In-vitro Preparation of Full Thickness Skin Equivalent

• Main Authors: Yi-Chau Huang, 黃怡超
• Other Authors: 林峯輝
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/57981419704137017599

博士 === 國立臺灣大學 === 醫學工程學研究所 === 94 === Abstract Fibroblasts produce a spectrum of necessary growth factors essential for growth and proliferation of a variety of cell types. First aim of this study was prepared the feeder gel with optimum fibroblast density and optimum conditioned medium that promoted keratinocyte proliferation without further differentiation for skin equivalent tissue engineering. The optimum cell density in collagen feeder gel for optimum selected medium preparation will be determined by checking the level of keratinocyte growth factor (KGF) and granulocyte/macrophage colony-stimulating factor (GM-CSF) in conventional medium. The results showed that the cell density of 1x105 cells/gel in the feeder gel is better to produce optimum selected medium. The conditioned medium is prepared by mixing the optimum selected medium and MCDB 153 medium together in different ratios for keratinocyte growth. The keratinocyte viability will be measured by MTT assay to determine the optimum conditioned medium. From the study, 67% conditioned medium was supposed the better medium for the keratinocyte proliferation. In this experiment, the optimum cell density in feeder gel to co-culture with keratinocyte is also determined as 1x105 cells/gel. Cytokeratin-10 and TUNEL stain will be used to check the cell differentiation and apoptosis, respectively. The results suggest that keratinocyte should not be cultured in post-confluent condition due to toward undesired apoptosis and differentiation. The result of cell viability from passages to passages shows that the optimum feeder gel plays more important role to the keratinocyte proliferation than that of optimum conditioned medium. Keratinocytes cultured with optimum feeder gel in 67% conditioned medium could effectively promote proliferation, inhibit apoptosis and prevent from differentiation. The combination of conditioned media and feeder gel to culture keratinocyte without external supplements can provide an inexpensive way for keratinocyte proliferation and construct an environment for real-time communication between the two cells. The results conclude that keratinocyte cultivation in feeder gel with modified medium should be feasible in the production of high quality keratinocyte for skin equivalents preparation. The second approach of preparing skin equivalents is regulation of proliferated and differentiated capacity. We investigated Ca2+ effects on the proliferation and differentiation using the primary keratinocytes model. Keratinocytes were incubated in DMEM (containing 1.2 mM Ca2+ concentration) or DK11 medium (containing 0.4 mM Ca2+ concentration) or K medium (containing 0.03 mM Ca2+ concentration). Cell viability was assessed with the MTT assay. Crystal violet assay was evaluated the proliferation rate and colony formation size of keratinocyte. Real-time PCR used to determine the terminal differentiated keratinocyte which expressed Caspase-14. Proliferation assays and real–time PCR were correlated with either proliferation or differentiation in cultured human skin epidermal keratinocytes. High Ca2+ concentration was inhibited the cell viability and proliferation rate of keratinocyte. Ca2+ also increased caspases-14 expression, and inhibited cell viability, and cell colony forming efficiency. These results are consistent with Ca2+ induction of the keratinocyte differentiation. Thus, the overall Ca2+ actions connote protective functions for the epidermis that appear to include the triggering or acceleration of the differentiation. At last, we designed a novel culture system included a self-designed 3-D collagen scaffold with different pore size and specific culture media for different culture stages. This skin equivalent culture model provides a new investigating system to study the role of extracellular matrix and growth factors including epidermal growth factor (EGF), keratinocyte growth factor (KGF), transforming growth factor beta 1 (TGF-beta1), in the cell-cell and cell-matrix interactions. Keratinocytes were seeded onto the dermal equivalent and incubated under submerged condition for 5 days then proceeding to air-liquid interface cultured either with or without EGF addition. In this study, EGF has a positive effect on the keratinocyte migration and proliferation in the submerged stage. However, when 10ng/ml of EGF was continual added in the air-lifted stage, a less organized and thin differentiated keratinocyte layers were found. Continual 10ng/ml of EGF addition in the air-lifted stage resulted in uneven cell-matrix interface, and disorganization of the suprabasal layers. On the contrary, in the air-lifted stage without excess EGF, the epithelium cells will stratify, differentiate, and form an epidermis completed with basal, spinous, granular, and cornified layers. The results showed that time scale modulation of EGF on keratinocyte cell behavior depend on the expression of paracrine or autocrine growth factors (e.g. KGF and TGF-beta1).