Objective

Drug toxicity remains a major issue in drug discovery and stresses the need for better predictive models. Here, we describe the development of a perfused renal proximal tubule cell (RPTC) model in Mimetas’ OrganoPlates® [1,2] to predict kidney toxicity. The OrganoPlate® is a microfluidic platform, which enables high-throughput culture of boundary tissues in miniaturized organ models. In OrganoPlates®, extracellular matrix (ECM) gels can be freely patterned in microchambers through the use of PhaseGuide technology. PhaseGuides (capillary pressure barriers) define channels within microchambers that can be used for extracellular matrix deposition or medium perfusion. The microfluidic channel dimensions not only allow solid tissue and barrier formation, but also perfused tubular epithelial vessel structures can be grown. The goal of developing a perfused RPTC model is to reconstruct viable and leak-tight boundaries for performing cytotoxicity, as well as transport and efficacy studies. SigmaRPTEC cells were seeded in the perfusion lane against the ECM gel and formed monolayers at the interface between lanes. Supported by perfusion, the cells grew against the inner wall of the medium channel to form a tubular structure. Healthy tubes in an OrganoPlate® are fully leak tight and may lose their barrier integrity upon compound exposure or induction of disease. Barrier integrity of the tubes is assessed by apical perfusion of 150kDa FITC labeled dextran and measuring fluorescence levels in the basal gel region (figure 5). Upon disruption of the barrier via drug-induced toxicity, FITC-dextran will leak towards the basal side, yielding a higher signal in the ECM compartment. Imaging of barrier integrity is performed with an ImageXpress® Micro XLS System (Molecular devices) allowing for real-time assessment of barrier disruption. Prior to compound exposure barrier integrity is assessed to exclude leaky tubes from the study. Fluorescence-based assays were used to study the activity of transporters. For example, the re-absorption of glucose by the SGLT2 transporter can be assessed by incubating the tubules with a fluorescent analogue of glucose (6-NBDG) from the apical side and measuring the increase of fluorescent signal in the cytoplasm. Fluorescent-probe based transport assays have been set up for both influx and efflux transporters with matching inhibitors/competitors, including the Pgp and MRP4 transporter. Human RPTEC (SA7K clone, Sigma MTOX1030) were grown against an ECM gel in a three channel OrganoPlate®, yielding access to both the apical and basal side. Confocal imaging revealed that the cells formed a tubular structure. The tubule stained positive for ZO-1 (tight junctions), acetylated tubulin and Ezrin (both polarization marker). Interestingly, cilia pointed in the direction of the lumen of tubules and Ezrin stained the microvilli on the apical side of the tubule. Tightness of the boundary against the ECM was assessed by diffusion of a dextran dye added to the lumen of the tubule. Boundaries of RPTEC showed leak-tight barriers and were maintained for several days. Addition of toxic compounds resulted in disruption of the barriers which could be monitored in time. Tubules started leaking depending on the concentration and the toxicity effect of the compounds. Furthermore, first fluorescent transport assays showed functional transport activity of in- and efflux transporters. The 3D proximal tubules cultured in the OrganoPlate™ are suitable for high-throughput toxicity screening, trans-epithelial transport studies, and complex co-culture models to recreate an in vivo-like microenvironment. 1. Trietsch, S. J., Israëls, G. D., Joore, J., Hankemeier, T. & Vulto, P. Microfluidic titer plate for stratified 3D cell culture. Lab Chip 13, 3548–54 (2013). 2. Moreno, E. L. et al. Differentiation of neuroepithelial stem cells into functional dopaminergic neurons in 3D microfluidic cell culture. Lab Chip 15,

Methods

SigmaRPTEC cells were seeded in the perfusion lane against the ECM gel and formed monolayers at the interface between lanes. Supported by perfusion, the cells grew against the inner wall of the medium channel to form a tubular structure. Healthy tubes in an OrganoPlate® are fully leak tight and may lose their barrier integrity upon compound exposure or induction of disease. Barrier integrity of the tubes is assessed by apical perfusion of 150kDa FITC labeled dextran and measuring fluorescence levels in the basal gel region (figure 5). Upon disruption of the barrier via drug-induced toxicity, FITC-dextran will leak towards the basal side, yielding a higher signal in the ECM compartment. Imaging of barrier integrity is performed with an ImageXpress® Micro XLS System (Molecular devices) allowing for real-time assessment of barrier disruption. Prior to compound exposure barrier integrity is assessed to exclude leaky tubes from the study. Fluorescence-based assays were used to study the activity of transporters. For example, the re-absorption of glucose by the SGLT2 transporter can be assessed by incubating the tubules with a fluorescent analogue of glucose (6-NBDG) from the apical side and measuring the increase of fluorescent signal in the cytoplasm. Fluorescent-probe based transport assays have been set up for both influx and efflux transporters with matching inhibitors/competitors, including the Pgp and MRP4 transporter.

Results

Human RPTEC (SA7K clone, Sigma MTOX1030) were grown against an ECM gel in a three channel OrganoPlate®, yielding access to both the apical and basal side. Confocal imaging revealed that the cells formed a tubular structure. The tubule stained positive for ZO-1 (tight junctions), acetylated tubulin and Ezrin (both polarization marker). Interestingly, cilia pointed in the direction of the lumen of tubules and Ezrin stained the microvilli on the apical side of the tubule. Tightness of the boundary against the ECM was assessed by diffusion of a dextran dye added to the lumen of the tubule. Boundaries of RPTEC showed leak-tight barriers and were maintained for several days. Addition of toxic compounds resulted in disruption of the barriers which could be monitored in time. Tubules started leaking depending on the concentration and the toxicity effect of the compounds. Furthermore, first fluorescent transport assays showed functional transport activity of in- and efflux transporters.

Conclusion

The 3D proximal tubules cultured in the OrganoPlate™ are suitable for high-throughput toxicity screening, trans-epithelial transport studies, and complex co-culture models to recreate an in vivo-like microenvironment. 1. Trietsch, S. J., Israëls, G. D., Joore, J., Hankemeier, T. & Vulto, P. Microfluidic titer plate for stratified 3D cell culture. Lab Chip 13, 3548–54 (2013). 2. Moreno, E. L. et al. Differentiation of neuroepithelial stem cells into functional dopaminergic neurons in 3D microfluidic cell culture. Lab Chip 15, 2419–28 (2015).