Cell culture interactomic engineering

细胞疗法现在已经成熟，细胞倍增并应用于治疗患者的应用数量正在蓬勃发展。要做到这一点，需要新一代的生化工程师，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，直接来自供体或患者本人的每种原代细胞培养物（PCC）具有非常特定的生化需求，这些需求尚未全部得到满足。通常，使用部分不确定性质的生物添加剂（BA），例如胎牛血清（FBS）。然而，在安全性和产品一致性方面，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单独作用因子已经被鉴定并且现在可用，但我们仍然缺少BA活性源的很大一部分。最有可能的是，这些复杂混合物的剩余效果在于次要的未知组分之间的协同作用。因此，该建议旨在确定BA中这些未识别的因子，它们相互作用于PCC，即BA/PCC相互作用组。为此，我们将结合联合收割机的一些方法：i）新的分馏方法，以分离这些混合物中包含的分子，其数量足以测试它们的效果; ii）实验的统计设计，以在最少的实验中测试最大可能数量的协同作用; iii）自动化流体处理站和显微镜，以在每个单一实验中准备最大数量的测试条件，并在非iv）细胞图像形态分析算法，基于2D小波变换和多元统计来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一个迭代的循环过程中进行，每一轮产生更精简的部分，这将使得有可能使用蛋白质组学来分析它们，以最终确定对特定细胞系统协同作用的新化合物的新组。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。在这些研究中积累的知识将在生物相互作用组数据库中构建。

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.