Establishment and characterization of an immortalized Z310 choroidal epithelial cell line from murine choroid plexus

Abstract

The choroid plexus plays a wide range of roles in brain development, maturation, aging process, endocrine regulation, and pathogenesis of certain neurodegenerative diseases. To facilitate in vitro study, we have used a gene transfection technique to immortalize murine choroidal epithelial cells. A viral plasmid (pSV3neo) was inserted into the host genome of primary choroidal epithelia by calcium phosphate precipitation. The transfected epithelial cells, i.e., Z310 cells, that survived from cytotoxic selection expressed SV40 large-T antigen throughout the life span, suggesting a successful gene transfection. The cells displayed the same polygonal epithelial morphology as the starting cells by light microscopy. Immunocytochemical studies demonstrate the presence of transthyretin (TTR), a thyroxine transport protein known to be exclusively produced by the choroidal epithelia in the CNS, in both transfected and starting cells. Western blot analyses further confirm the production and secretion of TTR by these cells. The mRNAs encoding transferrin receptor (TfR) were identified by Northern blot analyses. The cells grow at a steady rate, currently in the 110th passage with a population doubling time of 20-22 h in the established culture. When Z310 cells were cultured onto a Trans-well apparatus, the cells formed an epithelial monolayer similar to primary choroidal cells, possessing features such as an uneven fluid level between inner and outer chambers and an electrical resistance approximately 150-200 omega-cm(2). These results indicate that immortalized Z310 cells possess the characteristics of choroidal epithelia and may have the potential for application in blood-CSF barrier (BCB) research.

Fig. 1

Time course of pSV3neo-transfected murine choroidal epithelial cells. The primary choroidal epithelial cells were transfected with the plasmid pSV3neo and plated in the presence of cytotoxic agent G418. (A) Primary culture of choroidal epithelial cells. (B) Cell death of the majority of transfected cells 3 days following addition of G418 in culture medium. The cells floating on the surface (with a strong light reflection) were either dead or detached cells. (C) Appearance of the single colony of immortalized cells 2 weeks after G418 selection. (D) Expansion of the survived cells. The single colony was selected and expanded to yield independent choroidal epithelial cell line (Z310) at the 50th passage (×100). Note: the cells maintain the same polygonal cell type even at the 110th passage.

Fig. 2

Immunocytochemical staining of SV40 large T antigen in cultured Z310 cells. The cells were cultured on a glass coverslip and incubated with monoclonal anti-SV40 large T antigen antibody, followed by reaction with biotinylated goat anti-mouse IgG secondary antibody. The positively stain of SV40 large T was seen in nuclei (×200) at the 52nd passage.

Fig. 3

Growth patters of immortalized Z310 cells. (A) Cumulative growth curve following transfection with pSV3neo. The growth ‘crisis’ was seen between the 23rd and 26th passages. (B) Normal growth curves of Z310 cells at the 70th passage. (C) Normal growth curve of the primary choroidal cells.

Fig. 4

Immortalized choroidal Z310 cells possess cytosolic TTR by immunocytochemical staining. The cells were cultured on a glass coverslip and incubated with rabbit anti-rat TTR antiserum, followed by reaction with biotinylated secondary antibody. (A) Cultured Z310 cells show the positive stain for cytosolic TTR (×250). (B) Primary culture of rat choroidal epithelial cells was used as the positive control (×250).

Fig. 5

Western blot analyses of TTR in immortalized choroidal epithelial cells. Cytosolic proteins from the cells of selected colonies, choroid plexus tissues, and liver homogenates, as well as proteins in culture media, were electrophoresized on 8.5% SDS–polyacrylamide gel, followed by transferred on to a PVDF membrane. Immunoblots resulting from the reaction with anti-rat TTR antibody were visualized by ECL luminescence method. TTR migrates with apparent MW of 55 KDa. Lanes: (1) rat TTR standard; (2) primary cultured plexus cells; (3): clone 3-2 (developed into Z310 cell line); (4): clone 1–2; (5) culture medium for clone 3-2; (6) culture medium for clone 1–2; (7) choroid plexus tissues; (8) liver cytosolic fractions.

Fig. 6

Expression of TfR mRNA in immortalized choroidal epithelial cells by Northern blot analyses. Total cellular RNA was extracted from the cells of selected colonies, electrophoresed on 1.5% agarose gels, and hybridized with random-primed ³²P-labeled TfR cDNA probe. Lanes (1,2) Z310 cells; (3,4) primary choroidal cells.

Fig. 7

Paracellular passage of [¹⁴C]sucrose by immortalized Z310 cells or primary epithelial cells. Z310 cells or primary epithelial cells were cultured on Transwell-COL membranes in the inner chamber to confluence. [¹⁴C]sucrose was added into the outer chamber to a final concentration of 1 µM. Data represent means±S.D. (n = 3–5).