Khademhosseini Ali, Ferreira Lino, Blumling James, Yeh Judy, Karp Jeffrey M, Fukuda Junji, Langer Robert
Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Biomaterials. 2006 Dec;27(36):5968-77. doi: 10.1016/j.biomaterials.2006.06.035. Epub 2006 Aug 9.
Human embryonic stem (hES) cells are generally cultured as cell clusters on top of a feeder layer formed by mitotically inactivated murine embryonic fibroblasts (MEFs) to maintain their undifferentiated state. This co-culture system, which is typically used to expand the population of undifferentiated hES cells, presents several challenges since it is difficult to control cell cluster size. Large cell clusters tend to differentiate at the borders, and clusters with different sizes may lead to heterogeneous differentiation patterns within embryoid bodies. In this work, we develop a new approach to culture hES cells with controlled cluster size and number through merging microfabrication, and biomaterials technologies. Polymeric microwells were fabricated and used to control the size and uniformity of hES cell clusters in co-culture with MEFs. The results show that it is possible to culture hES cells homogeneously while keeping their undifferentiated state as confirmed by the expression of stem cell markers octamer binding protein 4 (Oct-4) and alkaline phosphatase (ALP). In addition, these clusters can be recovered from the microwells to generate nearly homogeneous cell aggregates for differentiation experiments.
人类胚胎干细胞（hES细胞）通常作为细胞团簇培养在由有丝分裂失活的小鼠胚胎成纤维细胞（MEF）形成的饲养层上，以维持其未分化状态。这种共培养系统通常用于扩大未分化hES细胞群体，但由于难以控制细胞团簇大小，存在几个挑战。大的细胞团簇往往在边界处分化，不同大小的团簇可能导致胚状体内部出现异质分化模式。在这项工作中，我们通过融合微制造和生物材料技术，开发了一种新的方法来培养具有可控团簇大小和数量的hES细胞。制造了聚合物微孔并用于控制与MEF共培养时hES细胞团簇的大小和均匀性。结果表明，可以均匀地培养hES细胞，同时通过干细胞标志物八聚体结合蛋白4（Oct-4）和碱性磷酸酶（ALP）的表达证实保持其未分化状态。此外，这些团簇可以从微孔中回收，以产生几乎均匀的细胞聚集体用于分化实验。
