Effect of Escin in Human Hair Follicular Organ Culture: Prolonged Anagen Phase

Conventional approach of hair biology is based on cell culture system, and ex vivo studies of human scalp hair follicles have shown many advantages in hair research, such as dynamic nature of chronobiology, endocrinology, immunology, metabolism, neurobiology, pharmacology, pigmentation and stem cell biology.1 Yet, high cost of maintaining organ culture system using expensive growth factors of serum-free culture medium to analyze biological parameters of hair follicle is still problematic. For example, R-spondin (R-spondin1, Sigma-Aldrich USA), a family of secreted proteins that act as potent agonists of the Wnt/β-catenin signaling pathway which is crucial for regulating embryonic development, maintaining cell proliferation, and homeostasis, has been used as culture additive, but it’s highly expensive for long term treatment in organoid culture period.2 Thus it’s favorable to screen the cost-effective, consistent experimental condition keeping hair follicle in its anagen phase long enough to study for evaluating therapeutic potential via Wnt/β-catenin signaling, etc.

Escin (beta-Escin Sigma-Aldrich USA), a natural mixture of triterpenoid saponins isolated from horse chestnut (Aesculus hippocastanum) seeds, is known to having vasoprotective, anti-inflammatory, anti-edematous and anti-nociceptive action, relatively inexpensive compare to R-spondin for long term organoid culture system.3

In this study, we investigated the cell viability effect of Escin in cultured human follicular (papilla) cells comparing with conventional R-spondin additive. We also measured the period of anagen phase in organ cultured hair follicular units.

Hair follicular units were easily obtained by hair pluck/pull test where gentle but firm pulling of hairs grasped between fingers. This non-invasive hair sampling was collected during the hair cosmetic efficacy test approved by Institutional Review Board of Chonnam National University Hospital (CNUH-2021-257). Each isolated intact hair follicles were placed into individual wells of a 96-well plate. Either Escin or R-spondin was added in Williams Eagle media (Thermo Fischer Scientific, USA) where L-glutamine (2 mM), insulin (10 µg/mL), hydrocortisone (10 µg/mL), penicillin/streptomycin, Fungizone (2.5 µg/mL) included.

A total of thirty-three hair follicular units placed in the wells to culture hair follicular (papilla) cells in the bottom, then removed hair follicular units at day 2, and its percentile survival ratio was measured at day 4 using water-soluble tetrazolium (WST) (Sigma-Aldrich, USA) for viability assay by measuring mitochondrial dehydrogenase activity, where viable cells convert into a soluble formazan dye in which absorbance at ~450 nm were assayed. For long-term morphology of anagen phase hair, two hundreds of hair follicular units were evaluated under microscopic findings. Characteristic morphology of anagen phase hair is a well-reserved intact root sheath compare to telogen hair showing no root sheath.

Cell viability (survival ratio) of Escin treating 10 to 50 µg/mL showed similar finding like R-spondin 0.5 µg/mL treatment in which two-fold increase with compare to no treatment. Interestingly, Escin of 10 to 50 µg/mL keep maintaining anagen phase hair morphology at day 24 more than 80% of hair follicles (Fig. 1). In general, it is known that the anagen phase morphology in organ cultured human hair follicles abruptly disappearing tendency after day 5.4

FIG. 1. (A) Cell survival ratio after Escin treatment at 10 to 50 µg/mL showed similar cell survival pattern like R-spondin 0.5 µg/mL treatment. (B) Anagen phase hair follicles were well-preserved with 10 to 50 µg/mL concentration of Escin from 8 days to 24 days. (C) Microscopic finding of anagen phase hair from the culture well showed intact root sheath (arrow) surrounding hair follicle.

Our data suggests that Escin could support hair growth by stimulating proliferation of follicular (papillary) cells as well as by elongating period of anagen phase, which may be useful as a cost-effective supplement to maintain follicle viability/anagen in organ culture. We suggest Escin can be considered as relatively inexpensive alternative growth factor in hair follicle organ culture system for long-term study.