| Summary: | 博士 === 國立臺灣大學 === 醫學工程學研究所 === 99 === Salivary glands locate at the start of digestive system and are responsible for the secretion of saliva, which has many functions in maintaining homeostasis of the oral cavity. However, radiotherapy and autoimmune diseases may cause dysfunction of these glands and lead in the desiccant oral environment and many side effects. An engineered auto-secretary device will be the ultimate solution for this condition. As a potential solution for patients to retrieve their lost salivary gland functions, tissue engineering of an auto-secretory device is profoundly needed. Applying tissue engineering principles to design an auto-secretory device is a potential solution for patients suffering loss of salivary gland function.
However, the largest challenge in implementing this solution is the primary culture of human salivary gland cells, because the cells are highly differentiated and difficult to expand in vitro. This situation leads to the lack of not only reports on the in vitro cell biology and physiology of human salivary gland cells but also proper cell source. Complexity of the gland explains the urgent demand for a reliable protocol to isolate and expand various gland cells that can be used for further study. This study aims to set up a stable protocol for primary cultivation of human salivary gland cells from surgery specimens and further introduce those cells into tissue engineering.
In the first part, respective optimal culture conditions for three types of gland cells were set up by medium selection and then identified the phenotypes from mRNA to protein level. Those cultured cells with different morphologies were isolated and expanded without complex mechanical processes and expensive techniques such as flow cytometry sorting. The harvested cells were acinar (PGAC) cells, myoepithelial (PGME) cells, and fibroblasts (FBs). The proposed protocol is simple with a high success rate to culture various gland cells, making it highly promising for use in future tissue engineering studies.
The second part discusses the aggregating behavior of PGAC cells. This study used the serum-free and low-calcium culture system to obtain PGAC cells from tissues with high purity in cell composition. This condition enables PGAC cells to continuously proliferate and retain the phenotypes of epithelial acinar cells to express secreting products (α-amylase) and function-related proteins (aquaporin-3, aquaporins-5, and ZO-1). Notably, when the cells reached confluence, 3-dimensional (3D) cell aggregates were observed in crowded regions. These self-formed cell spheres were termed post-confluence structures (PCSs). Unexpectedly, despite being cultured in the same media, cells in PCSs exhibited higher expression levels and different expression patterns of function-related proteins compared to the 2-dimensional (2D) cells. Translocation of aquoporin-3 from cytosolic to alongside the cell boundaries, and of ZO-1 molecules to the boundary of the PCSs were also observed. These observations suggest that when PGAC cells cultured on the 2D substrate would form aggregations and exhibit certain level of differentiating characteristics without the help of 3D scaffolds. This phenomenon implies that introducing 2D substrates instead of 3D scaffolds into artificial salivary gland tissue engineering is of potential.
Extending the phenomenon of PCS formation that was discussed in the second part, the third part aims to investigate if the culture substrates have the effects on controlling cells in this aggregating process. Under serum-free environment, primary human PGAC cells can be obtained. After reaching confluence, PGAC cells spontaneously form 3D cell aggregations, termed post-confluence structure (PCS), and change their behaviors. Poly (lactic-co-glycolic acid) (PLGA) has been widely used in the field of biomedical applications because of its biodegradable properties for desired functions. Nonetheless, the role of PLGA in facilitating PGAC cells to form PCS has seldom been explored to recover epithelial characteristics. In this study, PGAC cells were found to have a greater tendency to form PCS on PLGA than on tissue culture polystyrene (TCPS). By tracing cell migration paths and modulating E-cadherin activity with specific inhibitor or antibody, we demonstrated that the static force of homophilic interaction on surfaces of individual cells, but not the dynamics of cell migration, played a more important role in PCS formation. Thus, PLGA was successfully confirmed to support PGAC cells to form more PCS through the effects on enhancing E-cadherin expression, which is associated with FAK/ILK/Snail expression in PGAC cells. This result indicates that selective appropriate biomaterials may be potentially useful in generating 3D PCS on two-dimension (2D) substrate without fabricating a complex 3D scaffolds.
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