Introduction: Oral fibroblasts are the important cells for maintaining structural integrity of the oral mucosa. They can be activated and altered in various diseases like oral submucous fibrosis and other benign and malignant fibrous lesions. Fibroblast cell culture is an important in vitro tool allowing researchers to study the fibrotic diseases in detail and devise various therapeutic strategies. Different techniques of isolation of fibroblasts have been used so far and each has its own merits and demerits.

Objective: The objective of our study was to optimize the protocol of isolation of fibroblasts from oral buccal mucosa.

Methods: Primary cells were isolated from biopsy specimens of patients undergoing 3rd molar surgery. Tissue fragments were digested with trypsin for 10, 30 and 60 min., while some fragments were left as undigested explants. Primary cells were subcultured and maintained according to the standard protocols.

Results: By using combination of explant and enzymatic digestion for 10 min. we were able to isolate fibroblasts earlier by attachment of fibroblasts on 3rd day of culture and reaching 90% confluence on 14th day.

Conclusions: Our study revealed that combination of explant and enzymatic digestion for 10 min. is a faster method for isolation of oral fibroblasts as compared to longer time durations and undigested explants.

Keywords: Isolation of fibroblasts; Primary cell line; Explant-enzymatic technique