Serra Margarida, Leite Sofia B, Brito Catarina, Costa Júlia, Carrondo Manuel J T, Alves Paula M
Instituto de Biologia Experimental e Tecnológica/Instituto de Tecnologia Química e Biológica (IBET/ITQB), Oeiras, Portugal.
J Neurosci Res. 2007 Dec;85(16):3557-66. doi: 10.1002/jnr.21451.
Embryonal carcinoma (EC) stem cells derived from germ cell tumors closely resemble embryonic stem (ES) cells and are valuable tools for the study of embryogenesis. Human pluripotent NT2 cell line, derived from a teratocarcinoma, can be induced to differentiate into neurons (NT2-N) after retinoic acid treatment. To realize the full potential of stem cells, developing in vitro methods for stem cell proliferation and differentiation is a key challenge. Herein, a novel culture strategy for NT2 neuronal differentiation was developed to expand NT2-N neurons, reduce the time required for the differentiation process, and increase the final yields of NT2-N neurons. NT2 cells were cultured as 3D cell aggregates ("neurospheres") in the presence of retinoic acid, using small-scale stirred bioreactors; it was possible to obtain a homogeneous neurosphere population, which can be transferred for further neuronal selection onto coated surfaces. This culturing strategy yields higher amounts of NT2-N neurons with increased purity compared with the amounts routinely obtained with static cultures. Moreover, mechanical and enzymatic methods for neurosphere dissociation were evaluated for their ability to recover neurons, trypsin digestion yielding the best results. Nevertheless, the highest recoveries were obtained when neurospheres were collected directly to treated surfaces without dissociation steps. This novel culture strategy allows drastic improvement in the neuronal differentiation efficiency of NT2 cells, insofar as a fourfold increase was obtained, reducing simultaneously the time needed for the differentiation process. The culture method described herein ensures efficient, reproducible, and scaleable ES cell proliferation and differentiation, contributing to the usefulness of stem cell bioengineering.
源自生殖细胞肿瘤的胚胎癌（EC）干细胞与胚胎干细胞（ES）极为相似，是研究胚胎发育的宝贵工具。源自畸胎癌的人多能性NT2细胞系，在视黄酸处理后可被诱导分化为神经元（NT2-N）。为充分发挥干细胞的潜力，开发体外干细胞增殖和分化方法是一项关键挑战。在此，我们开发了一种用于NT2神经元分化的新型培养策略，以扩增NT2-N神经元、减少分化过程所需时间并提高NT2-N神经元的最终产量。NT2细胞在视黄酸存在下作为三维细胞聚集体（“神经球”）进行培养，使用小型搅拌生物反应器；可以获得均匀的神经球群体，其可转移至包被表面进行进一步的神经元筛选。与静态培养常规获得的数量相比，这种培养策略可产生更高数量且纯度更高的NT2-N神经元。此外，还评估了机械和酶法解离神经球以回收神经元的能力，胰蛋白酶消化产生的效果最佳。然而，直接将神经球收集到处理过的表面而不进行解离步骤时，回收率最高。这种新型培养策略可显著提高NT2细胞的神经元分化效率，产量提高了四倍，同时减少了分化过程所需时间。本文所述的培养方法确保了高效、可重复且可扩展的ES细胞增殖和分化，有助于干细胞生物工程的实用性。
