3D打印生物墨水以及肝组织结构打印过程

一、3D打印生物墨水的开发
1.凝胶因子的合成： 设计、合成具有不同氨基酸序列的短肽分子；
2.成胶条件的探究：调控凝胶因子的浓度、溶液pH值、离子强度、组装温度等探究并筛选可快速凝胶化的凝胶因子，并优化组装条件；
3. 凝胶性能调控：通过流变、原子力、万能拉力机等对凝胶的力学性能进行表征，并进一步通过优化组装条件，实现对凝胶力学性能的调控，以满足3D生物打印的要求；
4. 生物兼容性表征：通过CCK-8、活死标记以及流式细胞实验对水凝胶生物墨水的生物兼容性进行表征，进一步筛选可用于细胞培养的水凝胶生物墨水；
5. 3D打印条件的优化：通过调控打印速度、压力、墨滴大小等对打印条件进行优化，以满足不同组织对打印精度及成型性的要求。
二、功能肝类器官的体外构建
1. 类肝小叶结构的3D打印：通过对3D生物打印机的程序进行设定，打印具有类肝小叶结构的六棱柱水凝胶支架；
2. 肝细胞在水凝胶支架内3D培养：利用3D生物打印可对墨滴精细精准定位的特点，在类肝小叶结构的六棱柱水凝胶支架内不同区域分别种植肝细胞和血管内皮细胞，以实现不同细胞的分区域培养，通过CCK-8、免疫荧光染色等对支架内三维增殖的细胞进行表征

I. Development of 3D Printing Bioinks
1. Synthesis of gelators: Design and synthesize short peptide molecules with distinct amino acid sequences.
2. Investigation of gelation conditions: Adjust the concentration of gelators, solution pH, ionic strength, assembly temperature and other relevant parameters to explore and screen gelators capable of rapid gelation, and optimize the assembly conditions.
3. Regulation of gel performance: Characterize the mechanical properties of the hydrogel via rheology, atomic force microscopy (AFM), universal testing machine and other characterization techniques. Further optimize the assembly conditions to regulate the mechanical properties of the hydrogel to meet the requirements of 3D bioprinting.
4. Biocompatibility characterization: Evaluate the biocompatibility of the hydrogel bioink through CCK-8 assay, live/dead staining and flow cytometry experiments, and further screen hydrogel bioinks suitable for cell culture.
5. Optimization of 3D printing conditions: Regulate printing speed, printing pressure, droplet size and other parameters to optimize the printing conditions, so as to meet the requirements of printing accuracy and formability for different tissue types.

II. In vitro Construction of Functional Hepatic Organoids
1. 3D printing of hepatic lobule-like structures: Program the 3D bioprinter to fabricate hexagonal prismatic hydrogel scaffolds with hepatic lobule-like structures.
2. 3D culture of hepatocytes within hydrogel scaffolds: Leverage the precise positioning capability of 3D bioprinting to separately seed hepatocytes and vascular endothelial cells into different regions of the hexagonal prismatic hydrogel scaffolds with hepatic lobule-like structures, thereby achieving regional culture of different cell types. Characterize the three-dimensionally proliferated cells within the scaffolds via CCK-8 assay and immunofluorescence staining.