功能化及个性化的生物3D打印团队，任务1：3D组装-XDA16020802

研究内容及技术路线;
研究打印过程中多材料间的界面维持及培养过程中的动态变化。通过基质材料的改进以及管道化方案的改进探究后续培养过程中管道化的长期维持。在材料方面，重点完成弹性材料的开发和利于管道化的材料的研发。针对各器官通过对组织发育机理的理解，建立个性化的组织器官构建方案，完成器官构建过程中所需材料的研发和后续功能评价的研究，具体内容及路线如下：
（1）研究内容1：血管化组织构建：
根据材料剪切性能开发可打印且生物相容性好的生物墨水，通过打印溶芯去出牺牲材料以构建管道化组织，灌注细胞内皮化后形成可灌注血管，进一步在基质材料中加入血管化细胞或功能细胞以实现部分功能。
（2）研究内容2：体外构建肌肉组织
进行不同材料种类、细胞密度以及材料的不同浓度进行筛选，最终根据细胞活性、细胞伸展率、可打印性以及材料的孔径等因素选择一组最佳组合。
拟采用以明胶和纤维蛋白原为主体材料，借助3D打印技术，通过间隔打印的方法，获得体外肌肉组织，这样一来保证肌束的营养渗透率保证内部组织的存活，二来可以适当增大打印体积，之后进行培养最终得到功能性的成熟度较高的肌肉组织。
（3）研究内容3：类皮肤的构建
三层皮肤的构建
①皮下组织：采用胶原、海藻酸钠结合前体脂肪细胞，利用模型（PDMS，15 mm (length) ×15 mm (width) × 6 mm (height)），采用浇筑的方法，实现厚度为2mm的皮下组织的构建。
②真皮层：采用胶原结合成纤维细胞、血管内皮细胞和间充质干细胞，采取3D方法，实现厚度为3mm的真皮层的构建。
③表皮层：利用角质形成细胞均匀培养在构建的真皮层表面，通过气液培养14D的方式，角质形成细胞完成分化，实现表皮层的构建。
血管组织的构建：
①主血管的构建：采用3D打印以及灌流培养的系统，实现血管的构建。
②毛细血管网络的构建：首先在体外通过利用低黏附培养皿培养HUVEC细胞，使HUVEC在体外自组装成球状，最终通过因子的调控实现构建皮肤血管系统的网络化与成熟。
体系共培养：
培养基由成纤维细胞生长培养基和脂肪前体细胞分化培养基组成，采用1:1共培养法培养培养7D，将培养基加至真皮层。第7 d后，使用将人表皮角质形成细胞(HEKs)均匀滴加到真皮上层，通过气液培养的方式，实现表皮层分化成熟。

Research Content and Technical Route
This study focuses on the interfacial maintenance between multiple materials during the printing process and the dynamic changes during the cultivation process. We aim to explore the long-term maintenance of tubular structures in subsequent cultivation by improving matrix materials and tubularization schemes. In terms of materials, we will prioritize the development of elastic materials and materials conducive to tubularization. Based on the understanding of tissue development mechanisms for various organs, we will establish personalized tissue and organ construction schemes, and complete the research and development of materials required for organ construction and subsequent functional evaluation. The specific contents and routes are as follows:
(1) Research Content 1: Vasculature Tissue Construction
Develop printable and biocompatible bioinks based on the shear properties of materials. Construct tubular tissues by printing sacrificial cores and removing the sacrificial materials. Form perfusable blood vessels after seeding and endothelializing cells in the lumen, and further add vascularized cells or functional cells into the matrix materials to achieve partial biological functions.
(2) Research Content 2: In vitro Construction of Muscle Tissue
Screen different material types, cell densities and material concentrations, and finally select an optimal combination based on factors such as cell viability, cell spreading rate, printability and material pore size. It is planned to use gelatin and fibrinogen as the main materials, and utilize 3D printing technology with an interval printing method to obtain in vitro muscle tissue. This ensures the nutrient permeability of muscle bundles to maintain the survival of internal tissues, and also appropriately increases the printing volume. After subsequent cultivation, functional muscle tissues with high maturity can be finally obtained.
(3) Research Content 3: Construction of Skin-like Tissues
Construction of three-layer skin:
① Subcutaneous tissue: Use collagen and sodium alginate combined with preadipocytes, and use a PDMS mold (15 mm (length) ×15 mm (width) × 6 mm (height)) to construct a 2 mm-thick subcutaneous tissue via the casting method.
② Dermis layer: Use collagen combined with fibroblasts, vascular endothelial cells and mesenchymal stem cells, and use a 3D method to construct a 3 mm-thick dermis layer.
③ Epidermal layer: Culture keratinocytes uniformly on the surface of the constructed dermis layer, and allow keratinocytes to differentiate via air-liquid interface culture for 14 days, thereby completing the construction of the epidermal layer.
Construction of vascular tissues:
① Main blood vessels: Construct blood vessels via 3D printing and perfusion culture system.
② Capillary network: First culture human umbilical vein endothelial cells (HUVECs) in vitro using low-adhesion Petri dishes to allow HUVECs to self-assemble into spheroids in vitro, and finally achieve the networking and maturation of the skin vascular system via cytokine regulation.
Co-culture system:
The culture medium is composed of fibroblast growth medium and preadipocyte differentiation medium, and adopt a 1:1 co-culture method for 7 days, then add the medium to the dermis layer. On the 7th day, uniformly drop human epidermal keratinocytes (HEKs) onto the upper layer of the dermis, and achieve the differentiation and maturation of the epidermal layer via air-liquid interface culture.