Microscale control of stem cell signaling using cell patterning and perfusion

描述(由申请人提供)：在微尺度上控制细胞-细胞信号已直接导致基础细胞生物学和再生医学的进步。我们的目标是开发在体外调节细胞信号的技术，特别是专注于胚胎干细胞。虽然存在探测细胞间通讯的传统技术，但这些方法提供了对细胞培养微环境的不完全控制。许多新技术，如细胞图案化和微流控培养，已经产生了研究细胞与细胞相互作用的新方法。尽管这些技术取得了成就，但当我们试图将它们应用于干细胞生物学时，限制出现了。特别是，细胞图案化技术通常需要对基质进行图案化，这在研究增殖干细胞时是一个障碍。虽然微流控灌流培养被认为影响自分泌/旁分泌信号，但这一点还没有得到严格的证明，因此微流控灌流在调节可扩散信号方面的全部潜力还没有实现。我们相信，解决这些限制将不仅使研究干细胞信号的新方法成为可能，也将使研究其他细胞系统的信号转导成为可能。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，也是当前和未来疗法的组成部分。为此，我们开发了两种互补的技术来研究这些细胞：一种新的细胞模式方法和一种微流控灌流系统。我们的方法是开发微技术，它可以(1)利用微流控灌注室中的流体流动来调节可扩散信号，以及(2)生物倒装芯片细胞模式，以将祖细胞放置在特定的排列中，调节可扩散和旁分泌信号。我们将利用这些技术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)利用微流体灌流阵列通过控制细胞-细胞扩散信号来调控小鼠胚胎干细胞的自我更新和分化；(2)利用生物芯片研究传代过程中直接和扩散的细胞-细胞相互作用；(3)将这两种技术应用于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解干细胞生物学的奥秘，以便将干细胞用作疗法。在干细胞可以用于治疗疾病之前，我们必须了解如何在培养中繁殖它们，并用它们来创造各种组织。我们的技术为研究干细胞生物学提供了一个新的窗口，这将有助于确定控制它们行为的新方法。

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.