Name: the mouse isolated perfused kidney technique
Uploaded: 2016-11-17T14:00:00
Description: Watch this Scientific Journal Video about The Mouse Isolated Perfused Kidney Technique at JoVE.com

The authors would like to thank Hans-Joachim Schurek for invaluable scientific advice. The authors would like to thank Monique Carrel and Mich&#232;le Heidemeyer for excellent technical assistance, David Penton Ribas and Nourdine Faresse for a critical reading of the manuscript and Carsten Wagner and J&#252;rg Biber for the NaPi-2a antibody. This work was supported by the Swiss National Centre for Competence in Research "Kidney.CH" and by a project grant (310030_143929/1) from the Swiss National Science Foundation.

All procedures involving animals described in this manuscript were conducted according to Swiss law and approved by the veterinary administration of the Canton of Zurich, Switzerland.
1. Buffer Preparation
<ol>
	<li>Prepare solutions 1 - 4 and the antidiuretic hormone (ADH) solution (Table 1).</li>
	<li>Prepare the dialysis buffer (Table 1). 
	NOTE: This is the buffer used as the dialysis buffer during the perfusion. Later, the erythrocytes will be dilu...

<ol>
	<li>Carrel, A., Lindbergh, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+culture+of+whole+organs.">The culture of whole organs.</a> Science (New York, N.Y.). 81 (2112), 621-623 (1935).</li><li>Skutul, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> &#220;ber Durchstr&#246;mungsapparate Pfl&#252;ger's Archiv. 123 (4-6), 249-273 (1908).</li><li>Nizet, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolated+perfused+kidney:+possibilities,+limitations+and+results.">The isolated perfused kidney: possibilities, limitations and results.</a> Kidney Int. 7 (1), 1-11 (1975).</li><li>Weiss, C., Passow, H., Rothstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoregulation+of+flow+in+isolated+rat+kidney+in+the+absence+of+red+cells.">Autoregulation of flow in isolated rat kidney in the absence of red cells.</a> Am J Physiol. 196 (5), 1115-1118 (1959).</li><li>Schurek, H. J., Kriz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphologic+and+functional+evidence+for+oxygen+deficiency+in+the+isolated+perfused+rat+kidney.">Morphologic and functional evidence for oxygen deficiency in the isolated perfused rat kidney.</a> Lab Invest. 53 (2), 145-155 (1985).</li><li>Stolte, H., Schurek, H. J., Alt, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glomerular+albumin+filtration:+a+comparison+of+micropuncture+studies+in+the+isolated+perfused+rat+kidney+with+in+vivo+experimental+conditions.">Glomerular albumin filtration: a comparison of micropuncture studies in the isolated perfused rat kidney with in vivo experimental conditions.</a> Kidney Int. 16 (3), 377-384 (1979).</li><li>Schweda, F., Wagner, C., Kr&#228;mer, B. K., Schnermann, J., Kurtz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preserved+macula+densa-dependent+renin+secretion+in+A1+adenosine+receptor+knockout+mice.">Preserved macula densa-dependent renin secretion in A1 adenosine receptor knockout mice.</a> AJP Renal Physiol. 284 (4), F770-F777 (2003).</li><li>Moers, C., Smits, J. 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=Machine+perfusion+or+cold+storage+in+deceased-donor+kidney+transplantation.">Machine perfusion or cold storage in deceased-donor kidney transplantation.</a> N Engl J Med. 360 (1), 7-19 (2009).</li><li>Nicholson, M. L., Hosgood, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renal+transplantation+after+ex+vivo+normothermic+perfusion:+the+first+clinical+study.">Renal transplantation after ex vivo normothermic perfusion: the first clinical study.</a> Am J Transplant. 13 (5), 1246-1252 (2013).</li><li>Worner, M., Poore, S., Tilkorn, D., Lokmic, Z., Penington, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+low-cost,+small+volume+circuit+for+autologous+blood+normothermic+perfusion+of+rabbit+organs.">A low-cost, small volume circuit for autologous blood normothermic perfusion of rabbit organs.</a> Art Org. 38 (4), 352-361 (2014).</li><li>Kaths, J. M., Spetzler, V. N., 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=Normothermic+Ex+Vivo+Kidney+Perfusion+for+the+Preservation+of+Kidney+Grafts+prior+to+Transplantation.">Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation.</a> J Vis Exp. (101), e52909 (2015).</li><li>Song, J. J., Guyette, J. P., Gilpin, S. E., Gonzalez, G., Vacanti, J. P., Ott, H. C. <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 Med. 19 (5), 646-651 (2013).</li><li>Hall, A. M., Crawford, C., Unwin, R. J., Duchen, M. R., Peppiatt-Wildman, C. 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The mouse isolated perfused kidney is a tool for studying kidney function in a controlled environment ex vivo for 1 hr, bridging the gap between in vivo experiments in intact animals, which may be flawed by the impact of numerous systemic factors, and in vitro experiments in isolated nephron segments or cultured cells, which necessarily neglect the impact of intact organ structure on function. There is, to the authors&#39; knowledge, no alternative technique with which to perform this specific ...

The isolated perfusion of organs has been the subject of an ongoing effort among physiologists for many decades1. The technique enables the function of the organ, without systemic influences such as blood pressure, hormones, or nerves, to be studied. Carl Eduard Loebell is considered to be the first to have described the successful perfusion of an isolated kidney, in 18492. Since then, the perfusion apparatus has undergone significant refinement. Frey and Gruber introduced an artificial lung for oxygenation and pulsatile pumps for continuous perfusion2. While early researchers mainly studied the kidneys of large mammals-namely, pigs2 and dogs3-the first report of the use of rat kidneys, by Weiss et al., was a milestone in the study of small-mammal-organ perfusion4. Schurek et al. reported the necessity of adding mammalian erythrocytes to the perfusate if sufficient renal tubular oxygenation was to be achieved5. Critical for long-term experiments was the introduction of continuous dialysis of the buffer by the same research group6. In 2003, Schweda et al. were the first to report a functional mouse isolated perfused kidney (MIPK)7, later refined by Rahgozar et al.18 and Lindell et al.14.
While technically more challenging than the rat isolated perfused kidney, the use of the MIPK bears the advantage of enabling the use of a wide array of genetically altered mice. This paper presents the details of the authors&#39; method for perfusing isolated mouse kidneys for 1 hr. The method allows for the continuous assessment of renal flow rate, vascular resistance, hormone release, blood gas analysis, urine analysis, and the application of drugs. Following the procedure, kidneys could be processed for molecular and biochemical analysis, be fixed for microscopy, or transplanted into a recipient mouse (Figure 1).
<img alt="Figure 1" src="/files/ftp_upload/54712/54712fig1.jpg" /> 
Figure 1: Overview of Possible Input/Output to the Isolated Perfused Kidney. BGA: Blood gas analysis. <a href="http://cloudflare.jove.com/files/ftp_upload/54712/54712fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
This technique likely will receive increasing attention over coming years, as many innovative applications are being discussed with the dawn of prolonged normothermic kidney perfusion prior to transplantation (with or without the application of anti-rejection or genome-editing drugs)8,9, 10, 11, the bioengineering of whole kidneys from decellularized scaffolds12, and the application of high doses of fluorescent dyes for multiphoton imaging13. It is also an ideal model with which to study the role of specific genes during acute kidney injury14.
A step-by-step protocol is given to allow other laboratories to perform isolated mouse kidney perfusion successfully. First, the composition and preparation of the buffer is specified. Then, the surgery is described in detail and the critical steps are shown. Third, data is presented that are representative of a successful preparation: renal blood flow, vascular resistance, glomerular filtration rate , and fractional electrolyte excretion-all as functional measurements of viability-and transmission electron micrographs of the morphology of different nephron segments of perfused kidneys fixed after 1 hr of perfusion.

<table border="0" cellpadding="0" cellspacing="0" width="906">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Perfusion Circuit:</td>
			<td style="width:173px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Moist chamber 834/8</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-2901</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Cannular with basket and side port</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-2947</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Thermostat TC120-ST5&#160;</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-4544</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">ISM 827/230V Roller Pump Reglo Analogue</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-0114</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Reservoir jacketed for buffer solution 1L</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-3438</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Reservoir jacketed for buffer solution 0.5L</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-3436</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Pressure Transducer APT300&#160;</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-3862</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">TAM-D Plugsys Transducer</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-1793</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">SCP Plugsys servo controller</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-2806</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Windkessel&#160;</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-3717</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">HSE-USB data acquisition</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-3330</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Low-Flux Dialysator Diacap Polysulfone</td>
			<td style="width:173px;">B.Braun</td>
			<td align="right" style="width:119px;">7203525</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:447px;">PE-Tubing for aorta cannulation 1.19mm I.D. x 1.70mm O.D.</td>
			<td style="width:173px;">Scientific Commodities Inc.</td>
			<td style="width:119px;">BB31695-PE/8</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:447px;">Name</td>
			<td style="width:173px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Buffer reagents:</td>
			<td style="width:173px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Aminoplasmal 10%</td>
			<td style="width:173px;">B.Braun</td>
			<td align="right" style="width:119px;">134518064</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Sodium pyruvate</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">P2256-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">L-Glutamic acid monosodium salt hydrate</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">G1626-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">L-(-)-Malic acid sodium salt</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">M1125-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Sodium-L-Lactate</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">L7022-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">alpha-Ketoglutaric acid sodium salt</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">K1875-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">31434-1KG-R</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">NaHCO3</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5761-5KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">60130-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Urea</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">U5378-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Creatinine</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">C4255-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ampicillin</td>
			<td style="width:173px;">Roche</td>
			<td align="right" style="width:119px;">10835242001</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">MgCl2 * 6H2O</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">M2393-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">D-Glucose</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">G8270-1KG</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">CaCl2 * 6H2O&#160;</td>
			<td style="width:173px;">Riedel-de-Ha&#235;n</td>
			<td align="right" style="width:119px;">12074</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">NaH2PO4</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">S9638-500G</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Na2HPO4</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">S0876-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Antidiuretic Hormone dDAVP</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">V2013-1MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">FITC-Inulin</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Filter used for erythrocyte filtration</td>
			<td>Macherey-Nagel</td>
			<td style="width:119px;">MN 615</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">BGA Analysis:</td>
			<td style="width:173px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">ABL 80 flex</td>
			<td style="width:173px;">Radiometer Medical ApS</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Electron Microscope:</td>
			<td style="width:173px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Philips CM100 TEM</td>
			<td style="width:173px;">FEI</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

the mouse isolated perfused kidney technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

With the method described, isolated mouse kidneys can remain viable for at least 1 hr. We tested the tissue viability after 1 hr of continuous perfusion with functional (renal blood flow and vascular resistance, blood gas analysis of venous outflow, glomerular filtration rate, urinary fractional Na+ and K+ excretion, and urine osmolality) and morphological (transmission electron microscopy, TEM) methods in four kidneys of wildtype C57Bl/6 mice. Additionally, western ...

Watch this Scientific Journal Video about The Mouse Isolated Perfused Kidney Technique at JoVE.com

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

The overall goal of this protocol is to analyze the isolated kidney function in an ex vivo perfused mouse kidney. Excluding the influence of blood pressure, hormones, or the autonomic nervous system. This method can help answer key questions of Nefrology.Giving insides into isolated organ function and serving as an elegant tool to study acute kidney injury. The main advantage of the IPK is that organ function is studied in the absence of systemic influences. After preparing the mouse for surgery, using scissors, perform a medium laparotomy from the pubic crest to the sternum.First open the skin, and then the abdominal muscles. Next, remove the intestines and place them on the left side of the mouse, lateral from the abdomen. Now, free the bladder from connective tissue and expose both ureters and the urethra.For these procedures, place all the ligatures using 5-0 sutures. The first ligature to place is around the left ureter. Close this tightly with a double surgical knot.Next, place a ligature around the urethra using the same technique. Then, place a lasso ligature around the whole bladder, but leave it untied. With the first three sutures in place, use eye scissors to make a one millimeter long incision into the bladder.Into this incision, place a cannula made of two centimeters of PE-50 tubing. Now close the ligature that lassos the bladder, securing the cannula. With the bladder cannulated, now cut the left ureter and urethra distal from the ligatures.The bladder should now be only attached to the right ureter and should move freely. Next, remove the connective tissues and fat surrounding the abdominal aorta. Then place ligatures around the midpoint of the exposed aorta and just below the diaphragm between the superior mesenteric artery and the celiac trunk.Keep each of these loose as well. Next, place a ligature around the superior mesenteric artery. Now, place a third ligature around the aorta directly below the right renal artery and above the left renal artery.This is ligature number seven. Finally, put a ligature around the caudal vein package. Now place a clamp between ligature number seven and the branching of the left renal artery.Then, make a small incision in the aorta, caudal to ligature seven, taking care not to cut the dorsal wall and stretch the opening with a vessel dilator. Into the opening, insert a two centimeter pulled PE-50 cannula, pushing the tip just into the clamp. Then, open the clamp.Next, push the tip of the needle cranially until it reaches the junction of the right kidney artery and the aorta. Now close ligature seven, and then close the ligature at the midpoint of the abdominal aorta. Proceed by opening the chest with scissors by dissecting the diaphragm.Then, with a single cut, separate the aorta, vena cava, heart, and vegetative nerves. Now, start pressure control of the perfusion pump and maintain the mean pressure between 80 and 100 millimeters of mercury. Now, close the three remaining untied ligatures around the aorta and caudal vein package.Using scissors, free the right kidney from the surrounding connective tissue and release it from the adipose capsule. Cut the aorta proximally to the ligature between the superior mesenteric artery and the celiac trunk. Next, cut the superior mesenteric artery distally to the ligature around the superior mesenteric artery.Now, remove the kidney supporting vessel bundle, taking care not to cut into the vessels themselves. Then, cut the liver at the connection to the kidney, but leave a small part of the liver attached to it, so that the vena cava is kept open by it. The next step is to remove the kidney bundle from the mouse.Now, leave the kidney in the chamber and remove the mouse. At the connection between the liver and kidney, place a lasso ligature. Then, cannulate the vena cava with a venous line and close the lasso ligature.The kidney should now sit stably in the box. Venous outflow through the venous line should begin immediately. Urine outflow will begin after approximately three minutes.Peristalsis of the ureter should begin immediately and remain stable for at least one hour. Close the box. The preparation is now complete and should remain viable for at least an hour.After using the described method on C57 black 6 mice, the tissue viability of four kidneys was tested after an hour of continuous perfusion. Pressure was maintained at a continuous 100 millimeters of mercury. Perfusate flow and vascular resistance remained steady in all four kidneys.The glomerular filtration rate was assessed using FITC inulin clearance intended to increase over time but not significantly. Fractional excretion of sodium and potassium was assessed after 55 minutes of perfusion. These parameters increased compared to the in vivo situation.After 55 minutes, urinals molality did not rise above the osmolality of the venous outflow. The kidneys were then fixed and their ultrastructure was assessed with TEM. Glomeruli in S1 segments of proximal tubules appeared largely unaltered.Some, but not all S2, S3 segments of the proximal tubules in the thick ascending limbs that were not close to vascular bundles showed signs of organ damage. Upon closer inspection, distal convoluted tubules and collecting ducts did not show signs of necrosis. Overall, the kidneys mirrored what is reported for rat isolated perfused kidneys.Following this procedure, analysis methods like imaging or facts followed by protein or RNA analysis can be performed on the tissue to obtain further insights.

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Research

JoVE Journal

Biology

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermoforming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 &mu; (h x w x l) or round recesses with diameters of 300 &mu; and a depth of 300 &mu; (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed steril circulation loop that, in the simplest configuration, is additionaly comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

Optical Mapping of Langendorff-perfused Rat Hearts

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs[1, 2]. Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly[1]. The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods[1]. Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency[1, 3]. In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37&deg;C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS[4] (5 &mu;Mol/L) and briefly illuminated the preparation with 475&plusmn;15 nm excitation light. During a typical 2 second period of illumination, &gt;605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured with independent control over pulmonary arterial flow rate, flow rate waveform, airway pressure and left atrial pressure. Pulmonary vascular resistance is calculated based on multi-point, steady pressure-flow curves; pulmonary vascular impedance is calculated from pulsatile pressure-flow curves obtained at a range of frequencies. As now recognized clinically, impedance is a superior measure of right ventricular afterload than resistance because it includes the effects of vascular compliance, which are not negligible, especially in the pulmonary circulation. Three important metrics of impedance - the zero hertz impedance Z0, the characteristic impedance ZC, and the index of wave reflection RW - provide insight into distal arterial cross-sectional area available for flow, proximal arterial stiffness and the upstream-downstream impedance mismatch, respectively. All results obtained in isolated, ventilated and perfused lungs are independent of sympathetic nervous system tone, volume status and the effects of anesthesia. We have used this technique to quantify the impact of pulmonary emboli and chronic hypoxia on resistance and impedance, and to differentiate between sites of action (i.e., proximal vs. distal) of vasoactive agents and disease using the pressure dependency of ZC. Furthermore, when these techniques are used with the lungs of genetically engineered strains of mice, the effects of molecular-level defects on pulmonary vascular structure and function can be determined.

The following protocol outlines the process of isolating, ventilating and instrumenting mouse lungs to measure steady or pulsatile pulmonary vascular pressure-flow relationships in order to quantify the effects of blood flow, airflow, airway changes and vascular changes on right ventricular afterload.

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...