Three methods for allantois culture. Allantoises can be dissected from embryos from the HF to 4s stage and allowed to attach to glass, plastic, or filters for adherent cultures (top row), or they can be kept in suspension in rolling cultures (middle row) or hanging drops (bottom row). Asterisk indicates the proximal end of the allantois closest to the embryo. In adherent cultures the distal tip of the allantois attaches to the dish within 6 hours and a vascular plexus forms after 24 hours as indicated by staining with Flk1 (brown).31 Rolling cultures result in development of spheroids with 3 dimensional (3D) morphology, which can be stained whole as shown for Flk1 (blue) and VCAM (brown).19 Hanging drop cultures of allantoises also form 3D structures with a vascular plexus after 18 hours of culture. Image shows fluorescent staining of a whole mount allantois spheroid with α-SMA (red) and Pecam (green). After treatment with VEGF, the spheroid remodels into a single layer of Pecam-positive ECs surrounded by α-SMA–positive SMCs as shown in an optical section.111 Images a, b, and c are reprinted with permission from Downs et al,19 Arora et al,31 and Gentile et al,111 respectively.
