Perfusion of 3D culture enhances the phenotype of pluripotent stem cells in vitro, recapitulating aspects of the in vivo structure and phenotype of the teratocarcinoma. (a) Teratocarcinoma xenograft tumour derived from TERA2.cl.SP12 cells—teratocarcinomas derived from pluripotent stem cells display differentiated teratoma structures, neural rosette staining for Nestin and class III β tubulin (TUJ1) and cytokeratin 8-positive epithelial structures, and pluripotent embryonal carcinoma areas, which stain positively for Oct4. Scale bar is 100 µm. (b) Static culture—TERA2.cl.SP12 cells cultured in static conditions differentiate towards the neural lineage, but do not show the maintenance of any pluripotent areas. Scale bar is 50 µm. (c) Perfused culture—TERA2.cl.SP12 cells maintained in the perfused system showed both the maintenance of pluripotency and the formation of differentiated structures of both neural and epithelial identity, similar to the in vivo teratocarcinoma. This replicates both components of a germ cell tumour, notably aspects of the differentiated teratoma and undifferentiated stem cells in the embryonal carcinoma. Scale bar is 50 µm.