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，随着人口老龄化的加剧，我们需要寻找新的机会。它们的潜在应用范围从关节炎和骨质疏松症等骨科应用到帕金森病和阿尔茨海默病等神经退行性疾病，仅举几例。人体有一个稳定的干细胞来源，这些干细胞位于体内的壁龛中。从科学和临床的角度来看，成人干细胞最容易开发的来源之一是骨髓。骨髓相对容易接近，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，它们可以转变成许多不同的细胞类型，从而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利用的潜力和效力。我们最近（2011）证明，通过在一个独特的纳米图案表面（纳米，直径100纳米，深度100纳米，排列在方形晶格中）培养细胞，确实有可能在长时间培养中保持细胞处于多能状态，并扩大细胞数量。这些纳米图案的表面类似于蓝光光盘上的轨迹，事实上，我们的技术与光学介质的生产非常相似，在光学介质中，纳米图案可以以非常低的成本大量注射到聚合物光盘中。我们可以改变纳米模式的排列，从而调整干细胞对生长的反应，使其不发生谱（表型）漂移，并针对我们想要的组织进行所需的改变（称为分化）。这对于需要特定细胞变化（分化）的植入物（如髋关节置换术植入物）的设计具有重要意义。这方面的一个例子是，骨科植入物，其中细胞分化成骨骼是可取的，或者一个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。我们的技术规模将允许这一点。到目前为止，我们所使用的技术只允许我们探索非常有限数量的不同几何形状的干细胞相互作用（<10）。在这个提议中，我们将开发一个新的平台，其中一个样品将包含1000种不同的模式，从而使我们能够研究一个更大的纳米模式库及其影响干细胞命运的能力。从这些文库中将发现新的模式，我们将使用质谱法更详细地研究它们，以确定影响细胞命运的小分子。重要的是，要看到这些发现的真正好处，至关重要的是，我们能够大规模地使用材料，以维持用于再生医学或制药的干细胞的生长和扩张。如上所述，我们的技术与dvd和蓝光的生产非常相似，这意味着它可以使纳米图案表面的大规模生产具有成本效益。为了证明这种潜力，我们将把提取的骨髓干细胞扩大到500万个细胞，这是2008年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.