Characterization of arachnoidal cells cultured on three-dimensional nonwoven PET matrix.

PURPOSE: To culture physiologically functional primary arachnoidal cells on a suitable polymer substrate for an in-vitro model of the cerebrospinal fluid outflow pathway. METHODS: Primary cultures of arachnoidal cells were prepared within 24 hours post-mortem from brain tissue obtained from human cadavers at autopsy. Arachnoidal cells were characterized using immunocytochemistry and seeded onto needle punched non-woven poly(ethylene terephthalate)(PET) scaffolds. Metabolic rate, cell growth rate in log phase, morphologic assessment, immunocytochemistry, and protein analysis were used to characterize the cultures in both 2-D and 3-D-culture. Functional outflow assessment was performed using the Lucifer Yellow (LY) permeability assay and hydraulic conductivity (Lp) determination. RESULTS: Cells cultured on PET scaffold grew slightly slower than cells grown in 2-D-culture as measured by metabolic rate and growth rate, however, they often formed sheets that bridged between the adjacent scaffold filaments forming many junctional protein connections. LY permeability coefficients of 2-D cells were compared with cells from scaffolds, and were not significantly different (p > 0.05) for both culture conditions. Average Lp of cells from 2-D-culture and 3-D-scaffolds were compared and shown not to be significantly different. CONCLUSION: Based on the biochemical and functional analysis, it has been shown that cells cultured on 3D-PET scaffolds retained the same properties as cells from 2D-culture plates.

Characterization of cytoskeletal and junctional proteins expressed by cells cultured from human arachnoid granulation tissue.

BACKGROUND: The arachnoid granulations (AGs) are projections of the arachnoid membrane into the dural venous sinuses. They function, along with the extracranial lymphatics, to circulate the cerebrospinal fluid (CSF) to the systemic venous circulation. Disruption of normal CSF dynamics may result in increased intracranial pressures causing many problems including headaches and visual loss, as in idiopathic intracranial hypertension and hydrocephalus. To study the role of AGs in CSF egress, we have grown cells from human AG tissue in vitro and have characterized their expression of those cytoskeletal and junctional proteins that may function in the regulation of CSF outflow. METHODS: Human AG tissue was obtained at autopsy, and explanted to cell culture dishes coated with fibronectin. Typically, cells migrated from the explanted tissue after 7-10 days in vitro. Second or third passage cells were seeded onto fibronectin-coated coverslips at confluent densities and grown to confluency for 7-10 days. Arachnoidal cells were tested using immunocytochemical methods for the expression of several common cytoskeletal and junctional proteins. Second and third passage cultures were also labeled with the common endothelial markers CD-31 or VE-cadherin (CD144) and their expression was quantified using flow cytometry analysis. RESULTS: Confluent cultures of arachnoidal cells expressed the intermediate filament protein vimentin. Cytokeratin intermediate filaments were expressed variably in a subpopulation of cells. The cultures also expressed the junctional proteins connexin43, desmoplakin 1 and 2, E-cadherin, and zonula occludens-1. Flow cytometry analysis indicated that second and third passage cultures failed to express the endothelial cell markers CD31 or VE-cadherin in significant quantities, thereby showing that these cultures did not consist of endothelial cells from the venous sinus wall. CONCLUSION: To our knowledge, this is the first report of the in vitro culture of arachnoidal cells grown from human AG tissue. We demonstrated that these cells in vitro continue to express some of the cytoskeletal and junctional proteins characterized previously in human AG tissue, such as proteins involved in the formation of gap junctions, desmosomes, epithelial specific adherens junctions, as well as tight junctions. These junctional proteins in particular may be important in allowing these arachnoidal cells to regulate CSF outflow.