Manufacturing autologous myoblast for regenerative medicine applications
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METHODS PAPER
Manufacturing autologous myoblast for regenerative medicine applications Matthew Lee-Wing . David Szwajcer . Anthony Lockwood . Alanna Flynn . Karla Anjos . Marie Tulloch . Angeline Giftakis . Qingdong Guan
Received: 1 May 2020 / Accepted: 2 September 2020 Ó Springer Nature B.V. 2020
Abstract Background: Autologous myoblasts have been tested in the treatment of muscle-related diseases. However, the standardization of manufacturing myoblasts is still not established. Here we report a flask and animal-free medium-based method of manufacturing clinical-grade myoblast together with establishing releasing criteria for myoblast products under Good Manufacturing Practice (GMP). Methods: Quadriceps muscle biopsy samples were donated from three patients with myogenic ptosis. After biopsy Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10616-020-00420-9) contains supplementary material, which is available to authorized users.
samples were digested through enzymatic dissociation, the cells were grown in T175 flasks (passage 0) and hyperflasks (passage 1) in the animal-free SkGMTM-2 skeletal muscle cell growth medium containing 5% human platelet lysate for 15–17 days. The harvested cells were released based on cell morphology, cell dose, viability, sterility, endotoxin, mycoplasma and immunophenotype. Myotube differentiation was also evaluated. Results: 400 to 500 million myoblast cells were manufactured within 15 to 17 days by the end of passage 1, which met predetermined releasing criteria. The manufactured myoblast cells could differentiate and fuse into myotubes in vitro, with the possible trend that the
M. Lee-Wing A. Flynn Dept of Ophthalmology, University of Manitoba, Winnipeg, Canada
D. Szwajcer Q. Guan Dept of Internal Medicine, University of Manitoba, Winnipeg, Canada
D. Szwajcer K. Anjos M. Tulloch A. Giftakis Q. Guan Manitoba Centre for Advanced Cell & Tissue Therapy, Winnipeg, Canada
A. Lockwood Dept of Plastic Surgery, University of Manitoba, Winnipeg, Canada
D. Szwajcer K. Anjos M. Tulloch A. Giftakis Q. Guan (&) Cellular Therapy Laboratory, Manitoba Blood and Marrow Transplant Program, CancerCare Manitoba, MS773M, 820 Sherbrook St, Winnipeg R3A 1R9, MB, Canada e-mail: [email protected]
Q. Guan Dept of Immunology, University of Manitoba, Winnipeg, Canada
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Cytotechnology
donor age may impact the differentiation ability of myoblasts. Conclusions: The present study establishes a flask-based method of manufacturing myoblast in the animal-free medium together with releasing criteria, which is simple, robust, inexpensive and easily reproducible. This study will serve as the validation for a planned phase 1 clinical trial to assess the use of autologous myoblast transplants for the treatment of myogenic ptosis and other myogenic diseases. Keywords Myoblast Cell manufacturing Myogenic ptosis
Introduction The skeletal muscles make up 40–50% of the body mass. Aging decreases r
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