Name: epithelial cell repopulation and preparation of rodent extracellular matrix scaffolds for renal tissue development
Uploaded: 2015-08-10T14:00:00
Description: Watch this Scientific Journal Video about Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development at JoVE.com

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&#8217;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.

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
<ol>
	<li>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&#160;Triton X-100 for step 1.1.3):
	<ol>
		<li>Prepare 500 ml&#160;reverse osmosis water ...

<ol>
	<li>Uzarski, J. S., Xia, Y., Belmonte, J. C., Wertheim, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+strategies+in+kidney+regeneration+and+tissue+engineering.">New strategies in kidney regeneration and tissue engineering.</a> Current opinion in nephrology and hypertension. 23, 399-405 (2014).</li><li>Soto-Gutierrez, A., Wertheim, J. A., Ott, H. C., Gilbert, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+on+whole-organ+assembly:+moving+toward+transplantation+on+demand.">Perspectives on whole-organ assembly: moving toward transplantation on demand.</a> J Clin Invest. 122, 3817-3823 (2012).</li><li>Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+allogeneic+and+xenogeneic+tissue+as+a+bioscaffold+for+regenerative+medicine:+factors+that+influence+the+host+response.">Decellularized allogeneic and xenogeneic tissue as a bioscaffold for regenerative medicine: factors that influence the host response.</a> Ann Biomed Eng. 42, 1517-1527 (2014).</li><li>Miner, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renal+basement+membrane+components.">Renal basement membrane components.</a> Kidney Int. 56, 2016-2024 (1999).</li><li>Nichol, J. W., Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+Tissue+Engineering:+Engineering+Biological+Tissues+from+the+Bottom+Up.">Modular Tissue Engineering: Engineering Biological Tissues from the Bottom Up.</a> Soft matter. 5, 1312-1319 (2009).</li><li>Ott, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion-decellularized+matrix:+using+nature's+platform+to+engineer+a+bioartificial+heart.">Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart.</a> Nat Med. 14, 213-221 (2008).</li><li>Caralt, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+and+critical+evaluation+of+decellularization+strategies+to+develop+renal+extracellular+matrix+scaffolds+as+biological+templates+for+organ+engineering+and+transplantation.">Optimization and critical evaluation of decellularization strategies to develop renal extracellular matrix scaffolds as biological templates for organ engineering and transplantation.</a> American Journal of Transplantation. 15, 64-75 (2015).</li><li>Badylak, S. F., Taylor, D., Uygun, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-organ+tissue+engineering:+decellularization+and+recellularization+of+three-dimensional+matrix+scaffolds.">Whole-organ tissue engineering: decellularization and recellularization of three-dimensional matrix scaffolds.</a> Annu Rev Biomed Eng. 13, 27-53 (2011).</li><li>Orlando, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discarded+human+kidneys+as+a+source+of+ECM+scaffold+for+kidney+regeneration+technologies.">Discarded human kidneys as a source of ECM scaffold for kidney regeneration technologies.</a> Biomaterials. 34, 5915-5925 (2013).</li><li>Sullivan, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularization+methods+of+porcine+kidneys+for+whole+organ+engineering+using+a+high-throughput+system.">Decellularization methods of porcine kidneys for whole organ engineering using a high-throughput system.</a> Biomaterials. 33, 7756-7764 (2012).</li><li>Vishwakarma, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+natural+three-dimensional+goat+kidney+scaffold+for+the+development+of+bioartificial+organ.">Preparation of natural three-dimensional goat kidney scaffold for the development of bioartificial organ.</a> Indian journal of nephrology. 24, 372-375 (2014).</li><li>Ross, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cells+proliferate+and+differentiate+when+seeded+into+kidney+scaffolds.">Embryonic stem cells proliferate and differentiate when seeded into kidney scaffolds.</a> J Am Soc Nephrol. 20, 2338-2347 (2009).</li><li>Song, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+and+experimental+orthotopic+transplantation+of+a+bioengineered+kidney.">Regeneration and experimental orthotopic transplantation of a bioengineered kidney.</a> Nat Med. 19, 646-651 (2013).</li><li>Bonandrini, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recellularization+of+well-preserved+acellular+kidney+scaffold+using+embryonic+stem+cells.">Recellularization of well-preserved acellular kidney scaffold using embryonic stem cells.</a> Tissue Eng Part A. 20, 1486-1498 (2014).</li><li>Burgkart, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+kidney+matrix+for+perfused+bone+engineering.">Decellularized kidney matrix for perfused bone engineering.</a> Tissue Eng Part C Methods.2. 20, 553-561 (2014).</li><li>Ross, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+stem+cells+seeded+into+decellularized+rat+kidney+scaffolds+endothelialize+and+remodel+basement+membranes.">Mouse stem cells seeded into decellularized rat kidney scaffolds endothelialize and remodel basement membranes.</a> Organogenesis. 8, 49-55 (2012).</li><li>Uzarski, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-purpose+bioreactors+to+monitor+non-invasive+physical+and+biochemical+markers+of+kidney+and+liver+scaffold+recellularization.">Dual-purpose bioreactors to monitor non-invasive physical and biochemical markers of kidney and liver scaffold recellularization.</a> Tissue Eng Part C Methods. , (2015).</li><li>Brien, J., Wilson, I., Orton, T., Pognan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+Alamar+Blue+(resazurin)+fluorescent+dye+for+the+assessment+of+mammalian+cell+cytotoxicity.">Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity.</a> European journal of biochemistry / FEBS. 267, 5421-5426 (2000).</li><li>Loghman-Adham, M., Nauli, S. M., Soto, C. E., Kariuki, B., Zhou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalized+epithelial+cells+from+human+autosomal+dominant+polycystic+kidney+cysts.+American+journal+of+physiology.">Immortalized epithelial cells from human autosomal dominant polycystic kidney cysts. American journal of physiology.</a> Renal physiology. 285, F397-F412 (2003).</li></ol>

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...

An erratum was issued for <a href="http://www.jove.com/video/53271/epithelial-cell-repopulation-preparation-rodent-extracellular-matrix">Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development</a>.
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.&#160; 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).

Erratum: Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

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&#8217;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 &#8211; an inexpensive, non-cytotoxic, and non-invasive metabolic assessment previously used for cytotoxicity studies17&#8211; to provide an indication of cell viability and proliferation within the recellularized kidneys over time7.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TRITON X-100, Proteomics Grade, AMRESCO</td>
			<td>VWR</td>
			<td>M143-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate solution, BioUltra, for molecular biology, 20% in H2O</td>
			<td>Sigma-Aldrich</td>
			<td>05030</td>
			<td></td>
		</tr>
		<tr>
			<td>Peracetic acid solution, purum, ~39% in acetic acid (RT)</td>
			<td>Sigma-Aldrich</td>
			<td>77240</td>
			<td>Peracetic is flammable and corrosive. Prepare within a fume hood using appropriate personal protective equipment (e.g. gloves, goggles).</td>
		</tr>
		<tr>
			<td>200 proof ethanol</td>
			<td>VWR</td>
			<td>V1001TP</td>
			<td></td>
		</tr>
		<tr>
			<td>Sigmacote, siliconizing reagent for glass and other surfaces</td>
			<td>Sigma-Aldrich</td>
			<td>SL2</td>
			<td>For treatment of bioreactor reservoirs. Referred to in text as siliconizing reagent.</td>
		</tr>
		<tr>
			<td>DMEM/F12 media</td>
			<td>Life Technologies</td>
			<td>11320-033</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning cellgro Fetal Bovine Serum Premium, Mediatech</td>
			<td>Corning</td>
			<td>35-010-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution, 100X 10,000 I.U. Penicillin 10,000 &#181;g/mL Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express (1X), Phenol Red</td>
			<td>Life Technologies</td>
			<td>12605-028</td>
			<td>Referred to in text as cell dissociating enzyme.</td>
		</tr>
		<tr>
			<td>Trypan Blue Stain (0.4%)</td>
			<td>Life Technologies</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Resazurin sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>R7017</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex L/S Digital Drive, 600 RPM, 115/230 VAC</td>
			<td>Cole-Parmer</td>
			<td>EW-07522-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex L/S large cartridges for 07519-05 and -06 pump heads.</td>
			<td>Cole-Parmer</td>
			<td>EW-07519-70</td>
			<td>Referred to in text as large pump cartridge.</td>
		</tr>
		<tr>
			<td>Masterflex L/S 8-channel, 4-roller cartridge pump head.</td>
			<td>Cole-Parmer</td>
			<td>EW-07519-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight 6&#34; specimen forceps, serrated</td>
			<td>VWR</td>
			<td>82027-438</td>
			<td></td>
		</tr>
		<tr>
			<td>*Kidney perfusion bioreactor</td>
			<td>WilMad Labglass</td>
			<td>*Custom designs</td>
			<td>Bioreactors are produced as described by WilMad Labglass. The designs have been described in depth in a previous publication.</td>
		</tr>
		<tr>
			<td>Perfusion Circuit Components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>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)</td>
			<td>BD Biosciences</td>
			<td>381412</td>
			<td>Referred to in text as 24 gauge catheter.</td>
		</tr>
		<tr>
			<td>Masterflex PharMed BPT Tubing, L/S #14, 25&#39;</td>
			<td>Cole-Parmer</td>
			<td>HV-06508-14</td>
			<td>Referred to in text as peristaltic pump tubing.</td>
		</tr>
		<tr>
			<td>Peroxide-Cured Silicone Tubing, 1/16&#34;id X 1/8&#34;OD, 25 Ft/pack</td>
			<td>Cole-Parmer</td>
			<td>HV-06411-62</td>
			<td>Referred to in text as 1/16&#34; ID silicone rubber tubing.</td>
		</tr>
		<tr>
			<td>Masterflex platinum-cured silicone tubing, L/S 14, 25 ft.</td>
			<td>Cole-Parmer</td>
			<td>HV-96410-14</td>
			<td>Referred to in text as thick-walled 1/16&#34; ID silicone rubber tubing.</td>
		</tr>
		<tr>
			<td>VWR Silicone Tubing, 1/4&#34; ID x 0.5&#34; OD</td>
			<td>VWR</td>
			<td>89068-484</td>
			<td></td>
		</tr>
		<tr>
			<td>Acro 50 Vent Filters, Pall Life Sciences</td>
			<td>VWR</td>
			<td>28143-558</td>
			<td>Referred to in text as 0.2 micron vent filter.</td>
		</tr>
		<tr>
			<td>Cole-Parmer Luer Adapters, Male Luer Lock to 1/8&#34; ID, Nylon, 25/Pk</td>
			<td>Cole-Parmer</td>
			<td>T-45505-11</td>
			<td>Referred to in text as male Luer lock to 1/8&#34; barbed adapter.</td>
		</tr>
		<tr>
			<td>Cole-Parmer Luer Accessory, Male Luer Plug, Nylon, 25/Pk</td>
			<td>Cole-Parmer</td>
			<td>EW-45505-58</td>
			<td></td>
		</tr>
		<tr>
			<td>Female luer x female luer adapter, Nylon, 25/pk</td>
			<td>Cole-Parmer</td>
			<td>EW-45502-22</td>
			<td>Referred to in text as female Luer x female Luer adapter.</td>
		</tr>
		<tr>
			<td>Cole-Parmer Luer Accessory, Female Luer Cap, Nylon, 25/Pk</td>
			<td>Cole-Parmer</td>
			<td>EW-45502-28</td>
			<td></td>
		</tr>
		<tr>
			<td>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</td>
			<td>Careforde Healthcare</td>
			<td>10254821</td>
			<td>Smiths Medical (vendor) catalogue number is #MX4311L.</td>
		</tr>
		<tr>
			<td>Other Components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL BD Luer-Lok disposable syringe with BD Luer-Lok&#8482; tip.</td>
			<td>BD Biosciences</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm x 10 mm Easy Grip Culture Dish</td>
			<td>BD Biosciences</td>
			<td>353001</td>
			<td>Used to draw cell suspension into syringe for cell seeding.</td>
		</tr>
	</tbody>
</table>

epithelial cell repopulation and preparation of rodent extracellular matrix scaffolds for renal tissue development

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 &#176;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 &#181;M 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.

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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Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

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 ...

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Bioengineering

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the interface with a liquid core. The latter is removed post-printing, leaving behind a hollow tube. Integrating an additive manufacturing technique (like the one described here with tailor-made [bio]inks, which structurally and biochemically mimic the native extracellular matrix [ECM]) is an important step towards advanced tissue engineering. However, precise fabrication of well-defined structures requires tailored fabrication strategies optimized for the material in use. Therefore, it is sensible to begin with a set-up that is customizable, simple-to-use, and compatible with a broad spectrum of materials and applications. This work presents an easy-to-manufacture core/shell nozzle with luer-compatibility to explore core/shell printing of woodpile structures, tested with a well-defined, alginate-based scaffold material formulation.

Presented here is a simple-to-use, core/shell, three-dimensional bioprinting set-up for one-step fabrication of hollow scaffolds, suitable for tissue engineering of vascular and other tubular structures.

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...