Abstract：
Here, we describe methods for the production of adeno-associated viral (AAV) vectors by transient transfection of HEK293 cells grown in serum-free medium using orbital shaken bioreactors and the subsequent purification of vector particles. The protocol for expression of AAV components is based on polyethyleneimine (PEI)-mediated transfection of a three-plasmid system and is specified for production in milliliter-to-liter scales. After PEI and plasmid DNA (pDNA) complex formation, the diluted cell culture is transfected without a prior concentration step or medium exchange. Following a 7-day batch process, cell cultures are further processed using a set of methods for cell lysis and vector recovery. Methods for the purification of viral particles are described, including immunoaffinity and anion-exchange chromatography, ultrafiltration, as well as digital PCR to quantify the concentration of vector particles.
摘要：
这里,我们描述了通过使用轨道振荡生物反应器瞬时转染在无血清培养基中生长的HEK293细胞并随后纯化载体颗粒来生产腺相关病毒(AAV)载体的方法。用于表达AAV组分的方案基于聚乙烯亚胺(PEI)介导的三质粒系统的转染,并且被指定用于以毫升至升的规模生产。PEI和质粒DNA（pDNA）复合物形成后，在没有事先浓缩步骤或培养基交换的情况下转染稀释的细胞培养物。经过7天的批处理，使用一组用于细胞裂解和载体回收的方法进一步处理细胞培养物。描述了纯化病毒颗粒的方法。包括免疫亲和层析和阴离子交换层析,超滤,以及数字PCR来量化载体颗粒的浓度。
