Method for Primary Epithelial Cell Culture from the Rat Choroid Plexus

Background

The choroid plexus produces cerebrospinal fluid (CSF) and maintains chemical homeostasis in the extracellular fluid of the central nervous system. The choroid plexus is comprised of epithelial cells, connective tissue and blood vessels, and this structure is localized in all ventricular regions, including within the lateral, third and fourth ventricles. At each of these sites, the choroid plexus appears to differ relative to structure, function and factors produced and released into the ventricles (Johanson et al., 2005; Lun et al., 2015; Lallai et al., 2019). This tissue actively produces and secretes various signaling molecules, such as growth hormones, transthyretin and transferrin, into the brain and has been implicated in a wide range of functions related to brain development, aging, nutrient transport, endocrine regulation, and pathogenesis of neurodegenerative disorders. As such, dysfunction in the choroid plexus would potentially alter CSF composition and compromise brain health. As the field progresses, the vital function of factors derived from the choroid plexus will certainly continue to emerge, and these advances may then provide a foundation for novel approaches to treat neuropathology in humans.

Previous in vitro approaches to examine choroid plexus function have derived choroidal epithelial cell culture from rat, porcine, human and immortalized murine cells (Zheng and Zhao, 2002; Monnot and Zheng, 2013; Tenenbaum et al., 2013; Delery and MacLean, 2019). However, several considerations have been noted that limit the usefulness of prior techniques. First, immortalized cell lines do not appear to retain the properties of choroid plexus epithelial cells (CPEC) since they become very susceptible to changes in morphology and property in vitro (Angelow et al., 2004). Along these lines, we have found highly variable expression of the choroid plexus specific marker transthyretin across passages in the cell lines Z310 (obtained from Dr. Wei Zheng, Purdue University) and HCPEpiC (commercially available from ScienCell Research Laboratories), suggesting altered protein expression in the immortalized state compared to primary-derived tissues (Lallai and Fowler, unpublished findings). Next, since the location of the epithelial cells used to derive the cell lines may be from one or multiple ventricular locations, and given that different transcript expression and function is found in choroid plexus tissue among the ventricular locations (Lun et al., 2015; Lallai et al., 2019), the immortalized cell lines may not be representative of the specific subregion of the tissue of interest. Indeed, the prior approaches have focused on choroid plexus dissection solely derived from the lateral and/or fourth ventricular locations (e.g., as opposed to the smaller third ventricle), have combined choroid plexus from multiple locations into one sample for analysis, or have focused on species with larger yields of choroid plexus tissue (e.g., primate, porcine and human).

To overcome these challenges, we developed a modified protocol to derive primary culture of choroidal epithelial cells from rodents, which allows one to discern between choroid plexus tissue from third, lateral and fourth ventricles (Lallai et al., 2019). With this approach, we have been able to investigate the release of factors into the cell culture medium, thereby mimicking the physiological release of factors and vesicles into CSF of the brain. Of note, the current protocol also uses exosome-depleted fetal bovine serum (FBS) to allow for examination of extracellular vesicles released from the choroid plexus cells in culture conditions; this condition has been incorporated since non-depleted FBS has been shown to contain a variety of extracellular vesicles containing bovine-derived proteins, RNA and DNA, which may contaminate analyses and conclusions (Wei et al., 2016; Kornilov et al., 2018). Moreover, the cultures obtained from this protocol provide cells with distinct structural characteristics of epithelial cells and express the choroid plexus specific protein transthyretin (Lallai et al., 2019). In the following sections, we describe the methods to discretely dissect choroid plexus tissue from different ventricles and generate primary culture choroidal epithelial cells from rat. Further, recommendations and troubleshooting tips are also provided.