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

Preparação de Ilhotas para Transplante (Tx) <ol><li> Em microscópio invertido, mão pick-ilhotas usando um Pipetman P200 e ponta da pipeta em linha reta do ilhotas cultivadas em uma placa de 100mm. </li><li> Contagem de 100 ilhotas de cada vez e transferir para cada tubo de microcentrífuga (~ 500 ilhotas tubo / / mouse). </li><li> Permitir ilhotas para resolver a parte inferior dos tubos de microcentrífuga. </li><li> Desenhe uma das ilhotas pellet em um Pipetman P200 (fixado em 130ul) usando uma ponta da pipeta em linha reta de parede fina. </li><li> Coloque um adaptador de tubo de silicone sobre a ponta da seringa. Inserir um comprimento de tubulação PE50 no adaptador silicone. </li><li> Pendurar o Pipetman para o lado da capa e fita da tubulação PE50 à parede capô superior as ilhotas na ponta. Isso permitirá que as ilhotas de resolver apenas na ponta da pipeta. </li><li> Transferência das ilhotas na tubulação PE50 por lentamente discar o Pipetman e movendo as ilhotas na tubulação PE50, tomando cuidado para não expulsar os ilhéus a partir do final. </li><li> Faça uma torção no final chanfrada do tubo PE50. Mantendo a torção, desconecte o tubo adaptador de silicone do Pipetman e segura a torção com o adaptador silicone. </li><li> Coloque as ilhotas, no PE50 tubulação dobrada lado de silicone, tubo adaptador para baixo, em um 15 ml cônico com uma pipeta mL cortar 5 e tape o tubo PE50 sobre o lado da pipeta de 5 mL para evitar curling da tubulação PE50 enquanto centrifugação. </li><li> Centrifugar a conicals PE50/15mL a 1000 rpm e desligar centrífuga. (Nunca prepare mais de 10 preparações das ilhotas de uma só vez). </li><li> Coloque o tubo no gelo. </li></ol> Preparação de mouse para Transplante <ol><li> Anestesiar os ratos com isofluorano ou com Ketamina / Xylexene (ver protocolo de preparação). </li><li> Após anestesia tomou afeto, raspar o flanco esquerdo do mouse. </li><li> Swab pele de rato, centro-out, com swab iodopovidona e depois limpe com um algodão embebido em álcool etílico. </li><li> Localizar o rim esquerdo (logo à direita do baço). Fazer uma pequena incisão na pele, expondo o peritônio. </li><li> Fazer uma pequena incisão no peritônio expor o rim. Mantendo a pequena incisão irá ajudar na manutenção do rim levantadas e expostas. </li><li> Aplique uma leve pressão para ambos os lados da incisão, aumentar ou pop do rim para fora do mouse. </li><li> Manter a umidade do rim através da aplicação de soro fisiológico com um algodão com ponta cotonete. </li><li> Usando uma seringa de 23 ou 25 agulha, faça um pequeno arranhão no flanco direito do rim, a criação de um nick na cápsula renal, não muito profundas ou muito grande. </li></ol> Transplante de ilhotas Nota: Enquanto o mouse está sendo preparado para Tx, a segunda pessoa deve preparar o Hamilton parafuso drive-seringa transplante de ilhotas. <ol><li> Lentamente, retirar a mangueira do adaptador de silicone do tubo PE50, mantendo o kink na tubulação. </li><li> Coloque a extremidade oposta do tubo PE50 no tubo adaptador de silicone e colocar o tubo adaptador de silicone na ponta da pipeta anexado à seringa de vidro &quot;parafuso-drive&quot; Hamilton. Lentamente, liberte o kink na tubulação PE50, certificando-se que as ilhas não vazar. </li><li> Avançar a ilhotas lentamente até a ponta do tubo PE50 usando o &quot;parafuso&quot; mecanismo, mas manter uma pequena bolha de ar na frente das ilhotas na tubulação PE50. </li><li> Para o nick fez no rim, deslize cuidadosamente o tubo PE50 sob a cápsula, fazendo uma pequena bolsa. Tenha muito cuidado para não arrancar o rim ou punção através da cápsula. </li><li> Ela ajuda a manter a área e úmido de algodão cápsula com solução salina Gentamicina normais embebido ponta cotonete; uma cápsula seca vai rasgar facilmente. </li><li> Mova cuidadosamente o tubo em todas as direções, criando uma &quot;bolsa&quot; para as ilhotas transplantadas para descansar. Lembre-se de reaplicar o soro fisiológico adicional para manter a área úmida. </li><li> Sob a direção da pessoa que abriu o mouse e que tem colocado o tubo PE50 sob a cápsula renal, a segunda pessoa, que preparou o mouse e Hamilton seringa transplante, lentamente vai avançar ilhéus sob a cápsula, dentro do &quot;bolso&quot;, atrás de uma pequena bolha de ar emitido pela tubulação PE50, até que todas as ilhotas são transplantadas. </li><li> Lentamente, retirar a mangueira PE50, seque a área com um cotonete seco e cuidadosamente cauterizar o nick em lume brando. </li><li> Usando um cotonete de algodão seco ponta, certifique-se todas as sangramento parou. Uma vez que o sangramento parou, re-umedecer o rim com solução salina estéril, e gentilmente substituir o rim para o peritônio antes do fechamento do mouse com grampos de sutura e pele. </li></ol> Fechamento / Revival of Mouse <ol><li> Fechar o peritônio com um ponto em execução usando seda 5-0 w / a agulha C-6 19mm. </li><li> Forceps desenhar usando ambos os lados da incisão na pele junto. </li><li> Pele o grampo, juntamente com 2 ou 3 grampos. </li><li> Limpe a pele do mouse de qualquer sangue, usando um cotonete de algodão com ponta e saline. </li><li> Imediatamente tratar o mouse com uma injeção subcutânea de Flunixina e buprenorfina. </li><li> Posicione o mouse em uma gaiola, que é colocada sobre uma almofada de aquecimento ou abaixo de uma lâmpada de aquecimento, até que o mouse é totalmente ativo. </li><li> Remova os grampos de pele em duas semanas. </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>

Este protocolo fornece uma opção prática e eficiente para fornecer precisão e facilidade ilhotas ou células sob a cápsula renal de ratos diabéticos ou normal. A técnica de concentração e as ilhotas de granulação ou células ao tubo de final (PE50) utilizado para o transplante de células sob a cápsula renal oferece um método fácil e eficaz de transplante de células, reduzindo qualquer estresse para as células ou para o mouse.

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<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>
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<tr>
<td>Cidex solution</td>
<td>&nbsp;</td>
<td>Moore Medical</td>
<td>07535AK</td>
<td>for cleaning/santizing instruments</td>
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<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>
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<td>Cidex + 28 day soln</td>
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<td>Moore</td>
<td>35625AK</td>
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<td>Silicone tubing</td>
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<td>Spectrum Chromat.</td>
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<td>5/32&#x201D;OD x 1/32&#x201D; ID</td>
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<td>Instrument sterilizing container</td>
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<td>Straight Pipet Tips</td>
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<td>TipOne</td>
<td>1111-0810</td>
<td>sterile</td>
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<td>Glass syringe w/screw-drive</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>1001</td>
<td>&nbsp;</td>
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<td>Cauterizing tool</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS230</td>
<td>&nbsp;</td>
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<td>Needle Holder</td>
<td>&nbsp;</td>
<td>Moore Medical</td>
<td>41-067</td>
<td>&nbsp;</td>
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<td>5-0 silk</td>
<td>&nbsp;</td>
<td>Look</td>
<td>754B</td>
<td>for suture (6-C) </td>
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<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>
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transplantation of pancreatic islets into the kidney capsule of diabetic mice

Nosso protocolo foi desenvolvido para fornecer precisão e facilidade ilhotas ou células sob a cápsula renal de ratos diabéticos ou normal. Descobrimos que era mais fácil concentrar as ilhotas ou células em pelotas na tubulação de entrega final (PE50) utilizado para o transplante de células sob a cápsula renal. Esta técnica fornece a velocidade e facilidade, reduzindo qualquer estresse para as células ou para o mouse. Loading: Settled, escolhidos a dedo, ou células ilhotas peletizadas são cuidadosamente aspirado fora da parte inferior de um tubo de microcentrífuga de 1,5 mL usando um pipetteman p200 e uma reta, de parede fina ponta da pipeta. Um comprimento de tubos PE50 está ligado à ponta da pipeta usando um tubo de pequeno adaptador silicone. Células estão autorizados a resolver, na ponta, e depois são transferidos para o tubo PE50 lentamente por marcar o pipetteman. Uma vez que as células estão perto do fim do tubo PE50, uma torção é feita eo adaptador de tubo de silicone é colocado sobre o kink. A tubulação PE50 é transferido para a 15 mL uma pipeta de corte cônico contendo 5 mL, e os tubos PE50 é gravado sobre o lado da pipeta de 5 mL para evitar curling durante a centrifugação. Células podem chegar a 1.000 rpm e parou. Transplante: ratos são anestesiados destinatário, raspada e limpa. Uma pequena incisão é feita no flanco esquerdo do mouse e do rim está exposto. O rim, tecido adiposo, tecido e são mantidas úmidas com uma compressa de soro fisiológico. A extremidade distal do PE50 é anexado a uma seringa Hamilton movimentação do parafuso, contendo uma ponta de pipeta, usando o tubo adaptador silicone. Um nick pequeno é feito no lado do flanco direito do rim, não muito grande nem muito profundo. O fim chanfrada do tubo PE50, mais próximo das células, é cuidadosamente colocada sob a cápsula, a tubulação é movido suavemente para dar espaço ao mesmo tempo limpando soro fisiológico; uma cápsula seca pode rasgar facilmente. Uma pequena bolha de ar é fornecido sob a cápsula por lentamente marcação da unidade de rosca seringa. Ilhotas são entregues em seguida, lentamente por trás da bolha de ar. Homeostase renal uma vez que as ilhotas foram entregues é mantido e os knick é cauterizado com fogo baixo. O rim é colocado de volta na cavidade e do peritônio e da pele são suturados e grampeados. Ratos são imediatamente tratados com Flunixina quadrados e buprenorfina e colocado em uma jaula em uma almofada de aquecimento.

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

Nosso protocolo foi desenvolvido para fornecer precisão e facilidade ilhotas ou células sob a cápsula renal de camundongos. Células estão concentradas em pelotas na tubulação final utilizada para o transplante de células sob a cápsula renal. A facilidade desta técnica reduz o estresse nas células e mouse.

Transplante de ilhotas pancreáticas para a cápsula renal de ratos diabéticos

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

27.0K visualizações.  University of California, San Francisco - UCSF. Nosso protocolo foi desenvolvido para fornecer precisão e facilidade ilhotas ou células sob a cápsula renal de camundongos. Células estão concentradas em pelotas na tubulação final utilizada para o transplante de células sob a cápsula renal. A facilidade desta técnica reduz o estresse nas células e mouse.

Vídeo: 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.

Transplante de Medula Renal inteiro em Zebrafish Adulto

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

Biologia

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.

Transplante cardíaco heterotópico em ratos

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.

Transplante de intestino delgado em ratos

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.

Murino Procedimento de Transplante Renal

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.

Isolamento Islet humana Pancreatic: Parte II: Purificação e Cultura de Ilhotas Humanos

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.

Quantificação em situ da Missa das células beta pancreáticas em camundongos

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.

Assistida por computador visualização de grande escala e Quantificação da Missa das ilhotas pancreáticas, distribuição de tamanho e Arquitetura

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.

Transplante de células diretamente no Rim de Adulto 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 ...

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.

Dissecção do rim Zebrafish Adulto

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.

Transplante de células germinativas e Tecidos Testículo xeno em Ratos

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