Name: preparation of decellularized kidney scaffolds in rats
Uploaded: 2021-03-18T13:30:00
Description: Watch this Scientific Journal Video about Preparation of Decellularized Kidney Scaffolds in Rats at JoVE.com

This study was supported by a Biomedical Research Institute Grant from Pusan National University Hospital.

This study was approved by the administration of Pusan National University of Medicine and was conducted in accordance with ethical guidelines for the use and care of animals. (certificate no. 2017-119). Prior to any animal studies, institutional approval should be obtained. 
NOTE: All surgical and anesthetic instruments/equipment and reagents recommended for successful surgical presentation and imaging of abdominal organs are detailed in Table 1.
1. Preparation procedures ...

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	<li>Kawai, 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=Brief+report:+HLA-mismatched+renal+transplantation+without+maintenance+immunosuppression.">Brief report: HLA-mismatched renal transplantation without maintenance immunosuppression.</a> New England Journal of Medicine. 358 (4), 353-361 (2008).</li><li>Rana, D., Zreiqat, H., Benkirane-Jessel, N., Ramakrishna, S., Ramalingam, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+decellularized+scaffolds+for+stem+cell-driven+tissue+engineering.">Development of decellularized scaffolds for stem cell-driven tissue engineering.</a> Journal of Tissue Engineering and Regenerative Medicine. 11 (4), 942-965 (2017).</li><li>Fu, R. H., 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+and+recellularization+technologies+in+tissue+engineering.">Decellularization and recellularization technologies in tissue engineering.</a> Cell Transplantation. 23 (4-5), 621-630 (2014).</li><li>Hopkinson, 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=Optimization+of+amniotic+membrane+(AM)+denuding+for+tissue+engineering.">Optimization of amniotic membrane (AM) denuding for tissue engineering.</a> Tissue Engineering. Part C, Methods. 14 (4), 371-381 (2008).</li><li>Shupe, T., Williams, M., Brown, A., Willenberg, B., Petersen, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+the+decellularization+of+intact+rat+liver.">Method for the decellularization of intact rat liver.</a> Organogenesis. 6 (2), 134-136 (2010).</li><li>Petersen, T. H., 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-engineered+lungs+for+in+vivo+implantation.">Tissue-engineered lungs for in vivo implantation.</a> Science. 329 (5991), 538-541 (2010).</li><li>Fernandez-Perez, J., Ahearne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+decellularization+methods+on+extracellular+matrix+derived+hydrogels.">The impact of decellularization methods on extracellular matrix derived hydrogels.</a> Scientific Reports. 9 (1), 14933 (2019).</li><li>Naik, A., Griffin, M., Szarko, M., Butler, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+the+decellularization+process+of+an+upper+limb+skeletal+muscle;+implications+for+muscle+tissue+engineering.">Optimizing the decellularization process of an upper limb skeletal muscle; implications for muscle tissue engineering.</a> Artificial Organs. 44 (2), 178-183 (2020).</li><li>Crapo, P. M., Gilbert, T. W., 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=An+overview+of+tissue+and+whole+organ+decellularization+processes.">An overview of tissue and whole organ decellularization processes.</a> Biomaterials. 32 (12), 3233-3243 (2011).</li><li>Mason, C., Dunnill, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+brief+definition+of+regenerative+medicine.">A brief definition of regenerative medicine.</a> Regenerative Medicine. 3 (1), 1-5 (2008).</li><li>Guyette, J. P., 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+decellularization+of+whole+organs.">Perfusion decellularization of whole organs.</a> Nature Protocols. 9 (6), 1451-1468 (2014).</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> Nature Medicine. 19 (5), 646-651 (2013).</li></ol>

Various protocols have been used for decellularization of organs and other tissues. The optimal decellularization protocol should preserve the three-dimensional architecture of the extracellular matrix (ECM). In general, such protocols consist of lysing the cell membrane by physical processing or ionic solutions, dissociating the cytoplasm and nucleus from the ECM by enzymatic processing or detergents, and then removing cellular debris from the tissue3. Physical processes include scraping, solutio...

Cell culture techniques are applied for regeneration of functional tissues and organs to replace diseased or damaged organs. Allogenic organ transplantation is currently the most common treatment for irreversible organ damage; however, this approach requires the use of immunosuppression to prevent rejection of the transplanted organ. Moreover, despite advances in transplant immunology, 20% of transplant recipients may experience acute rejection within 5 years, and within 10 years after transplantation, 40% of recipients may lose their transplanted graft or die1.
Advances in tissue engineering technologies have yielded in a new paradigm for transplantation of new organs without immune rejection using differentiated stem cells. After stem cell differentiation, a scaffold, called a synthetic extracellular matrix, is needed to facilitate the generation of three-dimensional organs and enable the new tissue to thrive within the recipient. Scaffolds from decellularized native organs have advantages, including a more effective environment for establishment of cells and enhancement of stem cell proliferation, although these mechanisms have not been fully elucidated2. In particular, the kidney is a suitable organ for scaffold generation because it has abundant circulation and a niche for stem cell establishment. Additionally, because of the complex structure of the kidney, it is difficult to artificially regenerate kidneys for organ transplantation.
In this report, we introduce a method of developing vascularized scaffolds using decellularized organs in a rat model to facilitate future animal studies for tissue engineering purposes.

<table>
	<tbody>
		<tr>
			<td>1 cc syringe (inject probes and vehicle solutions</td>
			<td>Becton Dickinson</td>
			<td>305217</td>
			<td></td>
		</tr>
		<tr>
			<td>10-0 ethilon for vessel anastomosis</td>
			<td>Ethicon</td>
			<td>9032G</td>
			<td></td>
		</tr>
		<tr>
			<td>25 gauge inch guide needle(for vascular catheters)</td>
			<td>Becton Dickinson</td>
			<td>305145</td>
			<td></td>
		</tr>
		<tr>
			<td>3-0 PDS incision closure rat</td>
			<td>Ethicon</td>
			<td>Z316H</td>
			<td></td>
		</tr>
		<tr>
			<td>3-0 Prolene incision closure rat</td>
			<td>Ethicon</td>
			<td>8832H</td>
			<td></td>
		</tr>
		<tr>
			<td>3-0 silk spool vascular access/ligation in rat</td>
			<td>Braintree Scientific</td>
			<td>SUT-S 110</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 PDS incision closure mouse</td>
			<td>Ethicon</td>
			<td>Z773D</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 Prolene incision closure mouse</td>
			<td>Ethicon</td>
			<td>8831H</td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 silk spool vascular access/ligation in mouse</td>
			<td>Braintree Scientific</td>
			<td>SUT-S 106</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors to cut fascia/connective tissue</td>
			<td>Fine Science Tools</td>
			<td>14058-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Halsey needle holder</td>
			<td>Fine Science Tools</td>
			<td>12001-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Kelly Hemostat for rats: muscle clamp to minimize bleeding when cut</td>
			<td>Fine Science Tools</td>
			<td>13018-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethelyne 50 tubing, catheter tubing 100 ft</td>
			<td>Braintree Scientific</td>
			<td>.023&#34; &#215; .038&#8221;</td>
			<td></td>
		</tr>
		<tr>
			<td>Schwartz microserrefine vascular clamps</td>
			<td>Fine Science Tools</td>
			<td>18052-01 (straight)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>18052-03 (curved)</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors to cut skin</td>
			<td>Fine Science Tools</td>
			<td>14002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas-Tubingen Spring scissors for arteriotomy</td>
			<td>Fine Science Tools</td>
			<td>15003-08</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of decellularized kidney scaffolds in rats

Tissue engineering is a cutting-edge discipline in biomedicine. Cell culture techniques can be applied for regeneration of functional tissues and organs to replace diseased or damaged organs. Scaffolds are needed to facilitate the generation of three-dimensional organs or tissues using differentiated stem cells in vivo. In this report, we describe a novel method for developing vascularized scaffolds using decellularized rat kidneys. Eight-week-old Sprague-Dawley rats were used in this study, and heparin was injected into the heart to facilitate flow into the renal vessels, allowing heparin to perfuse into the renal vessels. The abdominal cavity was opened, and the left kidney was collected. The collected kidneys were perfused for 9 h using detergents, such as Triton X-100 and sodium dodecyl sulfate, to decellularize the tissue. Decellularized kidney scaffolds were then gently washed with 1% penicillin/streptomycin and heparin to remove cellular debris and chemical residues. Transplantation of stem cells with the decellularized vascular scaffolds is expected to facilitate the generation of new organs. Thus, the vascularized scaffolds may provide a foundation for tissue engineering of organ grafts in the future.

The gross morphology of rat kidneys was dark red (Figure 1A). After decellularization, the kidney became pale and translucent (Figure 1D). Residual genomic DNA was assessed with a commercial kit according to the manufacturer&#8217;s instructions, in decellularized kidney scaffolds and compared with that in native kidneys (control). Quantitative analysis confirmed that tissue genomic DNA was almost eliminated after decellularization. From 14 cases, the average DN...

Watch this Scientific Journal Video about Preparation of Decellularized Kidney Scaffolds in Rats at JoVE.com

Preparation of Decellularized Kidney Scaffolds in Rats

This protocol introduces a method to develop a scaffold using decellularized rat kidneys. The protocol includes decellularization and recellularization processes to confirm bioavailability. Decellularization is performed using Triton X-100 and sodium dodecyl sulfate.

Tissue engineering is a cutting-edge discipline in biomedicine. Cell culture techniques can be applied for regeneration of functional tissues and organs ...

This protocol makes it possible to develop vascularized scaffolds using kidneys in a rat model. The kidney is the most suitable organ for scaffolds of differentiated stem cells. This is a highly successful protocol for decellularization of the kidney.It effectively removes the nuclei with minimal destruction of the ECM ultrastructure. This technique can be applied to transplantation medicine and tissue engineering. Demonstrating the procedure will be Kim Joo Hyoung, an MD/PhD associate professor of plastic surgery from my laboratory.To begin, place the eight-week-old Sprague Dawley rat on a warming pad, and position a rectal thermometer probe in the rectum to monitor core temperature. After anesthetizing the rat, place it in a supine position, and secure the four limbs to the table with tape. Shave and clean the abdomen with germicidal soap.Apply 2%Betadine for at least one to two minutes, then wipe it off with a 70%ethanol solution. Repeat this sequence three times. Cover the operative field with a sterile, fenestrated drape.Make a vertical abdominal incision, and expose the left kidney, ureter, abdominal aorta, and inferior vena cava. Dissect the tissue just before cutting the pedicle. Prepare 50 milliliters of perfusion solution per rat by mixing PBS with approximately 10 units per milliliter heparin.Mix equal volumes of 8%PFA with PBS to make the 4%PFA solution. Extend the vertical abdominal incision cranially, making sure to draw the scissors away from the organs when cutting to avoid damage. Continue the incision through the rib cage, and then cut through the diaphragm by lifting the sternum.Retract the loose flap of skin out of the way with an Allis clamp, and free the heart by tearing any connective tissue with the forceps. Open the PBS line, and ensure that it is flowing before placing the needle into the left ventricle. Hold the heart gently with blunt forceps, and use a hemostat to control the needle.The needle should be inserted no more than a quarter inch. While supporting the heart with the needle and hemostat, locate the right atrium, and snip through it with iridectomy scissors. Rest the hemostat on the rat's body, and make sure the needle is still positioned inside the heart.Continue perfusing PBS for four minutes or longer if there is still blood visible in the kidney and liver. Harvest the left kidney with the abdominal aorta and inferior vena cava. Ligate the ureter, thoracic aorta, superior vena cava, and branches of the abdominal aorta.Keep the organ hydrated in DPBS in a 10-centimeter Petri dish. Cannulate the abdominal aorta and inferior vena cava with a 23-gauge catheter. To remove residual blood, connect the cannula with a peristaltic pump, and wash the organ with 500 milliliters of DPBS and 16 units per milliliter heparin for 90 minutes at five rpm and 37 degrees Celsius.To decellularize the kidney, perfuse it with 1%Triton X-100 for three hours and then with 0.75%SDS solution for six hours at a constant pressure of 40 milliliters of mercury. To remove residual SDS, perfuse the sample with 1%penicillin in distilled water for 18 hours and then with sterile DPBS and 16 units per milliliter heparin for 90 minutes. The gross morphology of rat kidneys was dark red.After decellularization, the kidney became pale and translucent. Residual genomic DNA was quantified in decellularized kidney scaffolds and compared with that in native kidneys, confirming that tissue genomic DNA was almost eliminated after decellularization. From 14 cases, the average DNA contents were 115.05 nanograms per microliter for the control and 1.96 nanograms per microliter for the decellularized scaffold.In total, 98.3%of DNA was removed. The three-dimensional structure was maintained, and acellular glomeruli were preserved in the cortical parenchyma. When inserting the needle into the left ventricle, it should be inserted no more than a quarter inch.Any further insertion may come out the other side. We have confidence that this technique will open a new prospect in the field of tissue engineering.

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