 4 particles/ml concentration was depicted as the optimum. Similarly, regarding the AD model of keratinocytes, the 1×10 4 particles/ml concentration has been shown to be optimum for repairing damaged skin, which may be attributed to several factors. A possible hypothesis is that the exosomes may become exceedingly concentrated at higher amounts and may not be adequately absorbed by the recipient cells, leading to decreased efficacy.
In conclusion, fibroblast-derived exosomes at a concentration of 1×10 4 particles/ml were the optimum to show efficaciousness in increasing skin epidermal barrier proteins, thus increasing the recovery rate of skin damage without causing cytotoxic effects in this study.
Based on these findings, we conclude that only fibroblast-derived exosomes at specific concentrations can be of clinical use for their prospective application in treating skin barrier dysfunction diseases like AD. To our knowledge, this was the first study of its kind to investigate how DF-derived exosomes recover skin barrier functions. This study might thus serve as a first step toward the development of fibroblast-derived, exosome-based treatments for AD clinically. Additionally, this study only used in vitro experiments to illustrate the effects of DF-derived exosomes; hence, we plan to conduct further