High throughput cell reprogramming by microfluidic jet injection

描述（由申请人提供）：最近出现的细胞重编程作为一种从体细胞产生诱导多能干细胞（iPS）的方法，由于它有可能绕过围绕传统胚胎干细胞的免疫学和伦理问题，在生物学和医学界引起了极大的兴奋。为了证明这项技术作为一种治疗和/或实验工具是有用的，人们必须首先开发一种能够以快速、有效和可重复的方式重新编程细胞的技术。最重要的是，人们必须找到目前用于这一过程的潜在致癌逆转录病毒的替代品。在本提案中，我们请求资金以进一步开发当前的，概念验证的细胞注射原型，并将其用作进行重编程研究的使能工具。该装置能够以高通量、高效的方式在细胞膜上输送皮升的材料。微流体系统利用微米级喷嘴将液体射流注入通过通道的细胞中。这种射流穿透细胞膜，不会导致细胞裂解，并且能够传递重编程体细胞所需的因子。通过进一步开发该设备，我们希望利用该系统以与病毒转染相当的速度和效率生产iPS细胞，同时避免诱变和毒性问题。基于微注射器的细胞重编程的有效性将使用已发表的检查iPS细胞的方法进行彻底测试。该系统产生的组织培养物也将用于动物研究，以验证重编程细胞的多能性。最终，我们将把该装置与我们的新型聚合物输送机制结合使用，作为进一步提高性能的手段。此外，该设备的定量、高通量特性将使我们能够对重编程过程本身的生物学方面进行研究。更具体地说，我们可以确定基因和因素的最佳组合将导致最大的重编程效率，以及单个基因在整个过程中的具体作用。由于传递系统的物理性质，我们的设备还可以探索使用蛋白质作为在iPS细胞生产中增强重编程（甚至取代DNA）的手段。在过去的几年里，胚胎干细胞已经彻底改变了再生医学领域；它们在制造整个器官和治疗多发性硬化症（MS）等遗传疾病方面的潜力，使它们成为研究人员的最爱。本文描述的细胞注射装置将具有通过对患者现有的成体细胞进行重编程，以快速、安全和有效的方式产生干细胞的能力。因此，该系统不仅可以规避围绕胚胎干细胞的伦理和技术问题，还可以避免与现有基于病毒的重编程技术相关的许多毒性和癌症风险。

DESCRIPTION (provided by applicant): This application addresses broad challenge area (14) Stem Cells and the specific challenge topic 14-EB-101 Synthetic Delivery Systems for Generating Pluripotent Stem Cells The recent advent of cell reprogramming as a means of producing induced pluripotent stem (iPS) cells from somatic cells has produced great excitement in the biological and medical communities due to its potential to circumvent the immunological and ethical issues surrounding traditional embryonic stem cells. For this technology to prove useful as a therapeutic and/or experimental tool one must first develop a technique that can reprogram cells in a rapid, efficient and repeatable manner. Most importantly, one must find alternatives to the potentially cancer-causing retroviruses that are currently used for this process. In this proposal, we request funding to further develop a current, proof of concept, cell injection prototype and to utilize it as an enabling tool to conduct reprogramming studies. The device is capable of delivering pico liters of materials across the cell membrane in a high throughput, efficient manner. The microfluidic system utilizes a micron-scale nozzle to inject a jet of liquid into cells passing through a channel. This jet pierces the cell membrane, without causing cell lysis, and is capable of delivering the factors necessary to reprogram somatic cells. By further developing this device, we hope to use the system to produce iPS cells at a rate and efficiency comparable to viral transfection, while avoiding the issues of mutagenesis and toxicity. The efficacy of micro-injector based cell reprogramming will be thoroughly tested using published methods of inspecting iPS cells. Tissue cultures produced by the system will also be used in animal studies for verification of pluripotency in reprogrammed cells. Eventually we will use the device in tandem with our novel polymeric delivery mechanism as a means of further enhancing performance. In addition, the quantitative, high throughput nature of the device will allow us to conduct studies on the biological aspects of the reprogramming process itself. More specifically, we can determine what optimal combination of genes and factors will result in maximum reprogramming efficiency as well as the specific role of individual genes in the overall process. Due to the physical nature of the delivery system, our device can also explore the use of proteins as a means of enhancing reprogramming (or even replacing DNA) in the production of iPS cells. Over the past years, embryonic stem cells have revolutionized the field of regenerative medicine; their potential for producing entire organs and tackling genetic disorders, such as Multiple Sclerosis (MS), has made them a favorite among researchers. The cell injection device described herein will have the capability of producing stem cells in a rapid, safe and efficient manner by reprogramming a patient's existing adult cells. The system can thus not only circumvent the ethical and technical issues that surround embryonic stem cells, but also avoids many of the toxicity and cancer risks associated with existing virus-based reprogramming techniques.