Name: microdissection of primary renal tissue segments and incorporation with novel scaffold-free construct technology
Uploaded: 2018-03-27T13:30:00
Description: Watch this Scientific Journal Video about Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology at JoVE.com

NIH Institutional Postdoctoral Training Grant, NIH-HL-007260

All animal surgical procedures described below were approved by the Institutional Animal Care and Use Committee (IACUC) at the Medical University of South Carolina prior to any animal surgeries or use of any animal tissues.
1. Murine Nephrectomy
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	<li>Don a surgical mask and bouffant cap to minimize the risk of contamination. Maintain sterility during set-up of the&#160;surgical area.
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		<li>Place non-fenestrated surgical drapes on the operating table.</li>
		<li>Open the pack of au...

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	<li>Jha, V., 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=Chronic+kidney+disease:+global+dimension+and+perspectives.">Chronic kidney disease: global dimension and perspectives.</a> Lancet. 382 (9888), 260-272 (2013).</li><li>2016 USRDS Annual Data Report: Epidemiology of Kidney Disease in the United States. U.S.R.D Available from: <a target="_blank" href="https://www.usrds.org/2016/download/v2_ESRD_16.pdf">https://www.usrds.org/2016/download/v2_ESRD_16.pdf</a> (2016)</li><li>Hart, 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=OPTN/SRTR+2015+Annual+Data+Report:+Kidney.">OPTN/SRTR 2015 Annual Data Report: Kidney.</a> American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 17, 21-116 (2017).</li><li>de Vries, E. F., Rabelink, T. J., van den Hout, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+the+Cost-Effectiveness+of+Delaying+End-Stage+Renal+Disease.">Modelling the Cost-Effectiveness of Delaying End-Stage Renal Disease.</a> Nephron. 133 (2), 89-97 (2016).</li><li>Lefebvre, P., Duh, M. S., Mody, S. H., Bookhart, B., Piech, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+economic+impact+of+epoetin+alfa+therapy+on+delaying+time+to+dialysis+in+elderly+patients+with+chronic+kidney+disease.">The economic impact of epoetin alfa therapy on delaying time to dialysis in elderly patients with chronic kidney disease.</a> Disease management : DM. 10 (1), 37-45 (2007).</li><li>Mennini, F. S., Russo, S., Marcellusi, A., Quintaliani, G., Fouque, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Economic+effects+of+treatment+of+chronic+kidney+disease+with+low-protein+diet.">Economic effects of treatment of chronic kidney disease with low-protein diet.</a> Journal of renal nutrition : the official journal of the Council on Renal Nutrition of the National Kidney Foundation. 24 (5), 313-321 (2014).</li><li>Moon, K. H., Ko, I. K., Yoo, J. J., Atala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kidney+diseases+and+tissue+engineering.">Kidney diseases and tissue engineering.</a> Methods. 99, 112-119 (2016).</li><li>Wobma, H., Vunjak-Novakovic, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Engineering+and+Regenerative+Medicine+2015:+A+Year+in+Review.">Tissue Engineering and Regenerative Medicine 2015: A Year in Review.</a> Tissue engineering. Part B, Reviews. 22 (2), 101-113 (2016).</li><li>Langer, R., Vacanti, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+tissue+engineering.">Advances in tissue engineering.</a> Journal of pediatric surgery. 51 (1), 8-12 (2016).</li><li>Jakab, 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=Tissue+engineering+by+self-assembly+and+bio-printing+of+living+cells.">Tissue engineering by self-assembly and bio-printing of living cells.</a> Biofabrication. 2 (2), 022001 (2010).</li><li>Fisher, M. B., Mauck, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+and+regenerative+medicine:+recent+innovations+and+the+transition+to+translation.">Tissue engineering and regenerative medicine: recent innovations and the transition to translation.</a> Tissue engineering. Part B, Reviews. 19 (1), 1-13 (2013).</li><li>Stewart, D. E., Garcia, V. C., Rosendale, J. D., Klassen, D. K., Carrico, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosing+the+Decades-Long+Rise+in+the+Deceased+Donor+Kidney+Discard+Rate+in+the+United+States.">Diagnosing the Decades-Long Rise in the Deceased Donor Kidney Discard Rate in the United States.</a> Transplantation. 101 (3), 575-587 (2017).</li><li>Mohan, 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=The+weekend+effect+alters+the+procurement+and+discard+rates+of+deceased+donor+kidneys+in+the+United+States.">The weekend effect alters the procurement and discard rates of deceased donor kidneys in the United States.</a> Kidney international. 90 (1), 157-163 (2016).</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 (24), 5915-5925 (2013).</li><li>Katari, 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=Renal+bioengineering+with+scaffolds+generated+from+human+kidneys.">Renal bioengineering with scaffolds generated from human kidneys.</a> Nephron. Experimental nephrology. 126 (2), 119 (2014).</li><li>Kim, S. S., Park, H. J., Han, J., Choi, C. Y., Kim, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renal+tissue+reconstitution+by+the+implantation+of+renal+segments+on+biodegradable+polymer+scaffolds.">Renal tissue reconstitution by the implantation of renal segments on biodegradable polymer scaffolds.</a> Biotechnology letters. 25 (18), 1505-1508 (2003).</li><li>Guimaraes-Souza, N. K., Yamaleyeva, L. M., AbouShwareb, T., Atala, A., Yoo, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+reconstitution+of+human+kidney+structures+for+renal+cell+therapy.">In vitro reconstitution of human kidney structures for renal cell therapy.</a> Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association. 27 (8), 3082-3090 (2012).</li><li>Kelley, 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=Tubular+cell-enriched+subpopulation+of+primary+renal+cells+improves+survival+and+augments+kidney+function+in+rodent+model+of+chronic+kidney+disease.">Tubular cell-enriched subpopulation of primary renal cells improves survival and augments kidney function in rodent model of chronic kidney disease.</a> American journal of physiology. Renal physiology. 299 (5), 1026-1039 (2010).</li><li>Czajka, C. A., Drake, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembly+of+prevascular+tissues+from+endothelial+and+fibroblast+cells+under+scaffold-free,+nonadherent+conditions.">Self-assembly of prevascular tissues from endothelial and fibroblast cells under scaffold-free, nonadherent conditions.</a> Tissue engineering. Part A. 21 (1-2), 277-287 (2015).</li><li>Rhett, J. M., Wang, H., Bainbridge, H., Song, L., Yost, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connexin-Based+Therapeutics+and+Tissue+Engineering+Approaches+to+the+Amelioration+of+Chronic+Pancreatitis+and+Type+I+Diabetes:+Construction+and+Characterization+of+a+Novel+Prevascularized+Bioartificial+Pancreas.">Connexin-Based Therapeutics and Tissue Engineering Approaches to the Amelioration of Chronic Pancreatitis and Type I Diabetes: Construction and Characterization of a Novel Prevascularized Bioartificial Pancreas.</a> Journal of diabetes research. 2016, 7262680 (2016).</li><li>Al-Awqati, Q., Oliver, 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=Stem+cells+in+the+kidney.">Stem cells in the kidney.</a> Kidney international. 61 (2), 387-395 (2002).</li><li>Aboushwareb, T., 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=Erythropoietin+producing+cells+for+potential+cell+therapy.">Erythropoietin producing cells for potential cell therapy.</a> World journal of urology. 26 (4), 295-300 (2008).</li></ol>

Methods used to engineer living renal tissue constructs vary widely with regard to both the type of cells and biomaterials utilized, and in many cases, are outdated or not well-characterized in the literature7. While many are using stem cell approaches or recapitulating individual components of the renal architecture in isolation, the prospect of artificially recreating an entire organ with over 26 different differentiated cell types from cellular suspensions is overwhelming to consider<sup class=...

Chronic kidney disease (CKD) is one of the current major public health challenges worldwide1. The prevalence of CKD in the United States is over 14% of the total population, with over 600,000 Americans suffering from the most severe form, end-stage renal disease (ESRD)2. The current treatment options available for those with ESRD include dialysis and kidney transplantation. Although approximately 25,000 patients undergo renal transplantation each year, a significant number of patients are added annually leading to a large disparity between those awaiting a life-saving organ and those receiving transplantation3. In addition to its serious negative effects on longevity and quality of life, dialysis is associated with an astonishing financial burden. In 2014, Medicare paid claims totaled over $30 billion for ESRD patients2. With a limited organ supply and no apparent downtrend in patients requiring dialysis, research efforts aimed at identifying alternative solutions to dialysis and transplantation are ever important. Even a relatively short delay in the need for dialysis increases a patient&#39;s number of quality-adjusted life years and productivity substantially while postponing dialysis-related costs4,5,6.
Solutions for functional tissue loss, like that in ESRD, are currently being studied in tissue engineering and regenerative medicine laboratories, with widely varied approaches ranging from scaffold-based organoid fabrication to whole organ engineering using decellularized organ structures for cellular implantation7,8,9,10,11. Recapitulating complex renal structures from marginal or discarded kidneys has only partially been investigated. In fact, nearly 20% of kidneys procured for transplantation are discarded for various reasons12,13. The functional renal tissue from these putative grafts could be utilized and incorporated into one or many tissue-engineered constructs. Prior studies have demonstrated the feasibility of working with these discarded organs, utilizing kidneys for the extra-cellular matrix for tissue engineering purposes14,15. However, few have used primary nephronal tissue from healthy kidneys for tissue-engineering purposes16,17,18.
One method previously described by Kim et al. involves isolation of renal &#34;segments&#34; from healthy rat kidneys, which were then seeded on polyglycolic acid (PGA) scaffolds for construct fabrication16. However, little information is given regarding exact dissection methodology, and segments were obtained from a combination of fine mincing and filtration. We describe a modification of this protocol, which similarly produces discrete renal segments with intact nephronal architecture, but instead relies on microdissection techniques. Nephrectomies are performed on living adult mice, after which the kidneys are transferred to the dissection microscope where the renal capsule is removed, and the tissue is further dissected. Small-bore 30&#189; G needles are used as cutting instruments and also as guides aiding in dissection, as the needle diameter is equal to the target diameter of the renal segments. The isolated, in this case murine, renal segments maintain viability in culture and incorporate with scaffold-free endothelial-fibroblast cellular constructs19. These constructs have previously been used to engineer other organs, including a bio-artificial pancreas20.

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			<td height="42" style="height:42px;width:173px;">Non-fenestrated Sterile Field</td>
			<td style="width:181px;">Busse Hospital Disposables</td>
			<td style="width:153px;">696</td>
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			<td height="42" style="height:42px;width:173px;">Fenestrated Sterile Field</td>
			<td style="width:181px;">Busse Hospital Disposables</td>
			<td style="width:153px;">697</td>
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			<td height="42" style="height:42px;width:173px;">Halsted Mosquito Forceps 5 Curved</td>
			<td style="width:181px;">Miltex</td>
			<td style="width:153px;">Mil-7-4</td>
			<td>&#34;Hemostat&#34; in manuscript</td>
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			<td height="63" style="height:63px;width:173px;">Extra Fine Graefe Forceps, Curved with teeth</td>
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			<td height="63" style="height:63px;width:173px;">Betadine Surgical Scrub with Pump, Povidone-iodine 7.5%</td>
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			<td style="width:153px;">67618-151-17</td>
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			<td height="42" style="height:42px;width:173px;">Sterile Cotton Gauze Pad (4&#34; x 4&#34;)</td>
			<td style="width:181px;">Fisher Healthcare</td>
			<td style="width:153px;">22-415-469</td>
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			<td height="42" style="height:42px;width:173px;">Penicillin/Streptomycin Solution, 100X</td>
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			<td style="width:153px;">30-002-Cl</td>
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			<td height="42" style="height:42px;width:173px;">Isoflurane, USP</td>
			<td style="width:181px;">Manufacturer: Piramal, Distributor: McKesson</td>
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			<td>Hair Removal Cream in text</td>
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			<td>Diluted to 70% Ethanol Solution</td>
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			<td style="width:153px;">130181</td>
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			<td style="width:181px;">Glad</td>
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			<td>Applied to Stereoscope</td>
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			<td height="21" style="height:21px;width:173px;">SZX16 Stereo Microscope</td>
			<td style="width:181px;">Olympus</td>
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			<td height="63" style="height:63px;width:173px;">Self-Supporting Dual-Light Pipe, 23&#34; L Gooseneck</td>
			<td style="width:181px;">Cole Parmer</td>
			<td style="width:153px;">EW-41720-60</td>
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			<td height="21" style="height:21px;width:173px;">Scalpel Handle #3</td>
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			<td style="width:153px;">Mil-4-7</td>
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			<td height="63" style="height:63px;width:173px;">Sterile Rib-Back Carbon Steel Blade, Blade Size 15</td>
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			<td style="width:153px;">371115</td>
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			<td height="21" style="height:21px;width:173px;">31 1/2 Gauge Needle</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:153px;">14-826F</td>
			<td style="width:199px;">Becton Dickinson 305106</td>
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			<td height="42" style="height:42px;width:173px;">Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td style="width:181px;">Corning</td>
			<td style="width:153px;">10-017-CV</td>
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			<td height="42" style="height:42px;width:173px;">Fetal Select 100% Bovine Serum</td>
			<td style="width:181px;">Atlas Biologicals</td>
			<td style="width:153px;">FS-0500-AD</td>
			<td></td>
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			<td height="42" style="height:42px;width:173px;">Normal Human Dermal Fibroblasts</td>
			<td style="width:181px;">Lonza</td>
			<td style="width:153px;">CC-2511</td>
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			<td height="63" style="height:63px;width:173px;">Human Adipose Microvascular Endothelial Cells</td>
			<td style="width:181px;">Sciencell Research Laboratories</td>
			<td style="width:153px;">7200</td>
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			<td height="21" style="height:21px;width:173px;">Surgical Loupes (2.5x)</td>
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			<td style="width:181px;">Lonza</td>
			<td style="width:153px;">CC-3131, CC-4126</td>
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			<td height="63" style="height:63px;width:173px;">EGM-2 (Endothelial Basal Medium with EGM-2 SingleQuots Added)</td>
			<td style="width:181px;">Lonza</td>
			<td style="width:153px;">CC-3156, CC-4176</td>
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			<td height="63" style="height:63px;width:173px;">Live/Dead Viability/Cytotoxicity Kit for Mammalian Cells</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:153px;">L3224</td>
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			<td height="42" style="height:42px;width:173px;">Anti-Cytokeratin-18 Antibody</td>
			<td style="width:181px;">Abcam</td>
			<td style="width:153px;">ab668</td>
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			<td height="42" style="height:42px;width:173px;">Goat anti-Mouse IgG, Alexa Fluor 633</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:153px;">A-21052</td>
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			<td height="42" style="height:42px;width:173px;">Goat anti-Rabbit IgG, Alexa Fluor 546</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:153px;">A-11010</td>
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			<td height="42" style="height:42px;width:173px;">Anti-Von Willebrand Factor Antibody</td>
			<td style="width:181px;">Abcam</td>
			<td style="width:153px;">ab6994</td>
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			<td height="63" style="height:63px;width:173px;">Albumin, Fluorescein isothiocyanate Conjugate</td>
			<td style="width:181px;">Sigma Aldrich</td>
			<td style="width:153px;">A9771-50MG</td>
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			<td height="21" style="height:21px;width:173px;">Hoescht 33342</td>
			<td style="width:181px;">BD Pharmingen</td>
			<td style="width:153px;">561908</td>
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			<td height="21" style="height:21px;width:173px;">Background Buster</td>
			<td style="width:181px;">Innovex Biosciences</td>
			<td style="width:153px;">NB306</td>
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microdissection of primary renal tissue segments and incorporation with novel scaffold-free construct technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

The protocol described produces approximately 50 renal segments per pyramidal 2 mm3 section of renal tissue. The renal segments that have been processed and imaged have tubular and glomerular components in differing proportions (see Figure 2). The intact segments were subjected to an assay in order to determine the viability of different segments once every 24 h for three days. Green-fluorescent calcein-AM is present with intracellular esterase act...

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Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

The overall goal of this microscopic dissection procedure is to isolate discrete segments of living renal tissue with intact renal architecture for tissue engineering applications. This method can help answer key questions in the field of tissue engineering such as can whole segments of isolated primary organ tissue be harvested sterilely and be kept alive in culture? The main advantage of this technique is the segments contain intact renal architecture as opposed to using cell lines to recreate a complex organ with over 26 different cell types.The implications of this technique extend towards therapy of end-stage renal disease because these constructs will improve filtration and may help patients stay off dialysis. During the surgery, wear proper surgical gear including a mask and a cap to minimize the risk of contamination. Likewise, make every effort to maintain sterility throughout.Start preparations by draping the operating table. Next, open the pack of autoclaved instruments onto the sterile drapes. They must include three small hemostats, fine forceps with teeth, fine forceps without teeth, and straight iris scissors.Cover the center of the operating table with a second non-fenestrated surgical drape. Prior to setting up the operating room, prepare 50 milliliters of antibiotic supplemented DBPS and aliquot five milliliters into a 15 milliliter tube. Now anesthetize an eight to 12-week-old C57 black six mouse and periodically monitor the level of anesthesia by assessing its pedal reflex with a firm toe pinch.Next, use a hair removal cream to remove all of the hair from the ventral torso area and rinse off the cream and hair with distilled water. Then transfer the mouse to the operating table and place it supine under the non-fenestrated drape. Now cut out a two centimeter square fenestration in the drape over the mouse's abdomen.Then scrub the exposed skin with povidone-iodine followed by 70%ethanol three times to sterilize the skin and complete the preparations for the surgery. Begin the surgery by using fine forceps with teeth and iris scissors. Make a three to four centimeter skin incision parallel to the midline one-half centimeter to the left of the midline.Next, grasp the peritoneum using fine forceps without teeth and incise with iris scissors to enter the abdominal cavity. Then apply hemostats to the superior and inferior lateral edges of skin and peritoneum to place tension laterally and enhance the exposure. Now delicately shift the intestinal contents to the right of the abdomen to expose the left kidney.Then gently place traction on the kidney to elevate it out of the abdomen. Now place a hemostat across the hilum of the kidney and use iris scissors to transect the ureter and renal vessels. Then transfer the kidney to the tube of 1%pen-strep DBPS on ice.Take this tube to a sterile culture hood for processing. There transfer the kidney to a 60 millimeter Petri dish and rinse it twice with fresh five milliliters of DPBS containing calcium and magnesium. After the second wash, suspend the kidney in fresh five milliliters of DBPS in a new 60 millimeter dish.Then prepare an additional Petri dish containing only five milliliters of DBPS for later use. Continue using sterile technique including sterile gloves, surgical mask, and bouffant to minimize contamination risks. Then drape the stage area and area surrounding the stereo microscope.Next, place plastic adhesive sheets on the microscope focus knobs and spray them with 70%ethanol. Then turn on the dual gooseneck illuminator to light the stage. Now onto the sterile drapes, open a package of sterilized instruments containing fine forceps without teeth, a scalpel handle, a number 15 scalpel blade, two straight hemostats, and two 30.5 gauge hollow-bore needles.Place the number 15 blade onto the scalpel handle and attach one hemostat to each needle adapter hub to create needle dissection instruments. Now transfer the two dishes, one with the kidney in DPBS and the other only DPBS, to the microscope area. Remove the lid on the kidney dish and focus the microscope on the anterior or posterior surface of the kidney at 3.2 to 4X magnification.Next, with the non-dominant hand, use fine forceps without teeth to pierce and pin the kidney against the dish at the inferior and superior poles of the kidney. Then with the dominant hand, use the number 15 blade to remove the translucent fibrous capsular layer from the exposed surface. Shave the most superficial 0.5 millimeters from the exposed surface of the kidney surface to remove the remainder of the capsule.Continue by using the number 15 blade to dissect an inverted pyramid of tissue from the de-capsulated area that is approximately two square millimeters. Then transfer the tissue into the clean dish of DBPS and discard the remainder of the kidney. Now focus on the dissected tissue and decrease the magnification to 1.5 to 2.0X.Using the two hemostat needle instruments as cutting tools, slice the tissue fragment into progressively smaller pieces until the tissue segments are the size of the needle tips or smaller. About 50 segments should be produced. This is a critical portion of the procedure.Each segment needs to closely approximate the diameter of the 30-1/2 gauge needle tip to avoid large segments which could hinder diffusion. Return the tissue to the tissue culture hood and remove the DPBS using a P1000 micropipette. Then add back five millimeters of DMEM supplemented with 10%fetal bovine serum and 1%pen-strep.Then culture the tissue or implant it within cellular constructs immediately. The viability of the intact renal segments produced by the described procedure was examined over three days in culture using a viability assay. Green fluorescent calcium AM is present with intracellular esterase activity indicative of living cells.Red fluorescent ethidium homodimer-1 is seen with loss of integrity of the plasma membrane. When the renal segments were embedded in scaffold-free endothelial fibroblast constructs, by day three they were incorporated with the cellular constructs to form intact structures. The constructs maintained their pre-vascular endothelial network shown by labeling with von Willebrand factor.Renal epithelial cells can be seen in green labeled by cytokertain-18. To test in-vitro renal functionality, the constructs were incubated with FITC-labeled albumin. While there was residual albumin found away from the segments of embedded renal tissue, there were also hotspots in the renal tubular epithelial cells.These hotspots are albumin that traverses intraluminally which is thought to represent albumin reuptake in the renal segment cellular construct. After watching this video, you should have a good understanding of how to isolate renal segments from murine kidneys for tissue engineering applications. Once mastered, this procedure can be done in one to 1-1/2 hours.Following this procedure, other methods including seeding cellularized constructs can be performed in order to answer other questions regarding renal construct fabrication and characterization. Don't forget that working with surgical instruments and pressurized oxygen cylinders can be extremely hazardous. Be careful when working with sharp objects and remember to anchor oxygen tanks to a lab bench.

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JoVE Journal

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