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

Lead Research Organisation: University of Oxford
Department Name: Engineering Science


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.


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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M017338/1 01/01/2017 30/06/2021
1848660 Studentship BB/M017338/1 01/01/2017 31/12/2021 Tong Chen
Description - During mesenchymal stem cell (MSC) transportation, cell death or damage may occur if cells become over-confluent or if metabolic waste reach high levels. It is therefore important to halt growth and reduce energy demand during transportation.
- Previous methods of transportation have faults - cells only last for 18 hours if preserved in saline. Wisconsin solution keeps MSCs alive for 3 days but we are unsure if differentiation capacity is maintained. Cells last longer in spheroid form in culture media + bovine serum (7 days). However, bovine serum cannot be in contact with the patient's bloodstream. I hope to extend the preservation time to 14 days or more, using blood free media.
- I have measured glucose consumption and lactate production of MSCs under normal conditions at 1 mM, 2.5 mM and 5.5 mM glucose. I estimated Km and Vmax from my data values.
- I have managed to greatly slow cell growth and reduce metabolism without causing cell death by modifying the ingredients in culture media.
- After 4 days exposure to the modified media, MSC proliferation is fully restored. After 8 days exposure to modified media (without disturbance), there is partial loss in proliferation rate, but most cells are still alive. I am about to test 12 days exposure.
Metabolic measurement of samples, experiments at low oxygen and differentiation tests to soon follow, plus additional experiments with primary MSCs.

Since Mar 2019 the outcomes have been:

- Low oxygen has no clear effect on MSC growth or metabolism.
- Perfusion culture of MSCs slows growth and metabolism.
- MSCs preserved in ITSE have higher oxidative stress vs fresh cells, but no change in autophagy.
- Neutralisation of oxidative stress by N-acetylcysteine does not improve viability or prevent morphological change (caused by preservation).
- MSC growth is slowed at 25C, and MSCs die at 4C in DMEM/FBS. We selected room temp (25C) as the temp to use for preservation.
- MSCs can be preserved for 12 days in ITSE medium (37'C and 5% CO2) with partial retention of growth and trilineage differentiation (adipogenic, osteogenic and chondrogenic).
- ITSE medium can be adapted to maintain physiological pH (7-8) at 0.04% CO2 simply by altering sodium bicarbonate concentration.
- After 12 days preservation of MSCs in ITSE at atmospheric conditions (25'C, 0.04% CO2), most cells are alive (>70%), and cells are still able to proliferate, but additional 3 days recovery time is needed compared to fresh cells.
- MSCs do not survive when preserved at atmospheric conditions in suspension as dispersed cells.
- MSCs cannot form spheroids in ITSE medium when we tried spheroid culture to improve viability.
- MSCs require extracellular pyruvate and cysteine from the culture media. MSCs produce Aspartic Acid, Proline, Tyrosine and Glutamic Acid (GC-MS metabolomics)

We will create MSC spheroids in DMEM first, and then transfer to ITSE medium for preservation. We will also repeat GC-MS with a higher number of cells in the absence of glucose and serum for more accurate measurements.
Exploitation Route The eventual goal is to produce a preservation/transportation media that keeps MSCs alive for 12 days or longer. This will consist of a sterile plastic container with MSCs suspended in preservation media, which the doctor can simply take out of the fridge and apply to the patient.
Sectors Pharmaceuticals and Medical Biotechnology

Title Metabolic measurements and assessment of cell growth 
Description Previous studies only have end point measurements of mesenchymal stem cell (MSC) growth and metabolism (at the end of passage, or after 48 hours). Previous methods of cell counting, such as trypan blue, CCK-8 and propidium iodide cannot be employed multiple times during cell growth. I have employed a non-destructive method of counting MSCs using a plate reader. This allows me to study how metabolism changes during proliferation. 
Type Of Material Cell line 
Year Produced 2018 
Provided To Others? No  
Impact More data is required before a notable impact can be made. 
Description Collaboration with Target Discovery Institute (Uni Oxford) to study MSC metabolomics with GC-MS 
Organisation University of Oxford
Department Target Discovery Institute (TDI)
Country United Kingdom 
Sector Academic/University 
PI Contribution Analysed spent media of mesenchymal stem cells (MSCs) to study amino acid and vitamin consumption.
Collaborator Contribution I learned how to prepare samples and analyse data for GC-MS from the target discovery institute.
Impact - At standard seeding densities (7500 cells/cm2) over a 72 hour period, human bone marrow mesenchymal stem cells consume cysteine and pyruvate, and produce aspartic acid, proline, tyrosine and glutamic acid. - The experiment will be repeated with higher cell densities (100,000 cells), and in the absence of glucose and serum to obtain more accurate results.
Start Year 2020
Description Extraction of mesenchymal stem cells from calf bone for culture using Oxford MEStar bioreactor 
Organisation Oxford MeStar Limited
Country United Kingdom 
Sector Private 
PI Contribution I extracted MSCs from calf bone, performed flow cytometry and trilineage differentiation studies to compare manual and bioreactor cultured cells.
Collaborator Contribution Oxford MEStar constructed the operated the bioreactor during the study.
Impact Bioreactor cultured MSCs adopt a more irregular morphology compared to manual cultured MSCs.
Start Year 2019
Description Use of John Radcliffe hospital for analysis of media glucose and lactate. 
Organisation John Radcliffe Hospital
Country United Kingdom 
Sector Hospitals 
PI Contribution N/A
Collaborator Contribution I send my spent media samples to the JR hospital for more accurate and cheaper measurement of glucose and lactate, using their bioanalyser.
Impact We have shown that: Cells have reduced metabolic activity during preservation in insulin based preservation medium (ITSE), shift towards oxidative phsophorylation Cell metabolism recovers when taken out of ITSE and transferred back to growth medium (DMEM/FBS), shift back to glycolysis Low oxygen has no clear effect on metabolic activity
Start Year 2019
Description Conference held by London Cell Cycle Club at the Francis Crick Institute 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact Attended conference at the Francis Crick Institute (24th Feb 2020). Gave poster presentation on my research to other students/professors who were there.
Year(s) Of Engagement Activity 2020