A method for identification of vocal fold lamina propria fibroblasts in culture

Abstract

Objective: Vocal fold biology research is emerging as a vital area of study in laryngology. One impediment is the lack of both commercially available vocal fold lamina propria fibroblasts and a constitutively expressed specific marker for fibroblasts. We present an in vitro technique that allows for identification of fibroblasts by ruling out the possibility of the cells belonging to other lineages that are found in vocal fold tissue.

Study design: An in vitro study.

Methods: Two primary vocal fold fibroblast cell lines and one immortalized vocal fold fibroblast cell line were cultured. Immunohistologic staining for alpha-actinin, cytokeratin 19, and von Willebrand factor was completed for the three fibroblast lines in addition to skeletal, endothelial, and epithelial cell lines. Cell type was differentiated by positive staining for alpha-actinin, cytokeratin 19, and von Willebrand factor.

Results: Fibroblast cultures did not express alpha-actinin, cytokeratin 19, and von Willebrand factor, whereas skeletal muscle, endothelial, and epithelial cultured cells expressed each respectively.

Conclusions: This simple rule-out methodology for fibroblast confirmation is an important step when establishing cell culture, and it establishes sound internal validity particularly in the early stages of this emerging area of study.

Figure 1

Skeletal cell negative (A) and positive (B) expression for α-actinin (20× magnification). Epithelial cell negative (C) and positive (D) expression for cytokeratin 19 (40× magnification). Endothelial cell negative (E) and positive (F) expression for vWF.

Figure 2

Primary fibroblast culture from a 63-year old: positive live cell presence in culture (A, B, and C). Cell cultures stained for α-actinin, cytokeratin 19, and vWF show a lack of staining in A+, B+, and C+, respectively. Negative controls for α-actinin, cytokeratin 19, and vWF show a lack of staining in A−, B−, and C−, respectively. All magnified at 20×.

Figure 3

Primary fibroblast culture from a 21-year old: positive live cell presence in culture (A, B, and C). Cell cultures stained for α-actinin, cytokeratin 19, and vWF show a lack of staining in A+, B+, and C+, respectively. Negative controls for α-actinin, cytokeratin 19, and vWF show a lack of staining in A−, B−, and C−, respectively. All magnified at 10×.

Figure 4

Immortalized fibroblast culture from a 21-year old: positive live cell presence in culture (A, B, and C). Cell cultures stained for α-actinin, cytokeratin 19, and vWF show a lack of staining in A+, B+, and C+, respectively. Negative controls for α-actinin, cytokeratin 19, and vWF show a lack of staining in A−, B−, and C−, respectively. All magnified at 20×.