A novel and simple method for culturing pericytes from mouse brain

Abstract

Pericytes play critical roles in the development, maturation and remodeling of blood vessels, and in the central nervous system (CNS), evidence suggests that pericytes also regulate blood flow and form an integral part of the blood-brain barrier. The study of this important cell type has been hampered by the lack of any pericyte-specific marker and by the difficulty of culturing pericytes in adequate numbers to high purity. Here we present a novel yet simple approach to isolate and culture large numbers of pericytes from the mouse CNS that nevertheless leads to very pure pericyte cultures. In our method, vascular cells obtained from adult mice brains are cultured initially under conditions optimized for endothelial cells, but after two passages switched to a medium optimized for pericyte growth. After growing the cells for 1-2 additional passages we obtained a largely homogeneous population of cells that expressed the pericyte markers NG2, PDGF -receptor, and CD146, but were negative for markers of endothelial cells (CD31), microglia (Mac-1) and astrocytes (GFAP). Under these conditions, pericytes could be grown to high passage number, and were maintained highly pure and largely undifferentiated, as determined by antigen expression profile and low levels of -SMA expression, a marker of pericyte differentiation. Furthermore, switching the cells from pericyte medium into DMEM containing 10% FBS promoted -SMA expression, demonstrating that high passage pericytes could still differentiate. Thus, we provide an alternative approach to the culture of CNS pericytes that is easy to establish and provides large numbers of highly pure pericytes for extended periods of time. This system should provide others working in the pericyte field with a useful additional tool to study the behavior of this fascinating cell type.

Prostacyclin Prevents Pericyte Loss and Demyelination Induced by Lysophosphatidylcholine in the Central Nervous System

“…Primary Cultures of Pericytes and Vascular Endothelial Cells-Brain pericytes were prepared from cerebral cortices obtained from female rats 3 weeks postnatally as described previously with modifications (19). The cerebral cortices were digested in a mixture of 1 mg/ml collagenase type 2 (Worthington) and 6.7 g/ml DNase (Sigma) in Dulbecco's modified Eagle's medium (DMEM) for 90 min at 37°C.…”

Defining Vascular Stem Cells

“…However, in this study, validation of pericyte identity was done by immunofluorescence for smooth muscle actin (SMA), which is obviously not a pericyte-specific marker. In fact, a recent study even showed that SMA is a negative marker for pericytes [39], with additional reference to another study that stated: ''few freshly isolated pericytes express this antigen, but with time in culture, almost 100% of cells express a-SMA'' [40].…”

Pericytes in Chronic Lung Disease

“…In humans, pericytes are easily isolated from various tissues, including the lung, using fluorescence-activated cell sorting with the gating strategy CD45-/CD31-/CD56-, CD146 high [10,11]. In mice, pericyte isolation and/or characterization has generally relied on culture methods using cells isolated from the heart [12], ear [13] and brain [14], or through the use of transgenic mice in which pericytes are labelled in the kidney [15], skeletal tissue [16], liver [17], lung [18,19,20] and brain [21]. Fluorescence-activated cell sorting is a rapid method and yields pure populations, which is not possible using culture methods alone.…”