Long-term, self-maintaining primary 3D cultures of mouse and human epithelial tissues for reduction and replacement of animals in research

该项目旨在传播和帮助应用最先进的体外3D系统，该系统是我在MRC-CU和Welcome Sanger研究所进行博士后研究期间开发的，并在我在gordon研究所/剑桥大学的实验室进行了优化，该系统允许直接从小鼠和人体组织中获得的原代和肿瘤上皮细胞的3D培养的生产，长期维护和扩展。避免任何先前的单细胞悬浮液来建立培养的步骤。它形成类组织结构的能力，以一种独特而简单的技术，结合了扩增的潜力，可扩展性和低成本（类似于任何细胞系培养的成本），以及生成模仿组织结构和类似生理细胞行为的复杂3D模型，使该系统对许多从事上皮组织或使用小鼠和人类原代细胞进行研究项目的实验室非常有用。它也引起了组织再生和细胞治疗研究人员的极大兴趣，因为它以高可靠性和高效率提供了几乎无限数量的原代上皮细胞，准备用于他们的实验研究项目。该系统可以培养和扩增来自多种上皮和内皮组织（食道、皮肤、膀胱、舌头、尿道、乳房、子宫、口腔粘膜等）的小鼠和人原代上皮细胞，并可以进行细胞反应、正常细胞和肿瘤细胞之间的细胞竞争机制、基因表达和蛋白质相互作用分析、癌症药物筛选和长期上皮-免疫细胞共培养试验等研究，具有很高的可靠性。与目前使用的其他方法相比，效率高，成本低。此外，该系统还可以作为祖细胞的可扩展资源，用于不同的目的（例如，比使用依赖于组织内细胞数量减少的传统方案产生更高产量的类器官，使用原代细胞的CRISP Cas9筛选测定，再生研究和细胞疗法）。该系统的另一个巨大优势在于其无传代特性，它避免了胰蛋白酶化的必要性，并允许将原代细胞在稳态培养中维持长达一年的时间，并进行实验，如长期药物治疗和克隆追踪分析，在整个实验过程中监测相同的培养（大多数实验室使用的当前协议无法实现）。这种创新的实验系统提供了在体外长时间进行类组织研究的能力，并使研究中使用的小鼠数量减少了40%至75%。它还极大地影响了动物替代，因为它允许生成和长期维持原代人类上皮3D培养物，使用患者来源的活检，最大限度地减少了在生物医学研究项目中使用小鼠模型的必要性。考虑到英国和世界上大部分实验室需要小鼠原代细胞来成功发展他们的研究项目，我相信，这种新技术的广泛实施将减少一半以上的研究目的牺牲的小鼠总数，同时保持他们的发现的稳健性和可重复性。

This project aims at disseminating and helping with the application of a state-of-the-art in vitro 3D system developed during my postdoctoral research at the MRC-CU and the Welcome Sanger Institute and optimised in my laboratory at the Gurdon Institute/University of Cambridge, which allows for the production, long-term maintenance and expansion of 3D cultures of primary and tumour epithelial cells, directly obtained from mouse and human tissues, avoiding any previous step of single cell suspensions to set up the cultures.Its ability to form tissue-like structures, in a unique and easy technique that combines the potential of amplification, scalability and a low cost, (similar to the cost of any cell line culture), with the generation of a complex 3D model that mimics tissue architecture and resembles physiological cell behaviour, makes this system tremendously useful for many laboratories working on epithelial tissues or using mouse and human primary cells for their research projects. It is also of great interest for researchers working on tissue regeneration and cell therapies, as it provides an almost unlimited number of primary epithelial cells with a high reliability and efficiency, ready to be used in their experimental research projects.The system allows the culture and amplification of mouse and human primary epithelial cells from several type of epithelial and endothelial tissues (oesophagus, skin, bladder, tongue, urethra, breast, uterus, oral mucosa, etc) and enables studies of cell responses, cell competition mechanisms between normal and tumour cells, gene expression and protein interaction analysis, cancer drug screening, and long-term epithelial-immune cell co-culture assays, with high reliability, efficiency and low cost, compared with other methods currently used.In addition, this system can also be used as an expandable resource of progenitor cells for different purposes (e.g., generation of organoids with higher yield than using traditional protocols that relies on the reduced number of cells within tissues, CRISP Cas9 screening assays using primary cells, regeneration studies and cell therapies).Another great advantage of the system relies on its passage-free feature, which avoids the necessity of trypsinization and allows for maintaining primary cells in a homeostatic culture for up to a year and perform experiments such as long-term drug treatments and clonal tracing analyses, monitoring the same culture during the entire length of the experiment (something impossible to achieve with the current protocols that most laboratories are using).This innovative experimental system provides a capability for tissue-like studies to be carried out in vitro during long periods of time and enables a Reduction in the number of mice used in research between 40% and 75%. It also greatly impacts animal Replacement as it allows the generation and long-term maintenance of primary human epithelial 3D cultures, using patient-derived biopsies, minimising the necessity of using mouse models in biomedical research projects.Given the great proportion of laboratories in the UK and rest of the world requiring mouse primary cells for the successful development of their research projects, I do believe, the wide implementation of this novel technique will reduce the total number of sacrificed mice for research purposes by more than half while maintaining the robustness and reproducibility of their findings.