UCSF Diyabet Merkezi. NIH UCSF DERC Islet Core. JDRF. LIFESCAN A.Ş. Johnson &amp; Johnson Company.

Nakli için Islets hazırlanması (Tx) <ol><li> Inverted mikroskop altında, P200 Pipetman ve 100mm plaka kültür adacıkları düz pipetlemeyin ucu kullanarak el almak adacıklar. </li><li> Kont, her mikrosantrifüj tüp (~ 500 adacık / tüp / fare) bir zaman ve transfer 100 adacık. </li><li> Adacıklar mikrosantrifüj tüplerin altına yerleşmek için izin ver. </li><li> Düz bir ince duvar pipetlemeyin ucunu kullanarak P200 Pipetman (130ul ayarlanır) içine bir adacık pelet çizin. </li><li> Şırınga ucu üzerinde bir silikon tüp adaptörü yerleştirin. PE50 boru uzunluğu silikon adaptörü içine yerleştirin. </li><li> Kaput duvara PE50 boru uç adacıklar daha yüksek bant kaput ve yan Pipetman asın. Bu adacıklar sadece pipet ucu yerleşmek için izin verecektir. </li><li> Yavaş yavaş sonuna adacıklar sınırdışı Pipetman arama ve PE50 tüp içine adacıklar hareket dikkatli olmak adacıklar PE50 tüp içine aktarın. </li><li> PE50 boru kesik sonunda bir kink olun. Kink korurken, Pipetman silikon adaptör boru bağlantısını kesin ve silikon adaptör ile kink güvenli. </li><li> Konik bir kesim 5 ml pipetlemeyin ve bant santrifüj ise PE50 boru kıvrık önlemek için 5 ml pipetlemeyin yan üzerinde PE50 tüp içine 15 ml, PE50 tüp, bükülmüş silikon adaptör boru tarafı, adacıklar yerleştirin. </li><li> 1000 rpm PE50/15mL conicals Santrifüj ve santrifüj kapatın. (Bir kerede 10&#39;dan fazla adacık hazırlıkları hazırlamak asla). </li><li> Buz tüpü yerleştirin. </li></ol> Nakli için Mouse hazırlanması <ol><li> Isofluorane veya Ketamin / Xylexene (bkz. hazırlık protokolü) ile fareleri anestezisi. </li><li> Anestezik etkileyebilir alındıktan sonra, farenin sol kanadını tıraş. </li><li> Swab fare Povidon İyot çubukla cilt, merkez, ve sonra bir ETOH bez ile silin. </li><li> (Dalak sadece sağ) sol böbrek bulun. Periton açığa, deride küçük bir kesi olun. </li><li> Böbrek açığa periton küçük bir kesi olun. Kesi küçük tutulması böbrek kaldırdı ve pozlanmış tutmada yardımcı olacaktır. </li><li> Kesi her iki taraf için hafif bir basınç uygulayın, artırma veya böbrek farenin dışarı pop. </li><li> Bir pamuk ile normal salin uygulayarak böbrek nemli tutun çubukla uçlu. </li><li> Bir şırınga 23 veya 25 iğne kullanarak, böbrek kapsülünde bir nick oluşturmak, böbrek sağ kanadında küçük bir çizik çok derin ya da çok büyük değil. </li></ol> Islets Nakli Not: fare Tx için hazırlanan edilirken, ikinci bir kişi Hamilton vida sürücü adacık nakli şırınga hazırlamalıdır. <ol><li> Hortumun kink tutarken silikon adaptör tüp PE50 tüp yavaşça çıkarın. </li><li> Silikon adaptör tüp ve yer &quot;vida-drive&quot; cam Hamilton şırınga bağlı pipet ucuna silikon adaptör tüp içine PE50 hortumun diğer ucunu yerleştirin. Yavaş yavaş, adalarında sızıntı olmadığını emin olun, PE50 boru kink bırakın. </li><li> &quot;Vida&quot; mekanizması kullanılarak PE50 hortumun ucu yavaş yavaş adacıklar Advance, ancak küçük bir hava kabarcığı PE50 tüp adacıklar önünde tutmak. </li><li> Nick böbrek içine küçük bir cep dikkatlice, kapsül altında PE50 boru slayt. Kapsül ile böbrek veya delinmesiyle oymak için çok dikkatli olun. </li><li> Normal tuzlu-Gentamisin ıslatılmış pamuklu uçlu alan ve kapsül nemli kalmasına yardımcı olur, kuru bir kapsülü kolayca gözyaşı olacak. </li><li> Yavaşça geri kalanı için nakledilen adacıklar için bir &quot;cep&quot; oluşturarak, tüm yönlerde boru hareket ettirin. Bölgenin nemli tutmak için ek olarak normal serum fizyolojik yeniden unutmayın. </li><li> Fare açtı ve kim, böbrek kapsülü altında fare ve Hamilton nakli şırınga hazırlanan ikinci kişi, PE50 tüp yerleştirilir kişinin yönetimi altında yavaş yavaş, &quot;cep&quot; içinde, kapsül altında adacıklar ilerleyecektir PE50 tüp tarafından verilen küçük bir hava kabarcığı arkasında, tüm adacıklar kadar nakledilmektedir. </li><li> Yavaşça, PE50 tüp kaldırmak için, kuru bir bez ile bölgeyi kuru ve düşük ısıda dikkatlice nick dağlamak. </li><li> Tüm kanama durdu emin olun ve kuru bir pamuk kullanarak çubukla uçlu. Kanama durduktan sonra, steril serum fizyolojik ile böbrek tekrar nemlendirin ve yavaşça dikiş ve cilt zımba ile fare kapanış önce periton içine böbrek yerine. </li></ol> Kapanış / Mouse Revival <ol><li> Periton 5-0 ipekle w / C-6 19mm iğne kullanarak çalışan bir dikiş ile kapatın. </li><li> Kullanarak forseps birlikte cilt insizyonu her iki tarafında çizin. </li><li> 2 veya 3 zımba ile birlikte cilt Zımba. </li><li> Herhangi bir kan fare Cildin temiz bir pamuk kullanarak sürüntü ve SALIN uçlue. </li><li> Hemen fareyi Flunixin ve Buprenorfin bir cilt altı enjeksiyonu ile tedavi. </li><li> Fare, fare tam aktif olana dek, bir ısıtma yastığı veya altında bir ısıtma lambası yerleştirilir bir kafes yerleştirin. </li><li> 2 hafta içinde cilt zımba çıkarın. </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>

Bu protokol, temiz ve kolay bir şekilde diyabetik veya normal farelerin böbrek kapsülü altında adacıklar veya hücreleri sunmak için pratik ve etkili bir seçenek sunmaktadır. Hücrelere herhangi bir aşırı stres ya da fare azaltırken hücrelerin nakli, böbrek kapsül altında hücre nakli için kullanılan son teslim boru (PE50) adacıklar veya hücreleri zenginleştirme ve peletleme tekniği kolay ve etkili bir yöntem sunmaktadır.

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		<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>
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transplantation of pancreatic islets into the kidney capsule of diabetic mice

Protokolü, temiz ve kolay bir şekilde diyabetik veya normal farelerin böbrek kapsülü altında adacıklar veya hücreleri sunmak için geliştirildi. Biz böbrek kapsülü altında hücre nakli için kullanılan son teslim boru (PE50) pelet içine adacıklar veya hücrelerin konsantre kolay olduğu bulundu. Bu teknik, hücrelere ya da fare herhangi bir aşırı stres azaltırken hem hız ve kolaylık sağlar. Yükleme: oturanlar, el adacıklar veya tohum hücreleri dikkatle P200 pipetteman ve düz, ince duvar pipet kullanarak 1,5 mL mikrosantrifüj tüp alt aspire, aldı. PE50 boru uzunluğu, küçük bir silikon adaptörü tüp kullanarak pipet ucu takılır. Hücreler ucu, yerleşmeye ve sonra yavaş yavaş pipetteman arama PE50 boru aktarılır izin verilir. PE50 boru sonuna yakın hücrelerin sonra, bir bükülme yapılır ve silikon adaptör boru kink üzerine yerleştirilir. PE50 tüp 15 ml içeren konik bir kesim 5 ml pipetlemeyin aktarılır ve PE50 tüp santrifüj sırasında kıvrık önlemek için 5 ml pipetlemeyin yan üzerinde bantlanmış. Hücreler 1.000 rpm ulaşmak için izin ve durdu. Nakli: Alıcı fareler anestezi, traş ve temizlenmelidir. Farenin sol kanadında küçük bir kesi yapılır ve böbrek maruz kalmaktadır. Böbrek, yağ ve doku normal tuzlu çubukla nemli tutulmalıdır. PE50 distal ucu silikon adaptörü boru kullanarak, pipet içeren, Hamilton vidalı tahrik şırınga eklenir. Küçük bir nick çok büyük ne de çok derin değil, böbrek sağ kanadında yan üzerinde yapılır. PE50 boru kesik uç, hücrelerin en yakın dikkatle kapsül altına yerleştirilir, tüp normal salin sürüntü sırasında yer açmak için hafifçe dolaştı; kuru bir kapsülü kolayca yırtılabilir. Kapsül altında küçük bir hava kabarcığı, yavaş yavaş şırınga vida sürücü arama tarafından teslim edilir. Adacıklar sonra yavaş yavaş hava kabarcığı arkasında teslim edilir. Adacıklar teslim edilmiştir böbrek homeostazı korunur ve ıvır düşük ısı ile koterize. Böbrek boşluğuna yerleştirilir ve periton ve cilt sütüre ve zımbalanmış. Fare hemen Flunixin ve Buprenorfin ² ile tedavi edilen ve bir ısıtma yastığı bir kafes yerleştirilir.

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

Protokolü, temiz ve kolay bir adacık veya hücreleri farelerin böbrek kapsülü altında sunmak için geliştirildi. Hücreler, böbrek kapsül altında hücrelerinin nakli için kullanılan son tüp pelet içine yoğunlaşmıştır. Bu tekniğin kolaylığı hücreleri ve fare stresi azaltır.

Diyabetik Fare Böbrek Kapsül içine, Pankreas Islets Nakli

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 Görüntüleme.  University of California, San Francisco - UCSF. Protokolü, temiz ve kolay bir adacık veya hücreleri farelerin böbrek kapsülü altında sunmak için geliştirildi. Hücreler, böbrek kapsül altında hücrelerinin nakli için kullanılan son tüp pelet içine yoğunlaşmıştır. Bu tekniğin kolaylığı hücreleri ve fare stresi azaltır.

Video: 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.

Yetişkin Zebra balığı Tüm Böbrek İliği Nakli

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

Biyoloji

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.

Farelerde Heterotopik Kalp Nakli

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.

Farelerde Küçük Bağırsak Nakli

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.

Murin Böbrek Nakli Prosedürü

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.

İnsan Pankreas Adacık İzolasyonu: Bölüm II: İnsan Islets saflaştırılması ve Kültür

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.

Farelerde, pankreatik beta-hücre Kütle yerinde Niceleme

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.

Bilgisayar destekli Büyük ölçekli Görselleştirme ve Pankreas Adacık Kütle, Boyut Dağılımı ve Mimarlık Niceleme

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.

Doğrudan Yetişkin Zebra balığı Böbrek Hücre Nakli

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.

Yetişkin Zebra balığı Böbrek diseksiyonu

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 Hücre Nakli ve Testis Dokusu Farelerde Xenografting

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