Integrated cell culture bioengineering

哺乳动物细胞培养在生产重组蛋白、疫苗、病毒载体以及干细胞和组织等细胞方面具有巨大的实用价值和潜力。这些细胞的培养过程非常复杂，因为它们的培养液通常由40多种昂贵的成分组成，而它们的细胞内部反应网络由数千个信号和代谢步骤组成，通常取决于细胞类型和时间。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)，一种未知的生物来源的混合物，以及一层饲养层细胞才能正常生长。尽管已经提出了许多补充剂来取代胎牛血清和饲养层，但它们往往不允许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)采用统计试验设计(DOE)方法对潜在因素组合的个体效应和协同效应进行了评价。2)发展了序贯FBS分级法，与DOE相结合，可以发现全新的活性因子及其组合。3)对(2)中得到的FBS组分进行了质谱分析，以确定支持这一活性的分子。4)自动显微镜和图像分析可以快速评估细胞对测试因素的反应，从而可以通过作为细胞状态的清晰指示器的形态参数来评估细胞生长，最有趣的是，细胞形状。5)正在设计流体处理站，以满足我们研究的特定需求。最后一种方法是使用最新技术建立数学模型的基因组规模的新陈代谢模型。基因组数据库和通过大规模线性优化估计的每个网络通量速率将与我们的实验平台相结合，形成完整的哺乳动物细胞培养过程的高通量评估体系。这项研究计划建议对这一创新的实验/理论平台进行开发、整合和改进，并将其应用于角质形成细胞的培养，用于治疗严重烧伤、糖尿病足部溃疡和口腔粘膜缺损。

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.