返回Google圖書搜尋

Egg Yolk Plasma, Gelled Egg Yolk Plasma and Egg White Combinations for Three Dimensional Cultures and Bioprinting of Salivary Glands

出版	McGill University Libraries, 2020
URL	http://books.google.com.hk/books?id=zSKSzgEACAAJ&hl=&source=gbs_api

註釋"Introduction: Human tissue engineering can improve tissue's availability for in vitro drug screening models or clinical implantations. Here, for tissue engineering, we investigated a new multi-compositional biomaterial based on the avian egg. More specifically, we worked on the egg yolk plasma (EYP), an isolated fraction from the egg's yolk (EY). We selected the EYP because of its low cost, availability, developmentally stimulating environment, gelation ability and translucency. In the biomaterial, --either in its liquid or gelled state, we explored human salivary gland survival in various EYP combinations and developed a new cell analysis method. We expect our results to stimulate further research in this biomaterial's potential applications for human tissue engineering. Materials and Methods: Centrifugation isolated EYP from the EY. SDS-Page and Bicinconinic assay characterized the biomaterial. Live/Dead stain stained cells and image analysis algorithms quantified survival, death and proliferation of human salivary acinar (NS-SV-AC cell line) and primary fibroblasts (HuSG-Fibro) in EYP. In addition to testing these cell parameters in EYP, we combined EYP with either media, NaOH or egg whites (EW) in attempts to create various medium-free cell-culturing systems. As a tool for histologic characterization of these culture combinations, we created the 3D-Cryo well insert. The insert made from a paraffin coated fragment of a gelatine pill enabled us to cryo-section the culture systems at 0 and 14 days. Chemical stain Sirius Red and immunohistochemistry (IHC) --using anti-Ki-67 antibody, stained sagittal section of the cells in the egg biomaterial systems. EYP's freezing at -20°C and thawing, gelled EYP (GEYP). We used GEYP+EW in cell overlay technique or GEYP in 3D bioprinting experiments. A rheometer measured GEYP's visco-elastic properties at 37°C and a 3D extrusion bioprinter conducted extrusion printing experiments. Results: In EYP, cells survived when combined to either media, NaOH or egg white (EW). Live/Dead assays demonstrated NS-SV-AC's optimal proliferation in EYP+Media 30:70, EYP+NaOH 0.0188M or EYP+EW (45:55). In the same conditions, most HuSG-Fibro remained alive but proliferated substantially slower. Freeze thaw transformed EYP into a gel and maintained gel like properties at 37°C. GEYP's stiffness depended on freezing duration. After 30days of freezing, the average shear elastic stiffness (G') equalled ~350Pa. 3D-Cryo well inserts permitted cell analysis over 14 days by staining cells in multiple commercial biomaterials, EYP and GEYP mixtures. It also analyzed manually extruded GEYP bio-inks containing fluorescent cells. Sagittal sections of cells in EYP showed cells sinking contrasting EYP+EW's ability to maintain cells above the well's base. In GEYP+EW and GEYP manually extruded structures, GEYP maintained cells in position preventing their sinking to the well's base. IHC showed constant Ki-67 expression over 14 days with NS-SV-AC cells only in EYP+Media, EYP+EW while a decrease in expression with GEYP+EW. HuSG-Fibro rarely expressed Ki-67. Conclusion: Under our optimized EYP combinations with NaOH or with EW or as GEYP+EW we created three new media free viable biomaterial mixtures. Furthermore, we validated both computer assisted and histologic analysis methods. Importantly, the egg based culture systems enable media-free cell survival and under certain circumstances form salivary acini like clusters"--