Microscale control of stem cell signaling using cell patterning and perfusion

描述（由申请人提供）：在微观尺度上控制细胞-细胞信号传导直接导致了基础细胞生物学和再生医学的进步。我们的目标是开发体外调节细胞信号的技术，特别是专注于胚胎干细胞。虽然存在用于探测细胞间通信的常规技术，但这些方法提供了对细胞培养微环境的不完全控制。一系列新技术，如细胞图案化和微流体培养，为研究细胞-细胞相互作用提供了新的方法。尽管这些技术取得了成就，但当我们试图将其应用于干细胞生物学时，局限性就会出现。特别地，细胞图案化技术通常需要图案化基底，这在研究增殖干细胞时是一个障碍。虽然微流体灌注培养已经被假定影响自分泌/旁分泌信号传导，但这尚未得到严格证明，因此微流体灌注用于调节扩散信号传导的全部潜力尚未实现。我们相信，解决这些限制将使新的方法，不仅研究干细胞信号，但在其他细胞系统的信号转导以及。我们对干细胞特别感兴趣，因为它们是发育和疾病过程的强大模型，也是当前和未来治疗方法的组成部分。为此，我们开发了两种互补技术来研究这些细胞：一种新的细胞模式和微流体灌注系统。我们的方法是开发微技术，可以（1）在微流体灌注室中使用流体流动来调节扩散信号，以及（2）生物倒装芯片细胞图案化以将祖细胞放置在定义的排列中，调节扩散和非细胞信号。我们将利用这些技术来研究小鼠胚胎干细胞的自我更新和向神经元的分化，以及人类胚胎干细胞的自我更新。我们的具体目标是（1）使用微流控灌注阵列来调节小鼠胚胎干细胞的自我更新和分化，通过控制细胞-细胞扩散信号，（2）研究直接和扩散的细胞-细胞之间的相互作用，在小鼠胚胎干细胞的自我更新传代过程中使用生物倒装芯片，和（3）应用这两种技术来研究人类胚胎干细胞的自我更新。该项目与人类健康的相关性在于破译干细胞生物学的奥秘，以便将干细胞用作治疗药物。在干细胞被用于治疗疾病之前，我们必须了解如何在培养中繁殖它们，并利用它们来创造各种组织。我们的技术为干细胞生物学提供了一个新的窗口，这将有助于确定控制其行为的新方法。

DESCRIPTION (provided by applicant): Controlling cell-cell signaling at the microscale has led directly to advances in fundamental cell biology and re-generative medicine. Our goal is to develop technology to modulate cell signaling in vitro, specifically focusing on embryonic stem cells. While conventional techniques exist for probing intercellular communication, these methods provide incomplete control over the cell-culture microenvironment. A host of new technologies, such as cell patterning and microfluidic culture, have resulted in new ways to study cell- cell interactions. Despite the achievements of these technologies, limitations arise as we try to apply them to stem cell biology. In particular, cell-patterning techniques typically require patterning the substrate, which is a hindrance when studying proliferating stem cells. While microfluidic perfusion culture has been postulated to affect autocrine/paracrine signaling, this has not been rigorously demonstrated, and thus the full potential of microfluidic perfusion for modulating diffusible signaling has not been realized. We believe that addressing these limitations will enable new ways of studying not only stem cell signaling, but signal transduction in other cell systems as well. We are particularly interested in stem cells because they are powerful models for developmental and disease processes, as well as components for current and future therapeutics. To this end, we have developed two complementary technologies to study these cells: a new way to pattern cells and a microfluidic perfusion system. Our approach is to develop microtechnology that can (1) uses fluid flow in microfluidic perfusion chambers to modulate diffusible signaling, and (2) bio-flipchip cell patterning to place progenitor cells in defined arrangements, modulating diffusible and juxtacrine signaling. We will use these technologies to study mouse embryonic stem cell self-renewal and differentiation to neurons, and human embryonic stem cell self- renewal. Our specific aims are to (1) use microfluidic perfusion arrays to modulate mouse embryonic stem cell self-renewal and differentiation by controlling cell-cell diffusible signaling, (2) study direct and diffusible cell-cell interactions during passaging in mouse embryonic stem cell self-renewal using bio-flipchips, and (3) apply both technologies to studying human embryonic stem cell self-renewal. ;; The relevance of this project to human health is in deciphering the mysteries of stem cell biology in order to use stem cells as therapeutics. Before stem cells can be used to treat disease, we must understand how to propogate them in culture and use them to create various tissues. Our technology provides a new window into stem cell biology that will help determine new ways to control their behavior.