Establishment of an in vitro brain barrier epithelial transport system for pharmacological and toxicological study

Abstract

An immortalized Z310 murine choroidal epithelial cell line was recently established in this laboratory. The purposes of this study were (1) to investigate the presence of tight junction (TJ) proteins in Z310 cells and (2) to develop a Z310 cell-based in vitro brain barrier transport model. Real-time RT-PCR studies revealed that Z310 cells possess mRNAs encoding ZO-1, -2, and -3, claudin-1, -2, -4, and -8, occludin, and connexin-32. Confocal microscopic analyses confirmed the presence of claudin-1 and ZO-1 in Z310 cells at cell-cell contact sites. When Z310 cells were grown on a two-chamber Transwell device, the [14C]sucrose permeability coefficient and transepithelial electrical resistance (TEER) across the cell monolayer were 6 x 10(-4) cm/min and 61 omega-cm2, respectively. To improve the tightness of Z310 barrier, the cells were cultured in astrocyte-conditioned medium (ACM), or in the presence of eicosapentaenoic acids (EPA, 10 microM), epidermal growth factor (EGF, 100 ng/mL), or dexamethasone (1 microM) in the growth medium. Treatment with ACM, EPA, EGF and dexamethasone significantly increased the TEER by 33%, 38%, 40%, and 50% above controls, respectively. However, only dexamethasone significantly reduced [14C]sucrose paracellular permeability (-231% of controls). These data suggest that Z310 cells possess the TJ proteins. The presence of dexamethasone in the growth medium improves the tightness of Z310 cell monolayer to the level better than that of the primary culture of choroidal epithelial cells. The Z310 cell-based in vitro model appears to be suitable for transepithelial transport study of drugs and toxicants.

Use of Z310 cells as an in vitro blood–cerebrospinal fluid barrier model: Tight junction proteins and transport properties

“…1A, 1B, and 1C). The results are consistent with our previous real-time RT-PCR and immunocytochemistry results (Shi and Zheng, 2005). However, claudin-2 was not detected in Z310 cells by Western blot (data not shown), although the transcripts were previously found in a low expression level by real-time RT-PCR (Shi and Zheng, 2005).…”

“…A cell monolayer was usually formed 3-5 days after seeding. For Z310 cells, the culture medium was the complete growth medium containing 1 μM dexamethasone (Shi and Zheng, 2005). For primary choroidal epithelial cells, the culture medium was DMEM containing 10% FBS, antibiotics, and 10 ng/ mL EGF.…”

“…The formation of cell monolayer was judged by three criteria: (1) the cells formed a confluent monolayer without visible spaces between cells under a light microscope; (2) the height of the culture medium in the inner chamber had to be at least 2 mm higher than that in the outer chamber for at least 24 h; and (3) a constant TEER value across the cell layer was obtained (90 ± 10 Ω.cm 2 for Z310 cells and 70 ± 5 Ω.cm 2 for primary choroidal epithelia) (Shi and Zheng, 2005). The cultures that reached the confluence were used in the transport studies.…”

“…To circumvent these limitations, an immortalized rat choroidal epithelial cell line, namely Z310 cells, has been created in this laboratory (Zheng and Zhao, 2002). The cells appear to possess tight barrier characteristics when cells are cultured on a permeable membrane (Shi and Zheng, 2005). Studies using real-time reverse-transcriptase (RT) polymerase chain reaction (PCR) reveal the presence of mRNAs encoding tight junction proteins such as ZO-1, -2, and -3, claudin-1, -2, -4, and -8, occludin, and connexin-32.…”

“…

AbstractImmortalized rat choroidal epithelial Z310 cells have the potential to become an in vitro model for studying transport of materials at blood-cerebrospinal fluid barrier (BCB) (Shi and Zheng, Brain Research 1057:37-48, 2005). This study was designed to demonstrate the presence of tight junction properties in Z310 cells and the functionality of Z310 monolayer in transport of selected model compounds.

…”