oa Adipose Tissue Derived-Mesenchymal Stromal Cell: Setting the ground for Clinical Grade Production at Sidra's GMP Facility

Adipose Tissue Derived-Mesenchymal Stromal Cell: Setting the ground for Clinical Grade Production at Sidra's GMP Facility1Asma Al-Sulaiti, 1 Moza Al- Khulaifi, 1Sara Al-Khawaga, 1Zohreh Tatari Calderone, 1Bella S. Guerrouahen, 1Chiara Cugno. 1 Clinical Research Center, Research Division, Translational Medicine, Sidra Medicine, Qatar. Correspondence: Dr. Chiara Cugno, MD, Director Clinical Research Center, Research Division, Translational Medicine Sidra Medicine, Qatar. Keyword list: MSC, Adipose tissue, SVF, GMP, ISCT criteria. Word count: 702Background: Among adult stem cells, mesenchymal stromal cells (MSCs) represent a highly examined population given their exceptional biological properties. The International Society for Cellular Therapy (ISCT) define human MSCs based on the following criteria: adherence to plastic in standard culture condition, expression of the surface molecules CD105, CD90, and CD73, and lack of expression for CD34, CD45, HLA-DR ( < 2%), CD14 or CD11b, CD79a or CD19, and finally capability to differentiate in vitro into osteocytes, chondrocytes, and adipocytes. The US National Institutes of Health database has reported 493 MSC-based clinical trials as of June 2015. Most clinical trials were implemented towards evaluating the biomedical potential of MSCs in treating graft versus host disease, hematological and inflammatory diseases diseases, organ transplantation, diabetes, and diseases in the liver, kidneys, and lungs, cardiovascular, bone and cartilage, neurological, and autoimmune disorders. Although multipotent MSCs are more commonly isolated from bone marrow (BM), adipose tissue-derived MSCs (AT-MSCs) represent a valid alternative source for MSCs due to their greater abundance, simple accessibility, and better immunomodulatory properties. Aim: We ultimately aim to develop the clinical-grade production of human AT-MSCs for Cellular Therapy at Sidra Good Manufacturig Practise (GMP) Facility. Our study represent the stepping stone for developing an MSC bank of high quality, reliable and well-characterized cells timely available for potential future clinical applications, such as Type 1 Diabetes (T1D). Objective 1 focuses on the isolation, expansion and characterisation of AT-MSCs. Objective 2 focuses on optimizing AT-MSCs production protocols to comply with the requirements of producing clinical-grade AT-MSCs. Methods: A) Sample collection: Adipose tissue is collected and obtained as waste material after liposuction procedures carried at Plastic Surgicenter, Doha. Aspirated fat is transferred to the Clinical Research Center at Sidra Medicine in sterile containers. B) Isolation and expansion of AT-MSCs: Both fat and blood/saline portion of lipoaspirate are used. AT-MSCs are isolated from the fatty portion, using a collagenase-based enzymatic method and from the blood / saline portion, using a non-enzymatic digestion. The isolated AT-MSCs are seeded and cultured in 75 cm2 flasks for expansion till passage 5. C) Phenotypic characterization, multilineage differentiation capacity and clonogenic assay: After reaching more than 95% confluence in the flasks at passage 1 (around 14 days in culture), phenotypic characterisation of MSCs is verified using a multi-color flow cytometry for the following specific antibodies: CD45, CD34, CD14, CD19, HLA-DR, CD105, CD73 and CD90. The adipogenesis and osteogenesis differentiation potential of AT-MSCs is also assessed at low passage. AT-MSCs are cultured under differentiating conditions in separate culture vessels. Cultures are processed for Oil Red O staining to evaluate adipogenesis differentiation (from day 7 to 14). Osteogenesis differentiation can be visualized after 21 days, using an Alizarin Red staining. To measure the clonogenic ability of AT-MSCs, Colony Forming Unit (CFU) is carried out using crystal violet staining. D) Translating AT-MSCs production to GMP facility: all protocols are optimized and standardized to comply the requirement of producing clinical-grade MSCs at Sidra Medicine GMP facility. The isolators (Biospherix XVIVO System) provides a strictly controlled environment that assures manufacturing of sterile, potent and uncontaminated products. It consists of modular sets of closed incubators and closed hoods, all integrated together as co-chambers and sub-chambers. The system is completely closed, with aseptic conditions throughout, and advanced controls as for GMP international standards. Results: AT-MSCs displayed a fibroblast-like morphology. The ISCT-recommended specific cell surface signature was verified: AT-MSCs were positive for CD105, CD90 and CD73 and negative for CD45, CD34, CD14, CD19 and HLA-DR. The biochemical assessment of adipogenic differentiation showed induced adipocytes with vacuoles containing fatty acids positive for Oil Red O staining. Alizarin Red staining showed induced osteocytes with calcium phosphate deposits. The stemness property of AT-MSCs was also confirmed. Conclusion: Recent studies have indicated MSCs as a powerful tool for several clinical applications. We are developing AT-MSCs clinical grade production at Sidra GMP Facility. We established standard operating procedures following the highest GMP international standards. Sidra GMP Facility is the first of its type in Qatar for cell therapy purposes, bringing future perspectives for the establishment of an AT-MSC bank from third-party healthy donors, after adequate screening for allogeneic donation, and a clinical trial in T1D to tackle the unmet need of preventative studies in this area.