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.
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Method Article
Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development
In This Article
Erratum Notice
Important: There has been an erratum issued for this article. View Erratum Notice
Summary
Abstract
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.
Introduction
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 ext....
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Protocol
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
- Prepare decellularization solutions. Prepare the following volumes of reagents for each kidney to be decellularized, plus one additional volume (e.g., for 4 kidneys, prepare 5,000 ml Triton X-100 for step 1.1.3):
- Prepare 500 ml reverse osmosis water (ROH2O). Note: Alternatively, deionized water may be used in steps where ROH2O is indicated.
- Prepare 1,000 ml Tr....
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Results
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 detergen.......
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Discussion
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 cul.......
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Disclosures
The authors have nothing to disclose.
Acknowledgements
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....
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 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. |
References
- Uzarski, J. S., Xia, Y., Belmonte, J. C., Wertheim, J. A. New strategies in kidney regeneration and tissue engineering. Current opinion in nephrology and hypertension. 23, 399-405 (2014).
- Soto-Gutierrez, A., Wertheim, J. A., Ott, H. C., Gilbert, T. W.
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Reprints and Permissions
Erratum
Formal Correction: Erratum: Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development
Posted by JoVE Editors on 3/03/2017. Citeable Link.
An erratum was issued for Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development.
The human RCTEC/RCTE cell stocks used here and in Caralt et al., 2015, Uzarski et al., 2015 were originally provided by Dr. Loghman-Adham (then at St. Louis University) to co-author Dr. Wandinger-Ness (University of New Mexico) through an MTA in 2001. The SV40 immortalized human RCTEC/RCTE cells were characterized as being of distal tubule cell line as detailed in Loghman-Adham et al., 2003. On the basis of recent short tandem repeat (STR) DNA sequencing (Performed by IDEXX BioResearch) of the earliest passages of the RCTEC/RCTE cell stocks it became evident the cells were of mixed lineage. Further analyses of PCR products using QIAxcel capillary electrophoresis demonstrated the presence of a canine product. The product was sequenced and established to be of canine origin. A set of canine specific STR markers were compared to the sample and showed that the sample had a genetic profile with 92% identity to the MDCK cell line. Later cell stocks that were used in the present publication were STR profiled and showed drift to 100% MDCK lineage. Despite being of canine origin, rather than human as was previously thought, MDCK is similarly a distal tubule epithelial cell line. For this reason, previous interpretation and conclusions drawn using these cells here and in Caralt et al., 2015, Uzarski et al., 2015 remain sound, but for purposes of rigor, reproducibility and experimental validation by others we report on this misidentification. This information will also be reported for listing on the International Cell Line Authentication Committee (ICLAC) database (http://iclac.org).
