Microfluidic-based high-efficiency cell fusion for studying nuclear reprogramming

描述(申请人提供)：我们建议开发一种新的微流控技术，高效地创建1,000‘S胚胎干细胞和体细胞之间的融合事件，以研究核重新编程中的早期事件。我们的设备使用三步加载程序和特殊的几何结构来高效地并排排列细胞，以创建大量正确配对的杂交细胞。我们正在开发这项技术来研究核重新编程，这是一个过程，通过重新启动体细胞的核程序，使该细胞回到更原始、更多能的状态。多能或全能细胞内的因子可以重新编程体细胞，目前的方法集中在将这些因子从一个细胞转移到另一个细胞的方法上。重新编程核移植的标准方法是将体细胞核移植到未受精卵中，这是一种繁琐的方法，而且仅限于少量细胞。细胞融合，即将体细胞与胚胎干细胞融合，合并细胞内容，也可以用于重新编程体细胞的核。与核移植相比，细胞融合具有更容易和更具可扩展性的潜力。阻碍这种方法广泛用于研究重编程的一个重要技术限制是，目前的细胞融合方法使用随机配对的细胞。这导致了少量需要的杂交融合(干细胞+体细胞)，在大量不需要的融合和未融合的细胞的背景下。为了从这种背景中提纯所需的融合，研究人员采取了抗生素选择步骤，使过程增加了几天，并阻止研究人员研究核重新编程中的早期事件。通过开发一种高效配对细胞的设备，我们正在解决创造杂交细胞的主要技术挑战，并相信我们的设备将对研究融合诱导的重新编程产生重大影响。

DESCRIPTION (provided by applicant): We propose to develop a new microfluidic technology to efficiently create 1,000's of fusion events between embryonic stem cells and somatic cells in order to study early events in nuclear reprogramming. Our device uses a three-step loading procedure and a special geometry to efficiently arrange cells side-by-side to create a large fraction of properly paired hybrid cells. We are developing this technology to study nuclear reprogramming, which is the process whereby a somatic cell's nuclear program is rebooted to bring that cell back into a more primitive, pluripotent state. Factors within pluripotent or totipotent cells can reprogram somatic cells, and the current approaches center on methods of transferring these factors from one cell to the other. The standard approach to reprogramming nuclear transfer, in which the somatic cell nucleus is transplanted into an unfertilized egg is tedious and limited to small numbers of cells. Cell fusion, whereby a somatic cell is fused to an embryonic stem cell, merging the cellular contents, can also be used to reprogram the somatic cell's nucleus. Cell fusion has the potential to be easier and more scalable than nuclear transfer. One significant technical limitation that prevents the widespread use of this approach for studying reprogramming is that current approaches to cell fusion use randomly paired cells. This results in a small number of desired hybrid fusions (stem cell + somatic cell) in a large background of unwanted fusions and unfused cells. In order to purify the desired fusions from this background, researchers undertake antibiotic selection steps that add days to the procedure and prevent researchers from studying early events in nuclear reprogramming. By developing a device to efficiently pair cells, we are addressing the dominant technological challenge in the creation of hybrids, and believe that our device will have significant impact for studying fusion-induced reprogramming.