Dermal fibroblasts were cultured from the skin tissue as
previously described [27]. Briefly, dermal specimens were
minced into small pieces of less than 0.5 mm in any dimension
in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma–
Aldrich, St. Louis, MO) with 10% fetal bovine serum (FBS)
(Sigma–Aldrich, St. Louis, MO). The tissue fragments were then
washed with medium and distributed into 60 mm 15 mm
Petri dishes. A sterile glass coverslip attached to the dish with a
drop of sterile silicone lubricant was used to immobilize the
tissue fragments. 3 mL of DMEM containing 10% FBS was added
to each dish and they were incubated at 37 8C in an atmosphere
of 5% CO2 and 95% relative humidity. The medium was replaced
every 5 days. After 4 weeks, the fibroblasts were released from
the dishes after treatment with trypsin (0.25%, w/v), transferred
to 75 cm2 culture flasks, and sub-cultured for experiments.