Fabrication of Kidney Proximal Tubule Grafts Using Biofunctionalized Electrospun Polymer Scaffolds

Abstract

The increasing prevalence of end-stage renal disease and persistent shortage of donor organs call for alternative therapies for kidney patients. Dialysis remains an inferior treatment as clearance of large and protein-bound waste products depends on active tubular secretion. Biofabricated tissues could make a valuable contribution, but kidneys are highly intricate and multifunctional organs. Depending on the therapeutic objective, suitable cell sources and scaffolds must be selected. This study provides a proof-of-concept for stand-alone kidney tubule grafts with suitable mechanical properties for future implantation purposes. Porous tubular nanofiber scaffolds are fabricated by electrospinning 12%, 16%, and 20% poly-ε-caprolactone (PCL) v/w (chloroform and dimethylformamide, 1:3) around 0.7 mm needle templates. The resulting scaffolds consist of 92%, 69%, and 54% nanofibers compared to microfibers, respectively. After biofunctionalization with L-3,4-dihydroxyphenylalanine and collagen IV, 10 × 10⁶ proximal tubule cells per mL are injected and cultured until experimental readout. A human-derived cell model can bridge all fiber-to-fiber distances to form a monolayer, whereas small-sized murine cells form monolayers on dense nanofiber meshes only. Fabricated constructs remain viable for at least 3 weeks and maintain functionality as shown by inhibitor-sensitive transport activity, which suggests clearance capacity for both negatively and positively charged solutes.

Keywords: polycaprolactone; regenerative medicine; renal replacement therapy; renal transport; tissue engineering.

Figure 1

Workflow of design and fabrication of the biofunctionalized electrospun polymer scaffolds for kidney proximal tubule grafts. a) A kidney contains 200 000–1 000 000 functional units, the nephrons. After blood filtration through the glomerulus, active secretion of metabolic waste products takes place between the proximal tubules and peritubular capillaries. While filtration can be replaced by hemodialysis, active secretion requires proximal tubule cells as part of advanced renal replacement therapies. b) Solution electrospinning was used to fabricate tubular scaffolds with different polymer concentrations. Two cell lines of proximal tubular epithelial cells were injected for luminal epithelialization to construct implantable kidney tubule drafts. c) Scaffold properties and cell behavior were examined regarding mechanical properties, epithelialization, long-term viability, and renal functionality.

Figure 2

Scaffold fabrication. a–c) Scanning electron microscopic images of tubular electrospun scaffolds made of 12%, 16%, and 20% w/v PCL. Scale bars: (a) 1 mm and (b,c) 100 μm. d) Comparison of scaffold wall thicknesses, measured on four locations in three sections of three different scaffolds. e) Comparison of scaffold fiber diameters, measured on three locations in three sections of three different scaffolds. Boxplots present the mean and 5–95 percentiles. *p < 0.05, **p < 0.01, ****p < 0.0001 using one-way ANOVA.

Figure 3

Mechanical scaffold properties. a) Experimental set-up for uniaxial tensile testing. b) Representative stress-strain curve for a 20% PCL scaffold with indicated tangent modulus and point at break. c) Comparison of tangent moduli (n = 3). d) Comparison of stress at break (n = 3). e) Comparison of strain at break (n = 3). Data are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 using one-way ANOVA.

Figure 4

Luminal epithelialization. iREC or ciPTEC-OAT1 was cultured on the luminal side of 12%, 16%, and 20% w/v PCL scaffolds. a) Double coating with L-DOPA and collagen IV improved cell adhesion and tight monolayer formation as proven by visible tight junction marker zona occludens-1 (ZO-1, green) and nuclei (blue). b–c) iREC formed monolayers only on 12% PCL scaffolds. ciPTEC-OAT1 grew on all scaffolds into monolayers. In the absence of ZO-1, the scaffold fibers became visible due to autofluorescence. d) Transverse cut: cells grew throughout the scaffold lumen. e–f) Both cell lines showed high viability through enzymatic calcein-AM conversion to calcein (green) and absent staining with cell-impermeant viability indicator ethidium homodimer-1 (red). Scale bars: (a) 100 μm, 50 μm in the high magnification image.

Figure 5

Cell functionality. ciPTEC-OAT1 cultured in 20% PCL scaffolds showed uptake of (left) 1 μM fluorescein via Organic Anion Transporter 1 (OAT1) and (right) 5 μM ASP⁺ via Organic Cation Transporter 2 (OCT2), which could be inhibited by 100 μM probenecid and 20 μM TPA⁺, respectively (n = 5). Scale bars: 100 μm. Data are expressed as mean ± SD. *p < 0.05, ***p < 0.0001 using Student’s unpaired t-test.