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

目的和目的-研究骨髓间充质干细胞（BMMSC）在扩增和分化的不同阶段的能量代谢、膜转运途径及其在不同环境条件下的修饰。设计一个合适的生物反应器和运输缓冲液，以保持BMMSCs在治疗前处于静止状态。研究背景和潜在影响- BMMSCs已在400多个临床试验中进行了研究，主要涉及组织修复或免疫系统疾病。最近，一项NEPTUNE研究正在研究它们用于减弱肾移植患者的免疫系统反应。尽管BMMSCs具有巨大的治疗潜力，但在其保存方面仍存在挑战。分离后，BMMSC只能存活很短的时间，因此在运输过程中必须将其冷冻在血清和冷冻保护剂中。目前，生理盐水（含和不含人血清白蛋白）已被研究作为BMMSCs在4 ℃下的替代保存介质。这避免了在治疗患者之前需要对细胞进行预处理。生理盐水已显示可维持细胞活力、增殖和分化潜力长达18小时。然而，需要将BMMSCs保存长达7天，以便它们可以在国际上运输。尽管已经在一系列不同的细胞类型中进行了代谢研究，但对BMMSCs的研究很少。一般来说，静止细胞被认为保持在低代谢状态，并产生较少量的活性氧（ROS），如在T淋巴细胞，人皮肤成纤维细胞（HDF）和胚胎干细胞中观察到的。在干细胞分化过程中，谷氨酰胺代谢增加，从糖酵解到氧化磷酸化的转变也被观察到，后者是由于线粒体的成熟。成骨细胞表现出更高水平的谷氨酰胺代谢，并有更大的O2需求相比，未分化的BMMSCs。BMMSC还依赖有氧糖酵解和氧化磷酸化来产生ATP。虽然对BMMSC的代谢进行了一些初步的研究，但缺乏对增殖和衰老过程中代谢活性的研究。研究自噬、转运蛋白表达和活性氧产生的变化将为开发合适的生物反应器和培养基以在4 ℃下保存和运输BMMSC提供深入了解，从而消除在应用前对这些细胞进行传代的需要。研究方法的新奇-在对人BMMSC进行研究之前，将从小牛腿收获原代牛BMMSC并培养约8代。对于每次传代，代谢活性和干细胞标志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在体内存在于缺氧条件下。这将使我能够发现如何BMMSCs的行为时，直接从体内环境中获得，如果在体外细胞培养和分化过程中发生任何变化，真实的时间聚合酶链反应技术（RT-PCR）将被用来研究葡萄糖转运蛋白1，葡萄糖转运蛋白3和单羧酸转运蛋白4（MCT-4）亚型在人类BMMSCs的细胞周期的不同阶段的表达。一系列的电极，荧光和放射性检测将被用来评估葡萄糖的摄取和消耗，总溶解氧消耗，自噬，谷氨酰胺代谢和活性氧在原代牛BMMSCs的生产。公司和合作者-该项目是在与牛津MEStar，从生物医学工程研究所（IBME）在牛津大学的分拆生物技术公司合作进行。他们专门为转化再生医学和医疗保健提供生物工艺工程解决方案。

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.