Department of Biochemical Engineering, University College London, London, United Kingdom.
PLoS One. 2012;7(12):e52246. doi: 10.1371/journal.pone.0052246. Epub 2012 Dec 19.
The capacity of milli and micro litre bioreactors to accelerate process development has been successfully demonstrated in traditional biotechnology. However, for regenerative medicine present smaller scale culture methods cannot cope with the wide range of processing variables that need to be evaluated. Existing microfabricated culture devices, which could test different culture variables with a minimum amount of resources (e.g. expensive culture medium), are typically not designed with process development in mind. We present a novel, autoclavable, and microfabricated scale-down device designed for regenerative medicine process development. The microfabricated device contains a re-sealable culture chamber that facilitates use of standard culture protocols, creating a link with traditional small-scale culture devices for validation and scale-up studies. Further, the modular design can easily accommodate investigation of different culture substrate/extra-cellular matrix combinations. Inactivated mouse embryonic fibroblasts (iMEF) and human embryonic stem cell (hESC) colonies were successfully seeded on gelatine-coated tissue culture polystyrene (TC-PS) using standard static seeding protocols. The microfluidic chip included in the device offers precise and accurate control over the culture medium flow rate and resulting shear stresses in the device. Cells were cultured for two days with media perfused at 300 µl.h(-1) resulting in a modelled shear stress of 1.1×10(-4) Pa. Following perfusion, hESC colonies stained positively for different pluripotency markers and retained an undifferentiated morphology. An image processing algorithm was developed which permits quantification of co-cultured colony-forming cells from phase contrast microscope images. hESC colony sizes were quantified against the background of the feeder cells (iMEF) in less than 45 seconds for high-resolution images, which will permit real-time monitoring of culture progress in future experiments. The presented device is a first step to harness the advantages of microfluidics for regenerative medicine process development.
毫微升生物反应器在传统生物技术中已成功地展示了其加速工艺开发的能力。然而，对于再生医学来说，目前较小规模的培养方法无法应对需要评估的广泛处理变量。现有的微制造培养设备虽然可以用最小量的资源（例如昂贵的培养基）来测试不同的培养变量，但它们通常不是为工艺开发而设计的。我们提出了一种新颖的、可高压灭菌的、微制造的缩小规模设备，用于再生医学的工艺开发。该微制造设备包含一个可重新密封的培养室，方便使用标准培养方案，与传统的小规模培养设备建立联系，以进行验证和放大研究。此外，该模块化设计可以轻松适应不同的培养基质/细胞外基质组合的研究。使用标准的静态接种方案，成功地在涂有明胶的组织培养聚苯乙烯（TC-PS）上接种了灭活的小鼠胚胎成纤维细胞（iMEF）和人胚胎干细胞（hESC）集落。该设备中包含的微流控芯片可以精确且准确地控制培养基流速和设备中的剪切应力。细胞在 300 µl.h(-1) 的灌流条件下培养两天，导致模型中的剪切应力为 1.1×10(-4) Pa。灌流后，hESC 集落对不同多能性标志物呈阳性染色，并保持未分化的形态。开发了一种图像处理算法，该算法可以从相差显微镜图像中定量共培养的集落形成细胞。使用高分辨率图像，不到 45 秒即可定量分析 hESC 集落的大小，该算法将允许在未来的实验中实时监测培养进展。所提出的设备是利用微流控技术用于再生医学工艺开发的第一步。
