Development of a Modular Automated System for Maintenance and Differentiation of Adherent Human Pluripotent Stem Cells
Development of a Modular Automated System for Maintenance and Differentiation of Adherent Human Pluripotent Stem Cells
复制标题
DOI：
10.1177/2472555217696797
复制
发表时间：
2017-09-01
期刊：
影响因子：
3.1
通讯作者：
Pebay, Alice
中科院分区：
文献类型：
--
作者：
Crombie, Duncan E.;Daniszewski, Maciej;Pebay, Alice
患者特异性诱导多能干细胞(IPSCs)在再生医学、疾病模型和药物发现方面具有巨大的发展潜力。然而，重新编程、维护和区分的过程是劳动密集型的，并受技术人员之间的差异影响。为了解决这些问题，我们建立和优化了方案，允许将重新编程的体细胞自动维护为IPSCs，从而能够使用定制的TECAN Freedom EVO大规模培养和传代人类多能干细胞(PSCs)。使用Miltenyi MultiMACS24分离仪进行脱机核切割，然后选择TRA-1-60阳性细胞。多能标记被评估以确认所产生的IPSCs的多能性。传代采用无酶解离方法。通过将人的PSCs分化为视网膜血统的细胞，获得了分化概念的证据。这种自动化方法的主要优势是能够增加样本大小，减少重新编程或分化过程中的变异性，并能够对人类PSC及其衍生物进行中高通量分析。随着使用干细胞的临床试验的出现，这些技术将变得越来越重要。
Patient-specific induced pluripotent stem cells (iPSCs) have tremendous potential for development of regenerative medicine, disease modeling, and drug discovery. However, the processes of reprogramming, maintenance, and differentiation are labor intensive and subject to intertechnician variability. To address these issues, we established and optimized protocols to allow for the automated maintenance of reprogrammed somatic cells into iPSCs to enable the large-scale culture and passaging of human pluripotent stem cells (PSCs) using a customized TECAN Freedom EVO. Generation of iPSCs was performed offline by nucleofection followed by selection of TRA-1-60-positive cells using a Miltenyi MultiMACS24 Separator. Pluripotency markers were assessed to confirm pluripotency of the generated iPSCs. Passaging was performed using an enzyme-free dissociation method. Proof of concept of differentiation was obtained by differentiating human PSCs into cells of the retinal lineage. Key advantages of this automated approach are the ability to increase sample size, reduce variability during reprogramming or differentiation, and enable medium- to high-throughput analysis of human PSCs and derivatives. These techniques will become increasingly important with the emergence of clinical trials using stem cells.