3.2. Co-Culture Models

To generate a model resembling more closely the in vivo scenario, possessing functional TJs, high TEER, and expression of specific transporters and enzymes, co-culture systems can be developed ^{[13][14][15][16][17]}[60,61,62,63,64]. To enhance the quality of BCSFB in vitro models, cell culture barrier models can be configured in complex co-culture systems, in which choroid plexus epithelial cells are grown on porous cell culture inserts alongside endothelial cells, mesenchymal (e.g., pericytes), and/or glial cells either cultivated on the bottom of a multi-well plate into which the insert is located (non-contact) or seeded on the opposite side of the inserts containing epithelial cells (leading to a so-called back-to-back contact co-culture).

3.3. 3D Cultures and Organoids

State-of-the-art three-dimensional (3D) culture systems, including matrix-based and matrix-free models, such as organoids and spheroids, have been recognized as the next advancement from static mono- and co-cultures. These 3D platforms with diverse features constitute promising alternatives to animal models and 2D cell culture systems in an in vitro tool to recapitulate the complex features of cerebral barriers ^[18][19][67,68]. In order to recapitulate the choroid plexus cytoarchitectural arrangement and a model resembling physiological conditions more closely, 3D cultures and organoids can be approached. Compared to other, less sophisticated in vitro counterparts, these models most accurately reflect the BCSFB properties and are valuable alternative tools to the use of animal subjects in CNS-oriented drug discovery programs.

3.3.1. 3D Explants and Cultured Cells in a Scaffold System

The simple design of appropriate cells cultured in a suitable scaffold or gel system, can be instrumental in the generation of high-throughput in vitro BCSFB models. In spite of few experimental experiences with the BCSFB, the currently available knowledge of BBB cells cultured in 3D platforms can be extended to the BCSFB field ^[20][21][22][69,70,71]. Three-dimensional explants of the choroid plexus can be generated from fragments of choroid plexuses, dissected from animals, human postmortem tissue, or human surgical samples cultured in suitable matrices, such as Matrigel^™ and other commercially available hydrogels ^[23][24][72,73]. Three-dimensional explant platforms of mouse, rat, and human choroid plexus have been well prepared and maintained in the culture ^{[23][25][26][27]}[72,74,75,76].

3.3.2. Organoids and Self-Organized 3D Models

Organoids, as defined by Huch et al., are 3D structures derived from either pluripotent stem cells or neonatal or adult stem/progenitor cells, in which cells spontaneously self-organize into properly differentiated functional cell types, and which recapitulates at least some function of the organ ^[28][77]. In the case of brain organoids, these accurate and versatile in vitro models reproduce several attributes of the brain barriers, including the expression of tight junctions, molecular transporters, and drug efflux pumps, and are critical tools for the study of brain barriers transport and the development of theranostics that can reach the CNS ^[29][30][78,79]. The potential to be scaled up to a high-throughput format, the ease of culture, and the miniature size nominate these multicellular organoids as robust, reliable, and predictive platforms to analyze and screen brain-penetrating compounds for the discovery of new and optimized treatment approaches for various neuropathologies. Organoids of the choroid plexus as in vitro research platforms, derived from human pluripotent stem cells (human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs)) recapitulate fundamental morphological and functional attributes of this organ ^[31][80]. These sophisticated models overcome the disadvantage of species-to-species differences and can improve theour understanding of the development/function of the human choroid plexus, which due to the lack of experimental access to this vital brain tissue, is still elusive.

3.3.3. Three-Dimensional Bioprinting Strategies

Three-dimensional bioprinting, as an additive manufacturing technology for modeling of user-defined biological samples, has emerged as a promising tool for the expansion of the BBB models. There is no known report of the in vitro 3D-bioprinted models of the BCSFB until now, but there are studied cases of the BBB ^{[32][33][34][35][36]}[84,85,86,87,88], which have the potential to be adapted by the researchers in the field of the BCSFB. In principle, major printing modalities of inkjet-based, extrusion-based, and light-assisted bioprinting can be exploited to establish models with a high level of heterogeneity and biomimicry, which possess great potential as drug screening platforms ^[32][84].

3.4. Dynamic Models and Microfluidic Platforms

To generate experimental conditions largely comparable to the in vivo environment, the impact of hydrostatic pressure and fluid-induced shear stress can be incorporated into in vitro model platforms. In the light of this phenomenon and with further technological advances, in addition to static models, dynamic systems and culture in microfluidic chamber devices have been evolved, in which a tunable shear stress is induced by a continuous flow of culture medium, in either pump-based or pumpless dynamic configurations ^[10][52]. Microfluidic device-based models, or so-called organ-on-a-chip systems, are dynamic models with a precisely controlled periodic physiological fluid flow that tend to augment the survival of three-dimensional cultures and organoid models, with improved nutrients/wastes and oxygen exchange, and more realistic dimensions and geometries. Microfluidics-based approaches, as next generation drug testing tools, rely on spatially resolved compartments joined together via microgrooves, allowing cell-to-cell interactions, precise control of the 3D cellular and extracellular matrix, and the flow of small amounts of fluids ^[18][67]. The integration of functional organ elements onto these structures enables the study of multi-organ interactions and dynamics of drug activities ^[37][90]. The rationale is that these emerging in vitro model platforms reconstitute the choroid plexus-mimetic microenvironment more accurately, which enables effective modeling of this tissue for (neuro)therapeutics development and research. Furthermore, the advancement in the 3D printing technology, nanofabrication, integrated sensors, and the versatility of human-derived stem cells have contributed largely to boost the field ^[38][39][91,92]. However, the system setup is sophisticated, time-consuming, and requires specialized equipment, significant resources, and technical skills compared to conventional static models, leading to low-throughput screening capabilities and hindering their broad application. Despite the widespread use of dynamic models, such as cone–plate apparatuses, microporous hollow fibers, and microfluidic-based devices in the generation of in vitro BBB models ^[40][41][93,94], their application and usefulness remain to be adapted for their BCSFB counterparts.

4. Available Cells

4.1. Cerebral Originating Cells

The application of human-origin primary cells as in vitro models is restricted due to ethical and technical issues. Therefore, small and large animals have been sources of primary choroid plexus epithelium for various studies. Primary cultures of choroid plexus epithelial cells have been established from various species, including mouse ^{[4][42][43][44][45]}[9,101,102,103,104], rat ^{[46][47][48][49][50]}[16,105,106,107,108], pig ^{[51][52][53][54][55][56][57][58][59][60][61]}[33,58,109,110,111,112,113,114,115,116,117], cow ^[62][63][118,119], sheep ^[64][65][120,121], rabbit ^[66][67][68][122,123,124], and non-human primates such as rhesus macaque ^[69][125]. Canine choroid plexus cells have been isolated as well, though being more challenging compared to other species ^[46][16]. Human primary choroid plexus epithelial cells can be obtained from aborted embryos, directly after surgical removal, or postmortem ^[3][70][71][3,126,127]. However, postmortem-derived human samples may have disturbed viability and functionality depending on the time elapsed after death and could be impacted by the health status of the subject in terms of infections, disorders, injuries, and medication history ^[3][72][73][3,128,129], and, due to limited applicability for functional assays and usual vectorial transport studies, are gradually abandoned by the researchers in the course of time. HCPEpiC from ScienCell Research Laboratories (also obtainable from other companies) are another human source for primary epithelial cells of the choroid plexus ^[74][75][130,131]. According to the manufacturer, further expansion of these cell populations for 15 doublings is guaranteed. However, multiple concerns are still inherited by this commercially available culture in terms of its origin, nature, and morphology [3]. The experimental procedure to isolate primary choroid plexus epithelial cells from different species and establish a pure cell culture devoid of contaminating cell types has been described comprehensively elsewhere and is not brought up here for the sake of brevity ^[4][76][77][9,132,133]. Principally, the freshly isolated primary cells retain the differentiated properties of the choroidal epithelium and exhibit a myriad of morphological- and biochemical-desired properties, yet the disadvantages are a small culture size, a limited proliferative potential, and a diminished viability over time after dissection. Therefore, prompt transfer to a suitable in vitro setting is crucial for establishing viable cultures ^[4][9]. Z310 and TR-CSFB cells, as rat immortalized cell lines carrying the simian virus 40 large T-antigen gene, are well-characterized choroid plexus epithelial cells that have found their way as suitable cells ^{[78][79][80][81][82][83][84][85]}[25,134,135,136,137,138,139,140]. TR-CSFB cells (five cell lines, TR-CSFB 1–5) exhibit a polygonal-shaped morphology comparable to primary cultured rat choroid plexus epithelial cells. Immunohistochemically, the TR-CSFB cell lines express TTR, apically located Na⁺, K⁺-ATPase, and the efflux transporters ABCB1/MDR1a, ABCC1/MRP1, and ABCG2/BCRP ^[86][141]. Murine choroid plexus carcinoma cell lines ECPC3 and ECPC4 are isolated from transgenic mice harboring the viral simian virus 40 large T oncogene under the transcriptional control of an intronic enhancer region from the human immunoglobulin heavy chain gene. These cells have shown acceptable stability in culture in a time span of a year ^[87][88][144,145]. The PCP-R cell line has been established based on primary porcine choroid plexus epithelial cells (PCPEC). The cell line exhibits a regular polygonal pattern, expresses junctional proteins, and develops morphologically dense TJs. When cultured on cell culture inserts, this cell line exhibits characteristic barrier properties of TEER above 600 Ω × cm² and a restricted permeability for macromolecular paracellular markers ^[89][90][143,148]. SCP, a sheep choroid plexus epithelial cell line, is another alternative option on hand ^{[8][91][92][93][94][95][96]}[13,149,150,151,152,153,154]. A variant of this ovine finite cell line (prepared from brain choroid plexus of Ovis aries) is listed in the American Type Culture Collection (ATCC) as cell line SCP No. CRL-1700^®, or as catalog number 89101302 in the European Collection of Authenticated Cell Cultures (ECACC).

4.2. Noncerebral-Based Cells (Surrogate Models)

Noncerebral-originating cells are considered surrogates in blood–CSF barrier modeling. Madin-Darby canine kidney (MDCK and MDCKII) cells, human colonic epithelial cell line Caco-2 cells, Ralph Russ canine kidney cells (RRCK), Lilly Laboratories Culture-Porcine Kidney 1 epithelial cells (LLC-PK1), and cells transfected with specific efflux transporters or pharmacologic targets can be employed as a surrogate cell-based model to assess the permeability of selected compounds in the presence and absence of overexpressed efflux transporters or to evaluate their mechanism of action and effectiveness ^{[97][98][99][100][101][102][103][104]}[159,160,161,162,163,164,165,166]. The spontaneously immortalized MDCK-MDR1 cell line expressing P-gp/ABCB1, MDCK2-ABCB1 cells expressing ABCB1, and LLC-PK1/BCRP cells expressing the efflux transporter ABCG2/BCRP are examples of surrogate cells, which can be used ^[105][167].

5. Models Validation Criteria

5.1. Barrier Morphology

Once confluent, an in vitro choroid plexus epithelial cell barrier represents a differentiated polarized morphology, whose ultrastructure can be assessed using standard imaging modalities, including immunofluorescence microscopy and TEM. The latter allows direct visualization of the cells, their organizational structure, and any possible defect or imperfection in the cellular monolayer. Nonetheless, only few studies have used morphology imaging of in vitro cellular barriers as a routine quality validation to assure a closeness to in vivo conditions ^[106][168].

5.2. Barrier Properties

The reproducible tightness of models can be ascertained by convenient measuring of the TEER. The TEER value is a well-acknowledged measure to appraise the ion permeability of cell layers, reflecting the passive conductance of the TJs to small inorganic electrolytes, and impedance analysis manifests the electrical capacitance of the barrier likewise. Since the TEER can be correlated to the amount and degree of complexity of functional TJs and the expression of microvilli and other membrane invaginations, a high TEER might reflect the resemblance to in vivo situations. As a non-invasive quantitative method, the TEER measurement provides the most selective approach towards evaluating barrier integrity ^{[59][107][108]}[115,169,170].

5.3. Exogenous Tracer Permeability

Exogenous tracers or inert paracellular flux markers compatible with analytical conditions can provide beneficial information on the permeability status of model barriers towards lipid insoluble organic compounds. However, one should be aware of potential side effects and avoid unnecessary interactions of the tracers with experimental elements. Exogenous tracers come in various physicochemical properties, ranging from proteins and polysaccharides to small polar compounds (such as, but not limited to, mannitol, sucrose, inulin, and dextran) ^[109][171]. Most routinely used protein markers are purified plasma proteins such as bovine albumin (~66 kDa), human albumin (~66 kDa), bovine fetuin (~49 kDa), and horseradish peroxidase (40 kDa). It should always be noted that the choice of the protein marker relies upon the biological process being studied and the experimental design. Still, it should be antigenically distinguished from the cells under study. A wide selection of dextran conjugates in a diverse molecular weight range (MW 3, 10, 40, 70, 150, 500, and 2000 kDa) is also available. Owing to satisfactory water solubility, low toxicity, limited immunogenicity, and biologically inertness, dextrans are among the most popular exogenous tracers used to determine barrier tightness.

5.4. Functional Junctional Proteins and Transporters

Analysis of the expression and localization of junctional proteins and relevant carriers/transporters and their accordance with the choroidal epithelium in vivo can reveal information regarding the quality index of the model. Depending on the individual experimental goal, the characterization of specific markers, enzymes, receptors, adhesion molecules, and specific proteins/polypeptides, can be performed. Immunostaining of cell-type-specific markers and junctional molecules leads to a qualitative confirmation of barrier integrity of an epithelial monolayer. To assess the efflux transporters’ activity, substrate accumulation assays are exploited. Various efflux inhibitors (such as cyclosporin A and MK-571) are used to perform such substrate accumulation assays, assuming that substrate-inhibitor pairs are appropriately matched for each efflux transporter. In this assay configuration, model cells are incubated with the desired substrate either with or without their respective inhibitors, and at the culmination of the experiment, normalized fluorescence of the cells is calculated. In the case of the functional expression of transporters, the uptake of substrate should be increased in the presence of a corresponding transporter inhibitor ^[110][178]. Directional transport: in another experiment variation, transporter activities can be evaluated using directional transport assays, in which inhibitors are only added on the side of the cells grown on cell culture inserts where directional transport is being assessed. Here, again, model cells are incubated with the desired substrate either with or without their respective inhibitors, and the fluorescence signal on the opposite filter side is measured. If efflux transporters are expressed functionally at apical or basolateral surfaces, then the directional transport of substrate in the presence of the corresponding inhibitor is enhanced ^[110][178].

5.5. Factors Critical to Cell Selection and Culture Conditions

Serum withdrawal also seems to impact choroid plexus cells to reach the full barrier function. Serum deprivation can tighten the epithelial monolayer and improve the cellular polarity ^[76][111][132,179]. However, the barrier-dismantling effect of serum is believed to be compartment-specific, meaning that it depends on the apical or basolateral exposure of the cell layer to serum. In the case of serum exposure to the apical (comparable to CSF side in vivo) surface of the choroid plexus epithelial cell monolayer, the barrier function is diminished vastly. On the other hand, when serum is applied to the basolateral (corresponding to the stroma/blood side in vivo) surface, the epithelial barrier function is scarcely affected ^[51][61][33,117]. Due to the fact that barrier properties of epithelia are modulated by cAMP-dependent pathways, the presence of membrane-permeable cAMP analogs such as 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) or the adenylate cyclase activator forskolin can have an augmenting effect on TEER values of choroid plexus epithelial cell layers ^{[51][112][113]}[33,180,181]. Corticosteroids (hydrocortisone, dexamethasone) application to in vitro models may increase the barrier tightness of the epithelial and endothelial cells monolayer. This phenomenon is proposed to be a consequence of the regulation of the expression and distribution of tight junction proteins upon corticosteroids treatment ^[114][182]. Dexamethasone, as a synthetic glucocorticoid, has been shown to improve barrier strength and can be exploited as a positive control to investigate the effect of various conditions on the barrier integrity ^{[115][116][117]}[183,184,185].

6. Applications in (Neuro)Therapeutics Development and Research

6.1. Permeability Screenings and Studies

Any rational drug discovery project dealing with candidates targeted to the brain or requiring exclusion from the CNS to prevent possible side effects should contemplate permeability measurements and prediction studies. Hence, profiling brain/CSF permeability of investigational novel molecular species at preliminary stages of the drug development track is game-changing. To this end, models can predict the CNS permeability of novel or known compounds in relation to both the route and rate.

6.2. Transport Mechanisms Studies and (Targeted)Drug Delivery

Thanks to their physicochemical nature, hydrophobic/lipophilic substances of a low molecular weight are capable of free diffusion across cell membranes. In contrast, as mentioned in previous sections, the unrestricted diffusion of hydrophilic chemical species is substantially hindered due to the effective closure and sealing of the paracellular shunt by TJs. Accordingly, access to the CSF is granted exclusively to those compounds that are transported actively by the corresponding transport systems in the plasma membrane of choroid plexus epithelial cells. Theoretically, therapeutic- and diagnostic agents that are effectively and successfully transported by the choroid plexus and remain to some extent unaffected by the metabolizing enzymes and efflux transporters are rapidly distributed throughout the CNS using the bulk flow of CSF. This is due to the fact that at the brain ventricles, the extracellular/interstitial fluid (ISF) and the CSF are separated from each other by the non-barrier/non-restrictive permeable layer of ependymal cells, leading to a direct continuity of these two components and the subsequent free exchange of substances within the extracellular space of CNS ^[118][193]. Models expressing the specific transporters found in choroid plexus epithelium could be harnessed to establish whether the permeation of a compound of interest is impacted by a specific carrier system (e.g., P-gp) and to provide comprehensive knowledge on the physiology and modulation of such transporters. From another point of view, several neurological disorders are assumed to be associated with the dysfunction of transporters in the brain, and the detailed region-specific knowledge of transporters and their interaction with investigational therapeutics can prove helpful. In the case that transporters govern the permeability or uptake of a test compound, Michaelis–Menten kinetics can be obtained using nonlinear regression analysis of the concentration dependence influx. In vitro models have proven advantageous and constructive in studies of thyroxine ^[119][194], leptin ^[120][195], taurine ^[68][124], ascorbic acid ^[56][112], creatinine ^[121][196], and the neuroactive flavonoid resveratrol ^[122][197] transport across epithelial cells of choroid plexus. The in vitro BCSFB models have also proven helpful in library screening and identification of cell specific penetrating peptides for the choroid plexus epithelium using phage display techniques ^[27][123][76,198]. Therapeutic and diagnostic agents can pass the BCSFB via the transcellular route by employing one of the possible pathways, which are passive diffusion, facilitated diffusion, or vesicular transfer or transcytosis mechanisms. Generally, cellular uptake mechanisms based on endocytosis/transcytosis are the preferred cell entry route for many compounds. The endocytosis process can be categorized into two broad divisions of phagocytosis and pinocytosis. While the former is restricted to specialized cell types, the latter occurs in all cell types and can be subdivided into macropinocytosis, clathrin-dependent endocytosis (CDE), and clathrin-independent endocytosis (CIE) ^[124][199]. Contrary to endocytosis, transcytosis mechanisms are not well understood. Endocytosis and transcytosis across choroid plexus epithelium, studied at the subcellular level, can shed light on mechanisms of targeted delivery of therapeutics across this target site. These studies may rely on endocytosis inhibitors (including, but not limited to, chlorpromazine, genistein, methyl-β-cyclodextrin, and potassium depletion) to provide information concerning the endocytic pathway of compounds under study.

6.3. Metabolites/Xenobiotics Transport(er) Regulation

The fate of drugs and xenobiotics metabolites, affiliated to several chemical classes, generated at choroidal epithelial cells and the relative contribution of various efflux pumps present at this barrier can also be investigated using these in vitro models. In addition, the potential effect of multiple metabolites, xenobiotics, and compounds on both influx into and efflux out of the CSF can be studied. Referring to static bicameral device models, efflux transport assays can be performed, and the efflux ratio is estimated by dividing the permeability value in the apical to basolateral (A−B) direction by the permeability value in the basolateral to apical (B−A) direction. With respect to efflux transport assays, the transport of the compounds under study is assessed in the presence of inhibitors of relevant transporter proteins and compared to the non-inhibited conditions. Namely, verapamil, GF120918, PSC833, and N-desmethyl-loperamide as inhibitors of ABCB1/P-gp, Ko143, and fumitremorgin C as an inhibitor of ABCG2/BCRPs, tariquidar and elacridar as inhibitors of both P-gp and BCRP, or MK571 as an inhibitor of various MRPs are examples that can be applied for such experiments ^[125][45]. As an alternative strategy, the inhibition of these efflux transporters potentially improves the pharmacokinetics of CNS therapeutic candidates. To this end, the efficacy of a range of compounds (mainly phytoestrogens) as modulators of BCRP/ABCG2 has been evaluated using in vitro BCSFB models ^[126][227]. Accordingly, genetic manipulation approaches, such as overexpression, knock-out, or knock-down of specific transporters can be approached.

6.4. In Vitro Molecular Verification of Pharmacological Activity

One of the main thrusts behind the development of in vitro BCSFB models has been to shed light on the pharmacological molecular mechanisms of compounds toward this interface. Using cell-based BCSFB models, in vitro activity, potency, and mechanisms of actions of investigational CNS therapeutics and compounds under study can be demonstrated in preclinical drug evaluations. Excerpts of case studies from distinct pharmacological categories describe investigations of the anti-inflammatory activity of synthetic matrix metalloproteinase (MMP) inhibitors ^[43][102], and events of receptor activation for drugs of potential abuse and hallucinogens have been studied ^[127][228].

6.5. (Neuro)Toxicological Studies

Harnessing appropriate in vitro BCSFB models, the ever-increasing concern regarding hazardous compounds, such as environmental toxins, metals, pesticides, solvents, as well as the broad field of study of neurotoxicants, can be the target of both exposure (amounts permeated) and impact (effects exerted) investigations. The models have proven valuable in evaluating the influx/efflux of (neuro)toxins, understanding the associated neuroprotective mechanisms, elucidating the alteration of the BCSFB function by toxic compounds, and the involvement of the BCSFB in neuroinflammation ^[128][59].

6.6. Pharmacological Interventions at the BCSFB

Transport data acquired through pharmacological interventive measures and modulation by hormones and drugs contribute to understanding the BCSFB homeostatic phenomena and barrier/transporting functions. In addition, establishing approaches for restoring aberrant choroid plexus and CSF dynamics during disease has been of great interest to researchers in pharmacological, neurological, and neurosurgical fields.

6.7. The BCSFB and Choroid Plexus as a Drug Target in Various Diseases

Choroid plexus breakdown has been proposed and hypothesized in a wide range of neurological conditions, including aging, Alzheimer’s disease, Parkinson’s disease, epilepsy, stroke, neoplasms, perhaps psychiatric disorders, intracranial hypertension, and certainly varying types of hydrocephalus. Therefore, targeting this tissue may offer opportunities to translate neurotherapeutics from the lab to the clinic, and provide insights toward rational drug therapy to restore barrier function ^{[129][130][131][132]}[261,262,263,264].