Improving transport and storage of viable mesenchymal stem cells through investigations into their energy metabolism

通过研究能量代谢来改善存活间充质干细胞的运输和储存

• 批准号: 1848660
• 金额: --
• 依托单位: University of Oxford
• 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-1848660
• 关键词: Improving; transport; storage; viable; mesenchymal

Aims and Objectives - Study the pathways of bone marrow mesenchymal stem cell (BMMSC) energy metabolism, membrane transport and their modification by different environmental conditions during different stages of expansion and differentiation.-Design a suitable bioreactor and transportation buffer to maintain BMMSCs in a quiescent state prior to treatment.Context of Research and Potential Impact - BMMSCs have been investigated in over 400 clinical trials, primarily involving tissue repair or immune system disorders. More recently, a NEPTUNE study is investigating their use for attenuating the immune system response in kidney transplant patients.Despite the enormous therapeutic potential of BMMSCs, challenges remain in their preservation. Following isolation, BMMSCs are only viable for a short period of time, so they must be frozen in serum and cryoprotectant agents during transportation. They are difficult to remove and may cause adverse events in patients.At present, saline (with and without human serum albumin) has been investigated as alternative preservation media for BMMSCs at 4 degree C. This circumvents the need for cells to be defrosted prior to treating the patient. Saline has been shown to maintain cell viability, proliferation and differentiation potential for up to 18 hours. However, there is a need to preserve BMMSCs for up to 7 days so they can be transported internationally.Although metabolic studies have been undertaken in a range of different cell types, little work has been carried out on BMMSCs. In general, quiescent cells are thought to remain in a state of hypometabolism and produce a lower amount of reactive oxygen species (ROS), as observed in T lymphocytes, human dermal fibroblasts (HDFs) and embryonic stem cells. During stem cell differentiation, increases in glutamine metabolism and shifts from glycolysis to oxidative phosphorylation have also been observed, the latter due to the maturing of mitochondria.Osteoblasts exhibit higher levels of glutamine metabolism and have greater O2 requirements compared to undifferentiated BMMSCs. BMMSCs also rely on both aerobic glycolysis and oxidative phosphorylation for ATP production. Although some initial work has been carried out on BMMSC metabolism, studies on metabolic activity during proliferation and senescence are lacking. Studying changes in autophagy, transporter expression and ROS production will provide insight into the development of a suitable bioreactor and medium for BMMSC preservation and transportation at 4 degree C, thus eliminating the need to defrost these cells prior to their application.Novelty of Research Methodology - Prior to study on human BMMSCs, primary bovine BMMSCs will be harvested from calf legs and cultured for approximately 8 passages. For each passage, metabolic activity and stem cell marker expression will be conducted at normal oxygen levels (21%) and hypoxia (0, 1, 2 and 5% oxygen), since BMMSCs reside in hypoxic conditions in vivo. This will enable me to discover how BMMSCs behave when obtained straight from an in vivo environment and if any changes occurred during in vitro cell culture and differentiation.The real time polymerase chain reaction technique (RT-PCR) will be used to study expression of the glucose transporter 1, glucose transporter 3 and monocarboxylate transporter 4 (MCT-4) isoforms at different stages of the cell cycle in human BMMSCs. A range of electrodes, fluorescence based and radioactive assays will be employed to assess glucose uptake and consumption, total dissolved oxygen consumption, autophagy, glutamine metabolism and ROS production in primary bovine BMMSCs.Companies and Collaborators - The project is undertaken in collaboration with Oxford MEStar, a spin-out biotechnology company from the Institute of Biomedical Engineering (IBME) at Oxford University. They specialise in providing bioprocess engineering solutions to translational regenerative medicine and healthcare.

目的和目标 - 研究骨髓间充质干细胞 (BMMSC) 能量代谢、膜运输的途径及其在不同扩增和分化阶段不同环境条件下的修饰。 - 设计合适的生物反应器和运输缓冲液，以在治疗前维持 BMMSC 处于静止状态。 研究背景和潜在影响 - BMMSC 已在 400 多项临床试验中进行了研究，主要涉及 组织修复或免疫系统紊乱。最近，一项 NEPTUNE 研究正在研究它们在减弱肾移植患者免疫系统反应方面的用途。尽管 BMMSC 具有巨大的治疗潜力，但其保存方面仍然存在挑战。分离后，BMMSC 只能在短时间内存活，因此在运输过程中必须将其冷冻在血清和冷冻保护剂中。它们很难去除，并可能对患者造成不良事件。目前，盐水（含或不含人血清白蛋白）已被研究作为 4 摄氏度下 BMMSC 的替代保存介质。这避免了在治疗患者之前对细胞进行解冻的需要。生理盐水已被证明可以维持细胞活力、增殖和分化潜力长达 18 小时。然而，需要将 BMMSC 保存长达 7 天，以便它们可以进行国际运输。尽管已经在一系列不同的细胞类型中进行了代谢研究，但针对 BMMSC 开展的工作却很少。一般来说，正如在 T 淋巴细胞、人真皮成纤维细胞 (HDF) 和胚胎干细胞中观察到的那样，静止细胞被认为保持代谢低下状态，并产生较低量的活性氧 (ROS)。在干细胞分化过程中，还观察到谷氨酰胺代谢增加以及从糖酵解向氧化磷酸化的转变，后者是由于线粒体的成熟所致。与未分化的 BMMSC 相比，成骨细胞表现出更高水平的谷氨酰胺代谢，并且具有更高的 O2 需求。 BMMSC 还依赖有氧糖酵解和氧化磷酸化来产生 ATP。尽管已经对 BMMSC 代谢进行了一些初步工作，但缺乏对增殖和衰老过程中代谢活性的研究。研究自噬、转运蛋白表达和 ROS 产生的变化将为开发适合 BMMSC 在 4°C 保存和运输的生物反应器和培养基提供深入见解，从而消除在应用前解冻这些细胞的需要。研究方法的新颖性 - 在对人 BMMSC 进行研究之前，将从小腿中收获原代牛 BMMSC 并培养约 8 代。对于每次传代，代谢活动和干细胞标记物表达将在正常氧水平（21%）和缺氧（0、1、2和5%氧）下进行，因为 BMMSC 存在于体内缺氧条件下。这将使我能够发现直接从体内环境获得的 BMMSC 的行为方式，以及在体外细胞培养和分化过程中是否发生任何变化。实时聚合酶链反应技术 (RT-PCR) 将用于研究人类 BMMSC 细胞周期不同阶段的葡萄糖转运蛋白 1、葡萄糖转运蛋白 3 和单羧酸转运蛋白 4 (MCT-4) 同工型的表达。将采用一系列电极、荧光和放射性测定来评估原代牛 BMMSC 中的葡萄糖摄取和消耗、总溶解氧消耗、自噬、谷氨酰胺代谢和 ROS 产生。 公司和合作者 - 该项目是与牛津 MEStar 合作开展的，牛津 MEStar 是生物医学工程研究所 (IBME) 的衍生生物技术公司 在牛津大学。他们专注于为转化再生医学和医疗保健提供生物过程工程解决方案。