Preadipocyte screening by laminin in porcine stromal vascular cell cultures.

Abstract

To determine if subpopulations of cells in stromal vascular (S-V) cultures could be segregated and separated based on affinity for laminin substratum. S-V cells were seeded and allowed to attach for various times; 4 hours was found to be optimal for cell attachment. Cultures were rinsed after 4 hours of seeding, and S-V cells were divided into three subpopulations based on affinity for laminin: (1) cells that did not attach to laminin; (2) cells that had a low affinity for laminin; and (3) cells that had a high affinity for laminin. After 24 hours, cultures were either stained for the AD-3 antigen (a marker for preadipocytes), C/EBP-alpha (a terminal differentiation marker), or C/EBP-delta (an early preadipocyte marker). Companion cultures were treated with various media for 9 days and stained with oil red-O. Cells with a high affinity for laminin had the highest proportion of AD-3 and C/EBP-alpha positive cells and the highest proportion of fat cells after treatment with insulin +/- dexamethasone. Cells with a low affinity for laminin had the highest proportion of C/EBP-delta cells and the highest proportion of fat cells after treatment with fetal bovine serum+dexamethasone, followed by insulin. These results indicate that differentiating preadipocytes adhere to laminin to a much greater degree than do non-preadipocytes. Therefore, laminin-coated dishes can be used to screen S-V cells to produce preadipocyte or fibroblast-enriched S-V cultures.