近幾年來，三維細胞培養已漸漸成為世界上細胞培養技術的主流概念，與傳統二維空間培養相較之下，三維的空間可以提供細胞類似生物組織的空間環境，且可以允許氧氣和養分在支架裡的孔洞間進行流通與傳遞，本實驗以天然瓊脂糖(Agarose)為材料主體，製備具有高度生物相容性的天然三維孔洞支架。天然高分子中，瓊脂糖(Agarose)具有良好機械性質且快速成膠的特性，因此常應用於組織工程相關領域研究中，但由於分子結構具剛性，所以有不易降解及細胞不易貼附等限制，本實驗會透過添加同樣為天然高分子的明膠(Gelatin)來改善材料與細胞的親和性，除了調控膠體結構的機械強度外，同時也賦予膠體具有可降解的特性，也大幅提升材料親水性、延展性及生物可應用性。
本實驗利用交聯劑N，N'-羰基二咪唑(N,N'-carbonyldiimidazole, CDI)，進行瓊脂糖(Agarose)與明膠(Gelatin)交聯反應，來彌補天然高分子機械性質不佳的缺點，並提升兩種高分子之間的作用力，來達到支架結構的穩定性。孔洞支架製備過程中，先將高分子在去離子水中加熱溶解並倒入模具，冷卻後轉變成膠態，透過簡易的凍乾法形成三維孔洞支架(300-500 μm)。
實驗結果可發現，製備的三維孔洞支架具有高孔隙率及足夠的孔洞大小，且交聯劑確實能提高支架的機械性質並穩定支架的結構，而細胞毒性測試也證明交聯反應後的支架不具有細胞毒性，說明支架可提供良好的環境供細胞生長、貼附、遷移、分化。將支架結合週期性壓縮細胞培養系統，由軟骨黏多醣蛋白染色與細胞分化基因分析可發現，說明添加TGF-β1能夠誘導支架內的脂肪幹細胞分化成軟骨細胞，且三維空間並搭配週期性壓縮培養的結果較二維平面(tissue culture plate)所培養的基因表現量佳。實驗證實製備的支架有利於大鼠脂肪幹細胞分化。

In recent years, three-dimensional cell culture has gradually become the popular concept of cell culture technology in the world. Compared with traditional two-dimensional culture, three-dimensional culture not only can provide an environment similar to biological tissues for cells but also can allow oxygen and nutrients to transfer in the pores of the scaffold. The goal of this study is to use natural agarose as the main material to prepare a natural three-dimensional porous scaffold with excellent biocompatibility. Among the natural polymers, agarose has good mechanical properties and short gelation time, which is often applied to research in tissue engineering. However, due to the rigid molecular structure, the materials are less degradable and have low attachment capacity for cells.
The study will improve the affinity of materials and cells by adding gelatin, which is also a natural polymer. The application of gelatin not only can regulate the mechanical strength of the colloid structure but also improves the degradability, hydrophilicity, malleability, and bio-applicability of the material.
In order to compensate for the poor mechanical properties of natural polymers, the project used N, N'- carbonyldiimidazole (CDI) as the cross-linking agent to carry out the cross-linking reaction between agarose and gelatin. The progress also increased the interaction between agarose and gelatin to get a stable structure. In the preparation of the porous scaffold, the polymer was dissolved in deionized water by heating and poured into the mold, and then the solution turned into a colloidal state after cooling, and a three-dimensional porous scaffold (pore size: 300-500μm) was formed through a simple freeze-drying method.
The results showed that the prepared three-dimensional pore scaffold had high porosity and sufficient pore size, and the cross-linking agent could improve the mechanical properties of the scaffold and stabilize the structure of the scaffold, and the cytotoxicity test also proved that the scaffold after cross-linking reaction didn’t have cytotoxicity, indicating that the scaffold can provide a good environment for cell growth, attachment, migration, and differentiation. According to proteoglycan staining and gene expression analysis, the addition of TGF-β1 could induce the adipose stem cells to differentiate into chondrocytes in the scaffold combining with a cyclic physical stimulation system. Also, the results of incubation in three three-dimensional pore scaffolds combining with a cyclic physical stimulation system had more gene expression than the incubation in a tissue culture plate. The experiment showed the developed scaffolds had the benefits to the differentiation of rat adipose stem cells.

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