This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.
This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 °C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 µM resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.
As the number of patients suffering from end-stage renal failure continues to increase, there is a severe and growing shortage in the number of donor kidneys available for transplantation. The inability to meet the demand of a continually rising number of candidates wait-listed for kidney transplantation has prompted research in kidney organ engineering with the ultimate goal of developing customized, implantable kidney grafts on demand1,2. Building functioning kidney tissue from a patient’s own cells would eliminate the need for lifelong immunosuppression, decrease the amount of time patients spend on dialysis waiting for a kidney transplant, and extend life-saving transplantation to more patients with chronic kidney disease.
The first step toward bioengineering a kidney tissue using patient-derived cells is to develop a scaffold that serves as a supportive substrate for renal parenchyma (e.g. tubular epithelial), stroma fibroblast, and vascular cell growth. Biomaterial scaffolds derived from natural organ extracellular matrices (ECMs) have several characteristics that make them desirable for use in tissue engineering, including their natural biological composition; appropriate macro- and microstructure to endow physiological function; and cellular biocompatibility, promoting cell adhesion, migration, and constructive tissue remodeling3. A promising method to produce scaffolds for renal tissue regeneration is through decellularization of allogeneic or xenogeneic kidneys that preserve much of the complex natural protein composition of the kidney ECM4, retain the inherent architectural intricacy of the organ, and overcome the difficulty associated with bottom-up engineering of thick cellularized tissues by providing a vascular supply to developing cells after scaffold recellularization5.
Perfusion decellularization is a process in which detergents, enzymes, or other cell-disrupting solutions are uniformly delivered through the vascular network of the organ6. This strategy has been established as an efficient process to derive acellular organ-based ECM scaffolds as three-dimensional (3D), biological templates for whole-organ engineering6-8, as evidenced by the development of acellular renal templates from discarded human kidneys9 and xenogeneic kidneys obtained from large-animal (e.g. pig10, goat11) and rodent sources12. In particular, the use of small animal models such as rodents requires fewer cells and culture media, which is especially helpful for organ recellularization studies in which cell numbers are usually limited, as is the case with stem cell-derived tissues. The goal of the described decellularization protocol is to produce an acellular renal ECM that can be used as a 3D scaffolding system for regeneration of kidney structures, including nephron tubules that are repopulated in the present example with human renal cortical tubular epithelial (RCTE) cells. We previously described our rigorous evaluation of an optimal, detergent-based rat kidney decellularization protocol7, which is more rapid (approximately one day) than other methods previously reported (Ross et al.- 5 days12, Song et al.- 4.5 days13), and exposes the organ to a considerably lower concentration (0.1%) of the denaturant sodium dodecyl sulfate (SDS) during decellularization than prior reports12-15.
A limited number of studies have described the use of rodent kidneys for decellularization and subsequent use as a 3D scaffold for cellular repopulation (reviewed elsewhere1)12-16. In this protocol, we provide a detailed description of our previously established, optimal decellularization strategy for producing acellular kidney scaffolds from Sprague Dawley rat kidneys7. Using custom-designed perfusion bioreactors capable of dual seeding and maintenance perfusion culture17, we recellularize the acellular kidney scaffolds with human RCTE cells, which consistently repopulate the tubular component in these decellularized matrices, proliferate, and survive in perfusion culture for over a week. We further demonstrate our use of the resazurin perfusion assay – an inexpensive, non-cytotoxic, and non-invasive metabolic assessment previously used for cytotoxicity studies17– to provide an indication of cell viability and proliferation within the recellularized kidneys over time7.
ETHICS STATEMENT:All procedures involving animals were performed according to guidelines approved by the Institutional Animal Care and Use Committee of Northwestern University.
1. Kidney Decellularization
2. Perfusion Bioreactor Assembly, Kidney Sterilization, and Preparation for Recellularization
3. Kidney Recellularization with Renal Cortical Tubular Epithelial Cells
4. Evaluation of Cell Viability and Proliferation using Resazurin Perfusion Assay
Kidneys sequentially perfused with water and dilute detergent solutions (1% Triton X-100, 0.1% SDS) according to a previously established, optimal decellularization protocol (see Figure 1A, B)7, become progressively more transparent over a 26 hr period, as shown in Figure 2A. The resulting acellular kidney scaffold is devoid of cells and retains a cohesive renal ECM supported by an intact renal capsule, which is undamaged following the perfusion protocol. By the final detergent perfusion (SDS), the kidney’s vascular network, and in particular the interlobar vessels, are prominently displayed in the decellularized scaffold, owing to the greater thickness of these blood vessels relative to the comparatively thin basement membrane of the nephron tubules (see Figure 2A, B). The entire organ is cleared of native cells, leaving behind the intact basement membrane network of glomeruli, tubules, and collecting ducts, and the ECM of decellularized blood vessels, including internal and external elastic laminae of cortical interlobular arteries (see Figure 2B, C). In addition to the larger vessels, the microvascular basement membranes within glomeruli also retain structural integrity (see Figure 2B, C).
Decellularized kidneys are stored in PBS at 4 °C to limit the natural hydrolytic deterioration of the renal ECM, and should be used within 2 weeks of decellularization. We have previously described in detail the design of a custom perfusion-based kidney bioreactor that is used for both seeding decellularized rodent kidney scaffolds, and long-term culture of the recellularized organs under flow17. For recellularization, a perfusion circuit composed of small-diameter silicone rubber and fatigue-resistant PharMed pump tubing is used to route culture medium from the bioreactor reservoir, to a peristaltic pump, and back to the inlet Luer acceptor to which the catheterized renal artery is connected at the inner face of the bioreactor head (see Figure 1C). After sterilizing the decellularized kidney by perfusion with a peracetic acid/ethanol mixture, the perfusion system is prepped for seeding by circulating standard culture medium (containing 10% FBS to improve cell-ECM adhesion) through the scaffold within the incubator.
The decellularized kidneys may now serve as acellular ECM templates for recellularization, which we have previously performed using induced pluripotent stem cell-derived endothelial cells for revascularization7. Here, we demonstrate the utility of this scaffold and perfusion bioreactor system for tubulogenesis using human-derived renal cortical tubular epithelial (RCTE) cells. RCTE cells are seeded into kidney scaffolds through the renal artery under a high-pressure perfusion protocol that has been described previously7,17.RCTE cells infused in this manner home primarily to the cortical regions of the kidney, where they preferentially repopulate the periglomerular tubules (see Figure 3A). Few cells embed within glomeruli at day 1 post-seeding, and by day 7, glomeruli are virtually devoid of cells. During perfusion culture, medium is changed every 2 days, at which point the resazurin perfusion assay is concurrently performed to provide comparative assessments of cell viability over time (see Figure 3B). As supported by the resazurin reduction results, RCTE cells proliferate within the 3D ECM, forming tightly organized, patent tubular structures by day 7. While the majority of these cells occupy the cortical regions of the renal ECM, after 7 days of antegrade perfusion culture many RCTE-lined tubules are present in the outer medullary and papillary tubules and collecting ducts (see Figure 4). After repopulation with RCTE cells and one week of perfusion culture, the transparency observed following decellularization is lost, and the recellularized kidney appears opaque and closer in appearance to its native state (see Figure 4).
Figure 1: Kidney Decellularization System and Protocol and Recellularization Perfusion Circuit. (A) Perfusion schedule of reagents for decellularization of rodent kidneys. The reagents used for decellularization, volumetric flow rate, and duration of each step are shown. (B) Perfusion decellularization set-up. Solutions are pumped unidirectionally from a reagent reservoir to a perfusate collection container through individual flow lines for each kidney undergoing decellularization. Up to four kidneys may be decellularized per peristaltic pump. (C) Perfusion circuit for seeding and culture of recellularized kidney scaffolds. Cells are loaded through a syringe connected directly upstream of the Luer inlet acceptor. Optional in-line sensors for monitoring pressure, dissolved oxygen (dO2), and pH may be placed upstream of bioreactor. The digital peristaltic pump may be controlled by computer, and negative pressure (partial vacuum) may be applied to aid ureteral seeding. (D) Components used to create perfusion lines for decellularization (B) and recellularization (C). (a) male Luer plug, (b) female Luer cap, (c) male Luer lock to 1/8” barbed adapter, (d) female Luer to female Luer adapter, (e) ¼” ID silicone tubing, (f) peristaltic pump tubing, g: thick-walled 1/16” ID silicone rubber tubing, (h) 1/16” ID silicone rubber tubing, (i) three-way stopcock, (j) coupler used to connect segments of tubing by combining two 1/8” barb to male Luer adapters (c) using a female Luer to female Luer coupler (d). Please click here to view a larger version of this figure.
Figure 2: Representative Results of Kidney Decellularization. Representative gross and microscopic images are shown of kidneys before and following decellularization. (A) Time lapse series of a kidney undergoing decellularization. Images are shown immediately following perfusion with each reagent specified. Following perfusion of 0.1% SDS (sodium dodecyl sulfate), preserved vascular network is visible. (B) Representative images of native or decellularized kidneys sectioned and stained with hematoxylin and eosin (H&E). The ECM architecture of microstructural features, including glomeruli, collecting ducts, and blood vessels, is well-preserved following decellularization. (C) Scanning electron micrographs comparing glomeruli and tubules in native (top row) and decellularized (bottom row) kidneys. Following cell removal, open tubules are prevalent throughout the kidney. Please click here to view a larger version of this figure.
Figure 3: Representative Results of Human Renal Cortical Tubular Epithelial Recellularization of Decellularized Rat Kidneys. Kidneys are recellularized through a high-pressure antegrade arterial perfusion technique that results in RCTE cell adhesion primarily to the renal tubules of the cortex. (A) Representative high-magnification images (20X) of hematoxylin and eosin-stained histological sections of recellularized scaffolds 1, 3, or 7 days after seeding. Top row shows that the cells occupy the periglomerular tubular space despite being injected through the arterial vasculature, indicating their preference for the former renal ECM niche. Yellow arrowhead points to an afferent arteriole with a lumen that is devoid of cells. Lower row shows cortical tubules where RCTE cells proliferate, with increasing cell density over one week, and form tightly packed tubular epithelial structures. (B) A small (10 ml) volume of resazurin-supplemented culture medium is recirculated through kidneys at the time of media changes. During 60 min of perfusion, cells reduce the oxidized resazurin compound (blue) to resorufin (red), which produces a highly fluorescent signal in proportion to the number of cells within the kidney. Consistent with the observed increase in cell density seen within the histological images, percent resazurin reduction increases over culture time with cell growth, providing a non-invasive indication of both cell viability and proliferation during maintenance culture. Results are presented as mean ± standard deviation (n = 4 recellularized kidneys). Please click here to view a larger version of this figure.
Figure 4: Representative Results: Comparison of Native, Decellularized, and Recellularized Kidneys. Low magnification histological images show the renal cortex, transition zone between the cortex and outer medulla, and medullary papilla regions in native kidneys (top row), decellularized (Decell; middle row), and 7-day RCTE-recellularized (7 Day Recell; bottom row) scaffolds. The dashed line indicates the approximate border between the renal cortex (C) and outer medulla (M). Gross images show a kidney in its native state after procurement, following decellularization, and 7 days following seeding of RCTE cells through the renal artery. Please click here to view a larger version of this figure.
The described decellularization protocol consistently produces a completely acellular kidney ECM that serves as a 3D template for culture of human renal cortical tubular epithelial cells (both proximal and distal tubule-derived), in addition to vascular endothelial cells7,17. The cannulated renal vasculature serves as the key feature for uniform delivery of reagents and cells throughout the scaffold within a bioreactor set-up, thus enabling the perfusion decellularization, cell seeding, long-term perfusion culture, and the resazurin perfusion protocols. As such, proper cannulation of the renal artery prior to organ perfusion is critical, and special care must be taken to ensure that the renal artery is not obstructed or damaged, and that the catheter is secured. The Sprague Dawley rats from which the kidneys are recovered must be systemically heparinized to avoid clotting within the vasculature during organ procurement, as intravascular clots cannot be removed, and may inhibit complete decellularization of the kidney.
The perfusion bioreactor used for seeding and perfusion culture of recellularized kidneys is designed to allow multiple seeding methods in situ17. In addition to the arterial injection technique described here, cells may be injected retrograde through the catheterized ureter or renal vein. Furthermore, the bioreactor body is fitted with a valve that permits application of negative pressure (partial vacuum; see Figure 1C), for ureteral seeding. Regardless of the seeding strategy utilized, perfusion of culture medium antegrade through the renal artery is critical for adequate nutrient (e.g. oxygen, glucose) delivery to seeded cells.
The described recellularization protocol demonstrates our use of decellularized renal matrices to serve as templates specifically for epithelial tubulogenesis. However, we have previously shown that the renal matrix also supports re-endothelialization of the retained vascular network and may be lined with human induced pluripotent stem cell-derived endothelial cells, which is an important observation for eventual long-term transplantation of recellularized kidneys in animal models by preventing thrombotic occlusion of the renal vasculature7. A current obstacle to the eventual scale-up of kidney recellularization protocols to large-animal kidney scaffolds is the substantially greater number of cells required to repopulate human-sized kidney scaffolds. Efficient seeding strategies, such as the high-pressure arterial injection technique described above, are critically needed to maximize the number of cells that engraft within the decellularized ECM. Given the heterogenous cellular composition of the native kidney, multiple seeding methods may ultimately be required to effectively repopulate the various extracellular renal niches with diverse cell types that collectively perform the numerous functions of the kidney, including filtration, reabsorption, concentration of urine, and hormone synthesis.
Finally, the resazurin perfusion assay demonstrated in this article provides a non-invasive, non-toxic assessment of cell viability and proliferation during long-term culture7,17,18. The assay provides instantaneous feedback on the metabolic state of cells growing within kidney scaffolds, and when regularly performed in between periodic medium exchanges, can be used to characterize cell proliferation over time. The resazurin reagent is inexpensive, the assay requires little time to perform (1 hr), and it is a more conservative analytical method to characterize cell proliferation or metabolic trends compared to histological evaluation, which requires terminal sacrifice of the recellularized scaffold. The resazurin perfusion assay can also be adapted for evaluation of cell populations during growth within other recellularized organs or tissues.
The authors have nothing to disclose.
The authors thank the support of the Zell Family Foundation. We recognize support from the Northwestern Memorial Foundation Dixon Translational Research Grants Initiative, the American Society of Transplant Surgeon’s Faculty Development Grant, and a Research Grant for the Young Investigator from the National Kidney Foundation of Illinois. We acknowledge support from the Robert R. McCormick Foundation. This work was also supported by NIDDK K08 DK10175 to J.A.W. Imaging and histology cores used for this research is supported by the Mouse Histology and Phenotyping Laboratory, Electron Probe Instrumentation Center (EPIC), and Simpson Querrey Institute Equipment Core at Northwestern University, and a Cancer Center Support Grant (NCI CA060553). The authors would like to acknowledge the Northwestern University Microsurgery Core for rodent kidney procurements. Evaluation of renal tubular epithelia morphology following recellularization conducted in the Fluorescence Microscopy Shared Resource supported by P30 CA118100.
Reagents | |||
TRITON X-100, Proteomics Grade, AMRESCO | VWR | M143-4L | |
Sodium dodecyl sulfate solution, BioUltra, for molecular biology, 20% in H2O | Sigma-Aldrich | 05030 | |
Peracetic acid solution, purum, ~39% in acetic acid (RT) | Sigma-Aldrich | 77240 | Peracetic is flammable and corrosive. Prepare within a fume hood using appropriate personal protective equipment (e.g. gloves, goggles). |
200 proof ethanol | VWR | V1001TP | |
Sigmacote, siliconizing reagent for glass and other surfaces | Sigma-Aldrich | SL2 | For treatment of bioreactor reservoirs. Referred to in text as siliconizing reagent. |
DMEM/F12 media | Life Technologies | 11320-033 | |
Corning cellgro Fetal Bovine Serum Premium, Mediatech | Corning | 35-010-CV | |
Penicillin-Streptomycin Solution, 100X 10,000 I.U. Penicillin 10,000 µg/mL Streptomycin | Corning | 30-002-CI | |
TrypLE Express (1X), Phenol Red | Life Technologies | 12605-028 | Referred to in text as cell dissociating enzyme. |
Trypan Blue Stain (0.4%) | Life Technologies | 15250-061 | |
Resazurin sodium salt | Sigma-Aldrich | R7017 | |
Equipment: | |||
Masterflex L/S Digital Drive, 600 RPM, 115/230 VAC | Cole-Parmer | EW-07522-20 | |
Masterflex L/S large cartridges for 07519-05 and -06 pump heads. | Cole-Parmer | EW-07519-70 | Referred to in text as large pump cartridge. |
Masterflex L/S 8-channel, 4-roller cartridge pump head. | Cole-Parmer | EW-07519-06 | |
Straight 6" specimen forceps, serrated | VWR | 82027-438 | |
*Kidney perfusion bioreactor | WilMad Labglass | *Custom designs | Bioreactors are produced as described by WilMad Labglass. The designs have been described in depth in a previous publication. |
Perfusion Circuit Components | |||
24 G x 0.75 in. BD Insyte Autoguard shielded IV catheter (0.7 mm x 19 mm) made of BD Vialon biomaterial. Has notched needle. (50/sp, 200/ca) | BD Biosciences | 381412 | Referred to in text as 24 gauge catheter. |
Masterflex PharMed BPT Tubing, L/S #14, 25' | Cole-Parmer | HV-06508-14 | Referred to in text as peristaltic pump tubing. |
Peroxide-Cured Silicone Tubing, 1/16"id X 1/8"OD, 25 Ft/pack | Cole-Parmer | HV-06411-62 | Referred to in text as 1/16" ID silicone rubber tubing. |
Masterflex platinum-cured silicone tubing, L/S 14, 25 ft. | Cole-Parmer | HV-96410-14 | Referred to in text as thick-walled 1/16" ID silicone rubber tubing. |
VWR Silicone Tubing, 1/4" ID x 0.5" OD | VWR | 89068-484 | |
Acro 50 Vent Filters, Pall Life Sciences | VWR | 28143-558 | Referred to in text as 0.2 micron vent filter. |
Cole-Parmer Luer Adapters, Male Luer Lock to 1/8" ID, Nylon, 25/Pk | Cole-Parmer | T-45505-11 | Referred to in text as male Luer lock to 1/8" barbed adapter. |
Cole-Parmer Luer Accessory, Male Luer Plug, Nylon, 25/Pk | Cole-Parmer | EW-45505-58 | |
Female luer x female luer adapter, Nylon, 25/pk | Cole-Parmer | EW-45502-22 | Referred to in text as female Luer x female Luer adapter. |
Cole-Parmer Luer Accessory, Female Luer Cap, Nylon, 25/Pk | Cole-Parmer | EW-45502-28 | |
Smiths Medical Large Bore Hi-Flo Stopcocks # MX4311L – 3-Way Hi-Flo Stopcock with Extended Male Luer Lock, Non-DEHP Formulation, Latex Free (LF), Lipid Resistant, Non-PVD, 50/cs | Careforde Healthcare | 10254821 | Smiths Medical (vendor) catalogue number is #MX4311L. |
Other Components | |||
5 mL BD Luer-Lok disposable syringe with BD Luer-Lok™ tip. | BD Biosciences | 309646 | |
35 mm x 10 mm Easy Grip Culture Dish | BD Biosciences | 353001 | Used to draw cell suspension into syringe for cell seeding. |