Assembly of Size Selective Multicellular Spheroids of Adipose-Derived Stem/Stromal Cells for Use in Regenerative Tissue Engineering: A Methods and Morphologic Study

for Abstract As regenerative scaffolds exhibit varying pore sizes, producing adipose-derived stem/stromal cell (ASC) spheroids of selective sizes to populate these pores may be helpful in autologous tissue engineering. Herein we present two protocols for the initial magnetic sorting of ASCs and the subsequent size selective assembly of ASC spheroids; as the critical quality attribute of ASC identity has been shown previously, only spheroid morphology will be studied. Paramagnetic microbeads (Pro-tein G ligand Dynabeads®, Thermo Fisher Scientific) were used to create single-core paramagnetic immunobeads (scPIBs) and dual core paramagnetic immunobeads (dcPIBs). The scPIBs were created by conjugating the paramagnetic microbeads to ASC-selective primary antibodies (mouse antihuman CD 44, CD73, CD90, CD105, BD Biosciences). The dcPIBs were created by initially conjugating the paramagnetic microbeads to secondary antibodies (mouse IgG) and then conjugating the secondary antibodies to the same ASC-selective primary antibodies. The scPIBs and dcPIBs were then admixed within 15 ml of fresh lipoaspirate respectively. The ASC-scPIBs and ASC-dcPIBs were then magnetically precipitated and subsequently cultured in low adherent conditions for five days. Within twenty minutes, scPIBs isolated an average of 1.2 million putative ASCs (8 x 104 cells per ml of lipoaspirate processed) and dcPIBs isolated an average of 1.3 million putative ASCs (8.7 x 104 cells per ml of lipoaspirate processed). Spheroids comprised of ASC-scPIBs were 19.3 µm (average, +/- 5 µm) and spheroids of the ASC-dcPIBs were 216.7 µm (average, +/- 25 µm). ASCs were magnetically precipitated from fresh lipoaspirate in twenty minutes and subsequently underwent self-assembly of small (approximately 20 µm) or large (approximately 220 µm) ASC spheroids over five days. These protocols may be useful in the rapid development of size-selectable ASC spheroids, which may be particularly useful for autologous toxicology, pharmacology, disease modeling, and tissue regeneration.