1] Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA. [2] Harvard-MIT Division of Health Sciences and Technology, Harvard Stem Cell Institute, Cambridge, Massachusetts, USA.
Department of Biomedical Engineering, University at Buffalo, The State University of New York, Buffalo, New York, USA.
Nat Protoc. 2014 Feb;9(2):233-45. doi: 10.1038/nprot.2014.002. Epub 2014 Jan 9.
Cell therapies enable unprecedented treatment options to replace tissues, destroy tumors and facilitate regeneration. The greatest challenge facing cell therapy is the inability to control the fate and function of cells after transplantation. We have developed an approach to control cell phenotype in vitro and after transplantation by engineering cells with intracellular depots that continuously release phenotype-altering agents for days to weeks. The platform enables control of cells' secretome, viability, proliferation and differentiation, and the platform can be used to deliver drugs or other factors (e.g., dexamethasone, rhodamine and iron oxide) to the cell's microenvironment. The preparation, efficient internalization and intracellular stabilization of ∼1-μm drug-loaded microparticles are critical for establishing sustained control of cell phenotype. Herein we provide a protocol to generate and characterize micrometer-sized agent-doped poly(lactic-co-glycolic) acid (PLGA) particles by using a single-emulsion evaporation technique (7 h), to uniformly engineer cultured cells (15 h), to confirm particle internalization and to troubleshoot commonly experienced obstacles.
细胞疗法为替代组织、破坏肿瘤和促进再生提供了前所未有的治疗选择。细胞疗法面临的最大挑战是无法控制移植后细胞的命运和功能。我们开发了一种通过在细胞内储存库中对细胞进行工程改造来控制细胞表型的方法，这些储存库可以持续释放改变表型的药物数天至数周。该平台可用于控制细胞的分泌组、活力、增殖和分化，并且该平台可用于将药物或其他因子（例如，地塞米松、若丹明和氧化铁）递送到细胞的微环境中。对于建立对细胞表型的持续控制，制备、高效内化和细胞内稳定约 1μm 的载药微球至关重要。在此，我们提供了一种通过单乳液蒸发技术（7 小时）生成和表征微米级药物掺杂聚乳酸-共-羟基乙酸（PLGA）颗粒的方案，使用该方案可以均匀地对培养细胞进行工程改造（15 小时），确认颗粒内化并解决常见的障碍。
