Engineering scalable immobilized cell culture systems for diabetes cellular therapy

干细胞研究和组织工程的进展已经导致复杂的三维（3D）组织构建的发展，其对于疾病建模以及组织替代具有巨大的希望。该研究计划的长期目标是开发可扩展的治疗性细胞生产和移植系统，特别是用于糖尿病细胞治疗。本项目的5年目标是优化水凝胶固定化培养胰岛样细胞团的方法。该项目的具体目标是：（1）通过微通道乳化微囊化ILC，（2）生成含血管化ILC的构建体，（3）基于氧传质模型优化血管几何形状，（4）研究氧张力和血管细胞对ILC分化的影响。首先，在我的实验室中开发的高度可扩展的微通道乳化过程将适于更好地控制微珠在酸化油中的停留时间。在评估细胞存活和功能后，微囊化ILC将在血管化cm尺度组织构建体中培养作为潜在的大囊化系统。这些血管通道将通过将牺牲材料嵌入含ILC的水凝胶支持基质中来产生。为此，将一种短效生物墨水3D打印到支持基质材料中，该材料将根据其血管生成潜力、机械性能和打印保真度进行选择。在支撑基质胶凝化之后，易消失的油墨将被抽空以产生中空通道。在ILC存活和功能评估之后，将所得组织构建体在灌注下培养数周以允许ILC成熟。为了优化血管网格几何形状，将开发预测不同配置结构中氧张力分布的数值模型。模型预测将进行比较，使用顺磁性氧敏感探头确定的实验氧浓度分布。这些研究将在向血管生成支持基质中加入或不加入血管内皮细胞的情况下进行，以研究它们与ILC的相互作用。这些研究将为设计用于体外药物测试以及最终移植的血管化生物人工胰腺铺平道路。该研究项目为培养高素质人才提供了肥沃的土壤，其中包括3名博士，1名硕士和5名本科生。这项研究计划预计将对生物医学界和加拿大人的健康产生深远的影响。研究结果的转化将通过与行业和临床医生的现有合作来加速。

Advances in stem cell research and tissue engineering have led to the development of complex three-dimensional (3D) tissue constructs that hold great promise for disease modelling as well as tissue replacement. The long-term goal of this research program is to develop scalable therapeutic cell production and transplantation systems, in particular for diabetes cellular therapy. The 5-year goal of this program is to optimize methods for hydrogel immobilized culture of islet-like cell clusters (ILCs). The specific aims of the 5-year program are to (1) microencapsulate ILCs by microchannel emulsification, (2) generate vascularized ILC-containing constructs, (3) optimize vessel geometries based on oxygen mass transfer models and (4) study the effects of oxygen tension and vascular cells on ILC differentiation. First, a highly scalable microchannel emulsification process developed in my laboratory will be adapted to better control bead residence time in acidified oil. After assessing cell survival and function, the microencapsulated ILCs will be cultured in vascularized cm scale tissue constructs as a potential macroencapsulation system. These vascular channels will be generated through embedded writing of sacrificial materials into ILC-containing hydrogel support matrices. To do so, a fugitive bioink will be 3D printed into the support matrix material, which will be selected based on its vasculogenic potential, mechanical properties and print fidelity. After gelation of the support matrix, the fugitive ink will be evacuated to create hollow channels. Following ILC survival and functional assessment, the resulting tissue constructs will be cultured under perfusion for several weeks to allow ILC maturation. To optimize vascular lattice geometries, a numerical model predicting oxygen tension profiles in constructs of different configuration will be developed. Model predictions will be compared to experimental oxygen concentration profiles determined using a paramagnetic oxygen-sensitive probe. These studies will be conducted with or without adding vascular endothelial cells into the vasculogenic support matrix to study their interactions with the ILCs. These studies will pave the way towards engineering a vascularized bioartificial pancreas used for in vitro drug testing as well as eventual transplantation. The proposed research program provides fertile grounds for training highly qualified personnel, which includes 3 PhDs, 1 Master's and 5 undergraduate trainees. This research program is expected to have far-reaching impact on the biomedical community and the health of Canadians. Translation of the research findings will be accelerated by existing collaborations with industry and clinicians.