Engineering Multicellular Spheroids for Tissue Engineering and Cell Therapy Applications

背景和动机：在过去的几十年里，基于细胞的生物测定已经从二维单层过渡到三维模型，因为三维模型更好地模拟了体内的生理条件。多细胞球体作为一种三维结构体，在组织工程、药物发现和细胞治疗等领域有着广阔的应用前景。多细胞球体是直径150-500微米的球形自组织细胞聚集体，由一个或多个细胞的单层悬浮液产生，使用诸如悬挂液滴等方法。这些方法通常是劳动密集型的、低产量的、耗时的，并且在形状和尺寸上产生非均匀的球体，这限制了它们的扩展应用。微流体技术通过提供精确的细胞操作和控制物理条件，均匀的尺寸分布，以及维持持续的灌注来调节细胞的营养和氧气供应，已经显示出克服球形形成中的一些技术障碍的希望。u型陷阱微流体和微井被探索形成球体。然而，它们的通量并不适用于临床规模，它们还需要复杂的球体回收机制，并且缺乏流体流动限制了营养物质或化学刺激的大量运输。目标和方法：我们的长期目标是实现基于表面声波（SAW）的微流控平台，用于组织工程、细胞治疗和疾病模型的多细胞球体的开发：1)实现用于多细胞球体制造的SAW设备；2) SAW工作参数的调研与定制；3)球体制造和涂层的可行性研究；4)开发用于组织工程和细胞治疗应用以及疾病建模的多细胞球体。将开发两个多用途SAW器件模块，用于使用软光刻技术和使用PDMS的复制模具技术进行单细胞和多细胞球体的“生物制造”和“应用”。研究了各种参数对细胞大小、稳定球体的形成、细胞活力和增殖的影响。SAW装置将用于促进多细胞球体结构中的血管化。采用逐层方法的适形涂层将用于诱导结构中的免疫调节特性。SAW将被用于共培养多个细胞系，以促进血管化或生产3D生物打印球体，作为骨组织工程的基石。影响：该提案为PI的实验室/组织芯片平台研究项目提供了新的研究流，其中9名学员将参与多学科研究。它提供了声学微流体平台，作为制造多细胞球体的成本效益解决方案，被认为是假组织，在设计昂贵且具有伦理挑战性的动物研究之前生成相关数据。

Background and motivation: In the past decades, cell-based bioassays have transitioned from 2-D monolayers to 3-D models, as the 3-D models better mimic the in-vivo physiological conditions. Multicellular spheroids are promising approaches as 3-D constructs for emerging applications in tissue engineering, drug discovery and cell therapy. Multicellular spheroids are spherical self-organizing cell aggregates ranging from 150-500µm in diameter, generated from a monolayer suspension of one or multiple cells, using methods such as hanging droplets among others. These methods are generally labor-intensive, low-yielded, time-consuming, and generate heterogeneous spheroids in shape and size which limits their scaled-up application. Microfluidics has shown the promise of overcoming some of the technical hurdles in spheroid formation by offering precise manipulation of cells and controlled physical conditions, homogenous size distribution, and maintaining continuous perfusion which regulates the nutrients and oxygen supply to cells. U-traps microfluidics and microwell are explored to form spheroids. However, their throughput is not applicable to clinical scales and they also need complex mechanisms for spheroids retrieval, and the lack of fluid flow limits the mass transport of nutrients or chemical stimuli. Objectives and methodology: Our long-term objective is to implement surface acoustic wave (SAW)-based microfluidic platforms for the development of multicellular spheroids for tissue engineering, cell therapy and to model diseases by 1) implementing SAW devices for multicellular spheroid fabrication; 2) Investigating and customizing working parameters of SAW; 3) Feasibility study for spheroid fabrication and coating; and 4) Developing multicellular spheroids for tissue engineering and cell therapy applications and disease modelling. Two multipurpose modules of SAW device will be developed for `biofabrication' and `applications' of mono and multi-cellular spheroids using soft lithography and replica mold techniques using PDMS. Effect of various parameters on size, formation of stable spheroids, cell viability and proliferation will be investigated. The SAW device will be used to promote vascularization in multicellular spheroid constructs. Conformal coating using layer-by-layer approach will be used to induce immunomodulatory properties in the construct. The SAW will be adopted for co-culturing multiple cell lines to promote vascularization or to produce 3D bioprintable spheroids as building block for bone tissue engineering. Impact: This proposal provides a new research stream to the PI's research program on lab/tissue-on-a chip platforms where 9 trainees will be involved in a multidisciplinary research. It delivers acoustic microfluidics platforms as cost-effective solution for the fabrication of multicellular spheroid considered as pseudo tissue to generate relevant data prior to designing costly and ethically challenging animal studies.