Drummond Nicola J, Singh Dolt Karamjit, Canham Maurice A, Kilbride Peter, Morris G John, Kunath Tilo
MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, The University of Edinburgh, Edinburgh, United Kingdom.
Cytiva, Cambridge, United Kingdom.
Front Cell Dev Biol. 2020 Nov 5;8:578907. doi: 10.3389/fcell.2020.578907. eCollection 2020.
Human pluripotent stem cells can be differentiated into midbrain dopaminergic (mDA) neurons by directing cells through a floor plate progenitor stage. The developmental identity of mDA neurons produced using floor plate protocols is similar to neurons, and this has improved the ability to model Parkinson's disease (PD) in a dish. Combined with the unlimited growth potential of pluripotent stem cells, mDA neural progenitor cell production can provide a scalable source of human dopaminergic (DA) neurons for diverse applications. However, due to the complexity and length of the protocols and inherent differences between cell lines, considerable variability of the final population of neurons is often observed. One solution to this problem is to cryopreserve committed mDA neural progenitor cells in a ready-to-use format. Creating a bank of cryopreserved mDA neural progenitor cells poised for neuronal differentiation could significantly improve reproducibility and facilitate collaborations. Here we have compared six (6) different commercial cryopreservation media and different freezing conditions for mDA neural progenitor cells differentiated from human embryonic stem cell (hESC) lines. Significant differences in cell recovery were observed at 24 h post-thawing, but no differences were observed immediately upon thawing. The presence of ROCK inhibitors improved cell recovery at 24 h for all cryopreservation media tested. A faster cooling rate of 1-2°C/min was significantly better than 0.5°C/min for all conditions tested, while rapid thawing at 37°C was not always superior to slow thawing at 4°C. Importantly, cryopreservation of mDA neural progenitor cells did not alter their potential to resume differentiation into mDA neurons. Banks of cryopreserved committed mDA neural progenitor cells provide a method to generate human DA neurons with reduced batch-to-batch variability, and establish a mechanism to share lineage-primed cells for collaborative research.
通过引导细胞经历底板祖细胞阶段，人类多能干细胞可分化为中脑多巴胺能（mDA）神经元。使用底板方案产生的mDA神经元的发育特性与体内神经元相似，这提高了在培养皿中模拟帕金森病（PD）的能力。结合多能干细胞无限的生长潜力，mDA神经祖细胞的产生可为多种应用提供可扩展的人类多巴胺能（DA）神经元来源。然而，由于方案的复杂性和长度以及细胞系之间的固有差异，最终神经元群体往往存在相当大的变异性。解决这个问题的一个方法是以即用型形式冷冻保存定向的mDA神经祖细胞。创建一批随时可用于神经元分化的冷冻保存的mDA神经祖细胞库，可显著提高可重复性并促进合作。在此，我们比较了六种不同的商业冷冻保存培养基以及从人类胚胎干细胞（hESC）系分化而来的mDA神经祖细胞的不同冷冻条件。解冻后24小时观察到细胞回收率存在显著差异，但解冻后立即未观察到差异。对于所有测试的冷冻保存培养基，ROCK抑制剂的存在提高了解冻后24小时的细胞回收率。对于所有测试条件，1 - 2°C/分钟的较快冷却速率明显优于0.5°C/分钟，而37°C快速解冻并不总是优于4°C缓慢解冻。重要的是，mDA神经祖细胞的冷冻保存并未改变其恢复分化为mDA神经元的潜力。冷冻保存的定向mDA神经祖细胞库提供了一种方法，可产生批次间变异性降低的人类DA神经元，并建立一种机制来共享预先定向的细胞用于合作研究。
