Integrated cell culture bioengineering

哺乳动物细胞培养在生产重组蛋白、疫苗、病毒载体和干细胞和组织等细胞方面具有巨大的效用和潜力。这些细胞的培养过程非常复杂，因为它们的培养基通常由40多种昂贵的成分组成，它们的细胞内部反应网络由数千个信号传导和代谢步骤组成，通常依赖于细胞类型和时间。许多原代培养，如造血细胞、肌肉细胞和皮肤细胞，仍然需要胎牛血清（FBS），一种未定义的生物来源混合物，以及一层喂养细胞才能正常生长。虽然已经提出了许多补充剂来取代FBS和饲料层，但它们通常不能产生具有治疗功能的细胞。我们的实验室专注于整合一套方法来研究和改善哺乳动物细胞的培养条件。1)采用实验统计设计（DOE）对潜在因素面板的个体效应和协同效应进行评价。2)开发了顺序FBS分馏，与DOE相结合，可以发现全新的活性因子及其组合。3)用质谱法分析(2)中获得的FBS馏分，以确定支持该活性的分子。4)自动化显微镜和图像分析快速评估细胞对被测因子的反应，允许评估细胞生长，最有趣的是，通过形态计量参数评估细胞形状，这是细胞状态的明确指标。流体处理站的设计是为了满足我们研究的具体需要。最后一种方法，6)基因组尺度代谢建模，利用最新技术建立数学模型。基因组数据库和各网络通量的大规模线性优化估计将与我们的实验平台相结合，形成一个完整的哺乳动物细胞培养过程的高通量评估系统。本研究计划拟对该创新实验/理论平台进行发展、整合和完善，并将其应用于严重烧伤、糖尿病足溃疡和口腔黏膜缺损的角化细胞培养。

Mammalian cell culture has enormous utility and potential for the production of recombinant proteins, vaccines, viral vectors and cells as such, like stem cells and tissues. Culture processes for these cells are very complex since their culture media is often made of more than 40 costly components, and their cell internal reaction network is composed of thousands of signalling and metabolic steps, often cell-type and time dependent. Many primary cultures, such as haematopoietic, muscle and skin cells, still need foetal bovine serum (FBS), an undefined mixture of biological source, as well as a layer of feeder cells to grow normally. Although many supplements have been proposed to replace FBS and feeder layers, they often don't allow the production of cells that are functional for therapy. Our lab specializes in integrating a set of approaches to study and improve culture conditions for mammalian cells. 1) Statistical design of experiments (DOE) is applied to evaluate the individual and synergistic effects of panels of potential factors. 2) Sequential FBS fractionation is developed which, combined to DOE, allows to discover entirely new active factors and combination thereof. 3) FBS fractions obtained in (2) are analysed by mass spectroscopy to identify molecules supporting this activity. 4) Automated microscopy and image analysis rapidly evaluate cell response to the tested factor, allowing the evaluation of cell growth, and most interestingly, cell shape through morphometric parameters which are clear indicators of the cell state. 5) Fluid handling station is being designed to address the specific needs of our research. A last approach, 6) Genome-scale metabolic modelling using the latest technologies to build mathematical models. Genomic databases and estimation of each network flux rate by large-scale linear optimization will be combined with our experimental platform to form a complete high-throughput evaluation system for mammalian cell culture processes. This research program proposes pursuing the development, integration and improvement of this innovative experimental/theoretical platform and applying it to the culture of keratinocytes used for the treatment of severe burn, diabetic foot ulcers and oral mucosa defects.