Identification of growth and attachment factors for the serum-free isolation and expansion of human mesenchymal stromal cells |
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| Authors: | Sunghoon Jung Arindom Sen Lawrence Rosenberg Leo A Behie |
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| Institution: | 1. Key Laboratory of Transplant Engineering and Immunology, NHFPC, Regenerative Medicine Research Center, West China Hospital, Sichuan University, Chengdu, PR China;2. Core Facility of West China Hospital, Sichuan University, Chengdu, PR China;3. Animal Lab Center, West China Hospital, Sichuan University, Chengdu, PR China;1. Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA;2. Stem Cell Program, Institute of Cell Engineering, The Johns Hopkins University School of Medicine, Edward D. Miller Research Building, Room 747, 733 N. Broadway, Baltimore, MD 21205, USA;3. Graduate Program of Molecular and Cellular Medicine, The Johns Hopkins University School of Medicine, USA;4. Division of Hematology, Department of Medicine, The Johns Hopkins University, Edward D. Miller Research Building, Room 747, 733 N. Broadway, Baltimore, MD 21205, USA;5. Department of Material Sciences and Engineering, The Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA;6. Institute for NanoBioTechnology, The Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA;1. Karnieli Ltd and ATVIO Biotech, Ticon, Israel;2. Pluristem Therapeutics, Matam Park, Haifa, Israel;3. Regenerative Medicine Clinical Center, Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina, USA;4. Hematology Branch, National Heart, Lung, and Blood Institute National Institute of Health, Bethesda, Maryland, USA;5. Cell Therapy Research & Technology Lonza Walkersville, Walkersville, Maryland, USA;6. Biological Industries Ltd., Beit Haemek, Israel;7. GE Healthcare Cell Therapy Division, Marlborough, Massachusetts, USA;8. Bioprocessing Technology Institute, Singapore;9. Stimlabs, Roswell, Georgia, USA;10. Irvine Scientific, Santa Ana, California, USA |
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| Abstract: | Background aimsEx vivo propagation of sparse populations of human mesenchymal stromal cells (hMSC) is critical for generating numbers sufficient for therapeutic applications. hMSC culture media have typically been supplemented with animal serum and, recently, human-sourced materials. However, these supplements are ill-defined and, thus, undesirable for clinical and research applications. Previously reported efforts to develop defined media for hMSC culture only resulted in slow or limited proliferation, and were unsuccessful in expanding these cells from primary cultures. Therefore a major step forward would be the identification of defined, serum-free culture conditions capable of supporting both the isolation and rapid expansion of hMSC.MethodsUsing classical approaches of medium development, we were able to identify a set of growth and attachment factors that allowed the serum-free isolation and expansion of hMSC from bone marrow.ResultsHeparin, selenium and platelet-derived growth factor (PDGF)-BB were found to be inhibitory for the growth of hMSC, whereas basic fibroblast growth factor (bFGF) was critical and worked synergistically with transforming growth factor (TGF)-β1 to allow significant cell expansion. Ascorbic acid, hydrocortisone and fetuin were also found to be important growth and attachment factors that, in conjunction with substrate-coating proteins, allowed the isolation of hMSC from primary culture and their subsequent expansion.ConclusionsWe report a defined medium formulation (PPRF-msc6), consisting of key recombinant and serum-derived components, for the rapid isolation and expansion of hMSC in the absence of serum. This work represents an important step forward for achieving an ideal, completely defined synthetic medium composition for the safe use of hMSC in clinical settings. |
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