Figure 4. Proliferation of ASCs within spheroids in serum free suspension culture.

(A) Light micrograph of an ASC spheroid grown in suspension culture in serum-free medium (HGFM-SF), with early evidence of polarization and directional growth. (B) H&E histology of the same spheroid demonstrates defined nuclei throughout a background of abundant ASC-derived extracellular matrix. (C) Fluorescent micrograph of the same section in (B) demonstrates a more fluorescent intense cell and matrix-dense ‘core’ relative to a growing ‘apical’ edge (toward the right of the spheroid). (D) Reveals a light micrograph of a section from the same spheroid that has been immunohistochemically stained for BrdU (brown nuclei), demonstrating viable, replicating ASCs within the center and edge of the structure. The spheroid was grown in serum-free medium for 8 days, and pulsed with BrdU for 24 hours prior to fixation and staining.