title: Enzymatic digestion improves the purity of harvested cerebral microvessels.
abstract: The harvest of intact cerebral microvessel yields could permit the in vitro characterization of mechanisms that underlie numerous vascular-linked central nervous system (CNS) phenomena. Here, we test (1) the effect of mild enzyme digestion on microvessel purity and yield; and then (2) the effect of variable centrifugation and filtration methods on microvessel yields. The brains of female Sprague-Dawley rats (4 weeks-old; n=38) were removed rapidly and homogenized. In Experiments 1 and 2, brain homogenates were incubated in DMEM or a solution of papain (2.5 U/ml), DNAse I (250 U/ml) and dispase II (1 U/ml) in DMEM for 15 min at 37 °C before microvessels were purified using differential (20% Ficoll) and then discontinuous (15/20% Dextran) centrifugation (@3500 × g) and collected with glass bead column filtration. Enzymatic digestion decreased microvessel yields (27 vs. 12 k/g tissue; p=0.053) but increased microvessel purity by decreasing adherent cells (p=0.002), which included NF-L(+) neurons (p<0.05) and GFAP+ astrocytes (p<0.001) and astrocyte endfeet (p<0.01). After one week in culture, >85% of harvested cells morphologically resembled microvessels and expressed the vascular proteins lectin and/or RECA-1. Finally, microvessels yields decreased when discontinuous centrifugation was omitted or nylon mesh filtration was employed. In summary, we found that digesting brain homogenates enzymatically could improve the purity of harvested microvessels that could be cultured for at least a week.
authors.affiliation: J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL, USA.
authors.forename: Vikram V
authors.initials: VV
authors.lastname: Munikoti
authors.forename: Lan B
authors.initials: LB
authors.lastname: Hoang-Minh
authors.forename: Brandi K
authors.initials: BK
authors.lastname: Ormerod
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002540:Cerebral Cortex / Q000098:blood supply*; D005260:Female; D007150:Immunohistochemistry; D055806:Microvessels; D051381:Rats; D017207:Rats, Sprague-Dawley; D020858:Tissue and Organ Harvesting / Q000379:methods*