2.2.1. Hair Follicle Isolation

Two different methods were used for obtaining human HFs. The first one consists in maintaining the hair follicle in its phase to investigate the three major phases of hair growth cycle (anagen, catagen, and telogen) and the second one permits inducing intermediate phases, in particular, to study intermediate anagen stages of the hair growth cycle. HFs were isolated from the human scalp according to Philpott's method [32].

For all our experiments, four donors were involved with two to three hair samples on the average per donor. For each hair sample, two to three sections were obtained. More precisely, for the first method, 32 anagen A1, 25 anagen A3, 26 catagen C1, 28 catagen C2, 31 telogen T1, and 41 telogen T3 HF sections were analyzed. For the second method, 9 early anagen D0, 18 intermediate anagen D3, and 11 catagen D6 HF sections were analyzed.

In the first approach, the HFs were isolated from the scalp at different hair growth cycle phases (anagen, catagen, and telogen) and maintained in culture in the William's E medium (W4128, Sigma-Aldrich, Saint-Louis, MO, USA) supplemented with 0.5% antibiotics, 2 mM L-glutamine (49420, Sigma-Aldrich), 10 ng/mL hydrocortisone (H-0396, Sigma-Aldrich), 10 µg/mL transferrin (T8158, Sigma-Aldrich), and 10 ng/mL selenite (S-5261, Sigma-Aldrich) for one day (named A1, C1 and T1, respectively) and for three days (named A3, C3, and T3, respectively) before analysis.

In the second approach, the different phases of the hair cycle (early anagen, intermediate anagen, and catagen) were induced in culture. To do so, early anagen HFs were isolated and selected from the scalp; one part was directly analyzed (D0, early anagen HFs), while the other part was maintained in culture for three days (D3, intermediate anagen HFs) and for six days (D6, catagen HFs) in the cell culture medium described above, supplemented with 0.5 µg/mL insulin (91077C, Sigma-Aldrich).
