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Title: Orthogonal potency analysis of mesenchymal stromal cell function during exvivo expansion Authors and affiliations

Danika Khong Matthew Li Amy Singleton Ling-Yee Chin Shilpaa Mukundan Biju Parekkadan All : Department of Surgery, Centre for Surgery, Innovation, & Bioengineering, Massachusetts General Hospital, Harvard Medical School and the Shriners Hospitals for Children, Boston, MA 02114, USA Biju: Department of Biomedical Engineering, Rutgers University and the Department of Medicine, Rutgers Biomedical and Health Sciences, Piscataway, NJ 08854, USA Biju: Harvard Stem Cell Institute, Cambridge, MA 02138, USA Biju: Corresponding author at: Department of Surgery, Center for Surgery, Innovation, & Bioengineering, Massachusetts General Hospital, Harvard Medical School and the ShrinersHospitals for Children, Boston, MA 02114, USA.

Aim: To investigate if age and age associated changes of adult bone marrow mesenchymal stromal cells can be used to predict bioactivity in biomanufacturing Adult bone marrow mesenchymal stromal cells (MSCs) have three significant characteristics, capability to regenerate bone, fat and cartilage, regulate the immune system and allow the growth of bone marrow hematopoietic stem cells (An immature cell that can develop into different blood cells) . These attributes make MSCs have a variety of transplant applications. MSCs can also multiply ex vivo (outside the body) allowing for large scale production. However MSCs produced vary significantly from batch to batch due to morphological differences. Although it has been shown that age negatively effects all three characteristics of MSCs through comparisons of varying aged donors, it is unknown whether this translates to MSCs aged through in vitro. In an in vitro model of MSCs growth the age and age-associated biophysical changes were observed, and corresponding effects on three characteristics were measured via assays. The information gathered allowed the scientists to hypothesize how differences in age and associated changes of size and shape can be utilised to produce specific MSCs Method 1. Isolation of MSCs via Inserting a Phosphate buffered saline centrifuging at 1500 rpm, changing the media then freezing the medium and harvesting MSC cells 2. passages creating MSCs were cultured and cryopreserved for 8 days each, 7 times, lasting a total of 56 days 3. The media holding MSCs replaced by Adipogenic induction media, after three days the media was changed to Adipogenic maintenance media. This was repeated another 2 times and on the last time the MSCs were cultured for seven days. All cells were in 37 degree, 5% CO2 incubators. Differentiation was analysed at the end 4. MSCs adipogenic differentiation were analyse by AAS using an oil red O working solution prepared.

5. MSCs ability to support Bone marrow cell (BMC) was analysed by culturing BMCs by themselves then in a 1-10 ratio with MSCs. Cells were co-cultured for seven days 6. MSCs then underwent Immuno-modulatory assays which determined the ability of MSCs in immune modulation using human peripheral blood monocular cells 7. The Immuno-phenotypic analysis was done via flow cytometry of MSCs with human monoclonal antibodies 8. MSCs were then counted using hemocytometer in order to get an equation for doubling times and levels. 9. Morphological attributes were then analysed by staining cells with Calcein AM then by dividing major by minor axis an aspect ratio was formed 10. Statistical analysis of all tests performed were done by Graph Pad prism. Two variables were tested and graphs were made with correlation coefficient and correlation determination used to highlight the level of relation between variables Major Results In vitro aging doesn’t effect MSCs immune-modulatory capacity Variability in MSC cell and nuclei morphology increases with increasing in vitro age in vitro age is a strong predictor of MSC adipogenic and hematopoietic-supporting potencies Low-density seeding expansion induce in vitro aging of human MSCs

It was determined that the MSCs age and biophysical properties could be used to predict the adipogenesis and hematopoietic supporting capabilities, yet it doesn’t effect the immunemodulatory capacity. This new found information can be used to lessen variation in batches of MSCs and can even be harnessed to create MSCs for specific purposes. This can in the future be vital in transplant surgeries. This information demonstrates the significance further research on MSCs could have, in order to accurately determine the abilities MSCs have which could lead to therapeutic use....