Photo-crosslinkable hydrogel and 3D rapid prototyping technology application for primary hepatocyte culture

近年來隨著3D印表機蓬勃發展，解決了組織工程支架在傳統工法上，製作困難且過程冗長的缺點，這項快速成型的技術，逐漸被應用到組織工程與再生醫學領域。由於肝臟器官包含多種細胞以及其臟器本身的複雜性，使得體外模型不易建立，且分離取得的原代肝細胞在短時間內即失去成熟肝臟基因的功能；本研究將上述技術應用於肝臟細胞的研究，由電腦預先設計好模型，將生物3D印表機做適當調整，接著將肝臟組織細胞包埋於膠體內，最後印出膠體支架模型。 以往作為支架常使用的熱塑性聚合物並不適用於包埋細胞，於是在材料上改用光固化高含水膠體作為支架主成分，在本研究中使用以明膠為主材料的光固化高含水膠體製作支架，明膠早已廣泛使用在藥學、食品、化妝品等各類產品上，研究發現也適用於細胞培養，但是明膠本身在攝氏37度下強度不足以作為組織支架主結構，必需進一步處理以提升其支撐強度。 本研究提出改良甲基丙烯酸修飾處理明膠的製程，取得高親水性光固化膠體，成為可以支撐組織細胞的支架，同時以核磁共振儀偵測光固化膠體水溶液，以確認取代反應的完成度。生物印表機改裝自開源社群的3D印表機，並解決原始設計以熱塑性聚合體材料無法包埋細胞的問題，重新設計出可以列印膠體的噴頭和光固化模組，建構完成可以列印製作軟組織支架的生物3D印表機。 在掃描式電子顯微鏡下觀察支架模型，最高精細度可達到300μm，且膠體支架具有許多孔隙，對於細胞貼附、養分擴散均有正面效果。本研究中的肝臟細胞支架的技術，肝臟細胞可以在膠體支架中順利生長，且在第五天仍繼續表現成熟肝臟基因。這項技術未來有潛力廣泛應用到軟組織支架、肝臟體外培養以及組織工程。3D printing moved from being theoretical to a reality, and in recent years 3D printers have become cheaper to produce. Nowadays, 3D bio-printing has been widely used in tissue engineering and regenerative medicine. This rapid prototyping technology provide a great opportunity to fabricate 3D scaffolds without problems such as restricted geometric shapes in traditional methods. In this research, we modified an open-source 3D printer project, originally printed thermoplastic polymer which was not support soft tissue and cell encapsulation. Thus, we designed a bio-printer including photo-crosslinking module and redesign syringe pump extruder of 3D printer. In the experiments, we used gelatin hydrogel, instead of thermoplastics, as main material of scaffold. Gelatin is commonly used in pharmaceutics, food, cosmetics, as well as cell culture. However, gelatin has poor mechanical properties at 37˚C to be the main structure of a tissue scaffold. Therefore, further processing is required to strengthen for gelatin. In this study, we demonstrated optimized methacrylation of gelatin forming a highly hydrophilic photo-crosslinkable hydrogel. The hydrogel is analyzed by NMR spectra to monitor degree of functionalization. Because of complexity of liver, it is hard to create in vitro liver model. Moreover, isolated primary hepatocytes rapidly lose its mature hepatic functions after culturing in 2D environment due to loss of ECM and intercellular interaction. We applied 3D bio-printing technology in creating hepatocyte laden hydrogel scaffold. Porous hydrogel scaffold was design by CAD software and printed by our well-calibrated 3D bio-printer reaching 300μm precision. Also scanning electron microscopy analysis was involved to confirm porous structure of the hydrogel scaffold offering cell attachment and nutrient dispersion well. In our research, primary hepatocytes expressed mature hepatic markers after 5 days cultured in our 3D hydrogel scaffolds. We believed this technology opens new possibilities for the application of 3D bio-printing in liver tissue engineering