Engineering Multicellular Spheroids for Tissue Engineering and Cell Therapy Applications

背景和动机：在过去的几十年中，基于细胞的生物测定已经从2-D单层过渡到3-D模型，因为3-D模型更好地模拟体内生理条件。多细胞球状体作为三维结构在组织工程、药物开发和细胞治疗等领域有着广阔的应用前景。多细胞球状体是直径为150-500µm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，使用方法如悬滴等。这些方法通常是劳动密集型的，低产量的，耗时的，并且产生形状和尺寸不均匀的球体，这限制了它们的放大应用。微流体技术已经显示出克服球体形成中的一些技术障碍的希望，其通过提供对细胞的精确操纵和受控的物理条件、均匀的尺寸分布以及维持调节对细胞的营养和氧气供应的连续灌注。探索U阱、微流体和微孔以形成球状体。然而，它们的吞吐量不适用于临床规模，并且它们还需要复杂的机制来进行球体回收，并且缺乏流体流动限制了营养物或化学刺激物的质量输送。 目标和方法：我们的长期目标是实现基于表面声波（SAW）的微流体平台，用于开发用于组织工程、细胞治疗和疾病模型的多细胞球体，具体包括：1）实现用于多细胞球体制造的SAW装置; 2）调查和定制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。将开发两个多用途SAW装置模块，用于使用软光刻和使用PDMS的复制模具技术的单细胞和多细胞球体的“生物制造”和“应用”。将研究各种参数对大小、稳定球状体的形成、细胞活力和增殖的影响。SAW器械将用于促进多细胞球体结构中的血管化。将使用逐层方法的保形涂层来诱导结构中的免疫调节特性。SAW将用于共培养多个细胞系，以促进血管化或产生3D生物打印球体作为骨组织工程的构建块。 影响力：该提案为PI的实验室/组织芯片平台研究计划提供了新的研究流，其中9名受训人员将参与多学科研究。它提供声学微流体平台作为制造被认为是伪组织的多细胞球体的成本效益解决方案，以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.