Vitrification and storage of oral mucosa epithelial cell sheets.

Shipping time and shipping delays might affect the quality of the stem cells based engineered "organs." In our laboratory, we have developed a limbal stem cell deficient (LSCD) rabbit model. To reverse the LSCD, we cultured oral mucosal epithelial cells for 2-3 weeks and engineered cultured autologous oral mucosa epithelial cell sheets (CAOMECS), which were grafted on the LSCD cornea. The purpose of this study was to vitrify CAOMECS and to store it until the CAOMECS can be grafted onto patients. CAOMECS were vitrified in LN2 for up to 204 days. We tested two different methods of vitrification with different solutions; however, CAOMECS were only viable when they were not stored in a vitrification solution; results were only reported from this CAOMECS. On the basis of hematoxylin and eosin staining, we showed that the CAOMECS morphology was well preserved after long-term storage in LN2 . Most of the preservation solutions maintained the CAOMECS phenotype (Ki67, proliferating cell nuclear antigen (PCNA), Beta-Catenin, ZO-1, E-Cadherin, CK3, CK4, CK13). The exception was the solution composed with ethylene glycol and Dimethyl sulfoxide (DMSO): this resulted in loss of DeltaN-p63 expression. DeltaN-p63 is an important marker for cell proliferation. The expression of proteins involved in cell-cell connection and the differentiation markers were maintained. Apoptosis was not detected in the thawed CAOMECS. We demonstrated that CAOMECS can be stored long-term in LN2 without affecting their morphology and phenotype.

Engineering, differentiation and harvesting of human adipose-derived stem cell multilayer cell sheets.

Aim: The study goals were to engineer and harvest scaffold-free undifferentiated/differentiated multilayer human adipose-derived stem cell (hADSC) cell sheets, in absence of treatment. Materials & methods: The hADSC are seeded in 35 mm culture dishes. At confluence or when multilayer cell sheets are formed, hADSC are treated with predefined differentiation culture media (adipocyte, chondrocyte and osteoblast). Results: Undifferentiated hADSC and differentiated adipocyte, osteoblast and chondrocyte hADSC multilayer cell sheets (hADSCmCS) have been harvested. Hematoxylin & eosin showed the formation of multilayer cell sheets. Undifferentiated hADSC multilayer cell sheets preserve their stem cell markers. Differentiated adipocyte, osteoblast and chondrocyte hADSCmCS expressed specific markers. Conclusion: This simple protocol opens possibilities to engineer scaffold-free hADSCm cell sheet to transplant them on damaged organs.

Feeder Cells Free Rabbit Oral Mucosa Epithelial Cell Sheet Engineering.

The optimal cell culture method of autologous oral mucosal epithelial cell sheet is not well established for a safe transplantation on to the patients' ocular surface. Animal serum and 3T3 mouse feeder cells are currently being used to stimulate the growth of the epithelial cells. However, the use of animal compounds can have potential side effects for the patient after transplantation of the engineered cell sheet. In the present study, we focused on engineering a rabbit oral mucosal epithelial cell sheet without 3T3 mouse feeder cells using a mix of Dulbecco's Modified Eagle Medium/Bronchial Epithelial Cell Growth Medium culture media (DMEM/BEGM). Autologous oral mucosal epithelial cell sheets, engineered with DMEM/BEGM feeder cell free culture media, were compared to those cultured in presence of serum and feeder cells. Using a DMEM/BEGM mix culture media, feeder cell free culture condition, autologous oral mucosal epithelial cells reached confluence and formed a multilayered sheet. The phenotype of engineered cell sheets cultured with DMEM/BEGM were characterized and compared to those cultured with serum and feeder. Hematoxylin and eosin staining showed the formation of a similar stratified multilayer cell sheets, in both culture conditions. The expression of deltaN-p63, ABCG2, PCNA, E-cadherin, Beta-catenin, CK3, CK4, CK13, Muc5AC, was similar in both culture conditions. We demonstrated that rabbit autologous oral mucosal epithelial cell sheet can be engineered, in feeder cell free conditions. The use of the DMEM/BEGM culture media to engineer culture autologous oral mucosa epithelial cell sheet will help to identify key factors involved in the growth and differentiation of oral mucosal epithelial cells.

Proteasomes in corneal epithelial cells and cultured autologous oral mucosal epithelial cell sheet (CAOMECS) graft used for the ocular surface regeneration.

PURPOSE: This study focuses on characterizing proteasomes in corneal epithelial cells (CEC) and in cultured autologous oral mucosal epithelial cell sheets (CAOMECS) used to regenerate the ocular surface. METHODS: Limbal stem cell deficiency (LSCD) was surgically induced in rabbit corneas. CAOMECS was engineered and grafted onto corneas with LSCD to regenerate the ocular surface. RESULTS: LSCD caused an increase in inflammatory cells in the ocular surface, an increase in the formation of immunoproteasomes (IPR), and a decrease in the formation of constitutive proteasome (CPR). Specifically, LSCD-diseased CEC (D-CEC) showed a decrease in the CPR chymotrypsin-like, trypsin-like and caspase-like activities, while healthy CEC (H-CEC) and CAOMECS showed higher activities. Quantitative analysis of IPR inducible subunit (B5i, B2i, and B1i) were performed and compared to CPR subunit (B5, B2, and B1) levels. Results showed that ratios B5i/B5, B2i/B2 and B1i/B1 were higher in D-CEC, indicating that D-CEC had approximately a two-fold increase in the amount of IPR compared to CAOMECS and H-CEC. Histological analysis demonstrated that CAOMECS-grafted corneas had a re-epithelialized surface, positive staining for CPR subunits, and weak staining for IPR subunits. In addition, digital quantitative measurement of fluorescent intensity showed that the CPR B5 subunit was significantly more expressed in CAOMECS-grafted corneas compared to non-grafted corneas with LSCD. CONCLUSION: CAOMECS grafting successfully replaced the D-CEC with oral mucosal epithelial cells with higher levels of CPR. The increase in constitutive proteasome expression is possibly responsible for the recovery and improvement in CAOMECS-grafted corneas.