Ex Vivo Expansion of Human Oral Mucosal Epithelial Cells: Serum Selection Is a Determinant Factor for Cell Proliferation, Differentiation and Gene Expression

Abstract

Purpose: Human oral mucosal epithelial cells (HOMECs) represent a readily available source of epithelial cells for ocular surface reconstruction and are easily obtained and expanded in vitro. In most protocols for in vitro culture of HOMECs, fetal bovine serum (FBS) is included as a nutritional supplement for the cells. It could be desirable to avoid animal derived components in cell cultures aimed at therapeutic purposes in humans. Therefore, we have investigated autologous serum (AS) as an alternative to FBS for expansion of HOMECs.

Methods: Human oral mucosal epithelial biopsies (HOMEBs) and blood as a source for preparation of AS under different conditions were obtained from donors. HOMEBs were in parallel cultured in media containing AS (DMEM/F12, 100 U/ml antibiotic, and 10% autologous human serum) or FBS (DMEM/F12, 100 U/ml antibiotic, 5% FBS, 2 ng/ml EGF, ITS (5 μg/ml insulin, 5 μg/ml transferrin and 5 ng/ml sodium selenite), 30 ng/ml cholera toxin A, 0.5% dimethylsulfoxid, 15μM hydrocortisone) at 37 C with 5% CO2 in a humidified atmosphere. HOMECs were harvested after 3 weeks and analyzed by qRT-PCR, immunohistochemistry, transmission electron microscopy (TEM).

Results: In the FBS containing medium HOMECs proliferated well. In the AS containing media the expansion of cells showed variations that seem to be related to the factors that generally are known to affect cell proliferation, differentiation and gene expression. In our study, these factors seem to reflect the nutritional status of the blood donors at the time the serum were taken. We used qRT-PCR analysis of MKI67, ABCG2, CK3, CK13, CK19, GJA1 (Connexin 43), OCLN, P53 and SOX9 genes, immunohistochemistry of related proteins, and transmission electron micrographs (TEM) to show these observations.

Conclusions: AS is an acceptable and safe serum supplement which eliminates the risk of transmitting prion diseases and inhibit xenogeneic immune response. However, the blood quality used for serum preparation of the AS seemed to be crucial for cell proliferation, differentiation and gene expression of HOMECs.

Keywords: 474 conjunctiva • 721 stem cells • 741 transplantation