Development and optimisation of novel bioprinting process for scalable production of tissue models using FRESH technique

研究背景英国每年有500多万人受到骨关节炎(OA)的影响，给当地经济和NHS带来了数十亿英镑的负担。尽管进行了50多年的研究，但目前还没有有效的药物可用于治疗骨性关节炎。体外模型被认为是目前药物开发中最常见的动物模型的伦理替代，但由于其固有的支架相关限制，即细胞活性差和细胞表型不稳定，仅提出了简单的模型，通常只有单一组织成分。因此，我们迫切需要新的技术平台来实现可重复性和集成化的人类相关骨关节炎体外模型。目的和目标我们的目标是开发基于自由形式可逆悬浮水凝胶嵌入(FRESH)技术的生物制造平台，适用于高通量生物制造，以生产标准的失效和可翻译的人类相关骨关节炎关节模型。具体目标包括：1.用于FRESH技术的生物墨水的表征和优化2.为人软骨细胞和人成骨细胞的单一和联合培养系统量身定制平台3.表征2中开发的组织模型的质量和表型。建立开发的模型的适用性，以产生标准化的、残废的和可翻译的人类相关的骨关节炎关节模型。潜在的应用和好处骨性关节炎的临床表现非常不同，有多个患者亚群。此外，早期骨性关节炎的发病机制仍然知之甚少，阻碍了早期诊断和疾病修正疗法的有效工具的开发。由于人体组织的可获得性有限，在过去的50年里，许多动物模型被用来研究疾病的发生和发展，以及测试新的治疗干预措施。虽然体内模型提供了自然发生的全关节疾病的反映，体外系统的多功能性，以及整合和开发3R哲学改进、减少和取代动物使用的潜力的愿望，使得疾病的体外建模非常可取。研究方法项目合作伙伴曼彻斯特生物凝胶公司(MBG)将一系列用于复杂生物系统的合成水凝胶产品商业化。该项目探索使用这些水凝胶来开发可扩展、标准化的软骨细胞和人类成骨细胞工程，用于单一和联合培养系统，以开发AO模型。本项目将探索一种新的3D制作技术--Fresh Print。这允许产生复杂和大的组织结构。学生将使用计算和实验相结合的方法来描述生物油墨和优化的新鲜印刷工艺。一旦工艺标准化，将开发出成骨细胞和软骨细胞的单细胞和共培养打印模型。将测试细胞模型的存活率和长期分化，以建立组织特定的表型发育。与EPSRC的战略和研究领域保持一致这个项目将对EPSRC的两个医疗技术挑战做出重要贡献：“开发未来疗法”和“优化治疗”。它将提供基本的工具(即体外模型)，以伦理和成本效益的方式确定未来的药物治疗，这肯定会导致更理想的治疗。在技术方面，该项目通过提供一个新的生物打印平台，在横切能力中涉及到“先进材料”和“未来制造技术”方面的新方法。

Context of Research More than 5 million people in the UK are affected by Osteoarthritis (OA) every year, placing a multi-billion-pound burden on the local economy and the NHS. Despite more than 50 years of research, no effective drug is commercially available for OA. In vitro models are deemed an ethical alternative of the animal models - the most common model currently - in drug development but only simple model, typically with single tissue component, have been proposed due to their inherent scaffold related limitations, i.e. poor cell viability and instable cell phenotype. There is therefore an urgent unmet need for new technological platforms that enable reproducible and integrative human relevant OA in vitro models.Aims and Objectives Our aim is to develop biofabrication platform based on Freeform Reversible Embedding of Suspended Hydrogels (FRESH) technology, suitable for high throughput biomanufacturing to produce standardisable and translational human relevant OA joint models.Specific objectives include:1. Characterisation and optimisation of bioinks developed by ManchesterBIOGEL used for FRESH technique 2. Tailor platform for human chondrocytes and human osteoblasts engineering for mono- and co-cultures systems 3. Characterise tissue models developed in 2 for their quality and phenotype. Establish applicability of developed models to produce standardisable and translational human relevant OA joint models. Potential application and BenefitsThe clinical presentation of OA is extremely heterogeneous, with multiple subsets of patients. Further, the pathogenesis of early OA remains poorly understood, hampering the development of effective tools for early diagnosis and disease-modifying therapeutics. Given the limited availability of human tissue, numerous animal models have been utilised over the past 50 years to study disease onset and progression, as well as to test novel therapeutic interventions. Whilst in vivo models offer a reflection of the naturally-occurring whole-joint disease, the versatility of an in vitro system, and the desire to incorporate and exploit the potential of the 3R philosophy of refining, reducing and replacing the use of animals makes in vitro modelling of the disease highly desirable. Research Methodology The project partner Manchester Biogel (MBG) commercialises a family of synthetic hydrogel products for complex biological systems. This project explores use of these hydrogels to develop scalable, standardise chondrocytes and human osteoblasts engineering for mono- and co-cultures systems for AO models development. A novel 3D fabrication technology, FRESH printing, will be explored in this project. This allows generation of complex and large tissue constructs. The student will characterise bioinks and optimised FRESH printing process using combination of computational and experimental approach. Once process is standardised, both on mono and co-culture cell printed models of osteoblast and chondrocytes will be developed. Cellular models will be tested for their survival and long-term differentiation, to establish tissue specific phenotypic development. Alignment to EPSRC strategies and research areas This project will critically contribute to two of the EPSRC healthcare technology challenges: "Developing Future Therapies" and "Optimising Treatment". It will provide fundamental tool (i.e. in vitro model) to identify future pharmacological therapy, in ethical and cost-effective manner, which will certainly lead to more optimal treatment. In the technological aspect, the project involves novel methods in "advanced materials" and "future manufacturing technologies" among the cross-cutting capabilities by providing a novel bioprinting platform.