Human-Derived Feeder Fibroblasts for the Culture of Epithelial Cells For Clinical use

Abstract

Aim: To investigate human oral mucosal fibroblasts (HOMF) and human limbal fibroblasts (HLF) as alternatives to murine 3T3 feeder fibroblasts currently used to support epithelial cell expansion for the treatment of limbal epithelial stem cell deficiency. Methods: HLF and HOMF were compared with 3T3s for their ability to support the culture of human limbal epithelial cells and human oral mucosal epithelial cells. Results: HOMF, but not HLF, were equivalent to 3T3s in terms of the number of epithelial population doublings achieved. Human limbal epithelial cells co-cultured with HOMF or 3T3s had similar expression of corneal and putative stem cell markers. Conclusion: HOMF are a suitable and safer feeder fibroblast alternative to 3T3s for the production of epithelial cells for clinical use.

Rat limbal niche cells can induce transdifferentiation of oral mucosal epithelial cells into corneal epithelial-like cells in vitro

“…At the beginning of our study, we evaluated the immunofluorescence of the oral mucosa, central cornea, and limbus to prove that only CK12 and Pax6 could be defined as cornea-specific markers, as previously reported [ 26 – 28 ]. We agree with previous studies [ 23 ] suggesting that CK3 cannot be considered a cornea-specific marker, and CK3/12 is a marker of epithelium differentiation. Furthermore, we also confirmed that oral mucosa and limbus express the same stem cell marker, ΔNp63α, as previously described [ 29 ].…”

“…We also noticed that 3T3 cocultured OMECs did not show as much negative expression, especially for Pax6, as cultured OMECs with no feeder layer. Other researchers have encountered a similar situation [ 23 ]; however, no evidence has indicated that 3T3 cells cocultured with OMECs can upregulate the expression of Pax6. Even so, coculturing with LNCs on a 3D Matrigel or in the Transwell system upregulated the expression of CK12 and Pax6 when compared with that in OMECs alone or cocultured with 3T3, which conflicts with a previous study [ 23 ].…”

“…As the most important component of the limbal microenvironment, limbal niche cells (LNCs) play a key role in the prevention of differentiation of limbal stem cells [ 20 – 22 ]. Oral epithelial cells and corneal epithelial cells originate from the same ectoderm; however, the only study that attempted to coculture OMECs and LNCs in vitro obtained unsatisfactory results [ 23 ]. In this study, we cocultured OMECs and LNCs with a new in-vitro model to observe whether LNCs can induce the transdifferentiation of rat OMECs to corneal epithelial-like cells.…”

The Characterization of Human Oral Mucosal Fibroblasts and Their Use As Feeder Cells in Cultivated Epithelial Sheets

“…The currently preferred cultivation method requires the use of xenobiotic 3T3 feeder cells from mouse embryonic fibroblasts in the culture system. Avoiding xeno-derived materials decreases the risk of unknown infections; recent studies have reported the production of stratified human corneal and oral mucosal epithelial sheets by coculturing with human MSCs, human dermal fibroblasts or hOMFs as substitutes for murine 3T3 feeder cells [ 17 , 42–45 ]. O'Callaghan et al .…”

“…Sourcing transplant cells from the oral mucosa also offers the advantages of quick healing and minimal invasiveness, and is a simple procedure that causes only slight discomfort for patients. Recently, hOMFs have been shown to support the expansion of epithelial cells as an alternative to murine 3T3 feeder fibroblasts [ 17 ]. To mediate the application of more sophisticated hOMFs in a clinical setting, we isolated hOMFs using the methylcellulose culture method, analyzed their characteristics, investigated whether hOMFs differentiate into corneal stroma keratocytes, and developed human corneal limbal epithelial sheets by coculturing with hOMFs as feeder cells in place of murine 3T3 feeder cells.…”