Decellularized rhesus monkey kidney as a three-dimensional scaffold for renal tissue engineering

Abstract

The goal of this study was the production of a decellularized kidney scaffold with structural, mechanical, and physiological properties necessary for engineering basic renal structures in vitro. Fetal, infant, juvenile, and adult rhesus monkey kidney sections were treated with either 1% (v/v) sodium dodecyl sulfate or Triton X-100 followed by quantitative and qualitative analysis. Comparison of decellularization agents and incubation temperatures demonstrated sodium dodecyl sulfate at 4 degrees C to be most effective in preserving the native architecture. Hematoxylin and eosin staining confirmed the removal of cellular material, and immunohistochemistry demonstrated preservation of native expression patterns of extracellular matrix proteins, including heparan sulfate proteoglycan, fibronectin, collagen types I and IV, and laminin. Biomechanical testing revealed a decrease in the compressive modulus of decellularized compared to fresh kidneys. Layering of fetal kidney explants on age-matched decellularized kidney scaffolds demonstrated the capacity of the scaffold to support Pax2+/vimentin+ cell attachment and migration to recellularize the scaffold. These findings demonstrate that decellularized kidney sections retain critical structural and functional properties necessary for use as a three-dimensional scaffold and promote cellular repopulation. Further, this study provides the initial steps in developing new regenerative medicine strategies for renal tissue engineering and repair.

FIG. 1.

Orientation of transverse section from kidneys of different age groups (fetal, infant, juvenile, and adult).

FIG. 2.

Decellularization of juvenile rhesus monkey kidney transverse sections. Kidney sections were incubated with a decellularization solution of 1% SDS or 1% Triton X-100 at either 4°C or 37°C for 10 days. Images of the same section were taken on days 0, 4, and 10 of the decellularization process. SDS, sodium dodecyl sulfate. Color images available online at www.liebertonline.com/ten.

FIG. 3.

Morphology. H&E stain of control (A, F, K) and decellularized adult rhesus monkey kidney sections. H&E of kidney decellularized with 1% SDS at 4°C (B, G, L) or 37°C (C, H, M), or 1% Triton X-100 at 4°C (D, I, N) or 37°C (E, J, O) for 10 days. Structurally intact glomerular basement membrane remained after decellularization with SDS at 4°C (L). Condensed ECM resulting from treatment with SDS at 37°C (H, M, arrows), and severe disruption of basement membrane from treatment with Triton X-100 (I, N, arrows); note residual cell nuclei (J, O, arrows). H&E, hematoxylin and eosin; ECM, extracellular matrix; g, glomeruli; bc, Bowman's capsule. Color images available online at www.liebertonline.com/ten.

FIG. 4.

Immunohistochemistry of control and decellularized kidney. Staining of ECM proteins in adult kidney sections (A–F), and after decellularization in 1% SDS (G–L) or 1% Triton X-100 (M–R) (20 ×). Decellularized tissues display the same localization of ECM proteins to glomeruli (g), tubules (t), and Bowman's capsule (bc) as control (no decellularization). Absence of collagen type I (C, I, O, arrows) and expression of HSPG (A, G, M) and fibronectin (B, H, N) in the glomerular basement membrane noted. Retention of distinct glomerular basement membrane (F, L, R, arrows) composition with co-localization of collagen type IV and laminin was more pronounced in SDS-decellularized tissue compared to Triton X-100. HSPG, heparan sulfate proteoglycan.

FIG. 5.

Biomechanical testing. Graph of compressive load versus extension before decellularization on day 0 and after complete decellularization on day 7 for one specimen. The slope (compressive modulus) is designated by linear lines tangent to the curve (A). Young's compressive modulus was calculated from the first 10% of the linear section of the compression curve for each specimen. Graph of the mean compressive modulus and corresponding standard error of the mean for day 0 and day 7 kidney is shown in megapascals (MPa) (B).

FIG. 6.

Layered decellularized kidney scaffold and kidney explant. Fetal kidney explant was layered on an age-matched decellularized kidney scaffold and cultured for 5 days. H&E-stained sections of the explant/scaffold reveal migration of cells (arrows) from the explant (e) into the decellularized scaffold (s).

FIG. 7.

Immunohistochemical staining of layered kidney scaffold and explant. Fresh and explant-cultured kidneys were stained for cytokeratin/vimentin or Pax2/WT1 for reference and renal structures—glomerulus (g), ureteric bud (ub), and S-shaped body (sb) are noted. Dotted line denotes boundary between scaffold (s) and explant (e). The rectangle in the 10 × image highlights the region magnified in the 40 × image. The nephrogenic zone of the explant is shown in direct contact with the scaffold, and arrows highlight populations of nucleated cells that have migrated into the scaffold cortex. DAPI, 4′,6-diamidino-2-phenylindole.