Development of a multifunctional, acoustofluidic 3D bioprinter with single-cell resolution

项目摘要 三维（3D）生物打印是一种快速新兴的技术，具有快速打印的潜力。 定制的功能性生物组织近年来，在修改传统的 3D生物打印的打印系统。然而，他们打印的组织往往缺乏分辨率和复杂性， 必须实现的基本功能，生理条件和各向异性的性质，发现在天然的 组织中因此，迫切需要开发下一代3D生物打印仪器， 以下三个关键技术限制：（1）无法控制印刷矩阵中的内部单元位置 材料体素并实现所需的单元-单元间距（即，单元邻近分辨率< 10 µm）， 从细胞水平确保适当的组织功能，并在3D中研究细胞-细胞相互作用 （2）不能打印具有高局部细胞密度（>109个细胞/mL）的组织，如在微环境中观察到的。 （3）不能用多尺度仿生细胞进行大尺度组织的无支架打印 体系结构（例如，细胞图案和排列），这对于获得期望的各向异性组织是必要的 属性和关键功能，取决于多尺度细胞的安排。在过去的十年里，我们 开发了一系列声流体（即，声学和微流体的融合）技术， 优秀的候选人，以解决上述瓶颈。特别是，我们最近开发了声流体 全息术，一种基于声学的、生物相容的和高分辨率的细胞操纵技术， 以在聚合之前图案化、旋转和浓缩细胞接种的基质材料。在此基础上 技术，在这个R 01项目中，我们建议开发和验证声流体3D生物打印原型， 打印功能组织，具有高细胞邻近分辨率（<10 µm）和复杂特征（如仿生 细胞结构、受控的各向异性特性和高细胞密度）， 可伸缩的方式。我们的声流体3D生物打印机将通过打印血管化肿瘤球体进行验证， 间质和各向异性的、神经支配的、血管化的骨骼肌组织。与目前的3D生物打印相比， 我们的声流体3D生物打印机将具有多种优势，包括：（1）能够控制内部 打印矩阵体素的单元位置，并实现高单元邻近分辨率（< 10 µm）;（2）能够打印 具有高局部细胞密度（>109个细胞/mL）和受控密度分布的组织;（3）打印组织的能力 具有多尺度细胞结构和控制组织的各向异性特性，而不使用支架; (4)高生物相容性（>95%活力）。有了这些优点，所提出的声流体3D生物打印 技术有可能大大超过目前的标准，并解决3D中未满足的需求 生物打印领域。我们预计，我们的声流体3D生物打印技术将具有巨大的价值， 生物医学研究社区致力于基础的体外和体内研究，癌症研究，细胞-细胞 相互作用研究、组织工程、再生医学和药物筛选。

Project Summary Three-dimensional (3D) bioprinting is a rapidly emerging technology that has the potential to quickly print customized, functional, biological tissues. In recent years, much progress has been made in modifying traditional printing systems for 3D bioprinting. However, their printed tissues often lack the resolution and complexity necessary to achieve the essential functions, physiological conditions, and anisotropic properties found in native tissues. Therefore, there is a critical need to develop next-generation 3D bioprinting instruments that address the following three key technologic limitations: (1) the inability to control internal cell positions in printed matrix material voxels and achieve the desired cell-cell spacing (i.e., cell proximity resolution < 10 µm) that is critical to ensure proper tissue functions from the cellular level and studying cell-cell interactions in the 3D microenvironments; (2) the inability to print tissues with high local cell densities (>109 cells/mL) as observed in vivo; and (3) the inability to perform scaffold-free printing of large-scale tissues with multiscale biomimetic cellular architectures (e.g., cell pattern and alignment), which are essential to achieve desired anisotropic tissue properties and key functions that depend on multiscale cell arrangements. Over the last ten years, we have developed a series of acoustofluidic (i.e., the fusion of acoustic and microfluidic) technologies, which are excellent candidates to address the bottlenecks above. In particular, we have recently developed acoustofluidic holography, an acoustics-based, biocompatible, and high-resolution cell manipulation technology that allows one to pattern, rotate, and concentrate cell seeded matrix materials before polymerization. Building upon this technology, in this R01 project, we propose to develop and validate an acoustofluidic 3D bioprinting prototype to print functional tissues with high cell proximity resolution (<10 µm) and complex features (such as biomimetic cellular architectures, controlled anisotropic properties, and high cell densities) in a biocompatible, fast, and scalable manner. Our acoustofluidic 3D bioprinter will be validated by printing vascularized tumor spheroids with stroma and anisotropic, innervated, vascularized skeletal muscle tissues. Compared to current 3D bioprinting instruments, our acoustofluidic 3D bioprinter will have multiple advantages including: (1) ability to control internal cell positions of printed matrix voxels and achieve high cell proximity resolution (< 10 µm); (2) ability to print tissues with high local cell densities (>109 cells/mL) and controlled density distributions; (3) ability to print tissues with multiscale cellular architectures and control tissues’ anisotropic properties without using scaffolds; and (4) high biocompatibility (>95% viability). With these advantages, the proposed acoustofluidic 3D bioprinting technology has the potential to significantly exceed current standards and address unmet needs in the 3D bioprinting field. We expect that our acoustofluidic 3D bioprinting technology will be of tremendous value to biomedical research communities working on fundamental in vitro and in vivo studies, cancer research, cell-cell interaction studies, tissue engineering, regenerative medicine, and drug screening.