1. Ex Vivo Expansion of Oral Mucosal Epithelial Stem Cells on Freeze Dried Amniotic Membrane for Potential Use in Ocular Surface Reconstruction Dina Kobtan MD, Hatem Kobtan MD FRCS Mervat Elansary MD, Nancy Elguindy MD The authors have no financial interest to disclose Cairo University, Egypt

2. Introduction  Stem cells (SCs) for the corneal epithelium reside in the basal layer of limbus (Nishida, 20003).  Limbal SCs (LSCs) are supported by a unique stromal microenvironment called the stem cell niche. Destructive loss of LSCs and/or dysfunction of their stromal environment render many corneas with a clinical entity called limbal stem cell deficiency (LSCD) (Grueterich et al, 2003).  Ex vivo cultivated corneal epithelial transplantation (CCET) has gained general acceptance as an effective treatment modality for LSCD (Koizumi et al, 2001; Shimazaki et al, 2002). Kobtan WCC VII 2015

3.  The ideal therapy for unilateral LSCD is the use of autologous CCET (using a minimal biopsy obtained from the contralateral healthy eye) (Schwab et al, 2000; Tsai et al, 2000).  In bilateral LSCD, allogenic limbus from living related or cadaveric donors needs to be transplanted (directly or after ex vivo expansion) onto the affected eye (Frucht-Pery et al, 1998). This necessitates long-term systemic immunosuppression to prevent graft rejection (Cauchi et al, 2008).  Cultured oral mucosal epithelial cells (OMECs) have been successfully utilized in the treatment of LSCD in a technique called cultivated oral mucosal epithelial transplantation (COMET) (Chu, 2000; Nakamura et al, 2004) Kobtan WCC VII 2015

4. Aim of the work The aim of the present study is to attempt to expand OMECs from oral biopsies on freeze-dried amniotic membrane (FD- AM). Kobtan WCC VII 2015

5. Materials and Methods  In the current study, oral biopsies were obtained from 3 potential COMET candidates after obtaining an informed consent. The oral biopsies were cultured as explants on FD denuded AM (dAM) without using feeder cells or airlifting for 24 days.  Epithelial outgrowth was assessed using phase contrast microscopy, haematoxylin and eosin (HE) staining, and immunohistochemistry (IHC) for keratin 3 (K3), keratin 12 (K 12) and p63. Kobtan WCC VII 2015

6. Explant culture. After careful dissection of each oral biopsy under a microscope, an explant (arrow head) was placed directly on the center of each AM fastened onto the culture insert. Cultures were submerged in culture media, incubated at 37°C and were maintained for 24 days. Outgrowth of ECs was assessed using light microscopy and IHC. Oral mucosal biopsy harvest. local anesthetic is injected into the donor site, an incision at the apex of the outline is made with a steel blade, a forceps is used to handle the tissue on the submucosa side of the graft. A: The resultant elliptical shaped raw area, B: Harvested oral mucosal tissue. AB

7. After 24 days in submerged culture, the cells grew into a healthy, confluent sheet (arrow head). Culture insert is indicated by the arrow. A. Phase contrast microscopy Kobtan WCC VII 2015 Results

8. Light microscopic examination of TS in the oral epithelial cultures on FD-dAM under submerged conditions on day 24 showed 2–5 layers of stratified epithelium (arrow head). The oral explant remained attached to the AM throughout the culture duration. The insert shows the oral explant after 24 days in culture. B. Haematoxylin and Eosin Kobtan WCC VII 2015

9. Positive control: Normal limbal epithelium shows positive immunostaining for K3 in all cell layers except the basal layer (Magnification x400). Cultured oral epithelium with positive cytoplasmic K3 immunostaining mainly in the superficial cell layer (Magnification x400). C. Immunohistochemistry for K3 Kobtan WCC VII 2015

10. Positive control: normal limbal epithelium shows positive immunostaining for K12 in all cell layers except the basal layer (Magnification x400) D. Immunohistochemistry for K12 Cultured oral epithelium with negative immuno-staining for K12 in all cell layers (Magnification x400) Kobtan WCC VII 2015

11. D. Immunohistochemistry for p63 Normal limbal epithelium shows positive immunostaining for p63 in the basal and suprabasal cell layers (Magnification x400). Cultured oral epithelium with positive p63 nuclear immuno- staining (Magnification x400). Kobtan WCC VII 2015

12. Conclusion  We have cultured oral mucosal epithelial cells (ECs) from biopsy-derived oral mucosal tissues on sterilized FD-AM. The use of the explant culture technique circumvented the dependence on feeder cells, which eliminates the risk of xenogeneic contamination.  We were able to observe nuclear expression of p63 (used here to identify the presence of ECs which had not terminally differentiated) within the cells of the stratified layers. K3, the marker of corneal and oral ECs was found to be expressed by cultured cells. This suggests that cell sheets derived from OMECs showed similar characteristics to normal corneal epithelium. Our cultured cells were negative for the corneal epithelium-specific K12 and hence do not acquire the corneal phenotype after culture.  We should investigate how long these cultivated oral ECs can maintain the ocular surface. Kobtan WCC VII 2015