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体技术，以有效地在胚胎干细胞和体细胞之间产生1000个融合事件，以研究核重编程的早期事件。我们的设备使用三步加载程序和特殊的几何形状来有效地排列细胞并排，以创建大部分正确配对的杂交细胞。我们正在开发这项技术来研究核重编程，这是一个过程，一个体细胞的核程序被重新启动，使细胞回到一个更原始的，多能的状态。多能或全能细胞内的因子可以对体细胞进行重编程，目前的研究主要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。重编程核移植的标准方法是将体细胞细胞核移植到未受精卵中，这种方法繁琐且仅限于少数细胞。细胞融合，即体细胞与胚胎干细胞融合，合并细胞内容物，也可以用来重新编程体细胞的细胞核。细胞融合有可能比核移植更容易和更可扩展。阻碍这种方法广泛应用于重编程研究的一个重要技术限制是，当前的细胞融合方法使用随机配对的细胞。这导致在大量不需要的融合和未融合细胞背景中产生少量所需的杂交融合（干细胞+体细胞）。为了从这种背景中纯化所需的融合体，研究人员进行了抗生素选择步骤，这增加了几天的时间，并阻止了研究人员研究核重编程的早期事件。通过开发一种有效配对细胞的设备，我们正在解决创造杂交的主要技术挑战，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.