背景:沃顿的果冻来源的间充质干细胞(WJ-MSCs)在再生医学中具有巨大的治疗潜力。因此,建立符合良好生产规范(GMP)的WJ-MSCs分离和培养方法是至关重要的。通过综合研究,包括实验室规模的实验到中试规模的研究,我们旨在制定标准化方案,确保WJ-MSCs生产的高产率和质量.
方法:首先,对用于分离WJ-MSCs的酶消化方法的参数进行了优化。这些参数包括酶浓度,消化时间,播种密度,和文化媒介。此外,对外植体法和酶消化法进行了比较分析。随后,WJ-MSCs的连续传代,特别是直到第9段,使用优化的方法进行评估。最后,制造工艺得到了发展和扩大,从实验室规模的烧瓶生产开始,发展到中试规模的细胞工厂生产。此外,进行了一项稳定性研究,以评估药品(DP)的储存和使用情况.
结果:酶消化方法的最佳参数为浓度为0.4PZU/mL胶原酶NB6,消化时间为3h,导致P0WJ-MSC的产量更高。此外,脐带组织重量与P0WJ-MSCs数量呈正相关。对不同浓度的人血小板裂解物的评价显示,2%和5%浓度导致相似水平的细胞扩增。比较分析显示,在初始传代期间,与外植体方法相比,酶消化方法表现出WJ-MSC的更快生长。传代2至5在整个连续传代过程中表现出更高的活力和增殖能力。此外,成功开发了从实验室规模到中试规模的可扩展制造工艺,确保生产高质量的WJ-MSC。DP的多次冻融循环导致降低的细胞活力和活细胞浓度。随后解冻和稀释DP导致两个指标的显着下降,尤其是在20-27°C下储存时。
结论:本研究为优化WJ-MSCs的分离和培养提供了有价值的见解。我们可扩展的制造工艺有助于大规模生产高质量的WJ-MSC。这些发现有助于基于WJ-MSC的疗法在再生医学中的进展。
BACKGROUND: Wharton\'s jelly-derived mesenchymal stem cells (WJ-MSCs) hold great therapeutic potential in regenerative medicine. Therefore, it is crucial to establish a Good Manufacturing Practice (GMP)-compliant methodology for the isolation and culture of WJ-MSCs. Through comprehensive research, encompassing laboratory-scale experiments to pilot-scale studies, we aimed to develop standardized protocols ensuring the high yield and quality of WJ-MSCs manufacturing.
METHODS: Firstly, optimization of parameters for the enzymatic digestion method used to isolate WJ-MSCs was conducted. These parameters included enzyme concentrations, digestion times, seeding densities, and culture media. Additionally, a comparative analysis between the explant method and the enzymatic digestion method was performed. Subsequently, the consecutive passaging of WJ-MSCs, specifically up to passage 9, was evaluated using the optimized method. Finally, manufacturing processes were developed and scaled up, starting from laboratory-scale flask-based production and progressing to pilot-scale cell factory-based production. Furthermore, a stability study was carried out to assess the storage and use of drug products (DPs).
RESULTS: The optimal parameters for the enzymatic digestion method were a concentration of 0.4 PZ U/mL Collagenase NB6 and a digestion time of 3 h, resulting in a higher yield of P0 WJ-MSCs. In addition, a positive correlation between the weight of umbilical cord tissue and the quantities of P0 WJ-MSCs has been observed. Evaluation of different concentrations of human platelet lysate revealed that 2% and 5% concentrations resulted in similar levels of cell expansion. Comparative analysis revealed that the enzymatic digestion method exhibited faster outgrowth of WJ-MSCs compared to the explant method during the initial passage. Passages 2 to 5 exhibited higher viability and proliferation ability throughout consecutive passaging. Moreover, scalable manufacturing processes from the laboratory scale to the pilot scale were successfully developed, ensuring the production of high-quality WJ-MSCs. Multiple freeze-thaw cycles of the DPs led to reduced cell viability and viable cell concentration. Subsequent thawing and dilution of the DPs resulted in a significant decrease in both metrics, especially when stored at 20-27 °C.
CONCLUSIONS: This study offers valuable insights into optimizing the isolation and culture of WJ-MSCs. Our scalable manufacturing processes facilitate the large-scale production of high-quality WJ-MSCs. These findings contribute to the advancement of WJ-MSCs-based therapies in regenerative medicine.