Fully animal-free manufacture of optimised growth factors: underpinning sustainable and scalable organoid culture for precision medicine, drug discovery and regenerative medicine applications

类器官是三维（3D）干细胞集群，它们聚集在一起，模拟单个器官内的微环境，无论是肝脏、肾脏、心脏、肠道还是其他特定器官。从本质上讲，它们可以被看作是微型的、简化的器官。它们的大小通常从几微米到5毫米不等，体内有多少不同的组织和器官，就有多少不同的类器官。类器官也可以用来模拟癌症和脑部疾病等疾病。通过控制所使用的特定干细胞的分化，可以形成这样一个多样化的类器官，这可以受到细胞从称为生长因子的蛋白质、3D细胞外基质（ECM）及其成分和类器官生长介质中的化学成分接收指导信号的影响。类器官有着非凡的前景：它们是一种真正具有颠覆性的技术，能够彻底改变我们对基础生物学的理解，彻底改变药物发现过程，以及对动物模型的依赖。也就是说，在实验室中用于培养类器官的方法需要改进和标准化，以允许更广泛的采用，特别是在药物发现中，并使类器官培养更容易和更可复制。需要特别关注的两个元素是生物活性蛋白生长因子和类器官生长的3D凝胶基质。基质是结构蛋白和生长因子的复杂混合物，目前是从小鼠肿瘤中提取的，因此迫切需要制造可重复性的合成替代品，并减少动物产品的使用。在我们之前的ukri资助的项目中，我们为类器官培养开发了优化的，高度定义的生长因子形式。大型跨国公司现在有兴趣批量购买这些产品，我们需要进行进一步的研发，以扩大生产规模，取消β -内酰胺类抗生素的使用（这与类器官的临床前应用不兼容），并引入ISO13485质量体系。此外，还有一种叫做Wnt3a的生长因子，它对许多类器官的生长至关重要，而且不容易获得。市面上可买到的少数几种具有可变的生物活性，价格异常昂贵（< 17000英镑/毫克），并且不是完全不含动物产品的生产过程。在上一个项目中，我们在解决这一问题所需的研发方面取得了良好的开端，并希望继续这项工作，利用蛋白质工程技术制造出高度生物活性，完全不含动物且具有成本效益的Wnt蛋白。

Organoids are 3-dimensional (3D) clusters of stem cells that come together and emulate the microenvironment within individual organs, whether that be liver, kidney, heart, gut or other specific organs. Essentially, they can be viewed as miniature, simplified organs. They typically range in size from a few micrometers to five millimeters and there are potentially as many different organoids as there are different tissues and organs in the body. Organoids can also be grown that mimic diseased such as cancer and brain disorders. Such a diverse range of organoids can be formed by controlling the differentiation of the specific stem cell used, which can be influenced by the cells receiving instructive signals from proteins called growth factors, the 3D extracellular matrix (ECM) and its components, and chemical moieties in the medium the organoids grow in.Organoids hold extraordinary promise: they are a truly disruptive technology capable of completely transforming our understanding of basic biology and revolutionising the drug discovery process, and its reliance on animal models.That said, methods used for growing organoids in the laboratory need to be improved and standardised to allow wider adoption, particularly in drug discovery, and to make organoid culture easier and more reproducible. The two elements that need particular focus are the bioactive protein growth factors and the 3D gel matrix in which organoids grow. The matrix, a complex mixture of structural proteins and growth factors, is currently extracted from mouse tumours so there is a pressing need to make synthetic alternatives for reproducibility and to reduce use of animal-products.In our previous UKRI-funded project we developed optimised, highly defined forms of growth factors for organoid culture. Large multinational companies are now interested in bulk purchase of these and we need to undertake further R&D to scale manufacture, remove the use of beta-lactam antibiotics (which are not compatible with pre-clinical applications of organoids) and introduce a quality system ISO13485.In addition, there is one growth factor, called Wnt3a, which is crucial for the growth of many types of organoids and is not easily sourced. The few commercially available have variable bioactivity, are exceptionally expensive (<£17,000 /milligram) and are not made in a full animal-product-free process.We made a promising start on the R&D needed to solve this problem in the last project and wish to continue this work, using protein engineering technologies to make a highly bioactive, totally animal-free and cost-effective Wnt protein.