UCSF Diabetes Center. NIH UCSF DERC Islet Core. JDRF. LifeScan Inc. Johnson &amp; Johnson Company.

Vorbereitung der Inseln für die Transplant (Tx) <ol><li> Unter einem inversen Mikroskop, hand-pick Inselchen mit einem P200 Pipetman und gerade Pipettenspitze aus den kultivierten Inseln in einem 100mm Platte. </li><li> Graf Inselchen 100 pro und Transfer in jede Mikrozentrifugenröhrchen (~ 500 Inseln / Rohr / Maus). </li><li> Lassen Inseln auf den Grund des Mikrozentrifugenröhrchen begleichen. </li><li> Zeichnen Sie eine kleine Insel Pellets in ein P200 Pipetman (eingestellt auf 130ul) mit einer geraden dünnwandigen Pipettenspitze. </li><li> Legen Sie einen Silikonschlauch-Adapter über die Spitze der Spritze. Legen Sie eine Länge von PE50 Rohr in die Silikon-Adapter. </li><li> Hängen Sie die Pipetman auf die Seite der Motorhaube und Band der PE50 Schlauch an der Haube Wand höher als die Inseln in der Spitze. Damit können die kleinen Inseln, nur in der Spitze der Pipette zu begleichen. </li><li> Übertragen Sie die Inseln in den PE50 Rohr durch langsames Wahl der Pipetman und Bewegung der kleinen Inseln in der PE50 Rohre, darauf achten, daß die Inseln aus dem Ende zu vertreiben. </li><li> Machen Sie einen Knick in das abgeschrägte Ende des PE50 Schlauch. Unter Beibehaltung der Knick, trennen Sie das Silikon-Adapter Schlauch von der Pipetman und sichern Sie den Knick mit dem Silikon-Adapter. </li><li> Legen Sie die kleinen Inseln in der PE50 Schläuche, geknickt Silikon-Adapter Schlauch nach unten in ein konisches 15 ml mit einem Schnitt 5 mL Pipette und Band der PE50 Schlauch über die Seite der 5 ml Pipette zu verhindern, Eisstockschießen des PE50 Schlauch während Zentrifugieren. </li><li> Zentrifugieren Sie die PE50/15mL konische zu 1000 Umdrehungen pro Minute, und schalten Sie Zentrifuge. (Never bereiten mehr als 10 Inselchen Vorbereitungen auf einmal). </li><li> Das Röhrchen auf Eis. </li></ol> Vorbereitung der Mouse for Transplant <ol><li> Anesthetize die Mäuse mit Isofluoran oder mit Ketamin / Xylexene (siehe Vorbereitung Protokoll). </li><li> Nach Betäubung entnommen beeinflussen, rasieren die linke Flanke der Maus. </li><li> Swab Haut von Mäusen, Center-out, mit Povidon Jod Tupfer und wischen Sie dann mit einem ETOH Tupfer. </li><li> Suchen Sie die linke Niere (knapp rechts von der Milz). Machen Sie einen kleinen Schnitt in der Haut, Aussetzen des Bauchfells. </li><li> Machen Sie einen kleinen Schnitt in das Bauchfell Freilegung der Niere. Keeping the Schnitt kleiner wird im Sinne der Niere erhöht und ausgesetzt zu helfen. </li><li> Tragen Sie einen leichten Druck auf beiden Seiten des Einschnitts, erhöhen oder Pop die Niere aus die Maus. </li><li> Halten Sie die Niere feucht durch die Anwendung physiologischer Kochsalzlösung mit einem Wattestäbchen Wattestäbchen. </li><li> Mit einer Spritze 23 oder 25 Gauge-Nadel, einen kleinen Kratzer auf der rechten Flanke der Niere, die Schaffung eines nick in die Nierenkapsel; nicht zu tief oder zu groß. </li></ol> Transplantation von Inselzellen Hinweis: Während sich die Maus für Tx vorbereitet wird, die zweite Person sollte die Hamilton Schraub-Laufwerk Inseltransplantats Spritze vorzubereiten. <ol><li> Entfernen Sie langsam die Silikon-Adapter Schlauch vom PE50 Rohr unter Beibehaltung der Knick im Schlauch. </li><li> Platzieren Sie das andere Ende des PE50 Rohr in die Silikon-Adapter Schlauch und platzieren Sie den Silikon-Adapter Schlauch an der Pipettenspitze an der &quot;Schraube-drive&quot; Glas Hamilton-Spritze. Langsam loslassen Knick in der PE50 Rohre darauf achten, dass die Inseln nicht austreten. </li><li> Schieben Sie die Inseln langsam an die Spitze des PE50-Schlauch mit der &quot;Schraube&quot;-Mechanismus, aber halten Sie eine kleine Luftblase in der Vorderseite des kleinen Inseln in der PE50-Schlauch. </li><li> Into the nick gemacht in der Niere, vorsichtig die PE50 Rohre unter der Kapsel, so dass eine kleine Tasche. Seien Sie sehr vorsichtig, nicht auf die Nieren-oder Punktion durch die Kapsel Beitel. </li><li> Es hilft, die Fläche und Kapsel feucht mit normaler Kochsalzlösung getränkt Gentamycin Wattestäbchen Wattestäbchen zu halten; einem trockenen Kapsel leicht reißen. </li><li> Bewegen Sie den Schlauch in alle Richtungen, wodurch ein &quot;Taschengeld&quot; für die transplantierten Inseln zur Ruhe. Denken Sie daran, zusätzliche physiologische Kochsalzlösung erneut, damit die Fläche feucht. </li><li> Unter der Leitung von der Person, die mit der Maus geöffnet hat und wer hat die PE50 Schlauch unter die Nierenkapsel platziert, die zweite Person, die die Maus und Hamilton Transplantation Spritze vorbereitet, wird langsam voran Inseln unter der Kapsel, in der &quot;Tasche&quot;, hinter einer kleinen Luftblase durch den PE50 Schläuche geliefert werden, bis alle Inseln verpflanzt. </li><li> Entfernen Sie langsam den PE50 Schläuche, trocknen Sie den Bereich mit einem trockenen Wattestäbchen und vorsichtig cauterize den nick mit geringer Wärmeentwicklung. </li><li> Mit einem trockenen Wattestäbchen Wattestäbchen, sicherzustellen, dass alle Blutung aufgehört hat. Sobald Blutung aufgehört hat, wieder befeuchten die Niere mit steriler Kochsalzlösung, und sanft ersetzen die Niere in die Bauchhöhle vor dem Schließen der Maus mit Naht und die Haut Heftklammern. </li></ol> Closing / Revival der Maus <ol><li> Schließen Sie das Bauchfell mit einer laufenden Stich mit 5-0 Seidenfäden w / a C-6 19mm Nadel. </li><li> Mit einer Pinzette ziehen beide Seiten der Hautschnitt zusammen. </li><li> Staple die Haut zusammen mit 2 oder 3 Klammern. </li><li> Reinigen Sie die Haut der Maus von Blut, mit einem Wattestäbchen Wattestäbchen und Saline. </li><li> Unmittelbar behandeln die Maus mit einer subkutanen Injektion von Flunixin und Buprenorphin. </li><li> Platzieren Sie die Maus in einen Käfig, der auf einem Heizkissen oder unter eine Wärmelampe gelegt wird, bis die Maus ist vollständig aktiv. </li><li> Entfernen Sie die Haut Heftklammern in 2 Wochen. </li></ol>

<ol>
	<li>Tang, Q., Henriksen, K. J., Bi, M., Finger, E. B., Szot, G., Ye, J., Masteller, E. L., McDevitt, H., Bonyhadi, M., Bluestone, 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=In+vitro-expanded+antigen-specific+regulatory+T+cells+suppress+autoimmune+diabetes.">In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes.</a> J Exp Med. 199, 1455-1465 (2004).</li><li>Rulifson, I. C., Szot, G. L., Palmer, E., Bluestone, 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=Inability+to+induce+tolerance+through+direct+antigen+presentation.">Inability to induce tolerance through direct antigen presentation.</a> Am J Transplant. 2, 510-519 (2002).</li><li>Lenschow, D. J., Zeng, Y., Hathcock, K. S., Zuckerman, L. A., Freeman, G., Thistlethwaite, J. R., Gray, G. S., Hodes, R. J., Bluestone, 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=Inhibition+of+transplant+rejection+following+treatment+with+anti-B7-2+and+anti-B7-1+antibodies.+Transplantation.">Inhibition of transplant rejection following treatment with anti-B7-2 and anti-B7-1 antibodies. Transplantation.</a> Transplantation. 60, 1171-1178 (1995).</li><li>Lenschow, D. J., Zeng, Y., Thistlethwaite, J. R., Montag, A., Brady, W., Gibson, M. G., Linsley, P. S., Bluestone, 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=Long-term+survival+of+xenogeneic+pancreatic+islet+grafts+induced+by+CTLA4lg.">Long-term survival of xenogeneic pancreatic islet grafts induced by CTLA4lg.</a> Science. 257, 789-792 (1992).</li></ol>

Dieses Protokoll bietet eine praktische und effiziente Möglichkeit für die sauber und einfach liefern Inseln oder Zellen unter die Nierenkapsel diabetischer oder normalen Mäusen. Die Technik der Konzentration und Pelletierung der Inseln oder Zellen in den letzten Förderschlauch (PE50) verwendet werden, um die Zellen unter die Nierenkapsel Transplantation bietet eine einfache und effektive Methode der Transplantation von Zellen bei gleichzeitiger Verringerung unnötigen Stress auf die Zellen oder die Maus.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Surgical gloves</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>11-394-95(sz)</td>
<td>&nbsp;</td>
<tr>
<td>Surgical scrub sponges</td>
<td>&nbsp;</td>
<td>Moore medical</td>
<td>42940AK</td>
<td>&nbsp;</td>
<tr>
<td>Forceps</td>
<td>&nbsp;</td>
<td>Miltex</td>
<td>6-114 and 6-26</td>
<td>2 with teeth, 2 straight</td>
</tr>
<tr>
<td>Dissecting scissors n=2</td>
<td>&nbsp;</td>
<td>Miltex</td>
<td>5-290</td>
<td>&nbsp;</td>
<tr>
<td>Oster razor (sz. 40 blade)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>01-305-10B</td>
<td>&nbsp;</td>
<tr>
<td>4x4 Sterile gauze pads</td>
<td>&nbsp;</td>
<td>Moore Medical</td>
<td>08252AK</td>
<td>&nbsp;</td>
<tr>
<td>Povidone Iodine pads</td>
<td>&nbsp;</td>
<td>Moore Medical</td>
<td>08486AK</td>
<td>&nbsp;</td>
<tr>
<td>Alcohol pads</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>14-819-2</td>
<td>&nbsp;</td>
<tr>
<td>Heating pad</td>
<td>&nbsp;</td>
<td>Moore Medical</td>
<td>42508AK</td>
<td>&nbsp;</td>
<tr>
<td>23G or 25G needles</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1" long</td>
</tr>
<tr>
<td>Cidex solution</td>
<td>&nbsp;</td>
<td>Moore Medical</td>
<td>07535AK</td>
<td>for cleaning/santizing instruments</td>
</tr>
<tr>
<td>PE50 polyethylene tubing</td>
<td>&nbsp;</td>
<td>BD</td>
<td>427411</td>
<td>PE50, 0.965mm O.D. x 0.58mm I.D.</td>
</tr>
<tr>
<td>Cidex + 28 day soln</td>
<td>&nbsp;</td>
<td>Moore</td>
<td>35625AK</td>
<td>&nbsp;</td>
<tr>
<td>Silicone tubing</td>
<td>&nbsp;</td>
<td>Spectrum Chromat.</td>
<td>123732</td>
<td>5/32&#x201D;OD x 1/32&#x201D; ID</td>
</tr>
<tr>
<td>Instrument sterilizing container</td>
<td>&nbsp;</td>
<td>Moore Medical</td>
<td>39074AK</td>
<td>&nbsp;</td>
<tr>
<td>Straight Pipet Tips</td>
<td>&nbsp;</td>
<td>TipOne</td>
<td>1111-0810</td>
<td>sterile</td>
</tr>
<tr>
<td>Glass syringe w/screw-drive</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>1001</td>
<td>&nbsp;</td>
<tr>
<td>Cauterizing tool</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS230</td>
<td>&nbsp;</td>
<tr>
<td>Needle Holder</td>
<td>&nbsp;</td>
<td>Moore Medical</td>
<td>41-067</td>
<td>&nbsp;</td>
<tr>
<td>5-0 silk</td>
<td>&nbsp;</td>
<td>Look</td>
<td>754B</td>
<td>for suture (6-C) </td>
</tr>
<tr>
<td>Cotton tipped swab</td>
<td>&nbsp;</td>
<td>Moore Medical</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>9mm autoclip stapler</td>
<td>&nbsp;</td>
<td>BD</td>
<td>7630</td>
<td>&nbsp;</td>
<tr>
<td>9mm staples</td>
<td>&nbsp;</td>
<td>BD</td>
<td>7631</td>
<td>&nbsp;</td>
<tr>
<td>9mm staple remover</td>
<td>&nbsp;</td>
<td>BD</td>
<td>7637</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
					</div>

transplantation of pancreatic islets into the kidney capsule of diabetic mice

Unser Protokoll wurde entwickelt, um sauber und einfach liefern Inseln oder Zellen unter die Nierenkapsel diabetischer oder normalen Mäusen. Wir fanden, dass es leichter war, die kleinen Inseln oder Zellen in Pellets in den letzten Förderschlauch (PE50) verwendet werden, um die Zellen unter die Nierenkapsel Transplantation zu konzentrieren. Diese Technik bietet sowohl Geschwindigkeit und Leichtigkeit bei gleichzeitiger Verringerung unnötigen Stress auf die Zellen oder die Maus. Loading: Settled, hob die Hand, Inseln oder pelletierten Zellen werden sorgfältig aus dem Boden eines 1,5 mL Mikrozentrifugenröhrchen mit einem p200 pipetteman und eine gerade, dünnwandige Pipettenspitze aufgenommen. Eine Länge von PE50 Schlauch wird die Pipettenspitze mit einem kleinen Silikon-Adapter Schlauch befestigt. Die Zellen sind absitzen, in die Spitze, und dann sind die PE50 Rohr durch langsames Wahl der pipetteman übertragen. Sobald die Zellen in der Nähe des Ende des PE50 Schläuche sind, ist ein Knick machte und die Silikon-Adapter Schlauch wird über den Knick gestellt. Der PE50 Schlauch wird in ein 15 ml konische mit einem Schnitt 5 ml Pipette übertragen, und die PE50 Schlauch wird über die Seite der 5 ml-Pipette mit Klebeband zu verhindern Eisstockschießen beim Zentrifugieren. Die Zellen sind erlaubt bis 1.000 Umdrehungen pro Minute erreichen und blieb stehen. Transplantation: Empfänger Mäuse betäubt, rasiert und gereinigt. Durch einen kleinen Schnitt an der linken Flanke der Maus gemacht und die Niere freigelegt. Die Niere, Fett und Gewebe feucht gehalten mit physiologischer Kochsalzlösung Tupfer. Das distale Ende des PE50 ist ein Hamilton Schneckenantrieb Spritze befestigt ist, mit einer Pipettenspitze, mit dem Silikon-Adapter Schlauch. Eine kleine nick ist auf der rechten Flanke Seite der Nieren gemacht, nicht zu groß noch zu tief. Die abgeschrägte Ende des PE50-Schlauch, am nächsten die Zellen, wird sorgfältig unter der Kapsel wird der Schlauch um sanft bewegt, um Platz zu machen, während swabbing physiologischer Kochsalzlösung; einem trockenen Kapsel kann leicht reißen. Eine kleine Luftblase unter der Kapsel durch langsames Wählen Sie die Spritze Schneckenantrieb geliefert. Inseln sind dann langsam hinter die Luftblase geliefert. Sobald die Inseln geliefert worden Nieren Homöostase erhalten bleibt und die knick ist bei niedriger Hitze verödet. Die Niere ist zurück in die Höhle gebracht und das Bauchfell und Haut werden vernäht und geheftet. Mäuse sind sofort mit Flunixin und Buprenorphin sq behandelt und in einem Käfig auf einem Heizkissen.

Watch this Scientific Journal Video about Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice at JoVE.com

Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

Unser Protokoll wurde entwickelt, um sauber und einfach liefern Inseln oder Zellen unter die Nierenkapsel von Mäusen. Die Zellen werden zu Pellets in den letzten Schlauch für die Transplantation der Zellen unter die Nierenkapsel verwendet konzentriert. Die Leichtigkeit dieser Technik reduziert den Stress auf die Zellen und der Maus.

Transplantation von Pankreas-Inseln in die Niere Capsule von diabetischen Mäusen

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice.  We found that it was easier ...

transplantation-pancreatic-islets-into-kidney-capsule-diabetic

Research

JoVE Journal

Biology

27.1K Aufrufe.  University of California, San Francisco - UCSF. Unser Protokoll wurde entwickelt, um sauber und einfach liefern Inseln oder Zellen unter die Nierenkapsel von Mäusen. Die Zellen werden zu Pellets in den letzten Schlauch für die Transplantation der Zellen unter die Nierenkapsel verwendet konzentriert. Die Leichtigkeit dieser Technik reduziert den Stress auf die Zellen und der Maus.

Serie: Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

Die Transplantation von Whole Kidney Marrow in Adult Zebrafisch

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

Forschung

Biologie

Heterotopic Heart Transplantation in Mice

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable for studying rejection and immune response now that newer transgenic and gene knockout mice are available, and a large number of immunologic reagents have been developed. The heart transplant model is less stringent than the skin transplant models, although technically more challenging. We have developed a modified technique and have completed over 1000 successful cases of heterotopic heart transplantation in mice. When making anastomosis of the ascending aorta and abdominal aorta, two stay sutures are placed at the proximal and distal apexes of recipient abdominal aorta with the donor s ascending aorta, then using 11-0 suture for anastomosis on both side of aorta with continuing sutures. The stay sutures make the anastomosis easier and 11-0 is an ideal suture size to avoid bleeding and thrombosis.
When making anastomosis of pulmonary artery and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s pulmonary artery. The left wall of the inferior vena cava and donor s pulmonary artery is closed with continuing sutures in the inside of the inferior vena cava after, one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s pulmonary artery are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis. In this article, we provide details of the technique to supplement the video.

The mouse heterotopic heart transplantation model has been proven by many investigators to be an important method for studying mechanisms of rejection and immune response. However, the techniques involved are still challenging. By modifying standard techniques we have had success with more than 1000 transplants.

Heterotope Herztransplantation in Mäuse

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable ...

Small Bowel Transplantation In Mice

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive medications, infection prophylaxis, and suitable patient selection helped improve actuarial graft and patient survival rates for all types of intestine transplantation. Patients with irreversible intestinal failure and complications of parenteral nutrition should now be routinely considered for small intestine transplantation. However, Survival rates for small intestinal transplantation have been slow to improve compares increasingly favorably with renal, liver, heart and lung. The small bowel transplantation is still unsatisfactory compared with other organs. Further progress may depend on better understanding of immunology and physiology of the graft and can be greatly facilitated by animal models. A wider use of mouse small bowel transplantation model is needed in the study of immunology and physiology of the transplantation gut as well as efficient methods in diagnosing early rejection. However, this model is limited to use because the techniques involved is an extremely technically challenging. We have developed a modified technique. When making anastomosis of portal vein and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s portal vein. The left wall of the inferior vena cava and donor s portal vein is closed with continuing sutures in the inside of the inferior vena cava after, after one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s portal vein are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis.  In this article, we provide details of the technique to supplement the video.

The mouse small bowel transplantation model has been recognized as an important tool to study mechanismes of immune rejection and screen new immunosuppressive drugs. However, this model is limited to use because the techniques involved is an extremely technically challenge. Now we introduce the modified technique.

Dünndarmtransplantation In Mice

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive ...

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

Murine Renal Transplantation

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristics, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004 and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. As described in Part I: Digestion and Collection of Pancreatic Tissue, human pancreas was trimmed, cannulated, perfused, and digested. After collection and at least 30 minutes of incubation in UW solution, the tissue was loaded in the cell separator (COBE 2991, Cobe, Lakewood, CO) for purification 3. Following purification, islet yield (expressed as islet equivalents, IEQ), tissue volume, and purity was determined according to standard methods 9. Isolated islets were cultured in CMRL-1066 media (Mediatech, Herndon, VA), supplemented with 1.5% human albumin, 0.1% insulin-transferrin-selenium (ITS), 1 ml of Ciprofloxacin, 5 ml o f 1M HEPES, and 14.5 ml of 7.5% Sodium Bicarbonate in T175 flasks at 37&deg;C overnight culture before islets were transplanted or used for research.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.

Menschliche Inselzellen der Bauchspeicheldrüse Isolation: Part II: Reinigung und Kultur der Menschenrechte Islets

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

In situ Quantification of Pancreatic Beta-cell Mass in Mice

Tracing changes of specific cell populations in health and disease is an important goal of biomedical research. The process of monitoring pancreatic beta-cell proliferation and islet growth is particularly challenging. We have developed a method to capture the distribution of beta-cells in the intact pancreas of transgenic mice with fluorescence-tagged beta-cells with a macro written for ImageJ (rsb.info.nih.gov/ij/). Following pancreatic dissection and tissue clearing, the entire pancreas is captured as a virtual slice, after which the GFP-tagged beta-cells are examined. The analysis includes the quantification of total beta-cell area, islet number and size distribution with reference to specific parameters and locations for each islet and for small clusters of beta-cells. The entire distribution of islets can be plotted in three dimensions, and the information from the distribution on the size and shape of each islet allows a quantitative and qualitative comparison of changes in overall beta-cell area at a glance.

In situ Quantification of Pancreatic Beta-cell Mass in Mice

The following protocol outlines the process of pancreatic dissection for virtual slice imaging, and the subsequent quantification of all GFP-tagged beta-cells in the entire pancreas.

In situ Quantifizierung von Pankreas-Beta-Zellmasse in Mäuse

Tracing changes of specific cell populations in health and disease is an important goal of biomedical research. The process of monitoring pancreatic ...

Computer-assisted Large-scale Visualization and Quantification of Pancreatic Islet Mass, Size Distribution and Architecture

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), and delta-cells (somatostatin) that are embedded in the exocrine tissues and comprise 1-2% of the entire pancreas. There is a close correlation between body and pancreas weight. Total beta-cell mass also increases proportionately to compensate for the demand for insulin in the body. What escapes this proportionate expansion is the size distribution of islets. Large animals such as humans share similar islet size distributions with mice, suggesting that this micro-organ has a certain size limit to be functional. The inability of large animal pancreata to generate proportionately larger islets is compensated for by an increase in the number of islets and by an increase in the proportion of larger islets in their overall islet size distribution. Furthermore, islets exhibit a striking plasticity in cellular composition and architecture among different species and also within the same species under various pathophysiological conditions. In the present study, we describe novel approaches for the analysis of biological image data in order to facilitate the automation of analytic processes, which allow for the analysis of large and heterogeneous data collections in the study of such dynamic biological processes and complex structures. Such studies have been hampered due to technical difficulties of unbiased sampling and generating large-scale data sets to precisely capture the complexity of biological processes of islet biology. Here we show methods to collect unbiased "representative" data within the limited availability of samples (or to minimize the sample collection) and the standard experimental settings, and to precisely analyze the complex three-dimensional structure of the islet. Computer-assisted automation allows for the collection and analysis of large-scale data sets and also assures unbiased interpretation of the data. Furthermore, the precise quantification of islet size distribution and spatial coordinates (i.e. X, Y, Z-positions) not only leads to an accurate visualization of pancreatic islet structure and composition, but also allows us to identify patterns during development and adaptation to altering conditions through mathematical modeling. The methods developed in this study are applicable to studies of many other systems and organisms as well.

Novel computer-assisted methods of large-scale procurement and analysis of immunohistochemically stained pancreatic specimens are described: (1) Virtual Slice capture of the entire section; (2) Mass analysis of large-scale data; (3) Reconstruction of 2D Virtual Slices; (4) 3D islet mapping; and (5) Mathematical analysis.

Computer-assisted Großflächige Visualisierung und Quantifizierung der Inselzellen der Bauchspeicheldrüse Masse, Größenverteilung und Architektur

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), ...

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.

Die Transplantation von Zellen direkt in die Niere von Adult Zebrafisch

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.

Präparation des Adult Zebrafisch 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 ...

Germ Cell Transplantation and Testis Tissue Xenografting in Mice

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies showed that microinjection of germ cells from fertile donor mice into the seminiferous tubules of infertile recipient mice results in donor-derived spermatogenesis and sperm production by the recipient animal2. The use of donor males carrying the bacterial &beta;-galactosidase gene allowed identification of donor-derived spermatogenesis and transmission of the donor haplotype to the offspring by recipient animals1. Surprisingly, after transplantation into the lumen of the seminiferous tubules, transplanted germ cells were able to move from the luminal compartment to the basement membrane where spermatogonia are located3. It is generally accepted that only SSCs are able to colonize the niche and re-establish spermatogenesis in the recipient testis. Therefore, germ cell transplantation provides a functional approach to study the stem cell niche in the testis and to characterize putative spermatogonial stem cells. To date, germ cell transplantation is used to elucidate basic stem cell biology, to produce transgenic animals through genetic manipulation of germ cells prior to transplantation4,5, to study Sertoli cell-germ cell interaction6,7, SSC homing and colonization3,8, as well as SSC self-renewal and differentiation9,10.
Germ cell transplantation is also feasible in large species11. In these, the main applications are preservation of fertility, dissemination of elite genetics in animal populations, and generation of transgenic animals as the study of spermatogenesis and SSC biology with this technique is logistically more difficult and expensive than in rodents. Transplantation of germ cells from large species into the seminiferous tubules of mice results in colonization of donor cells and spermatogonial expansion, but not in their full differentiation presumably due to incompatibility of the recipient somatic cell compartment with the germ cells from phylogenetically distant species12. An alternative approach is transplantation of germ cells from large species together with their surrounding somatic compartment. We first reported in 2002, that small fragments of testis tissue from immature males transplanted under the dorsal skin of immunodeficient mice are able to survive and undergo full development with the production of fertilization competent sperm13. Since then testis tissue xenografting has been shown to be successful in many species and emerged as a valuable alternative to study testis development and spermatogenesis of large animals in mice14.

Protocols for germ cell transplantation and testis tissue xenografting are described. Germ cell transplantation results in donor-derived spermatogenesis in recipient testes and represents a functional reconstitution assay for identification of spermatogonial stem cells (SSCs). Testis tissue xenografting reproduces testis development and spermatogenesis of various donor species in recipient mice.

Germ Cell Transplantation und Hodengewebe in Mäusen Xenografting

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies ...