Measurement of Hyaluronic Acid Amounts Using ELISA from Cultured Human Skin Fibroblasts

Kim, Seong Jin; Lee, Su Ah; Lee, Je Hyuk; Yun, Sook Jung; Lee, Jee Bum; Lee, Seung Chul; Won, Young Ho

Abstract

BACKGROUND: Assay of glycosaminoglycans or proteoglycans from skin is complicated due to individual methods where the measurements are highly specialized. The results from these different methods are not able to be compared and there is a large variance.
OBJECTIVE
It seems reasonable that a major glycosaminoglycan in the skin, hyaluronic acid, might be ideal as a representative instead of the whole components of glycosaminoglycan. To develop a simple and reliable assay method, the in vitro cell culture system was selected to reduce time and variety of data. The usefulness of the ELISA method, using hyaluronic acid binding protein (HA-ELISA), was evaluated.
METHODS
The amount of hyaluronic acid synthesis was measured under a standardized protocol for cultured human skin fibroblasts from the elderly and neonates, as well as the NIH 3T3 mouse fibroblast cell line. To see whether this screening method (HA-ELISA) could be time-saving and reliable under in vitro conditions, some well-known stimulants for glycosaminoglycan synthesis such as retinol, retinyl palmitate, polyethoxyretinide retinamide and hydroxyproline were treated.
RESULTS
The production of hyaluronic acid was influenced by both culture condition and source of fibroblasts. The level of quantity showed different patterns due to factors such as culture period, serum in the medium and cell proliferation rate. We found that stable levels of hyaluronic acid assay from culture supernatant were obtained by delaying the sampling time after 24 hours of treatment with stimulants.
CONCLUSION
For a reliable quantitative assay, either NIH 3T3 mouse fibroblasts or neonate fibroblasts were suitable. The culture condition and time of harvest should be determined first to estimate the stable kinetics of hyaluronic acid synthesis. This in vitro test protocol can be used as an additional evaluation system towards a potential agent for dermal connective tissue, while further efforts are still mandatory to correlate the confounding factors of in vitro and in vivo.