Abstract
Ocular surface reconstruction (OSR) using tissue-engineered cultivated oral mucosal epithelial cell sheets (COMECS) is a promising newly developed treatment for patients with severe ocular surface disease. Until now, this technique has used exogenic and undefined components such as mouse-derived 3T3 feeder cells and fetal bovine serum. To minimize associated risks of zoonotic infection or transmission of unknown pathogens and so establish a safe and effective protocol for the next generation of treatment modality, we developed a novel technique for the COMECS protocol, using a feeder-free and serum-free (FFSF) culture system. Following this new protocol, COMECS exhibited 4–5 layers of well-stratified and differentiated cells, and we successfully produced functional COMECS that included holoclone-type stem cells. Immunohistochemistry confirmed the presence of markers for cell junction (ZO1, Desmoplakin), basement membrane assembly (Collagen 7, Laminin 5), differentiation (K13, K3), proliferation (Ki67) and stem/progenitor cells (p75) in the FFSF COMECS. When transplanted to the ocular surfaces of rabbits, the tissue survived for up to 2 weeks. This study represents a first step toward assessing the development of functional FFSF COMECS for safe and ideal OSR.
Highlights
The currently preferred COMET method requires the use of xenobiotic materials, such as mouse-derived 3T3 feeder cells and fetal bovine serum (FBS), in the culture system
We have previously reported that p75, a low-affinity neurotrophin receptor, is a potential marker for oral mucosal epithelial stem/progenitor cells[18], there have been no reports to date regarding the development of functional cultivated oral mucosal epithelial stem/progenitor cell sheet using a defined feeder-free and serum-free (FFSF) culture system
We calculated total cell number for cultivated oral mucosal epithelial cell sheets (COMECS) from the same donor in both conditions and found that this was increasing as compared to Control COMECS (10.5 ± 2.1 × 105 vs 7.5 ± 1.4 × 105, p < 0.1, N = 4). These results indicate that the FFSF culture system maintained the proliferative potential of human oral mucosal epithelial cells as well as the previous conventional method
Summary
The currently preferred COMET method requires the use of xenobiotic materials, such as mouse-derived 3T3 feeder cells and fetal bovine serum (FBS), in the culture system This raises major clinical concerns about the risk of transmission of zoonotic infection or unknown pathogens[17]. This is the case when expanding cells ex vivo for clinical application, which increases the risk of transmission of diseases such as bovine spongiform encephalitis or other unknown infections In light of these issues, the development of feeder-free and serum-free (FFSF) culture systems seems ideal for the generation of COMET. In this context, the use of human oral mucosal epithelial stem/progenitor cells in tissue-engineered cultivated grafts is another key issue, as the ability to identify stem cell populations is of great clinical value.
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