A novel characterisation and separation technique for pluripotent human embryonic and hematopoeitic stem cells

要实现干细胞及其衍生物在再生医学疗法或药物开发的高通量筛选平台中所承诺的益处，就必须开发纯化细胞的工具。理想情况下，这种工具应该是非侵入性的，具有比当前方法分离几个数量级的能力。纯化治疗性蛋白的工业系统为大规模生物处理提供了先例。迄今为止，绝大多数这些都集中在柱色谱法，以达到所需的纯度水平。然而，细胞的大小，灵敏度和复杂性对下游工程师提出了特殊的挑战，这些挑战不太可能通过传统柱色谱技术的任何进一步发展或修改来解决。因此，需要新的技术。该提案旨在开发一种新颖，简单，可扩展和商业上有用的技术，用于分离/纯化人类干细胞，而不考虑其来源组织。这将通过使用原子力显微镜来定义人类胚胎和成人造血干细胞表面的地形和电荷分布，然后在计算模型中使用这些信息来设计互补表面来实现。然后，表面原型将用于活细胞的小规模实验工作，以证明它们可逆结合和分离细胞的能力以及随后的生存能力。通过关注干细胞表面的电荷和地形，该项目将产生一种基于吸附的分离技术，比目前的色谱技术更适合细胞纯化。成人和胚胎干细胞群体的使用将证明该技术在当前和未来的临床和研究应用中的实用性。

Realising the promised benefits of stem cells and their derivatives in regenerative medical therapies or in high throughput screening platforms for drug development necessitates the development of tools to purify cells to homogeneity. Ideally, such tools should be non-invasive with a capacity for separation several orders of magnitude beyond current methods. Industrial systems for the purification of therapeutic proteins provide precedents for large scale bioprocessing. To date, the vast majority of these have focused on column chromatography to achieve desired levels of purity. However, the size, sensitivity and complexity of cells present particular challenges to the downstream engineer which are unlikely to be solved by any further evolution or modification of traditional column chromatographic techniques. Consequently, novel techniques are needed. This proposal intends to develop a novel, simple, scalable and commercially useful technique for the separation/purification of human stem cells irrespective of their tissue of origin. This will be achieved by using Atomic Force Microscopy to define the topography and electrical charge distribution on the surfaces of human embryonic and adult haematopoietic stem cells followed by the use of this information in computational models to design complimentary surfaces. Surface prototypes will then be used in small-scale experimental work with living cells to demonstrate their ability to reversibly bind and separate cells and their subsequent viability. By focusing on both charge and topography of stem cell-surfaces the project will produce an adsorption-based separation technology more specifically suited for cell purification than current chromatographic techniques. The use of both adult and embryonic stem cell populations will exemplify the utility of this technology to both current and future clinical and research applications.