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Specific exosomes as novel media component to enhance manufacturing cell lines.

特定的外泌体作为新的培养基成分来增强细胞系的生产。

基本信息

批准号：
1916135
负责人：
金额：
--
依托单位：
University College London
依托单位国家：
英国
项目类别：
Studentship
财政年份：
2017
资助国家：
英国
起止时间：
2017 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=studentship-1916135
关键词：
Specific exosomes novel media component

项目摘要

Characterisation and quantification of exosomes of manufacturing cell lines with a prefered phenotype (growth / productivity). Analysing the function of those exosomes by applying them in a quantitative manner on cells with contray phenotypes (slow to fast growth, low to high productivity and vice versa).Generating cell lines which stably expresses exosomes loaded with a defined set of RNA/Protein and applying those exosomes to CHO cells to validate exosome triggered effects on phenotype.The standard procedure in cell line development to select cells with the required attributes for manufacturing takes up to 6 months from DNA to clonal cell line. Reducing this period of time and improving product quantity as well as quality will save laboratory costs and help to reduce the overall project timelines and risks of failure. During the last decade an increasing number of publications propose that exosomes are able to influence the characteristics of up taking cells. Considering this the exosomes could be employed to shift the averaged behaviour of cell populations towards preferred characteristics like growth or productivity. To investigate potential effects, exosomes of cell lines with different phenotypes will be characterised, quantified and applied to cell lines with contrary phenotypes. The development of specific exosomes which drive cell phenotypes towards preferred behaviour enables the design of a novel tool box to enhance the cell line development on the molecular level in real time. Furthermore it extends our knowledge how to produce and purify exosomes on manufacturing scale which can have major impact for the exosome usage as a therapeutic format.First, fundamental baseline understanding will be created by isolating from CHO pools cells that are either a) rapid growing or b) slow growing. These suspension-adapted cells will be propagated in shake flasks for 2 weeks and then resulting exosome product concentrated using tangiential flow filtration using techniques already available in the UCL Department of Biochemical Engineering. Exosomes from rapid growing cells will then be added to slow growing cells in culture (and vice versa) to determine whether exosomal signalling can direct cell behaviour. A matrix of experimental conditions that assesses timing, dosage and number of exosome doses will explore the effect strength over time in culture. A second set of experiments assessing CHO cells that have a) high productivity versus b) low productivity will be undertaken, whereby exosome product isolated from high producers will be used as a cell culture supplement for low producing cells (and vice versa). A range of measurements will be used to characterise exosomes, including nanoparticle number, size, concentration, RNA, DNA, protein.Generating CHO cells that stably express reporter exosomes (generated using a commercial kit) where exosomes are loaded with a defined set of RNAs/proteins that elicit known responses will provide validation that exosomes are correctly targeting and regulating cell behaviour. Ambr15 will be used in fed-batch mode to validate the preliminary studies. During the second half of the project, a similar approach to that listed above will be undertaken to assess the effect of cell state-specific exosomes on adherent HEK-293 cells.

具有优选表型（生长/生产率）的生产细胞系的外泌体的表征和定量。通过将这些外泌体以定量方式应用于具有相反表型的细胞来分析这些外泌体的功能（从慢到快的增长，产生稳定表达载有一组确定的RNA/RNA复合物的外来体的细胞系。蛋白质并将这些外泌体应用于CHO细胞以验证外泌体触发的对表型的影响。从DNA到克隆细胞系的生产需要长达6个月的时间。缩短这段时间并提高产品数量和质量将节省实验室成本，并有助于缩短整个项目的时间表和失败风险。在过去的十年中，越来越多的出版物提出外泌体能够影响摄取细胞的特征。考虑到这一点，外泌体可以用于将细胞群体的平均行为转向优选的特征，如生长或生产力。为了研究潜在的影响，将对具有不同表型的细胞系的外泌体进行表征、定量并应用于具有相反表型的细胞系。驱动细胞表型朝向优选行为的特定外泌体的开发使得能够设计新颖的工具箱以在真实的时间中在分子水平上增强细胞系开发。此外，它扩展了我们如何以生产规模生产和纯化外泌体的知识，这对于外泌体作为治疗形式的使用具有重大影响。首先，通过从CHO池中分离a）快速生长或B）缓慢生长的细胞来建立基本的基线理解。这些悬浮适应细胞将在摇瓶中繁殖2周，然后使用UCL生物化学工程系已有的技术，使用有形流过滤浓缩所得外泌体产物。然后将来自快速生长细胞的外泌体添加到培养物中的缓慢生长细胞中（反之亦然），以确定外泌体信号传导是否可以指导细胞行为。评估外泌体剂量的时间、剂量和数量的实验条件矩阵将探索培养物中随时间的效应强度。将进行评估具有a）高生产力对比B）低生产力的CHO细胞的第二组实验，由此从高生产者分离的外来体产物将用作低生产细胞的细胞培养补充物（反之亦然）。将使用一系列测量来确定外泌体，包括纳米颗粒数量、大小、浓度、RNA、DNA、蛋白质。产生稳定表达报告外泌体（使用商业试剂盒产生）的CHO细胞，其中外泌体装载有引发已知反应的一组确定的RNA/蛋白质，将提供外泌体正确靶向和调节细胞行为的验证。Ambr 15将以补料分批模式用于验证初步研究。在项目的后半部分，将采用与上述方法类似的方法来评估细胞状态特异性外泌体对贴壁HEK-293细胞的影响。