Presence of macrophages within cardiac fibroblast cultures and methods for their removal

Abstract

Many assessments of cardiac fibroblast (CFb) culture purity are based mainly on positive immunostaining for vimentin (vim) (or α smooth muscle actin [SMact] for myofibroblasts [myoFb]) and negative staining for endothelial cells. However, macrophages (MΦ) reside within normal cardiac tissues and can differentiate from activated monocytes. Incomplete blood removal and rapid (Ca++-independent) adherence to tissue culture plastic may allow MΦ to preside in CFb cultures. Furthermore, MΦ can also express vim and SMact and can exhibit fibroblast-like morphology and mobility in culture. The objective of this study was to characterize MΦ levels in CFb cultures from adult mouse hearts and to develop methods for improving CFb culture purity. Hearts were retrograde perfused to remove excess blood and enzymatically (Liberase Blendzyme IV) digested. Myocytes were removed by low-speed centrifugation. The presence of MΦ within CFb-containing fractions was evaluated by RT-PCR after 30-min pre-plating in cold Ca++Mg++-free buffer containing 2mM EDTA and 0.5% BSA and/or anti-CD11b magnetic cell sorting. Expression levels of MΦ scavenger receptor 1 (MSR1) and CD68 (expressed in tissue MΦ) were each reduced by at least 5-fold in the CFb fractions after pre-plating. After CD11b sorting, these values were reduced by at least 10-fold. These results demonstrate that MΦ are present in CFb isolations and provide two approaches for reducing their numbers. Since MΦ may modulate myoFb differentiation and express many proteins in common with CFb, their presence in CFb cultures may complicate the interpretation of studies of CFb function. Supported in part by a NSF Fellowship and an AHA grant.