Live functional assays reveal longitudinal maturation of transepithelial transport in kidney organoids.

Kidney organoids derived from hPSCs have opened new opportunities to develop kidney models for preclinical studies and immunocompatible kidney tissues for regeneration. Organoids resemble native nephrons that consist of filtration units and tubules, yet little is known about the functional capacity of these organoid structures. Transcriptomic analyses provide insight into maturation and transporter activities that represent kidney functions. However, functional assays in organoids are necessary to demonstrate the activity of these transport proteins in live tissues. The three-dimensional (3D) architecture adds complexity to real-time assays in kidney organoids. Here, we develop a functional assay using live imaging to assess transepithelial transport of rhodamine 123 (Rh123), a fluorescent substrate of P-glycoprotein (P-gp), in organoids affixed to coverslip culture plates for accurate real-time observation. The identity of organoid structures was probed using Lotus Tetragonolobus Lectin (LTL), which binds to glycoproteins present on the surface of proximal tubules. Within 20 min of the addition of Rh123 to culture media, Rh123 accumulated in the tubular lumen of organoids. Basolateral-to-apical accumulation of the dye/marker was reduced by pharmacologic inhibition of MDR1 or OCT2, and OCT2 inhibition reduced the Rh123 uptake. The magnitude of Rh123 transport was maturation-dependent, consistent with MDR1 expression levels assessed by RNA-seq and immunohistochemistry. Specifically, organoids on day 21 exhibit less accumulation of Rh123 in the lumen unlike later-stage organoids from day 30 of differentiation. Our work establishes a live functional assessment in 3D kidney organoids, enabling the functional phenotyping of organoids in health and disease.

Bioengineered Kidney Models: Methods and Functional Assessments.

Investigations into bioengineering kidneys have been extensively conducted owing to their potential for preclinical assays and regenerative medicine. Various approaches and methods have been developed to improve the structure and function of bioengineered kidneys. Assessments of functional properties confirm the adequacy of bioengineered kidneys for multipurpose translational applications. This review is to summarize the studies performed in kidney bioengineering in the past decade. We identified 84 original articles from PubMed and Mendeley with keywords of kidney organoid or kidney tissue engineering. Those were categorized into 5 groups based on their approach: de-/recellularization of kidney, reaggregation of kidney cells, kidney organoids, kidney in scaffolds, and kidney-on-a-chip. These models were physiologically assessed by filtration, tubular reabsorption/secretion, hormone production, and nephrotoxicity. We found that bioengineered kidney models have been developed from simple cell cultures to multicellular systems to recapitulate kidney function and diseases. Meanwhile, only about 50% of these studies conducted functional assessments on their kidney models. Factors including cell composition and organization are likely to alter the applicability of physiological assessments in bioengineered kidneys. Combined with recent technologies, physiological assessments importantly contribute to the improvement of the bioengineered kidney model toward repairing and refunctioning the damaged kidney.

Prospect of in vitro Bile Fluids Collection in Improving Cell-Based Assay of Liver Function.

The liver plays a pivotal role in the clearance of drugs. Reliable assays for liver function are crucial for various metabolism investigation, including toxicity, disease, and pre-clinical testing for drug development. Bile is an aqueous secretion of a functioning liver. Analyses of bile are used to explain drug clearance and related effects and are thus important for toxicology and pharmacokinetic research. Bile fluids collection is extensively performed in vivo, whereas this process is rarely reproduced as in the in vitro studies. The key to success is the technology involved, which needs to satisfy multiple criteria. To ensure the accuracy of subsequent chemical analyses, certain amounts of bile are needed. Additionally, non-invasive and continuous collections are preferable in view of cell culture. In this review, we summarize recent progress and limitations in the field. We highlight attempts to develop advanced liver cultures for bile fluids collection, including methods to stimulate the secretion of bile in vitro. With these strategies, researchers have used a variety of cell sources, extracellular matrix proteins, and growth factors to investigate different cell-culture environments, including three-dimensional spheroids, cocultures, and microfluidic devices. Effective combinations of expertise and technology have the potential to overcome these obstacles to achieve reliable in vitro bile assay systems.

Tubular bile duct structure mimicking bile duct morphogenesis for prospective in vitro liver metabolite recovery.

BACKGROUND: Liver metabolites are used to diagnose disease and examine drugs in clinical pharmacokinetics. Therefore, development of an in vitro assay system that reproduces liver metabolite recovery would provide important benefits to pharmaceutical research. However, liver models have proven challenging to develop because of the lack of an appropriate bile duct structure for the accumulation and transport of metabolites from the liver parenchyma. Currently available bile duct models, such as the bile duct cyst-embedded extracellular matrix (ECM), lack any morphological resemblance to the tubular morphology of the living bile duct. Moreover, these systems cannot overcome metabolite recovery issues because they are established in isolated culture systems. Here, we successfully established a non-continuous tubular bile duct structure model in an open-culture system, which closely resembled an in vivo structure. This system was utilized to effectively collect liver metabolites separately from liver parenchymal cells. RESULTS: Triple-cell co-culture of primary rat hepatoblasts, rat biliary epithelial cells, and mouse embryonic fibroblasts was grown to mimic the morphogenesis of the bile duct during liver development. Overlaying the cells with ECM containing a Matrigel and collagen type I gel mixture promoted the development of a tubular bile duct structure. In this culture system, the expression of specific markers and signaling molecules related to biliary epithelial cell differentiation was highly upregulated during the ductal formation process. This bile duct structure also enabled the separate accumulation of metabolite analogs from liver parenchymal cells. CONCLUSIONS: A morphogenesis-based culture system effectively establishes an advanced bile duct structure and improves the plasticity of liver models feasible for autologous in vitro metabolite-bile collection, which may enhance the performance of high-throughput liver models in cell-based assays.

Efficient functional cyst formation of biliary epithelial cells using microwells for potential bile duct organisation in vitro.

Establishing a bile duct in vitro is valuable to obtain relevant hepatic tissue culture systems for cell-based assays in chemical and drug metabolism analyses. The cyst constitutes the initial morphogenesis for bile duct formation from biliary epithelial cells (BECs) and serves the main building block of bile duct network morphogenesis from the ductal plate during embryogenesis in rodents. Cysts have been commonly cultured via Matrigel-embedded culture, which does not allow structural organisation and restricts the productivity and homogeneity of cysts. In this study, we propose a new method utilising oxygen permeable honeycomb microwells for efficient cyst establishment. Primary mouse BECs were seeded on four sizes of honeycomb microwell (46, 76, 126, and 326 µm-size in diameter). Matrigel in various concentrations was added to assist in cyst formation. The dimension accommodated by microwells was shown to play an important role in effective cyst formation. Cytological morphology, bile acid transportation, and gene expression of the cysts confirmed the favourable basic bile duct function compared to that obtained using Matrigel-embedded culture. Our method is expected to contribute to engineered in vitro liver tissue formation for cell-based assays.

Alteration of pancreatic carcinoma and promyeloblastic cell adhesion in liver microvasculature by co-culture of hepatocytes, hepatic stellate cells and endothelial cells in a physiologically-relevant model.

In vitro models of the liver microvasculature, especially with respect to cancer cell extravasation, should include not only endothelial and cancer cells but also surrounding cells to mimic the physiological situation. To this end, in the present study, we established a physiologically-relevant hierarchical co-culture model by stacking layers of primary rat hepatocytes (Hep), hepatic stellate cells embedded in collagen gel (LX-2) and endothelial cells (HUVECs) on a specially designed oxygen-permeable polydimethylsiloxane PDMS bottom plate. The model was used to investigate the role and contribution of each of the three cell types in pancreatic cancer and promyeloblast cell adhesion. In particular, we showed an increase in albumin production by the primary hepatocytes and in the consumption of the produced vascular endothelial growth factors (VEGFs). Furthermore, in co-culture, the HUVECs exhibited a mature vascular endothelial and non-inflamed phenotype, as evidenced by Stabilin-1, lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), intercellular adhesion molecule (ICAM-1), and vascular adhesion protein-1 (VAP-1) expression. The HUVECs were also successfully activated with an inflammatory cytokine and their ICAM-1 response was found to be higher in monoculture compared to co-culture. Additionally, the adhesion of MiaPaCa-2 pancreatic cancer cells and HL60 promyeloblasts was tested in both cases (i.e.: activation or not by an inflammatory cytokine). It has been found that their adhesion was always reduced in the co-culture model. These results highlight the importance of integrating hepatic stellate cells in the design of biomimetic models of the hepatic endothelial barrier.