Background

The blood-brain barrier (BBB) forms a nearly impregnable wall preventing the distribution of numerous blood-borne middle-sized and macromolecules (middle/macromolecules) and low-molecular-weight drugs into the human brain (Sanchez-Cano et al., 2021; Segarra et al., 2021). As such, it often imposes a frustrating barrier to brain drug delivery efforts, thus rendering central nervous system (CNS) diseases among the most difficult to treat pharmaceutically. Brain microvascular endothelial cells (BMECs), which work with the support of pericytes and astrocytes, make up the critical components of the BBB. The pericytes are located in the immediate vicinity of the BMECs, while the astrocytes wrap them through their endfeet. This structural integrity is essential for BBB functions (Ballabh et al., 2004; Abbott et al., 2006; Daneman et al., 2015). The two primary functional characteristics of the BBB are tight/adherens junctions and efflux transporters. The former tightly seals BMEC plasma membranes to limit paracellular transport of various substances, including middle/macromolecules, while the latter are localized on the vascular side of BMECs and actively pump out their substrates into circulation (Ballabh et al., 2004; Abbott et al., 2010). Therefore, as part of efforts to create effective therapeutic approaches to CNS diseases, it is necessary to develop BBB-permeable drug delivery system (DDS) carriers that can overcome this barrier.

From that point of view, receptor-mediated transcytosis (RMT) occurring at the BBB has gained significant attention (Lajoie et al., 2015; Fang et al., 2017). Physiologically, after binding to their middle/macromolecule ligands at the blood side of BMECs, RMT receptors undergo endosomal vesicle formation to travel to the brain side, where they eventually release their cargo into the brain via exocytosis (Freskgård et al., 2017; Kouhi et al., 2021; Zhou et al., 2021). One intriguing idea for facilitating the development of brain DDS carriers involves taking advantage of those RMT processes, as exemplified by antibodies targeting transferrin or insulin receptors expressed in BMECs (Boado et al., 2016; Sonoda et al., 2018), resulting in an ongoing urgent need for effective and high-throughput evaluations of their BBB permeabilities. To this end, there have been a variety of next-generation in vitro BBB models based on microphysiological systems (MPSs), including BBB-on-a-chip, organoids, and spheroids (Cho et al., 2017; Wevers et al., 2018; Simonneau et al., 2021). These models feature high functionality derived based on their reproduction of the structure and surrounding microenvironment of the BBB in vivo (specifically, MPSs).

As part of efforts to facilitate the development of such models, we have been working on human conditionally immortalized cell-based multicellular spheroidal BBB models (hiMCS-BBB models) consisting of human conditionally immortalized BMECs (HBMEC/ci18 cells) (Kamiichi et al., 2012; Ito et al., 2019), astrocytes (HASTR/ci35 cells) (Furihata et al., 2016; Kitamura et al., 2018), and brain pericytes (HBPC/ci37 cells) (Umehara et al., 2018). These cells carry immortalization genes that enable them to extensively proliferate, while their status can be changed simply by varying the cell culture temperature (33 °C for growth and 37 °C for differentiation — for details, please refer to our above-cited previous papers).

In hiMCS-BBB models, HASTR/ci35 cells shape the central core, which is covered by an HBPC/ci37 cell layer. That layer is further surrounded by an HBMEC/ci18 cell monolayer serving as the BBB (Kitamura et al., 2021, 2022). This structural feature allows us to perform several BBB functional analyses, such as transporter activity assessments, and immune cell recruitments (Kitamura et al., 2021, 2022). In particular, the models exhibit RMT functions that are sufficiently high for use in evaluating the BBB permeability of antibodies and peptides (Kitamura et al., 2022). Additionally, the immortalized cells used are both scalable and easy to handle, and neither special equipment nor techniques are required to produce the models (as described herein). Owing to all these advantages, hiMCS-BBB models are expected to have great potential for use in developing novel middle/macromolecule-based DDS carriers.

In this paper, we describe the method used for constructing hiMCS-BBB models together with the culture methods for the human immortalized BBB cells. In alignment with that, we will also introduce an example of a middle/macromolecule permeability assay performed using hiMCS-BBB models. Through this publication, we aim not only to share hiMCS-BBB models among the BBB community but also to provide some technical clues that may contribute to the development of MCS-BBB models using other cell sources, such as primary or iPS-derived BBB cells, although some modifications may be necessary.