Enzymatic digestion improves the purity of harvested cerebral microvessels

• Published in: Journal of neuroscience methods 2012-05, Vol.207 (1), p.80-85
• Main Authors: Munikoti, Vikram V., Hoang-Minh, Lan B., Ormerod, Brandi K.
• Format: Article
• Language: English
• Subjects: Adherent cells; Animals; Brain; Cell Culture Techniques - methods; Central nervous system; Centrifugation; Cerebral Cortex - blood supply; Deoxyribonuclease; Dextran; Digestion; Enzymes; Female; Filtration; Fractionation; Glass bead column; Glass beads; Immunohistochemistry; Lectin; Lectins; Microvessels; Nervous system; Neurons; Nylon; Papain; Rats; Rats, Sprague-Dawley; RECA-1; Tissue and Organ Harvesting - methods
• ISSN: 0165-0270; 1872-678X
• DOI: 10.1016/j.jneumeth.2012.03.011

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 (2.5U/ml), DNAse I (250U/ml) and dispase II (1U/ml) in DMEM for 15min 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. 12k/g tissue; p=0.053) but increased microvessel purity by decreasing adherent cells (p=0.002), which included NF-L+ neurons (p