Development of nanopatterned substrates for the delivery of high quality stem cells

干细胞在再生医学中的应用具有巨大的潜力，随着人口日益老龄化，我们需要寻找新的机会。它们的潜在用途从骨科应用（如关节炎和骨质疏松症）到神经退行性疾病（如帕金森氏症和阿尔茨海默氏症），仅举几例。人体有一个恒定的干细胞来源，位于体内的壁龛。从科学和临床的角度来看，成人干细胞最受欢迎的来源之一是骨髓。骨髓相对容易获取，干细胞可以很容易地从提取的细胞群中分离出来。然而，直到最近，一个主要的障碍是干细胞不能在培养中长时间培养并保持其再生潜力（多能性）。干细胞的潜力在于，它们可以变成许多不同的细胞类型，因此可以根据需要帮助修复损伤，这意味着它们的特征（表型）在培养中是不稳定的。因此，当我们培养干细胞时，它们很快就会失去我们想要开发的潜力和效力。我们最近（2011年）证明，通过在独特的纳米图案表面上培养细胞，（纳米孔，直径为100 nm，深度为100 nm，这些纳米图案化的表面类似于蓝光上的轨迹，射线盘和实际上我们的技术非常类似于光学介质的生产，其中纳米粒子可以以非常低的成本大量注塑成聚合物盘。我们可以改变纳米粒子的排列，从而调整干细胞对生长的反应，而不会发生轮廓（表型）漂移，并将所需的变化靶向我们想要的组织（称为分化）。这对需要特定细胞变化（分化）的植入物（如髋关节置换植入物）的设计具有重要意义。这方面的一个例子是，骨科植入物，其中细胞分化成骨是理想的，或者一个可能更有潜力的领域是大量干细胞的生长。因此，一个关键的研究目标是获取患者的干细胞，在实验室中将它们培养到有用的数量，然后将它们放回患者体内以激发再生。我们的技术规模的扩大将允许这一点。到目前为止，我们使用的技术只允许我们探索干细胞相互作用的非常有限的不同几何形状（<10）。在这项提案中，我们将开发一个新的平台，其中单个样本将包含1000种不同的模式，从而使我们能够研究更大的纳米粒子库及其影响干细胞命运的能力。从这些文库中，我们将识别新的模式，并使用质谱法对其进行更详细的研究，以识别影响细胞命运的小分子。重要的是，要看到这些发现的真实的好处，至关重要的是，我们能够将用于再生医学或制药的干细胞生长和扩增的材料扩展到大面积。如上所述，我们的技术与DVD和蓝光的生产非常相似，这意味着它有助于纳米图案化表面的成本效益大规模生产。为了证明这种潜力，我们将把提取的骨髓干细胞扩增到500万个细胞，这是2008年证明的用于完全组织工程气管的细胞数量。

The use of stem cells in regenerative medicine holds great potential and with an increasingly aging population, we need to look for new opportunities. Their potential use span from orthopaedic applications such as arthritis and osteoporosis to neurodegenerative disorders such as Parkinson's and Alzheimer's, to name a few. The body has a constant source of stem cells located in niches within the body. From a scientific and clinical point of view, one of the most exploited sources for adult stem cells is the bone marrow. The bone marrow is relatively easy to access and stem cells can be easily isolated from the extracted cell population.However, until recently, a major hurdle is that the stem cells cannot be cultured for extended periods in culture and maintain their regenerative potential (multipotent). The very potential of stem cells, that they can change into many different cell types and so help repair damage on demand, means that their profile (phenotype) is unstable in culture. Hence, as we culture stem cells they soon lose the very potential and potency we want to exploit. We have recently (2011) demonstrated that by culturing the cells on a uniquely nanopatterned surface (nanopits, 100 nm in diameter and 100 nm deep, arranged in a square lattice) it is indeed possible to keep the cells in the multipotent state in prolonged culture as well as expand the number of cells.These nanopatterned surfaces resemble the tracks on a Blu-Ray disc and indeed our technology is very similar to the production of optical media where nanopatterns can be injection moulded into polymer discs in high volumes and at a very low cost. We can change the arrangement of the nanopatterns, thereby tuning the stem cell response to growth without profile (phenotype) drift and to target desired changes to tissues we want (known as differentiation). This is has important implications on the design of implants (like a hip replacement implants) where a specific cell changes (differentiation) is desirable. An example of this is again, orthopaedic implants where differentiation of cells to bone is desirable, or an area with perhaps even more potential is the growth of large numbers of stem cells. Thus, a key research goal is to take a patient's stem cells, grow them in the laboratory to useful numbers, and then place them back into the patient to spark regeneration. Scale up of our technology will allow this. The technology we have used so far has only allowed us to explore the stem cell interaction to a very limited number of different geometries (<10). In this proposal we will develop a new platform where a single sample will contain 1000 different patterns thereby allowing us to investigate a much larger library of nanopatterns and their ability to influence the fate of the stem cells. From these libraries new patterns will be identified and we will investigate them in more detail using mass spectrometry to identify small molecules influencing the cell fate.Importantly, to see real benefit of these discoveries, it is vital that we are able to scale the materials used to large areas to sustain the growth and expansion of stem cells used for regenerative medicine or pharma. As described above, our technology is very similar to the production of DVDs and Blu-Rays, which means that it lends itself to a cost effective mass production of the nanopatterned surfaces. To demonstrate this potential, we will expand extracted bone marrow stem cells to 5 million cells, the number of cells used for the fully tissue engineered trachea demonstrated in 2008.