Migration of CD34⁺ cells in mixed spheroids is dynamic.

<p>CB and BM CD34⁺ cells were co-cultured with simple or mixed spheroids for 24 hours. The supernatant was removed and the spheroids were washed and maintained in culture for more 48 hours. Phase contrast microscopy of CB CD34⁺ cells emigrating from mixed spheroids at 24 hours (A) and 48 hours (B). Number above scale bar represents the value (in micrometers) of both scale bars. Percentage of CB and BM CD34⁺ cells that migrated out from mixed and simple osteo-induced spheroids was determined by flow cytometry (E). Unlabeled cells were co-cultured with mixed spheroids for 24 hours and then the supernatants were removed and the spheroids were washed. CFSE labeled CD34⁺ cells were added to these spheroids and the co-cultures were maintained for an additional 48 hours. Representative FACS analysis showing CD34⁺ cells that were CFSE⁻ or CFSE⁺ in supernatants (C) and spheroids (D) after 48 hours of co-culture. (F) Histogram showing the percentage of CD34⁺ cells that were positive or negative for CFSE in the supernatant or spheroids after 24 (open bar) and 48 hours (grey bar). Data represent average percentages (± SEM) of CFSE⁺ and CFSE⁻ among CD34⁺ cells in one experiment with duplicates. Similar results were obtained in a second independent experiment.