Cell culture interactomic engineering

细胞疗法现在已经成熟，细胞增殖和用于治疗患者的应用数量正在迅速增加。要做到这一点，就需要能够持续生产大量高质量细胞的新一代生化工程师。这些生产过程的挑战之一是，每个原代细胞培养物（PCC），直接来自供体或患者本人，具有非常特定的生化需求，仍然不能全部满足。通常，部分性质不明确的生物添加剂（BA），如胎牛血清（FBS）被使用。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。然而，尽管这些添加剂中的大多数强单独作用因素已经被确定并且现在已经可用，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作用在于次要的未知成分之间的协同相互作用。因此，本提案旨在识别BA中相互作用于PCC的未识别因素，BA/PCC相互作用。为此，我们将结合多种方法：i)新的分馏方法，以分离这些混合物中包含的分子，其数量足以测试其效果；Ii)实验的统计设计，以最少的实验数量检验尽可能多的协同效应；iii)自动化流体处理站和显微镜，在每个单次实验中准备最多的测试条件，并通过长时间的无损方式对其进行分析；iv)基于二维小波变换和多元统计的细胞图像形态学分析算法，以监测细胞生长和形态学，以确保保持细胞类型的完整性。这些技术的组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化细胞的影响。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被组织在一个生物相互作用数据库中。

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.