利用微流體技術探討維度對心肌母細胞及脂肪幹細胞之分化影響

Abstract

在生醫材料影響細胞行為的眾多物理性質中，維度是一較少被論及之性質。因為過往之技術頃向製作出孔洞大小形狀不一之支架，造成眾多因素影響細胞行為。在這份研究中，我們利用微流體技術製作出具有均一圓球狀孔洞大小之三維明膠細胞支架以模仿細胞在生物體內的生長環境，並比較細胞行為在三維及二維環境下的差異，實驗中所用的細胞為二種具有分化能力的細胞: H9c2老鼠心肌母細胞與人類脂肪幹細胞 (hASC)。 H9c2為一同時具骨骼肌與心肌細胞特徵的細胞株，能夠進一步分化成具有此二種肌肉特徵的細胞。因此在這部分的實驗中，我們旨在探討維度上的差異會有利於何種肌肉特徵的表現。首先我們透過Live/Dead螢光染色確定此支架能讓細胞維持良好生存率，接著我們探討細胞的生長速率、型態、分化率、Connexin 43染色定量還有基因表現。結果發現: H9c2在三維空間中傾向分化成骨骼肌，而在二維空間中傾向分化成心肌細胞，不過維度對心肌分化的影響遠小於化學因素所能造成的影響。 hASC具有多種分化能力，包括肌肉、硬骨、軟骨及脂肪分化等等，我們透過觀察細胞型態、增殖速率、鈣沉積或是脂滴染色以及基因表現探討維度對於脂分化及硬骨分化的影響。結果指出，三維的結構較有利於脂肪分化而不利於硬骨分化。未來將深入探討這樣的現象是由何種因素導致，並且進一步研究軟骨及肌肉的分化。

Dimensionality is a physical cue that has been discussed less frequently among the various physical cues that influence cell behaviors. In this study, we took the advantage of microfluidics to fabricate three-dimensional (3D) gelatin-based scaffolds to mimic the natural circumstances of cell growth in an organism. We further compared the differences of cellular behaviors between 2D and 3D conditions. The models used in the presented study are H9c2 rat cardiac myoblast and human adipose-derived stem cell (hASC), both of which have differentiation ability. H9c2 is able to differentiate into skeletal or cardiac myocytes. Therefore, in this series of experiments, we aimed at investigating how dimensionality contributes to different types of muscle differentiation. We have found that H9c2 cells tended to fuse into multi-nucleated skeletal myotubes in 3D scaffold and differentiated into cardiac cells on 2D plane. However, the influence of dimensionality on cardiac differentiation was far less than that of chemical stimulation. hASC possesses multipotent differentiation ability toward osteogenic, adipogenic, myogenic and chondrogenic lineages. Specifically, we investigated osteogenic and adipogenic differentiation capability of hASC under different dimensional conditions. According to our results, the 3D environment supported adipogenesis and the 2D condition promoted osteogenesis. Our future work will focus on finding the factors which influence differentiation capability on the matrixes in different dimensionality. Furthermore, the myogenesis and chodrogenesis will also be investigated.