Engineering Multicellular Spheroids for Tissue Engineering and Cell Therapy Applications

背景和动机：在过去的几十年里，基于细胞的生物检测已经从二维单层模型过渡到三维模型，因为三维模型更好地模拟了体内的生理条件。多细胞球体是一种很有前途的三维结构，在组织工程、药物发现和细胞治疗中具有新兴的应用前景。多细胞球体是直径在150-500微米之间的球形自组织细胞聚集体，由一个或多个细胞的单层悬浮产生，方法包括悬挂液滴等。这些方法通常是劳动密集型的，产量低，耗时长，并且产生形状和大小不均的球体，这限制了它们的放大应用。微流控技术通过提供对细胞的精确操纵和可控的物理条件、均匀的大小分布以及维持调节细胞营养和氧气供应的持续灌流，显示出克服球体形成中的一些技术障碍的前景。探索了U形圈闭、微流体和微井来形成球体。然而，它们的吞吐量不适用于临床规模，而且它们还需要复杂的机制来恢复球体，并且缺乏液体流动限制了营养物质或化学刺激的质量传输。目标和方法：我们的长期目标是实现基于声表面波(SAW)的微流体平台，用于开发用于组织工程、细胞治疗和疾病建模的多细胞球体，方法是：1)实现用于多细胞球体制造的声表面波设备；2)研究和定制SAW的工作参数；3)球体制造和涂层的可行性研究；以及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SAW器件的多用途模块，用于单细胞和多细胞球体的生物制造和应用，使用软光刻和使用PDMS的复制模制技术。不同的参数对大小、稳定的球体的形成、细胞活力和增殖的影响将被研究。SAW装置将用于促进多细胞椭圆形结构中的血管形成。使用层层方法的保形涂层将被用来诱导构建物中的免疫调节特性。SAW将用于多个细胞系的联合培养，以促进血管形成或生产可生物打印的三维球体，作为骨组织工程的构建块。影响：这项建议为PI的实验室/组织芯片平台研究计划提供了一个新的研究流，其中9名受训人员将参与多学科研究。它提供声学微流控平台，作为成本效益高的解决方案，用于制造被视为伪组织的多细胞球体，以便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.