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

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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.