Serra Margarida, Brito Catarina, Leite Sofia B, Gorjup Erwin, von Briesen Hagen, Carrondo Manuel J T, Alves Paula M
Instituto de Tecnologia Química e Biológica-Universidade Nova de Lisboa/Instituto de Biologia Experimental e Tecnológica (ITQB-UNL/IBET), Apartado 12, 2781-901 Oeiras, Portugal.
Ann Anat. 2009 Jan;191(1):104-15. doi: 10.1016/j.aanat.2008.09.005. Epub 2008 Nov 1.
Adult pluripotent stem cells are a cellular resource representing unprecedented potential for cell therapy and tissue engineering. Complementary to this promise, there is a need for efficient bioprocesses for their large scale expansion and/or differentiation. With this goal in mind, our work focused on the development of three-dimensional (3-D) culture systems for controlled expansion of adult pancreatic stem cells (PSCs). For this purpose, two different culturing strategies were evaluated, using spinner vessels: cell aggregated cultures versus microcarrier technology. The use of microcarrier supports (Cytodex 1 and Cytodex 3) rendered expanded cell populations which retained their self-renewal ability, cell marker, and the potential to differentiate into adipocytes. This strategy surmounted the drawbacks of aggregates in culture which were demonstrably unfeasible as cells clumped together did not proliferate and lost PSC marker expression. Furthermore, the results obtained showed that although both microcarriers tested here were suitable for sustaining cell expansion, Cytodex 3 provided a better substrate for the promotion of cell adherence and growth. For the latter approach, the potential of bioreactor technology was combined with the efficient Cytodex 3 strategy under controlled environmental conditions (pH-7.2, pO2-30% and temperature-37 degrees C); cell growth was more efficient, as shown by faster doubling time, higher growth rate and higher fold increase in cell concentration, when compared to spinner cultures. This study describes a robust bioprocess for the controlled expansion of adult PSC, representing an efficient starting point for the development of novel technologies for cell therapy.
成体多能干细胞是一种细胞资源，在细胞治疗和组织工程方面具有前所未有的潜力。与此前景相补充的是，需要有高效的生物工艺来实现其大规模扩增和/或分化。出于这一目标，我们的工作聚焦于开发用于成年胰腺干细胞（PSC）可控扩增的三维（3-D）培养系统。为此，使用旋转培养瓶评估了两种不同的培养策略：细胞聚集培养与微载体技术。使用微载体支架（Cytodex 1和Cytodex 3）可得到扩增的细胞群体，这些细胞群体保留了自我更新能力、细胞标志物以及分化为脂肪细胞的潜力。这种策略克服了培养中聚集体的缺点，因为明显不可行的是，聚集在一起的细胞不会增殖并失去PSC标志物表达。此外，所获得的结果表明，尽管这里测试的两种微载体都适合维持细胞扩增，但Cytodex 3为促进细胞黏附和生长提供了更好的底物。对于后一种方法，在受控环境条件（pH - 7.2、pO2 - 30%和温度 - 37摄氏度）下，将生物反应器技术的潜力与高效的Cytodex 3策略相结合；与旋转培养相比，细胞生长更高效，表现为倍增时间更快、生长速率更高以及细胞浓度增加倍数更高。本研究描述了一种用于成年PSC可控扩增的强大生物工艺，是开发细胞治疗新技术的一个有效起点。
