Why is AUTOSTEM important? This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no 667932. The development of a fully automated platform for the manufacture of stem cells for cell therapies Shibani Ratnayake1, Panagiota Moutsatsou1, Alvin W. Nienow 1,2 , Christopher J. Hewitt 1, Qasim A. Rafiq 1 , 3, Mariana P. Hanga1 1. School of Life and Health Science, Aston University, Aston triangle, Birmingham, B4 7ET, UK. 2. School of Chemical Engineering, University of Birmingham, Edgbaston, Birmingham, UK. 3. Department of Biochemical Engineering, University College London, Gower Street, London, UK. What is AUTOSTEM? AUTOSTEM is an EU H2020 project that is dedicated to developing a closed, automated, sterile pipeline for large-scale production of clinically relevant human mesenchymal stem cells (hMSCs). The aim is to facilitate high quality consistent stem cells at lower cost that will be beneficial to patients as new stem cell therapies. Why is AUTOSTEM important? Stem cells are emerging as a treatment for several diseases including diabetes, heart disease and Parkinson’s. However, current manufacturing methods are time consuming, expensive and will be unable to satisfy patient demand. AUTOSTEM will enable the production of high quality stem cell at low cost for cell therapy.   The AUTOSTEM Process Figure 1: Schematic representation of the AUTOSTEM process How does AUTOSTEM work? Bone marrow is taken from the hip bone of a donor using a novel closed suction device that is being developed by the project. The samples are then transferred to an automated platform where stem cells will be isolated using novel antibodies and expanded using novel bioreactor technology. The product will be delivered to patients in a closed device. The whole process is completed with minimal human intervention for the production of high quality stem cells. Aston Project aims: To optimise and manufacture MSCs on a large scale using different bioreactors and selection of optimal bioreactor for the automated platform. Methods: Cell expansion of MSCs Mobius CellReady (at 1L) Solohill Plastic microcarriers 5000 cm2 surface area 6000 cells/cm2 pH 7.3-7.4 11 day culture 50% medium exchange at day 4 then every other day Full harvest Cell counts Metabolite analysis Imaging Figure 2: Schematic representation of the culture process and conditions. Results and discussion: MSCs at p4 day 6 in culture Growth Kinetics Cell Characterisation Growth Kinetics Specific growth rate (h-1) 0.01 Population doubling time (h) 63 Cumulative population doublings 4 Fold increase 18 Figure3: Live/Dead staining of MSCs cultured on Plastic microcarriers in the Mobius CellReady. Images were acquired at day 6 in culture using fluorescence microscopy (Leica, UK).Live cells are recognized by intercellular esterase activity, by the enzymatic conversion of non-fluorescent cell-permeable Calcein AM to fluorescent green Calcein. Ethidium homodimer enters cells with damaged membranes and binds to nucleic acids whereby producing a red fluorescence in dead cells. Metabolite analysis Figure 7: CFU-F efficiency of MSCs before and after inculcation in the bioreactor. B A Table 1: Growth kinetics of MSC over a 11 day culture period. MSCs were expanded successfully in the bioreactor over 11 days with 4 cumulative population doublings and with a fold increase of 18. However, more optimisation is being done to increase the cell yield at harvest. Cell harvest Figure 8: A) Adipogenesis,B ) Osteogenesis of MScs after bioreactor harvest. Cells were differentiated for 12-14 days and stained for adipocytes using oil red O and Bone mineralisation stained in black (with silver nitrate) and Alkaline Phosphatase stained in red Figure 4: Glucose and lactate concentrations of spent medium from 11 days. Daily samples were taken and glucose and lactate concentrations of the medium were measured before and after medium exchanges using an Accutrend Plus blood test meter (Roche Diagnostics, USA) A B Viability 96% Live 545x106 Dead 20x106 Total 565x106 Diameter(µm) 13.1 Cell counts Cell Aggregation Figure 9: Increased level of aggregation cells after 9 days in culture with predominance around the probes . Addition of microcarriers prior to aggregation combined with gradually increasing agitation speed can minimise aggregation Figure 6: A) Cell detachment from microcarriers after 20 minutes exposure to TrypLE. B) Cell viability and cell numbers post harvesting. Figure 5: Cell counts from a 11 day culture. Two independent samples of cell-microcarrier suspension were taken 2 hours post inoculation at day 0 and then at day 6,8 and 11 preharvest and post-harvest for cell counts. Cells were lysed from the microcarriers using Reagent A100 and then stabilised by Reagent B and counted using the NucleoCounter® NC-3000 (Chemometec, Denmark) following the Reagent A100+B protocol according manufacturers instructions. When microcarriers were agitated at high speeds there was successful cell detachment without compromising cell viability. Conclusion and future work: The MSCs were successfully grown on microcarriers at a 1L scale over a 11 day period while retaining their potency post processing. More optimisation is being done by investigating the feeding regime, microcarrier addition mid culture as well as expansion time of cells to increase the final cell yield. Once the optimisation is complete, the process will be introduced to the automated platform. Partners: References: 1. Heathman,T.R.J.,Rafiq,Q.A.,Chan,A.K.C.,Coopman,K.,Nienow, A.W., Kara, B., Hewitt, C.J.(2016) Characterization of human mesenchymal stem cells from multiple donors and the implications for large scale bioprocess development ,Biochemical Engineering Journal,108,14-23. 2. Heathman, T. R. J., Stolzing, A., Fabian, C., Rafiq, Q. A., Coopman, K., Nienow, A. W., Hewitt, C. J. (2016). Scalability and process transfer of mesenchymal stromal cell production from monolayer to microcarrier culture using human platelet lysate. Cytotherapy, 18(4), 523-535.