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 samples were digested through enzymatic dissociation, the cells were grown in T175 flasks (passage 0) and hyperflasks (passage 1) in the animal-free SkGM^TM-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 pre-determined releasing criteria. The manufactured myoblast cells could differentiate and fuse into myotubes in vitro, with the possible trend that the 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.

背景：自体成肌细胞已被测试用于治疗肌肉相关疾病。然而，制造成肌细胞的标准化仍未建立。在这里，我们报告了一种基于烧瓶和无动物培养基的制造临床级成肌细胞的方法，并在良好生产规范 (GMP) 下建立了成肌细胞产品的发布标准。方法：四头肌活检样本来自三名肌源性上睑下垂患者。活检样品通过酶解消化后，细胞在 T175 烧瓶（第 0 代）和无动物 SkGM ^{TM 的}超烧瓶（第 1 代）中生长-2 骨骼肌细胞生长培养基，含有 5% 人血小板裂解物，可培养 15-17 天。基于细胞形态、细胞剂量、活力、不育性、内毒素、支原体和免疫表型释放收获的细胞。还评估了肌管分化。结果：在第 1 代结束时的 15 至 17 天内制造了 400 至 5 亿个成肌细胞，符合预定的释放标准。制造的成肌细胞在体外可以分化融合成肌管，可能存在供体年龄影响成肌细胞分化能力的趋势。结论：本研究建立了一种在无动物培养基中制造成肌细胞的基于烧瓶的方法以及释放标准，该方法简单、稳健、廉价且易于重现。