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. 2020 Jan-Apr;24(1):68-75.
doi: 10.4103/jomfp.JOMFP_282_19. Epub 2020 May 8.

Isolation, culture and characterization of primary cell lines of human buccal mucosal fibroblasts: A combination of explant enzamytic technique

Ritiha Patil  1 , Alka D Kale  1 , Deepa R Mane  1 , Dhanashree Patil  2
Affiliations

Isolation, culture and characterization of primary cell lines of human buccal mucosal fibroblasts: A combination of explant enzamytic technique

Ritiha Patil et al. J Oral Maxillofac Pathol. 2020 Jan-Apr.

Abstract

Background: The cell culture technique has become a routine and a popular method for its wide applications in the field of cell biology and biotechnology and in medical research. Isolation of primary cells over the cancer cells is an essential component of cell culture technology as they are the reliable source to understand normal physiological, morphological and molecular process of human cells. As fibroblasts are the prominent cells of the connective tissue of oral mucosa, many disease entities and histogenesis are linked to fibroblasts VSports手机版. Culture of oral fibroblast cells helps the oral biologists and researchers to study the morphological and molecular process in the oral diseases. .

Aim: The aim of our experiment is to isolate and culture the human buccal mucosal fibroblast cells from healthy individuals using a combination of explant-enzymatic method and characterization of the cells by short tandem repeat (STR) profiling. V体育安卓版.

Materials and methods: The tissue samples were collected from healthy individuals undergoing routine impacted third molar extraction. A combination of explant-enzymatic technique was used for the isolation from the tissue samples. The cells were further subcultured, maintained and stored as per the standard protocols. Thus, to confirm the oral fibroblasts of human origin and its uniqueness, they were characterized using STR profiling V体育ios版. .

Results and conclusion: Using the combination technique, we were successful in isolating the cells at a faster rate by detachment of cells on day 3 and confluency on day 10. The morphological assessment and STR profiling further confirmed that the isolated cell lines resemble human fibroblast cells VSports最新版本. .

Keywords: Cell lines primary culture; enzymatic technique; explant culture; human buccal mucosal fibroblast; short tandem repeats. V体育平台登录.

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Conflict of interest statement

There are no conflicts of interest.

Figures

Figure 1
Figure 1
Photomicrograph of images (a-d) depicting steps involved in the tissue processing like collection of sample in sterilized Eppendorf tube containing dish containing the culture media (a), process of mincing of tissue sample in the laminar air flow chamber with proper arrangement of sterilized instruments (b), tissue samples are minced or processed in a glass Petri dish using a BP blade No. 22 into small pieces of 1 mm × 1 mm in size (c and d)
Figure 2
Figure 2
Photomicrograph of images showing the isolation and primary outgrowth of the cells from the explant tissue. Cells migrating and outgrowing from the explant tissue on day 2 (a). Round clump of cells seen indicating the outgrowth of the explant tissue and multiplication of cell lines on day 3 (b). Small spindle-shaped fibroblast cells (F1) with mixture of spherical dividing cells (c). Cultures showing majority of F1-shaped fibroblasts with few F2- and F3-shaped cells (d). The fibroblasts reach the confluency around day 10 with mostly F1-shaped fibroblast cells (e and f)
Figure 3
Figure 3
Linear bar graph describing the growth characteristics of primary cell lines of human buccal mucosal fibroblast where the number of cells dividing (X) was plotted against the number of days (Y)
Figure 4
Figure 4
Morphological changes of fibroblasts noted during culture. Predominant spindle-shaped fibroblasts (F1) (a). Mixture of cells with few number of both stellate-shaped (F3) and epithelioid-shaped fibroblasts (F2) (b)
See this image and copyright information in PMC

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