UCSF 당뇨병 센터. NIH UCSF DERC 아일릿 코어. JDRF. LIfeScan 주식 회사 존슨 앤 존슨 회사입니다.

이식 아일 레츠의 준비 (TX) <ol><li> 거꾸로 현미경, P200 pipetman와 100mm 접시에 교양 아일 레츠에서 바로 pipet 팁을 사용하여 직접 선택할 아일 레츠. </li><li> 카운트는 각 microcentrifuge 관 (~ 500 아일 레츠 / 튜브 / 마우스)로 시간과 전송에 100 아일 레츠. </li><li> 아일 레츠는 microcentrifuge 튜브의 바닥에 정착하도록 허용합니다. </li><li> 스트레이트 얇은 벽을 pipet 팁을 사용하여 P200 pipetman (130ul에서 설정) 하나의 아일릿 펠렛을 그립니다. </li><li> 주사기 팁 통해 실리콘 튜브 어댑터를 넣으십시오. 실리콘 어댑터에 PE50 튜브의 길이를 넣습니다. </li><li> 팁에서 아일 레츠보다 후드 벽에 PE50 튜브 높은 후드와 테이프의 측면에 pipetman을 만요. 이것은 아일 레츠는 피펫의 팁을에 정착하실 수 있습니다. </li><li> 천천히 끝에서 아일 레츠를 추방하지 pipetman를 다이얼링하고 PE50 튜브로 아일 레츠 이동, 조심 됨으로써 PE50 튜브로 아일 레츠를 전송합니다. </li><li> PE50 관의 beveled 결국 꼬임을 만듭니다. 꼬임을 유지하면서, pipetman에서 실리콘 어댑터 튜브를 분리하고 실리콘 어댑터와 꼬임을 확보. </li><li> 컷 5 ML의 pipet과 테이프 centrifuging 동안 PE50 튜브의 컬링 방지하기 위해 5 ML의 pipet의 측면을 통해 PE50 튜브와 원뿔 15 ML로, 아래로 PE50 튜브, kinked 실리콘 어댑터 튜브 측면에서 아일 레츠를 놓습니다. </li><li> 1,000 RPM에 PE50/15mL conicals을 원심 분리기 및 원심 분리기를 끄십시오. (한 번에 10 개 이상의 아일릿 준비를 준비하지 마십시오.) </li><li> 얼음 플레이스 튜브. </li></ol> 이식을 위해 마우스를 준비 <ol><li> isofluorane 또는 케타민을 / Xylexene (준비 프로토콜을 참조)과 함께 생쥐를 마취. </li><li> 마취가 영향을 찍은 후, 마우스의 왼쪽 측면을 면도. </li><li> 면봉의 Povidone 요오드 면봉와 마우스의 피부 센터 아웃 후 ETOH 면봉로 닦아. </li><li> 왼쪽 신장 (비장의 우측) 찾​​습니다. 복막을 노출, 피부에 작은 절개를합니다. </li><li> 신장을 노출 복막의 작은 절개를합니다. 절개가 작고 유지하면 신장이 제기 및 노출 유지하는 데 도움이됩니다. </li><li> 절개의 양면에 약간의 압력을 적용, 늘리거나 마우스의 신장을 열어. </li><li> 목화와 함께 정상적인 생리를 적용하여 신장 촉촉한 유지 면봉 스쳐. </li><li> 주사기 23 또는 25 게이지 바늘을 사용하여 신장 캡슐에 동전을 만드는, 신장의 오른쪽 측면에 작은 흠집을, 너무 깊거나 너무 크지는 않습니다. </li></ol> 아일 레츠의 이식 참고 : 마우스가 TX 준비되는 동안, 두 번째 사람은 해밀턴 나사 드라이브 아일릿 이식 주사기를 준비한다. <ol><li> 튜브의 꼬임을 유지하면서 천천히 PE50 튜브에서 실리콘 어댑터 튜브를 제거합니다. </li><li> 실리콘 어댑터 튜브 및 장소 &quot;스크류 드라이브를&quot;유리 해밀턴 주사기에 부착된 피펫 팁에 실리콘 어댑터 튜브에 PE50 튜브의 반대 끝에 놓습니다. 천천히 제도가 누설되지 않도록하고, PE50 튜브의 꼬임을 풀어주고있다. </li><li> &quot;나사&quot;메커니즘을 사용하여 PE50 튜브의 끝에 천천히 아일 레츠를 사전지만, PE50 튜브에있는 아일 레츠 전면에 작은 공기 방울을 유지. </li><li> 별명은 신장에서 만들어진로, 신중하게 작은 주머니를 만드는 캡슐에서 PE50 튜빙을 슬라이드. 캡슐을 통해 신장이나 펑쳐링 가우지하지 정말 조심 해야해. </li><li> 그것은 정상적인 생리 - Gentamycin 절어 목화 면봉을 밀고있는 지역과 캡슐 촉촉한을 유지하는 데 도움이, 건조 캡슐 쉽게 눈물 것입니다. </li><li> 부드럽게 휴식 이식 아일 레츠위한 &quot;주머니&quot;를 만들어, 모든 방향으로 튜빙을 이동합니다. 지역 촉촉한 유지하는 추가적인 정상 생리을 다시 적용해야합니다. </li><li> 마우스를 개설하고 누가, 신장 캡슐에서 마우스 및 해밀턴 이식 주사기를 준비 두 번째 사람, PE50 튜브를 삽입한 가지고있는 사람의 지시에 따라 천천히, &quot;포켓&quot;내부, 캡슐 아래 아일 레츠를 사전에합니다 PE50 관에 의해 전달되는 작은 기포 뒤에, 모든 아일 레츠까지 이식하고 있습니다. </li><li> 천천히, PE50 튜브를 제거 마른 면봉으로 영역을 건조하고 신중하게 낮은 열로 닉 부식. </li><li> 마른 면봉을 사용하면 모든 출혈이 멈추었 확인, 면봉을 밀고. 출혈이 중지되면, 무균 식염수에 신장을 다시 축축하게하다, 부드럽게 봉합하고 피부 스테이플로 마우스를 폐쇄하기 전에 복막에 신장을 교체하십시오. </li></ol> 휴관일 / 마우스의 리바이벌 <ol><li> 5-0 실크 봉합에게 w / C - 6 19mm 바늘을 사용하여 실행 스티치로 복막을 닫습니다. </li><li> 사용 포셉 함께 피부 절개의 양쪽 측면을 그립니다. </li><li> 함께 2 ~ 3 스테이플 스테이플과 함께 피부. </li><li> 목화를 사용하는 모든 혈액의 마우스의 피부를 청소 면봉과 살랑 스쳐E. </li><li> 즉시 Flunixin 및 Buprenorphine의 피하 주사와 마우스를 취급. </li><li> 마우스가 완전히 활성화 때까지 가열 패드 또는 아래 가열 램프에 위치 새장에 마우스를 놓습니다. </li><li> 이주의 피부 스테이플을 제거합니다. </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>

이 프로토콜은 깔끔하게 쉽게 당뇨병이나 정상적인 생쥐의 신장에 따라 캡슐 아일 레츠이나 세포를 전달하기위한 실용적이고 효율적인 옵션을 제공합니다. 신장 캡슐에서 세포를 이식하는 데 사용되는 최종 배달 튜브 (PE50)로 아일 레츠이나 세포를 집중하고 pelleting의 기술은 세포 또는 마우스 어떠한 부당한 스트레스를 줄이면서 세포를 이식의 간단하고 효과적인 방법을 제공합니다.

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		<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

우리 프로토콜이 정상적으로 쉽게 당뇨병이나 정상적인 생쥐의 신장에 따라 캡슐 아일 레츠이나 세포를 제공하기 위해 개발되었습니다. 우리는 신장 캡슐에서 세포를 이식하는 데 사용되는 최종 배달 튜브 (PE50)에 쥐똥에 아일 레츠이나 세포를 집중하는 것이 더 쉽습 것으로 나타났습니다. 세포 또는 마우스 어떠한 부당한 스트레스를 줄이는 한편이 기술은 속도와 편의를 모두 제공합니다. 로딩 : 해결 된거 같고, 손이 아일 레츠 또는 pelleted 세포 신중하게 p200 pipetteman와 스트레이트, 얇은 벽을 피펫 팁을 사용하여 1.5 ML의 microcentrifuge 관의 하단을 aspirated있다 골랐어요. PE50 관의 길이는 작은 실리콘 어댑터 튜브를 사용하여 피펫 팁에 첨부되어 있습니다. 세포 팁에 정착하고, 다음 천천히 pipetteman를 다이얼링하여 PE50 튜브로 전송됩니다 허용됩니다. 세포 PE50 튜브의 끝에 가까이되면, 꼬임이 만들어이며 실리콘 어댑터 튜브는 꼬임을 통해 배치됩니다. PE50 관은 15 ML 포함 원뿔 절단 5 ML의 pipet로 전송되며, PE50 튜브는 centrifuging 동안 컬링 방지하기 위해 5 ML의 pipet의 측면 지워졌을 것입니다. 전지는 1,000 RPM에 도달 허용하고 중지합니다. 이식 :받는 사람의 쥐가 anesthetized 있으며, 면도하고, 청소. 작은 절개는 마우스의 좌측에서 만들어이며 신장이 노출됩니다. 신장, 지방, 그리고 조직은 정상적인 생리와 젖은 면봉 보관됩니다. PE50의 말초 끝은 실리콘 어댑터 튜브를 사용하여, 피펫 팁을 포함, 해밀턴 스크류 드라이브 주사기에 첨부되어 있습니다. 작은 별명은 큰하거나 너무 깊이도 아니라, 신장의 오른쪽 측면 사이드에 이루어집니다. PE50 관의 beveled 끝은 세포 가까운 조심스럽게 캡슐 아래에 위치, 튜브는 정상적인 생리를 보라고하면서 공간을 만들기 위해 부드럽게 주위에 이동, 건조 캡슐 쉽게 눈물하실 수 있습니다. 작은 공기 방울이 서서히 주사기 스크류 드라이브를 다이얼링하여 캡슐에 따라 제공됩니다. 아일 레츠 그런 다음 천천히 기포 뒤에 전달됩니다. 일단 아일 레츠가 배달되었습니다 신장 항상성 유지되고 장식용은 낮은 열로 불로 지졌죠 있습니다. 신장은 구멍으로 다시 배치되고 복막과 피부 봉합하고 잘라입니다. 마우스 즉시 Flunixin와 Buprenorphine 평방 대우 및 난방 패드에있는 새장에 배치됩니다.

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

우리 프로토콜이 정상적으로 쉽게 생쥐의 신장에 따라 캡슐 아일 레츠이나 세포를 제공하기 위해 개발되었습니다. 세포 신장 캡슐에서 세포를 이식에 사용되는 최종 튜브에 쥐똥에 집중되어있다. 이 기법의 용이성은 세포와 마우스에 스트레스를 줄여줍니다.

당뇨병 마우스의 신장 캡슐으로 췌장 아일 레츠의 이식

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 ...

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Research

JoVE Journal

Biology

27.0K 조회수.  University of California, San Francisco - UCSF. 우리 프로토콜이 정상적으로 쉽게 생쥐의 신장에 따라 캡슐 아일 레츠이나 세포를 제공하기 위해 개발되었습니다. 세포 신장 캡슐에서 세포를 이식에 사용되는 최종 튜브에 쥐똥에 집중되어있다. 이 기법의 용이성은 세포와 마우스에 스트레스를 줄여줍니다.

비디오: 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.

성인 Zebrafish의 전체 신장 마로우의 이식

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

연구

생물학

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.

마우스의 Heterotopic 심장 이식

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.

마우스에 작은 창자 이식

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 신장 이식 절차

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.

인간 췌장 아일릿 절연 : 2 부 : 인간 아일 레츠의 정화와 문화

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.

마우스의 췌장 베타 세포 질량의 원위치 부량에서

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.

컴퓨터를 이용한 대규모 시각화 및 췌장 아일릿 질량, 크기 유통과 건축의 부량

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.

직접 성인 Zebrafish의 신장에 세포의 이식

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.

성인 Zebrafish의 신장의 해부

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.

배아 세포 이식 및 Testis 조직 마우스에 Xenografting

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