Optimisation and scale-up of nanovibrational stimulation to control the differentiation of adult stem cells

纳米振幅振动(称为纳米敲击)先前已被证明可以刺激间充质干细胞(MSCs)的成骨分化。具体地说，1 kHz的振动和30 nm+的振动足以在体外驱动成骨和矿化。骨是仅次于血液的第二大移植组织，由于临床上需要现成的自体骨移植替代材料，目前这些细胞正被开发为一种潜在的骨修复疗法。目前的生物反应器系统每批能够刺激4000-5000万个细胞，但提供未来的临床试验将需要每批数十亿个细胞。这个项目将研究纳米振动是否可以放大，以及这种刺激方法是否可以用来产生其他类型的分化细胞。优化振动参数(频率、幅度、对细胞的作用力)将使这些问题得以探索，并朝着新的临床细胞疗法取得进一步进展。该项目的目的是确定特定MSC分化表型的最佳值，并测试这些条件是否可以规模化提供，以使技术从实验室转移到临床环境。现有的生物反应器专注于1 kHz，30 nm的振动，但这些参数如何与机械传导信号联系在一起，以及是否可以通过改变振动条件(如软骨细胞和心肌细胞)产生其他MSC前体细胞，目前尚不清楚。在本项目中，将开发一种新型的振动生物反应器，以准确测试不同机械载荷下MSCs的分化潜力。对MSCs和相关的机械敏感细胞类型的体外分析将研究频率如何与诱导的细胞张力和硬度有关，同时检查每个MSC谱系的细胞标志物的上调。如果可以产生新的表型，那么最优的振动条件将在新的可伸缩细胞培养系统中应用和测试，如中空纤维生物反应器和细胞堆，以展示治疗细胞制造的潜力。

Nano-amplitude vibration (dubbed 'Nanokicking') has previously been shown to stimulate osteogenic differentiation of mesenchymal stem cells (MSCs). Specifically 1 kHz vibration with 30 nm+ vibration is sufficient to drive osteogenesis and mineralisation in vitro. Bone is the second most transplanted tissue (behind blood) and now these cells are currently being developed as a potential therapy for bone repair due to the clinical need for off-the-shelf replacements for bone autograft. The current bioreactor system capable of stimulating 40-50 million cells per batch but supplying future clinical trials will require billions of cells per batch. This project will examine whether nanovibration can be scaled up and whether this method of stimulation can be used to generate other differentiated cell types. Optimising the vibration parameters (frequency, amplitude, force on cells) will enable these questions to be explored and further progress towards new clinical cell therapies to be made. The aim of the project is to determine optimal values for specific MSC differentiation phenotypes and test if these conditions can be delivered at scale to enable technology transfer from the lab to a clinical environment.The existing bioreactor is focused on 1 kHz, 30 nm vibration but it is not well understood how these parameters tie into mechanotransductive signalling and whether other MSC progenitors could be generated by altering the vibration conditions (e.g. chondrocytes and myocytes). During this project a new vibration bioreactor will be developed to accurately test the differentiation potential of MSCs under varying mechanical loading. In vitro analysis of MSCs, and related mechanosensitive cell types, will investigate how frequency relates to induced cellular tension and stiffness whilst examining the upregulation of cellular markers for each MSC lineage. If new phenotypes can be produced then the optimal vibration conditions will be applied and tested in new scalable cell culture systems such as hollow fibre bioreactors and cell stacks to demonstrate potential for therapeutic cell manufacture.