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

Abstract

Immortalized 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, 2005) [Shi, L.Z., Zheng, W., 2005. Establishment of an in vitro brain barrier epithelial transport system for pharmacological and toxicological study. Brain Research 1057, 37-48]. 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. Western blot analyses revealed the presence of claudin-1, ZO-1, and occludin in Z310 cells. Transmission electron microscopy showed a "tight junction" type of structure in the sub-apical lateral membranes between adjacent Z310 cells. Real-time RT-PCR revealed that Z310 cells expressed representative transporters such as DMT1, MTP1, TfR, p-glycoprotein, ATP7A, ZnT1, ABCC1, Oat3, OCT1 and OB-Ra. Moreover, Z310 cells cultured in a two-chamber Transwell device possessed the ability to transport zidovudine (anionic drug), thyroxine (hormone), thymidine (nucleoside), and leptin (large polypeptide) with kinetic properties similar to those obtained from the in vitro model based on primary culture of choroidal epithelial cells. Taken together, these data indicate that the Z310 BCB model expresses major tight junction proteins and forms a tight barrier in vitro. The model also exhibits the ability to transport substances of various categories across the barrier.

Figure 1

Presence of claudin-1, ZO-1, and occludin proteins in Z310 cells by Western blot. Cell homogenates were immunoprecipitated and electrophoresized. Tissue extracts were directly electrophoresized. Results were not qualitative. (A). Claudin-1: lane 1, kidney extract as the positive control (40 μg); lane 2-3, Z310 cell immunoprecipitates. (B). ZO-1: lane 1-2, Z310 cell immunoprecipitates; lane 3, rat choroid plexus tissue extract as the positive control (40 μg). (C). Occludin: lane 1-2, Z310 cell immunoprecipitates; lane 3, liver extract as the positive control (40 μg).

Figure 2

Presence of “tight junction” type of structure between adjacent Z310 cells by TEM. Panel A illustrates a cell-to-cell contact site at confluence by high magnification (8000×). Z310 cells were grown on a collagen-precoated polyester filter membrane in the inner chamber of a Transwell apparatus and fixed with 2.5% glutaraldehyde. The arrowhead indicates a “tight junction” type of structure in the subapical region along adjacent lateral membranes. Panel B illustrates the presence of microvilli on apical surface of Z310 cells prior to the formation of monolayer barriers. The arrows indicate the microvillus structures on the surface of Z310 cells (1000×).

Figure 3

Transport kinetics of [¹⁴C]AZT efflux at BCB. Parallel sets of Transwells with similar TEER values (for Z310 model, TEER value was 90 ± 10 Ω.cm²; for primary CP model, TEER value was 70 ± 5 Ω.cm²) were used for either [¹⁴C]sucrose (A) or [¹⁴C]AZT (B). C_R/C_D represents the ratio of the chemical concentrations in the receiver chamber to that in the donor chamber. Linear ranges were decided by statistical analyses of linear model fitting as well as linear regression. The resulting slopes (ΔC_R/C_D·Δt) were used in Eq. 1 for calculation of permeability coefficients (P_B or P_T), the values of which are presented in Table 3.

Figure 4

Transport kinetics of [³H]thymidine and [¹²⁵I]thyroxine influx at BCB. Same sets of Transwells were used for co-transportf of [³H]thymidine and [¹⁴C]sucrose or of [¹²⁵I]thyroxine and [¹⁴C]sucrose transport studies. Panel A presents the transport of sucrose. Panel B and C depicts the transport kinetics of thymidine and thyroxine, respectively. The values of permeability coefficients are presented in Table 3.

Figure 5

Transport kinetics of [¹²⁵I]leptin influx at BCB. (A) [¹⁴C]inulin. (B) [¹²⁵I]leptin. Same sets of Transwells were used for both [¹²⁵I]leptin and [¹⁴C]inulin transport studies. The values of permeability coefficients are presented in Table 3.