Epidermal Cells Scaling-up In Bioreactor And Delivered To Wound

Object:Scaling-up of Epidermal cells for therapeutic purpose and delivered them to the wound.Methods:The use of the bioreactor in the construction of the tissue-engineered skin is an imperative requisite yet there is no optimization research relevant to the design of it.This study conducted a computational fluid dynamic modeling of several types of the perfusion bioreactor designed for tissue engineering skin. The distribution of velocity, pressure, turbulent dynamics, and the turbulent dissipation rate of the fluid in the bioreactor were observed and the mean particle residence times were calculated under two configuration and two perfusion modes. The fluid pattern in the cylinder-shaped perfusion chamber with two inlets and two outlets exhibited more homogeneous and better mass transfer performance than that of cubic shaped perfusion chamber and the cylinder-shaped perfusion chamber with one inlet and one outlet. Further more, when used in parallel, the double inlets-outlets cylinder-shaped perfusion chamber manifested a different fluid dynamic features comparing to the single inlet-outlet cylinder-shaped perfusion chamber, the distal chamber received less fluid than the proximal chamber to the inlet in the single inlet-outlet cylinder-shaped perfusion chamber, whereas the fluid pattern was identical in the double inlets-outlets chambers in parallel. We hence proposed that the double inlets-outlets cylinder-shaped perfusion chamber is a better design for the perfusion bioreactor for skin tissue engineering.Epidermal cell plays key role in construction of the tissue engineered skin and the treatment of the burn or chronic wound in that it functions epithelialization. Autologous originated epidermal cells are the ideal source for healing wound, however, the technique for harvesting and scaling-up the cells in limited duration impeded its use. This hindrance limited the production of the tissue engineered skin and the cultured autologous epidermal cell sheet. Therefore, we studied the optimization techniques for the harvesting of the epidermal cells, the influence of ECM on the adhesion of the cells, reforming of the medium and the epidermal cell culture methods, bioreactor cultured epidermal cells and the vehicle for delivery the cells to the wound.First, we optimized the digestion process of the primary epidermal cell under the dynamic condition of three dimensional gyration to procure the quantity of the cells. Second, we studied the influence of different ECM on the adhesive ability of the cells and found that the repeated freeze-thawing of HaCat and keratinocytes generated ECM had more inclination to adhesion and select the keratinocytes, the fibroblast ECM had the adhesive effect on the multiple cell types in epidermis, the collagen IV ECM had the lower effect of adhesion on the epidermal cells. Medium contained 5%FCS and 10%FCS improved the adhesion of the cells in epidermis and kept them alive in the culture process. When serum deprived, few epidermal cells were found adhesion to the culture plate. There was no obvious synergetic effect of the serum and the ECM, indicated the effect of the adhesion of each shared different mechanism. Besides, we also noticed that when the primary epidermal cells grown well when the fibroblast existed in the plate, and when deprived, the growth of the epidermal cell became slow. If the sufficient culture area, the medium contained 5%FCS could maintain the fibroblast survival in the culture and did not complete with the keratinocytes, and, for the purpose of clinical use, the fibroblast in the culture might improve the healing effect of the epidermal cells. These results led us to premise that the low serum medium may improve the adhesion of the epidermal cells and survive small quantity of fibroblast in the culture system when scaling up in the bioreactor. Because the adhesion effect of the serum on the cells was independent to the ECM, there would be no need to modify the microcarrier by the ECM for improvement of the adhesion. Third, epidermal cells are adhesion-dependent cells. The sufficient surface should be provided when culture the cells in the stirred bioreactor. This could be approached by culturing the cell on the microcarrier. The choice of the microcarrier should take the clinical aim of the cultured cells-for the treatment of wound into consideration. Therefore, in this study, we chose the bio-degradable commercial gelatin microcarrier Cultispher G) for the epidermal cutlture. In the initialization phase, the epidermal cells were adhered to the microcarriers under serum medium condition without stirring. After that the intermittent stirring was started from 35rpm and increased gradually to 55rpm, meanwhile the stationary interval was gradually curtailed. We hence successfully cultivated the epidermal cells in a large mount in the limited duration. The cells adhered on the surface of the microcarriers or grew into the inner part of the microcarriers. The epidermal cells synthesized collagen under this culture condition observed by Massion triple-staining. But we also found that the cells grew in the bioreactor were not better than that grew in static condition in terms of the population doubling time, which indicated that the large number of the cells harvested from the bioreactor were achieved by increasing the culture surface and the culture volume. Fourth, uncultured epidermal cells and cultured epidermal cells both can be applied to the wound. Non-stratified epidermis may harbor more epidermal stem cells and thus has more capability in epithelialization. Improper method and inappropriate vehicle for delivery may impair the vitality of the cells while the other delivery technique may be too sophisticated for the extensive clinical use. The fibrin glue was employed in this study as a vehicle to deliver the epidermal cell to the wound. The cells were glued to the surface of the wound in the same time the fibrin formed a protective layer on the wound. The results shown that, through animal experiment, the epithelialization of the wound was completed within two weeks when the cells used. In the contrary, the epithelialization delayed more than two weeks when the fibrin glue was applied without the cells. When the fresh uncultured epidermal cells or the microcarrier loaded cells cultivated in the bioreactor were used, they shared the similar characteristic in histological morphology, thickness of the new-formed epidermis, distribution of the epidermal stem cells and the proliferation profiles of the epidermal cell in the wound. When the epidermal cells cultivated in the static condition were applied, the new formed epidermis were thinner, contained less epidermal stem cells and had lower proliferation profile. The wound healed by scar with incomplete epithelialization when only the fibrin glue was applied to the wound surface. No malignancy was found when the microcarrier loaded cells was subcutaneous injected within the experimental duration. The microcarrier aggregated into a mass in vivo and gradually degraded. Many cells and the neo-vascularizations were found in the injected cell-loaded microcarrier.Conclusions:Different techniques for harvesting and the culturing of the epidermal cells were studied in this study. The harvest methods of the epidermal cells were optimized and the scaling up of the epidermal cells in bioreactor was realized. The efficacy of the fresh uncultured epidermal cells and the microcarrier loaded epidermal cells on the wound healing was confirmed through animal experiment.