Use of synthetic serum-free medium for culture of human dermal fibroblasts to establish an experimental system similar to living dermis

Abstract

In this study, we sought to establish a defined experimental system for fibroblast growth similar to that of the living dermis. To this end, we evaluated the growth and biochemical characteristics of fibroblasts cultured with serum-free HFDM-1, a finely tuned synthetic medium for human fibroblast culture. Three culture conditions were used to grow fibroblasts obtained from primary culture: (1) culture with Dulbecco's modified Eagle medium (DMEM) plus 10 % fetal bovine serum (serum-supplemented DMEM), (2) culture with DMEM (serum-free DMEM), and (3) culture with HFDM-1 (HFDM-1), and fibroblast morphology, growth, collagen type I production, and lipid composition were analyzed. Fibroblasts grown in HFDM-1 maintained cell numbers at nearly 100 % from days 14 to 21 and produced more collagen type I than cells grown in serum-supplemented and serum-free DMEM. Arachidonic acid (20:4) and total polyunsaturated fatty acids were lower in cells grown in serum-free DMEM and HFDM-1 than in serum-supplemented DMEM. These results suggested that HFDM-1 recapitulated growth conditions in the dermis better than traditional, serum-supplemented DMEM. In addition, the controlled chemical composition of HFDM-1 eliminated a potential source of variability in cell culture conditions.

Fig. 1

Experiment schedule

Fig. 2

a Morphology of the fibroblasts in serum-supplemented DMEM, serum-free DMEM, and HFDM-1 on day 7, 10, and 14. b Morphology of the fibroblasts in serum-supplemented DMEM, serum-free DMEM, and HFDM-1 on day 17 and 21

Fig. 3

Cell growth of the fibroblasts in serum-supplemented DMEM, serum-free DMEM, and HFDM-1. Number of the fibroblasts on day 0 was defined as 100 %, and Y axis was defined as log2 of % of day 0. Error bar means SD (N = 9)

Fig. 4

Collagen type I production per fibroblast cell of fibroblasts in serum-supplemented DMEM, serum-free DMEM, and HFDM-1. The concentration of collagen type I in each sampled culture supernatant minus that in each medium itself divided by the cell number. This value was defined as the collagen type I production of one fibroblast. *0.01 < p < 0.05, **p < 0.01; no asterisk means no significant difference (after applying for Bonferroni correction). Error bar means SD (N = 9)

Fig. 5

Percent compositions of fatty acids of the fibroblasts in serum-supplemented DMEM, serum-free DMEM, and HFDM-1. *0.01 < p < 0.05, **p < 0.01; no asterisk means no significant difference (after applying for Bonferroni correction). Error bar means SD. EFA essential fatty acid, TLC thin layer chromatography, WC whole cell (N = 9)