The Feasibility to Isolate and Expand Tympanic Membrane Squamous Epithelium Stem Cells from Scarred Perforation Margins

Hypothesis:The scarred rim of chronic tympanic membrane (TM) perforation contains keratinocytes with potential for regeneration while maintaining their morphological and genetic characteristics.Background:The squamous epithelium of the TM has a good regeneration capacity. Successful isolation and expansion of human TM keratinocytes (hTMKR) was reported from a full, en-bloc, healthy TM.Methods:Trimmed margins of the TM perforation (harvested during tympanoplasty) underwent enzymatic digestion (collagenase or trypsin) and were seeded either with serum-containing medium (SCM) or keratinocyte serum-free medium (KSFM) and progenitor cell growth medium (PR) (KSFM:PR, 1:1). Gene expression analysis by real-time qRT-PCR was used to compare between human TM cells derived from scarred perforation margins (hTMKR), normal human skin keratinocytes (NhSKR), and human fibroblasts.Results:Twelve patients were included in the study. In 9 of 12 cases (75%) single-cell isolation with fibroblastic or epithelial cell morphology (or both) was achieved. Cells seeded with KSFM:PR yielded epithelial morphology (hTMKR) while SCM culturing resulted in a fibroblastic morphology (hTMFib). Gene expression analysis revealed significant higher expression of VCAN (p=0.002) and FOXC2 (p=0.015) at the mRNA levels (normal hTMKR markers) in hTMKR compared to NhSKR. In addition, a comparison of gene expression between hTMKR and hTMFib revealed significantly higher levels of both VCAN (p=0.045) and SLC6A14 (p=0.036) among hTMKR.Conclusion:For the first time, we developed a protocol to isolate hTMKR from scarred TM perforation margins. Furthermore, we succeeded in achieving tissue expansion that preserved the characteristic of healthy TM cells. This study bridges "regenerative medicine" approach with clinical and surgical objectives.