Rapid and Stable Formation Method of Human Astrocyte Spheroid in a High Viscous Methylcellulose Medium and Its Functional Advantages.

Astrocytes, a type of glial cell in the brain, are thought to be functionally and morphologically diverse cells that regulate brain homeostasis. Cell immortalization is a promising technique for the propagation of primary human astrocytes. The immortalized cells retain their astrocytic marker mRNA expression at lower levels than the primary cells. Therefore, improvement of the differentiation status is required. The use of a 3D formation technique to mimic structural tissue is a good strategy for reflecting physiological cell-cell interactions. Previously, we developed a spheroid formation method using highly viscous methyl cellulose (MC) medium. In this study, we applied this formation method to the well-established immortalized human astrocyte cell line HASTR/ci35. Stable HASTR/ci35 spheroids were successfully formed in MC medium, and laminin deposition was detected inside of the spheroids. Their functional markers were enhanced compared to conventional spheroids formed in U-bottom plates. The inflammatory response was moderately sensitive, and the ability to support neurite growth was confirmed. The HASTR/ci35 spheroid in the MC medium demonstrated the differentiation phenotype and could serve as a potent in vitro model for matured astrocytes.

Generation of a Human Conditionally Immortalized Cell-based Multicellular Spheroidal Blood-Brain Barrier Model for Permeability Evaluation of Macromolecules.

There is an urgent need for the development of brain drug delivery carriers based on middle-sized or macromolecules, to which in vitro blood-brain barrier (BBB) models are expected to contribute significantly through evaluation of BBB permeability. As part of efforts to develop such models, we have been working on human conditionally immortalized cell-based multicellular spheroidal BBB models (hiMCS-BBB models), and we herein introduce the model development protocol. Briefly, astrocytes are first seeded in an ultra-low attachment 3D cell culture plate, to make the central core (Day 0). Next, pericytes are added over the core, to form an outer layer (Day 1). Then, brain microvascular endothelial cells are further added to each well, to create the outmost monolayer serving as the BBB (Day 2). Finally, the spheroids cultured for two days (on Day 4) can be used for assays of interest (e.g., antibody permeability assays). Neither special equipment nor techniques are required to produce hiMCS-BBB models. Therefore, the protocol presented here will not only facilitate the model sharing among the BBB community but also provide some technical clues contributing to the development of similar MCS-BBB models using other cell sources, such as primary or iPS-derived BBB cells. Graphical abstract.

Human Immortalized Cell-Based Blood-Brain Barrier Spheroid Models Offer an Evaluation Tool for the Brain Penetration Properties of Macromolecules.

Blood-brain barrier (BBB)-permeable middle- or macromolecules (middle/macromolecules) have recently attracted significant attention as new drug delivery carriers into the human brain via receptor-mediated transcytosis (RMT). During the development process of such carriers, it is necessary to thoroughly evaluate their human BBB permeability levels. In such evaluations, our recently established human immortalized cell-based multicellular spheroidal BBB models (hiMCS-BBB models) have shown high potential. However, the specifics of those capabilities have yet to be elucidated. Therefore, in this study, we characterize the ability of the hiMCS-BBB models to evaluate RMT-mediated BBB penetration properties of middle/macromolecules. More specifically, we began by validating transferrin receptor (TfR)-mediated RMT functionalities using transferrin in the hiMCS-BBB models and then examined the BBB permeability levels of MEM189 antibodies (known BBB-permeable anti-TfR antibodies). The obtained results showed that, as with the case of transferrin, temperature-dependent uptake of MEM189 antibodies was observed in the hiMCS-BBB models, and the extent of that uptake increased in a time-dependent manner until reaching a plateau after around 2 h. To further expand the evaluation applicability of the models, we also examined the BBB permeability levels of the recently developed SLS cyclic peptide and observed that peptide uptake was also temperature-dependent. To summarize, our results show that the hiMCS-BBB models possess the ability to evaluate the RMT-mediated BBB-permeable properties of antibodies and peptides and thus have the potential to provide valuable tools for use in the exploration and identification of middle/macromolecules showing excellent BBB permeability levels, thereby contributing powerfully to the development of new drug delivery carriers for transporting drugs into the human brain.

Development, Characterization and Potential Applications of a Multicellular Spheroidal Human Blood-Brain Barrier Model Integrating Three Conditionally Immortalized Cell Lines.

In vitro blood-brain barrier (BBB) models are essential research tools for use in developing brain-targeted drugs and understanding the physiological and pathophysiological functions of the BBB. To develop BBB models with better functionalities, three-dimensional (3D) culture methods have gained significant attention as a promising approach. In this study, we report on the development of a human conditionally immortalized cell-based multicellular spheroidal BBB (hiMCS-BBB) model. After being seeded into non-attachment culture wells, HASTR/ci35 (astrocytes) and HBPC/ci37 cells (brain pericytes) self-assemble to form a spheroid core that is then covered with an outer monolayer of HBMEC/ci18 cells (brain microvascular endothelial cells). The results of immunocytochemistry showed the protein expression of several cellular junction and BBB-enriched transporter genes in HBMEC/ci18 cells of the spheroid model. The permeability assays showed that the hiMCS-BBB model exhibited barrier functions against the penetration of dextran (5 and 70 kDa) and rhodamine123 (a P-glycoprotein substrate) into the core. On the other hand, facilitation of 2-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)-2-deoxyglucose (2-NBDG; a fluorescent glucose analog) uptake was observed in the hiMCS-BBB model. Furthermore, tumor necrosis factor-alpha treatment elicited an inflammatory response in HBMEC/ci18 cells, thereby suggesting that BBB inflammation can be recapitulated in the hiMCS-BBB model. To summarize, we have developed an hiMCS-BBB model that possesses fundamental BBB properties, which can be expected to provide a useful and highly accessible experimental platform for accelerating various BBB studies.

A Human Immortalized Cell-Based Blood-Brain Barrier Triculture Model: Development and Characterization as a Promising Tool for Drug-Brain Permeability Studies.

Brain microvascular endothelial cells (BMEC), together with astrocytes and pericytes, form the blood-brain barrier (BBB) that strictly restricts drug penetration into the brain. Therefore, in central nervous system drug development, the establishment of an in vitro human BBB model for use in studies estimating the in vivo human BBB permeability of drug candidates has long been awaited. The current study developed and characterized a human immortalized cell-based BBB triculture model, termed the "hiBBB" model. To set up the hiBBB model, human immortalized BMEC (HBMEC/ci18) were cocultured with human immortalized astrocytes (HASTR/ci35) and brain pericytes (HBPC/ci37) in a transwell system. The trans-endothelial electrical resistance of the hiBBB model was 134.4 ± 5.5 (Ω × cm2), and the efflux ratios of rhodamine123 and dantrolene were 1.72 ± 0.11 and 1.72 ± 0.45, respectively, suggesting that the hiBBB model possesses essential cellular junction and efflux transporter functions. In BBB permeability assays, the mean value of the permeability coefficients (Pe) of BBB permeable compounds (propranolol, pyrilamine, memantine, and diphenhydramine) was 960 × 10-6 cm/s, which was clearly distinguishable from that of BBB nonpermeable compounds (sodium fluorescein and Lucifer yellow, 18 × 10-6 cm/s). Collectively, this study successfully developed the hiBBB model, which exhibits essential BBB functionality. Taking into consideration the high availability of the immortalized cells used in the hiBBB model, our results are expected to become an initial step toward the establishment of a useful human BBB model to investigate drug penetration into the human brain.

Differentiated HASTR/ci35 cells: A promising in vitro human astrocyte model for facilitating CNS drug development studies.

Astrocytes have shown longstanding promise as therapeutic targets for various central nervous system diseases. To facilitate drug development targeting astrocytes, we have recently developed a new conditionally immortalized human astrocyte cell line, termed HASTR/ci35 cells. In this study, in order to further increase their chances to contribute to various astrocyte studies, we report on the development of a culture method that improves HASTR/ci35 cell differentiation status and provide several proofs related to their astrocyte characteristics. The culture method is based on the simultaneous elimination of serum effects and immortalization signals. The results of qPCR showed that the culture method significantly enhanced several astrocyte marker gene expression levels. Using the differentiated HASTR/ci35, we examined their response profiles to nucleotide treatment and inflammatory stimuli, along with their membrane fatty acid composition. Consequently, we found that they responded to ADP or UTP treatment with a transient increase of intracellular Ca2+ concentration, and that they could show reactive response to interleukin-1ß treatments. Furthermore, the membrane phospholipids of the cells were enriched with polyunsaturated fatty acids. To summarize, as a unique human astrocyte model carrying the capability of a differentiation induction properties, HASTR/ci35 cells are expected to contribute substantially to astrocyte-oriented drug development studies.

Cancer-type organic anion transporting polypeptide 1B3 is a target for cancer suicide gene therapy using RNA trans-splicing technology.

Cancer-type organic anion transporting polypeptide 1B3 (Ct-OATP1B3) has been identified as a cancer-specific transcript in various solid cancers, including colorectal cancer. Given its excellent cancer-specific expression profile, we hypothesized that Ct-OATP1B3 could represent a promising target for cancer-specific expression of the suicide gene, herpes simplex virus 1 thymidine kinase (HSV-tk), via a spliceosome-mediated RNA trans-splicing (SMaRT) approach. SMaRT technology is used to recombine two RNA molecules to generate a chimeric transcript. In this study, we engineered an RNA trans-splicing molecule carrying a translation-defective HSV-tk sequence (RTM44), which was capable of inducing its own trans-splicing to the desired Ct-OATP1B3 pre-mRNA target. RTM44 expression in LS180 cells resulted in generation of Ct-OATP1B3/HSV-tk fusion mRNA. A functional translation start site contributed by the target pre-mRNA restored HSV-tk protein expression, rendering LS180 cells sensitive to ganciclovir treatment in vitro and in xenografted mice. The observed effects are ascribed to accurate and efficient trans-splicing, as they were absent in cells carrying a splicing-deficient mutant of RTM44. Collectively, our data highlights Ct-OATP1B3 as an ideal target for the HSV-tk SMaRT suicide system, which opens up new translational avenues for Ct-OATP1B3-targeted cancer therapy.

A New Conditionally Immortalized Human Fetal Brain Pericyte Cell Line: Establishment and Functional Characterization as a Promising Tool for Human Brain Pericyte Studies.

While pericytes wrap around microvascular endothelial cells throughout the human body, their highest coverage rate is found in the brain. Brain pericytes actively contribute to various brain functions, including the development and stabilization of the blood-brain barrier (BBB), tissue regeneration, and brain inflammation. Accordingly, detailed characterization of the functional nature of brain pericytes is important for understanding the mechanistic basis of brain physiology and pathophysiology. Herein, we report on the development of a new human brain pericyte cell line, hereafter referred to as the human brain pericyte/conditionally immortalized clone 37 (HBPC/ci37). Developed via the cell conditionally immortalization method, these cells exhibited excellent proliferative ability at 33 °C. However, when cultured at 37 °C, HBPC/ci37 cells showed a differentiated phenotype that was marked by morphological alterations and increases in several pericyte-enriched marker mRNA levels, such as platelet-derived growth factor receptor ß. It was also found that HBPC/ci37 cells possessed the facilitative ability of in vitro BBB formation and differentiation into a neuronal lineage. Furthermore, HBPC/ci37 cells exhibited the typical "reactive" features of brain pericytes in response to pro-inflammatory cytokines. To summarize, our results clearly demonstrate that HBPC/ci37 cells possess the ability to perform several key brain pericyte functions while also showing the capacity for extensive and continuous proliferation. Based on these findings, it can be expected that, as a unique human brain pericyte model, HBPC/ci37 cells have the potential to contribute to significant advances in the understanding of human brain pericyte physiology and pathophysiology.