Cell culture interactomic engineering

细胞疗法现在正在走向成熟，细胞增殖并用于治疗患者的应用程序的数量正在蓬勃发展。要做到这一点，需要新一代生化工程师，能够持续生产大量最高质量的细胞。这些生产过程的挑战之一是，每个直接来自捐赠者或患者本人的原代细胞培养(PCC)都有非常具体的生化需求，这些需求仍然没有全部得到满足。通常，使用部分不确定性质的生物添加剂(BA)，如胎牛血清(FBS)。然而，在安全和产品一致性方面，使用这些BA的潜在风险很高，必须避免。尽管如此，尽管这些添加剂中的大多数强烈的单独作用因子已经被识别出来，并且现在可以获得，但我们仍然缺少BA活性来源的很大一部分。最有可能的是，这些复杂混合物的剩余效应在于次要的、未知的成分之间的协同作用。因此，这项建议旨在确定BA中这些在相互作用中作用于PCC的不明因素，BA/PCC互动。为此，我们将结合一些方法：i)新的分离方法，以足够的数量分离这些混合物中的分子，以测试其效果；ii)实验的统计设计，以在最少的实验中测试尽可能多的协同效应；iii)自动化流体处理站和显微镜，在每个单独的实验中准备最多的测试条件，并在较长的时间内以无损的方式分析它们；iv)细胞图像形态分析算法，基于2D小波变换和多元统计，以监测细胞生长和形态，以确保维持细胞类型的完整性。这些技术的结合将在迭代循环过程中进行，每一轮产生更精简的组分，这将使使用蛋白质组学分析它们成为可能，最终确定对特定细胞系统起协同作用的新化合物组。在第一个项目中，将探讨FBS的相互作用组和蛋白质组对人成肌细胞和角质形成细胞的影响。稍后，将研究其他生物混合物和细胞系统。在这些研究期间积累的知识将被构建在生物相互作用组数据库中。

Cell therapy is now coming of age and the number of applications where cells are multiplied and applied to treat patients is blooming. To do so, a new generation of biochemical engineers, able to consistently produce large number of cells of the highest quality, is needed. One of the challenges of these production processes is that each primary cell cultures (PCC), coming directly from a donor, or the patient himself, has very specific biochemical needs that are still not all met. Often, biological additives (BA) of partially undefined nature, such as foetal bovine serum (FBS) are used. However, the potential risks, both in terms of safety and product consistency, of using these BA are high and must be avoided. Still, despite the fact that most of the strong individually-acting factors in these additives have been identified and are now available, we are still missing a good part of BA activity source. Most probably, the remaining effect of these complex mixtures lies in synergistic interactions between minor, unknown components. This proposal therefore aims at identifying these unidentified factors in BA that act on PCC in interaction with each other, BA/PCC interactomes. To do so, we will combine a number of approaches: i) new fractionation methods to separate the molecules comprised in these mixtures in quantities sufficient to test their effects; ii) statistical design of experiment to test in the least number of experiments the largest possible number of synergies; iii) automated fluid handling stations and microscopes to prepare the largest number of test conditions in each single experiment and analyse them in a non-destructive manner through long time period, and iv) cell image morphology analysis algorithms, based on 2D wavelet transform and multivariate statistics to monitor both cell growth AND morphology to ensure cell type integrity is maintained. The combination of these technologies will be performed in an iterative cyclic process, each round generating fractions that are leaner, which will make it possible to analyse them using proteomics to ultimately identify new groups of new compounds acting synergistically on specific cell systems. In a first project the interactome and proteome of FBS will be probed for its effect on human myoblast and keratinocytes. Later, other biological mixtures and cell systems will be studied. The knowledge accumulated during these studies will be structured in a Biological Interactome Database.