For those and other reasons, experts usually reply, “It depends,” when asked about the best transfection strategies for gene therapy programs based on adenoassociated virus (AAV). As a senior bioprocess application scientist at Corning Life Sciences, Ann Rossi Bilodeau often receives questions from researchers about which approaches they should apply: Should they use suspension or adherent cell culture systems? Is one transfection reagent preferable to another? How long should a transfection process run? Bilodeau explains that which strategy to use “depends greatly on what you’re trying to achieve in the end, and there’s not just one right or wrong approach. From cell lines to platforms, facilities, and individual researchers, many factors can influence transfection — and a process should be optimized to account for all of them.” Best practices are worth exploring, however, as Bilodeau and experts from reagent supplier Polyplus-transfection demonstrate herein.
The primary question with transfection is how to do it. AAV-based gene therapy research generally relies on chemically mediated transfection. Compared with complex and expensive processes such as electroporation and microinjection, chemical transfection is easy to use and thus considered to be the gold standard in cost-efficient upscaling.
Cassie-MariePeigné, a scientific support specialist at Polyplus-transfection, explains, “Most AAV therapies involve human embryonic kidney (HEK)293 cells, which are transfected easily using chemical reagents. When developed specifically for viral vector production, such reagents are as good as it gets in terms of reproducibility and robustness, negating the need for more expensive methods of physical transfection.”
“It takes time to optimize transfection for adherent cells and for cells grown in suspension,” says Alengo Nyamay’antu, a scientific communication specialist at Polyplus-transfection. “The important thing is that you optimize.”
如果研究人员确定哪些平台choose, Peigné suggests considering timelines and scales. “Adherent platforms are conventional methods to generate good titers quickly, and such processes are already well established in manufacturing facilities.” Adherent-culture vessels might be limited to scale-out rather than scale-up processes, which can present challenges for labor and manufacturing footprints. But options such as Corning CellSTACK, HYPERStack, and CellCube platforms can help increase yields and decrease labor requirements compared with single-layer vessels, especially when configured as closed systems. Whereas suspension tanks and transfection reagents can facilitate scale-up, such platforms require additional time to optimize for cell viability and quality, especially when working with suspension-adapted cells.
请记住，规模双向发展 - 缩小可能与扩展一样重要。Bilodeau说：“使用可伸缩的容器和转染试剂可帮助您相对轻松地在尺度上移动。如果您需要返回并进行优化，则可以小规模对流程进行故障排除，然后再将其恢复到制造业。通常，拥有良好的过程比简单地最大化生产更为重要。”
Researchers also should feel comfortable asking for support. Suppliers such as Polyplus-transfection and Corning Life Sciences can help scientists select the right products for their objectives. Peigné says, “If you’re starting a new process, it’s always helpful to contact suppliers to ensure that you’re beginning with an optimal combination of platform, media, reagents, and other factors.” Suppliers also can help determine “that you’re operating with the correct guidelines beforehand. Knowing where to look can save considerable time.”
了解您的协议：Follow a transfection protocol or guidelines for use. Sometimes seeding densities and times in culture before transfection must be adapted for different cell types and applications, provided that cells are dividing actively at the time of transfection.
Mitigate Detachment:If adherent cells detach from a vessel surface in response to protein or viral vector expression or because of toxicity, seeding cells on specific surfaces (e.g., Corning CellBIND surface) before transfection can enhance attachment.
优化试剂和原材料：Be sure to use only high-qualityplasmid preparationsthat are free from proteins, RNA, and endotoxins.
调整plasmid DNAand the试剂与DNA比率在你的过程,以增加其transfection efficiency.
细胞毒性sometimes occurs after transfection. If toxicity is observed, then decrease your reagent-to-DNA ratio or the amount of plasmid DNA used in your process. (Doing so is especially important if the protein that is expressed is toxic.) It might also be necessary to change culture medium four to six hours after transfection.
安·罗西·比洛多（Ann Rossi Bilodeau）is senior bioprocess applications scientist at Corning Life Sciences, 2 Alfred Road, Kennebunk, ME 04043; 1-207-985-3111.Alengo Nyamay’antuis a scientific communication specialist, andCassie-MariePeignéis a scientific support specialist at Polyplus-transfection SA, 75 Rue Marguerite Perey, 67400 Illkirch-Graffenstaden, France; 33-3-90-40-61-80.
CellCUBE, CellBIND, CellSTACK, and HYPERStack are registered trademarks of Corning Incorporated. PEIpro is a registered trademark of Polyplus-transfection.