Consequences of adjusting cell density and feed frequency on serum-free expansion of thymic regulatory T cells |
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| Institution: | 1. School of Biomedical Engineering, University of British Columbia, Vancouver, Canada;2. British Columbia Children''s Hospital Research Institute, Vancouver, Canada;3. Michael Smith Laboratories, University of British Columbia, Vancouver, Canada;4. Department of Surgery, University of British Columbia, Vancouver, Canada;5. Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada;6. Department of Molecular Oncology, British Columbia Cancer Research Institute, Vancouver, Canada;7. Department of Chemical and Biological Engineering, University of British Columbia, Vancouver, Canada;1. Dirección Técnica de Investigación, Desarrollo e Innovación, Instituto Nacional de Investigación en Salud Pública “Leopoldo Izquieta Pérez\"- INSPI,Guayaquil, Ecuador;2. Escuela de Medicina, Colegio de Ciencias de la Salud, Universidad San Francisco de Quito USFQ, Quito, Ecuador;3. Instituto de Investigaciones en Biomedicina iBioMed, Universidad San Francisco de Quito USFQ, Quito, Ecuador;4. Mito-Act Research Consortium, Quito, Ecuador;5. Biología, Colegio de Ciencias Biológicas y Ambientales COCIBA, Universidad San Francisco de Quito USFQ, Quito, Ecuador;6. PhD Program in Biomedicine, Faculty of Medicine, Universidad de los Andes, Santiago, Chile;7. Biología, Pontificia Universidad Católica del Ecuador PUCE, Quito, Ecuador;8. Escuela de Medicina Veterinaria, Colegio de Ciencias de la Salud, Universidad San Francisco de Quito USFQ, Quito, Ecuador;9. Sistemas Médicos SIME, Universidad San Francisco de Quito USFQ, Quito, Ecuador;1. School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing, China;2. Daxing Research Institute, University of Science and Technology Beijing, Beijing, China;3. Cell Therapy Laboratory, First Hospital of Hebei Medical University, Shijiazhuang, China;4. Department of Immunology, Basic Medical College, Hebei Medical University, Shijiazhuang, China;1. Graduate Program for Collective Health, Faculty of Health Sciences, University of Brasilia, Brasilia, Brazil;2. Oswaldo Cruz Foundation, Rio de Janeiro, Brazil;3. Departament of Hemotherapy and Cell Therapy, Israelita Albert Einstein Hospital, São Paulo, Brazil;4. Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil;5. Postgraduate Program in Health Sciences and Technologies, Faculty of Ceilandia, University of Brasilia, Brasília, Brazil;6. Collective Health School, Faculty of Ceilandia, University of Brasilia, Brasília, Brazil;1. Beijing Key Laboratory of Traditional Chinese Medicine Basic Research on Prevention and Treatment for Major Diseases, Experimental Research Center, China Academy of Chinese Medical Sciences, Beijing, China;2. School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing, China;3. Daxing Research Institute, University of Science and Technology Beijing, Beijing, China;1. University of Vermont College of Medicine, Burlington, Vermont, USA;2. Department of Immunology, Duke University, Durham, North Carolina, USA;3. Marcus Center for Cellular Cures, Duke University, Durham, North Carolina, USA;4. Department of Pathology, Duke University, Durham, North Carolina, USA;5. Department of Neurosurgery, Duke University, Durham, North Carolina, USA;6. Department of Medicine, University of Wisconsin Carbone Cancer Center, Madison, Wisconsin, USA;7. IMPACT-Center for Interventional Medicine for precision and advanced cellular therapy, Universidad de los Andes, Santiago, Chile;8. Cells for Cells and Consorcio Regenero, Chilean Consortium for Regenerative Medicine, Santiago, Chile;9. Department of Medicine, Section of Hematology, University of Verona, Verona, Italy;10. Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada;11. Anesthesiology and Pain Medicine, University of Ottawa, Ottawa, Ontario, Canada;12. Clinical Epidemiology, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada;13. Regenerative Medicine, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada;14. Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden;15. Department of Cellular Therapy and Allogeneic Stem Cell Transplantation, Karolinska University Hospital, Stockholm, Sweden;16. Department of Internal Medicine, Stem Cell Program and Institute for Regenerative Cures, University of California Davis, Sacramento, California, USA;17. UF Scripps Biomedical Research, Jupiter, Florida, USA;18. Laboratory of Pulmonary Investigation, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil;19. The First Affiliated Hospital, Soochow University Institutes for Translational Medicine, Suzhou, China;20. Institute of Health Sciences, Chinese Academy of Sciences, Shanghai, China;21. UMR U1236-MICMAC, Immunology and Cell Therapy Lab, Rennes University Hospital, Rennes, France;22. Osteoarthritis Research Program, Division of Orthopedic Surgery, Schroeder Arthritis Institute, University Health Network, Toronto, Canada;23. Krembil Research Institute, University Health Network, Toronto, Canada;24. Institute of Biomedical Engineering, University of Toronto, Toronto, Canada;25. Department of Medicine, Division of Hematology, University of Toronto, Toronto, Canada;26. Department of Biomedicine, University Hospital Basel, University of Basel, Basel, Switzerland |
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| Abstract: | BackgroundGiven the promising results from phase 1/2 clinical trials of therapy involving regulatory T cells (Tregs), it is critical to develop Treg manufacturing methods that use well-defined reagents.MethodsSeeking to maximize expansion of human thymic Tregs activated with anti-CD3/CD28 antibody-coated beads and cultured in serum-free medium, the authors investigated the effect of adjusting process parameters including cell density and cell concentration, and feeding strategy on Treg yield and quality.ResultsThe authors found that levels of expansion and viability varied with cell density on the day of restimulation. Tregs restimulated at low cell densities (1 × 105 cells/cm2) initially had high growth rates, viability and FOXP3 expression, but these parameters decreased with time and were less stable than those observed in cultures of Tregs restimulated at high cell densities (5 × 105 cells/cm2), which had slower growth rates. High-density expansion was associated with expression of inhibitory molecules and lower intracellular oxygen and extracellular nutrient concentrations as well as extracellular lactate accumulation. Experiments to test the effect of low oxygen revealed that transient exposure to low oxygen levels had little impact on expansion, viability or phenotype. Similarly, blockade of inhibitory molecules had little effect. By contrast, replenishing nutrients by increasing the feeding frequency between 2 days and 4 days after restimulation increased FOXP3, viability and expansion in high-density cultures.ConclusionThese data show the previously undescribed consequences of adjusting cell density on Treg expansion and establish a Good Manufacturing Practice-relevant protocol using non-cell-based activation reagents and serum-free media that supports sustained expansion without loss of viability or phenotype. |
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