Department of Micro- and Nanotechnology, Technical University of Denmark, Denmark.
Department of Micro- and Nanotechnology, Technical University of Denmark, Denmark.
Acta Biomater. 2018 Jan;65:174-184. doi: 10.1016/j.actbio.2017.10.047. Epub 2017 Nov 10.
One of the fundamental steps needed to design functional tissues and, ultimately organs is the ability to fabricate thick and densely populated tissue constructs with controlled vasculature and microenvironment. To date, bioprinting methods have been employed to manufacture tissue constructs with open vasculature in a square-lattice geometry, where the majority lacks the ability to be directly perfused. Moreover, it appears to be difficult to fabricate vascular tissue constructs targeting the stiffness of soft tissues such as the liver. Here we present a method for the fabrication of thick (e.g. 1 cm) and densely populated (e.g. 10 million cells·mL) tissue constructs with a three-dimensional (3D) four arm branch network and stiffness in the range of soft tissues (1-10 kPa), which can be directly perfused on a fluidic platform for long time periods (>14 days). Specifically, we co-print a 3D four-arm branch using water-soluble Poly(vinyl alcohol) (PVA) as main material and Poly(lactic acid) (PLA) as the support structure. The PLA support structure was selectively removed, and the water soluble PVA structure was used for creating a 3D vascular network within a customized extracellular matrix (ECM) targeting the stiffness of the liver and with encapsulated hepatocellular carcinoma (HepG2) cells. These constructs were directly perfused with medium inducing the proliferation of HepG2 cells and the formation of spheroids. The highest spheroid density was obtained with perfusion, but overall the tissue construct displayed two distinct zones, one of rapid proliferation and one with almost no cell division and high cell death. The created model, therefore, simulate gradients in tissues of necrotic regions in tumors. This versatile method could represent a fundamental step in the fabrication of large functional and complex tissues and finally organs.
Vascularization within hydrogels with mechanical properties in the range of soft tissues remains a challenge. To date, bioprinting have been employed to manufacture tissue constructs with open vasculature in a square-lattice geometry that are most of the time not perfused. This study shows the creation of densely populated tissue constructs with a 3D four arm branch network and stiffness in the range of soft tissues, which can be directly perfused. The cells encapsulated within the construct showed proliferation as a function of the vasculature distance, and the control of the micro-environment induced the encapsulated cells to aggregate in spheroids in specific positions. This method could be used for modeling tumors and for fabricating more complex and densely populated tissue constructs with translational potential.
设计功能性组织，最终设计器官的基本步骤之一是能够制造具有受控脉管系统和微环境的厚且高密度的组织构建体。迄今为止，已经采用生物打印方法来制造具有开放脉管系统的组织构建体，其具有正方形晶格几何形状，其中大多数不具备直接灌注的能力。此外，似乎难以制造针对诸如肝脏的软组织的硬度的血管组织构建体。在这里，我们提出了一种制造具有三维（3D）四臂分支网络和软组织范围内的硬度（1-10kPa）的厚（例如 1cm）和高密度（例如 1000 万个细胞·mL）组织构建体的方法，可以直接在流体平台上长时间（>14 天）进行灌注。具体来说，我们使用水溶性聚（乙烯醇）（PVA）作为主要材料和聚（乳酸）（PLA）作为支撑结构共打印 3D 四臂分支。选择性去除 PLA 支撑结构，然后使用水溶性 PVA 结构在定制的细胞外基质（ECM）中创建针对肝脏硬度的 3D 血管网络，并封装肝细胞癌（HepG2）细胞。这些构建体直接用诱导 HepG2 细胞增殖和形成球体的培养基进行灌注。通过灌注获得了最高的球体密度，但是总体而言，组织构建体显示出两个明显的区域，一个区域快速增殖，一个区域几乎没有细胞分裂且细胞死亡很高。因此，所创建的模型模拟了肿瘤中坏死区域的组织中的梯度。这种多功能的方法可能代表制造大型功能性和复杂组织并最终器官的基本步骤。
在软组织范围内具有机械性能的水凝胶中的血管化仍然是一个挑战。迄今为止，已经采用生物打印方法来制造具有开放脉管系统的组织构建体，其具有正方形晶格几何形状，大多数情况下不进行灌注。本研究显示了具有 3D 四臂分支网络和软组织范围内的硬度的高密度组织构建体的创建，该构建体可以直接灌注。包裹在构建体中的细胞随着脉管距离的增加而增殖，并且微环境的控制诱导包裹的细胞在特定位置聚集在球体中。该方法可用于模拟肿瘤，并用于制造具有转化潜力的更复杂和高密度的组织构建体。
