Bu araştırma Avrupa toplumun yedinci çerçeve programı (FP7/2007-2013) fon hibe sözleşme numarası 305436 altında aldı STELLAR.

The local medical ethical committee and ethical advisory board of the European consortium (STELLAR) approved the research and collection of human transplant grade kidneys discarded mainly for surgical reasons. Research consent was given for all kidneys.
1. Preparations for Cell Culture
<ol>
	<li>Prepare a pool of platelet lysates.
	<ol>
		<li>Store the platelets that have expired for less than 2 d in -80 &#176;C until use (for a maximum of 1 year). 
		NOTE: The platelets were originally sourced from a commercial vendor. Hospital surplus material distributed by the local blood bank were obtained.</li>
		<li>Thaw the platelets of at least 5 donors (preferably 10) O/N at 4 &#176;C.</li>
		<li>Pool the platelets in a large sterile bottle, transfer to conical centrifuge tubes and spin down for 10 min at 1,960 x g at 4 &#176;C. 
		NOTE: The volume of platelets depends on the number of donors and the amount of hospital surplus material per donor.</li>
		<li>Pipette the supernatant to blood bags (50 mL/bag) through the barrel of a 50 mL syringe. Store at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Preparation of cell culture medium.
	<ol>
		<li>Defrost one bag of platelet lysates in a water bath at 37 &#176;C.</li>
		<li>Add 1 L (i.e. 2 bottles) of Minimum Essential Medium - alpha modification (alphaMEM), 5 mL 200 mM glutamine, and 20 mL of penicillin (5,000 U/mL)/streptomycin (5,000 &#181;g/mL) (pen/strep) to the bag.</li>
		<li>Incubate for 3 h at 37 &#176;C.</li>
		<li>Shake the bag for degelling by gently tapping on the bag when the bag is resting on a flat surface.</li>
		<li>Filter the 5% platelet lysates medium by pulling the lysates through the filter of the transfusion system with a sterile 50 mL syringe.</li>
		<li>Aliquot the medium in 50 mL tubes and store in -80 &#176;C until use. 
		NOTE: Every new batch of platelet lysates is tested in cell culture and compared to the previous batch by growing cells of interest (bmMSCs or kPSCs) to confluency in both batches. In cell numbers and viability, the kPSCs or bmMCs grown in the new batch should not differ more than 10%, and the marker expression of CD73, CD90, CD105 and CD31, CD34 and CD45 as analyzed by flow cytometry should not differ. The methods of cell culture are described in section 6 of the protocol (Culture of kPSC).</li>
	</ol>
	</li>
</ol>
2. Preparations for Cell Harvest
<ol>
	<li>Preparation of solutions.
	<ol>
		<li>Prepare the plain medium by adding 10 mL of pen/strep to 500 mL (1 bottle) of Dulbecco&#39;s Minimum Essential Medium (DMEM)-F12.</li>
		<li>Prepare the washing medium by adding 50 mL of Normal Human Serum (NHS) and 10 mL of pen/strep to 1 bottle of DMEM-F12.</li>
		<li>Prepare the enzyme-stock buffer by adding 2.9 mL 1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1.2 mL NaHCO3 2.5% [w/v] and 160 &#181;L CaCl2 (1 M) to 160 mL University of Wisconsin solution.</li>
		<li>Prepare the collagenase solution by slowly adding 20 mL of enzyme stock buffer to the bottle of GMP-grade collagenase containing &#62;2,000 U/bottle collagenase. Dissolve at 4 &#176;C for approximately 40 min. Gently shake several times during the dissolving period.</li>
		<li>Prepare the freeze medium by adding 5 mL dimethyl sulfoxide (DMSO) to 45 mL NHS.</li>
	</ol>
	</li>
	<li>Preparation of perfusion tray (Figure 1A).
	<ol>
		<li>Clean the flow cabin and cover with surgical drapes. Heat a water bath to approximately 40 - 45 &#176;C to heat the perfusion tray to 37 &#176;C. Put on surgical gowns and surgical gloves.</li>
		<li>Connect a sterilized perfusion tray to the water bath to preheat the perfusion tray with warm water. Make sure that there is no air in the perfusion tray by starting with the perfusion tray in the vertical position. If necessary add more water to the water bath to prevent air infusion into the perfusion tray.</li>
		<li>Place the LS25 tube in the pump. Leave both sterile ends of the tube in the flow cabinet. Place a Kocher&#39;s forceps on one end of the tube and allow to rest on the bottom of the perfusion tray. Place a sterile gauze on top of the tube to prevent obstruction. Attach a Luer connector on the other tube end.</li>
		<li>Add approximately 100 mL of plain medium into the perfusion tray and start flushing the tube on a pump speed of 100 mL/min.</li>
	</ol>
	</li>
</ol>
3. Kidney Cell Isolation
<ol>
	<li>Preparation of the kidney.
	<ol>
		<li>Remove the kidney from the double sterile bags and place on the sterile perfusion tray (Figure 1B). Remove the perirenal adipose tissue and kidney capsule with scissors and gentle tearing (Figure 1C) and identify the renal artery on the aortic patch as shown in Figure 1D.</li>
		<li>Cannulate the renal artery with the accessory spike, fix with a sterilized tie-rip, and attach to the pump tube end with the Luer connector while the pump is on. If the tubes are not completely filled, first add more medium to the tray and flush tubes by starting the pump (Figure 1E - F). Flush the kidney with plain medium via the pump with a flow of 100 mL/min.</li>
	</ol>
	</li>
	<li>Collagenase treatment and kidney cell isolation.
	<ol>
		<li>Add collagenase (20 mL, &#62;2,000 U) and DNase (2.5 mL, 1 mg/mL) to the perfusion tray (Figure 1G). Turn the kidney from time to time gently by hand. Wait until the kidney becomes soft and the perfusion liquid becomes less transparent (approximately 30 - 40 min).</li>
		<li>Gently massage the kidney (Figure 1H) until a cell suspension is obtained (Figure 1I). Remove non-digested material and the spike in the renal artery.</li>
	</ol>
	</li>
	<li>Washing and collection of kidney cells.
	<ol>
		<li>Add 50 mL of the NHS to the cell suspension. Drain the cell suspension from the perfusion tray by putting the pump at low flow and placing the tube first connected to the kidney above the 50 mL tubes (Figure 1J).</li>
		<li>Centrifuge at 300 x g for 7 min at 4 &#730;C and remove the supernatant. Wash the pellets with washing medium containing 10% NHS and again centrifuge at 300 x g for 7 min. Repeat washing once more. Measure the volume of the cell pellets and add an equal volume of cell culture medium.</li>
		<li>Either cryopreserve the cells from here by adding freezing medium 1:1 to the cell suspension and store in cryogenic vials according to standard protocols, or continue to culture the cells (section 4). 
		NOTE: The cell suspension contains cell clumps and requires extra steps to obtain single cells, which results in an increase in cell death. Therefore, the cells are kept in suspension and are not counted at this stage.</li>
	</ol>
	</li>
</ol>
4. Cell Culture of Crude Kidney Cells
<ol>
	<li>Add approximately 1 mL of the cell suspension to 24 mL of alphaMEM 5% platelet lysates (cell culture medium) in a T175 cell culture flask (Figure 1K) and culture at 37 &#176;C, 5% CO2. Remove the medium and cell debris after 2 d&#160;from the adherent cells and refresh the medium with 25 mL of cell culture medium.</li>
	<li>Refresh the culture medium twice a week by removing half of the medium (12.5 mL) and adding fresh medium (12.5 mL) using aseptic techniques.</li>
	<li>Culture until confluent (usually 5 - 7 d) (Figure 1L). Remove the medium, wash twice with PBS and trypsinize the cells by adding 5 mL trypsin to each T175 flask for 5 min at 37 &#176;C. Wash in culture medium and centrifuge at 490 x g for 5 min and remove the supernatant.</li>
</ol>
5. Cell Enrichment for NG2 by Magnetic Cell Separation (Figure 1M)
<ol>
	<li>Prepare the cell separation buffer according to manufacturer&#39;s protocol.</li>
	<li>Resuspend the trypsinized kPSCs in 10 mL cell separation buffer. Centrifuge at 490 x g for 5 min, discard the supernatant and resuspend the cells in 300 &#181;L cell separation buffer.</li>
	<li>Add 100 &#181;L FcR blocking reagen/5 x 107 cells, add 100 &#181;L anti-melanoma (NG2) beads per 5 x 107 cells, and incubate for 30 min at 4 &#176;C. Separate the NG2-positive fraction according to manufacturer&#39;s protocol. Add 10 mL of medium and centrifuge cells for 5 min at 490 x g.</li>
	<li>Count the cells using a B&#252;rker counting chamber according to manufacturer&#39;s protocol. Seed the cells in a culture flask (approximately 4 x 103 cells/cm2) and incubate. 
	NOTE: Seed the cells in the following concentrations: &#60;250,000 in a T25 flask, 250,000 - 500,000 in a T75 flask, and 700,000 - 800,000 in a T175 flask. After magnetic cell enrichment, a positive fraction of approximately 1 - 2% is isolated. However, as this step is cell enrichment and not cell sorting, other cell types will be present in the culture (Figure 1N). After several passages (usually around 4 - 5), only a homogenous kPSC-population is present (Figure 1P).</li>
</ol>
6. Culture of kPSCs
<ol>
	<li>Refresh the medium twice a week by removing half of the medium and replace it with freshly thawed culture medium (Figure 1N). 
	NOTE: The kPSCs tend to be more viable and proliferative when only half of the medium is refreshed.</li>
	<li>Passaging of kPSCs. 
	NOTE: Passage the kPSCs at either 90 - 100% confluency, or when 3D structures appear (see Figure 1O), or when there hasn&#39;t been cell growth for more than a week. The latter two particularly might occur at early passages after cell enrichment. In this case, after passaging, the cells will usually start to grow again in monolayer culture (Figure 1P).
	<ol>
		<li>Remove the medium from the 90 - 100% confluent cells (keep the medium for later use). Wash the flask twice with PBS (1 mL in T25, 5 mL in T75, 10 mL in T175). Add trypsin (0.5 mL in T25, 2 mL in T75, 5 mL in T175) and incubate for 5 min at 37 &#176;C. Add the old medium to the flask and resuspend gently.</li>
		<li>Transfer to a 15&#160;or 50 mL tube (depending on the volume) and centrifuge at 490 x g for 5 min. Count the viable cells and plate &#60;250,000 in a T25, 250,000 - 500,000 in a T75, or 700,000 - 800,000 in a T175 (approximately 4 x 103 cells/cm2).</li>
	</ol>
	</li>
	<li>Test the kPSCs at passage 5 or 6 (e.g., scratch assay, section 7). 
	NOTE: Characterize the propagated cells by flow cytometry using standard protocols. Marker expression of NG2, PDGFR-&#946;, CD146, CD73, CD90, CD105, HLA-ABC should all be positive and CD31, CD34, CD45 and HLA-DR should be negative. Test for mycoplasma, bacteria and fungi in the culture medium according to standard protocols of the clinical microbiology lab. Test the kPSCs functionally in a kidney epithelial wound scratch assay. Experiments are usually performed with sterile, flow cytometry-confirmed homogeneous kPSC populations with wound healing capacity between passage 6-8.</li>
	<li>Cryopreserve the kPSCs by adding 0.5 mL cryopreservation medium to 0.5 mL cell suspension (2 million cells per mL of culture medium) using standard protocols. 
	NOTE: After thawing, seed the kPSCs at a higher density (106&#160;cells in a T175).</li>
</ol>
7. Functional Test of kPSCs: Kidney Epithelial Wound Scratch Assay
<ol>
	<li>Culture the kPSCs in a 6-well plate at a density of 200,000 cells/well in 2 mL culture medium. After 48 h of culture at 37 &#176;C, 5% CO2, collect the supernatant. This is the conditioned medium.</li>
	<li>Seed the HK-2 (proximal tubular epithelial cells)17 in 2 mL of Proximal Tubular Epithelial Cell (PTEC) medium consisting of a 1:1 ratio of DMEM-F12 supplemented with insulin (5 mg/mL), transferrin (5 mg/mL), selenium (5 ng/mL), hydrocortisone (36 ng/mL), triiodothyrinine (40 pg/mL), epidermal growth factor (10 ng/mL) and pen/strep, in a density of 500,000 cells per well in a 6-well cell culture plate.</li>
	<li>Culture for 48 h at 37 &#176;C, 5% CO2. Remove the cell culture medium of the HK-2s and create a scratch wound in the monolayer of HK-2s by making a scratch with the tip of a yellow pipette point from the top to the bottom of the well. Wash the HK-2s with PBS and add the conditioned medium of the kPSCs or control medium. Mark on the bottom of the plate the area to be imaged. 
	NOTE: The HK-2s should be confluent. Image two areas per well.</li>
	<li>Image the scratch at 4, 8, 12 and 24 h at the same marked position with an inverted bright-field microscope.</li>
	<li>Measure the scratch area in Image J using the polygon selection tool to determine the percentage of closure.</li>
</ol>

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D.G.L., M.A.E., ve T.J.R. bir patent başvurusu ile ilgili bu araştırma ile ilişkilidir. Diğer yazarlar ifşa gerek yok.

Perivasküler hücreleri pankreas, yağ, kıkırdak ve böbrek9,15,16da dahil olmak üzere birçok farklı insan solid organ, izole edilmiştir. Pek çok yöntem, ancak, küçük parçalara ve daha sonra sindirim enzimleri ile tedavi doku örnekleri temel alır. Ayrıca, bu genellikle klinik sınıf ürünleri ile gerçekleştirilir. Bu bu stratejileri daha az doğrudan klinik çeviri için uygun klinik-grade hücrelerinin büyük miktarlarda gerekli nerede yapar.
İşte insan kPSCs bir roman yalıtım yöntemini perfüzyon klinik sınıf enzimler ve malzemelerle dayalı tüm organlar için göstermektedir. Bizim Merkezi18klinik uygulamada için kullanılmakta olan klinik adacıkları of Langerhans yalıtım protokolden adapte iletişim kuralıdır.
Bu ilk klinik sınıf yöntem nereye kPSCs büyük miktarlarda elde edilebilir. Değişkenlik hücre verim büyük ölçüde bağımlı verici. Ancak, ne zaman biz şu anda üç farklı bağış ham hücre süspansiyon bir kısmını elde hücre verim yaygınlaştırılması, teorik olarak, 2.7 x 1012 kPSCs donör başına ortalama bir verim elde edilebilir. MSC tedavisi genellikle 1-2 x 106 hücreler/kg vücut ağırlığı42 hücre infüzyon oluşur, bu hücre sayıları birden fazla hastada allojenik tedavisi için yeterli şunlardır.
Yalıtım yordamdaki bir kritik adım collagenase sindirim süresi kadardır. Sindirim süresi çok kısa olduğu durumlarda hangi kültür için daha zor olacak büyük kümeleri doku kalır. Perfüzyon dönem çok uzun olduğunda, artan hücre ölümü gözlenen. Bu nedenle, böbrek yumuşak ve sıvı daha az şeffaf olmaya başladığı anda böbrek hafifçe masaj ve collagenase tedavi kesilmelidir.
Başka bir önemli adım NG2 hücre zenginleştirme sonra hücrelerin kültürdür. NG2 hücre zenginleştirme bazen sonra Perivasküler hücreleri çoğalırlar veya 3D yapılar büyümeye başlar başlamaz. Bu durumda, hücreleri trypsinized ve reseeded olduğunda, hücreleri genellikle monolayer kültüründe büyümeye başlayacak.
Malzeme başlangıç olarak, esas olarak cerrahi nedenlerle atılan insan nakli sınıf böbrekler kullanılmıştır. Bu büyük fibroz olmadan fonksiyonel organları vardır. Bu nispeten nadir olduğu gibi bu bir sınırlama olabilir kabul ve organ kaynak elde etmek zor. Explanted böbrekler başka bir kaynağı olabilir; Ancak, bağlı olarak böbrek explantation nedeni, bu böbrekler daha fazla fibrozis ve böylece myofibroblasts içerebilir ve myofibroblasts izole ve Perivasküler hücreler yerine kültürlü olabilir bu yana bu nedenle özen gösterilmelidir.
KPSCs izole olarak bu protokolü göstermek ile organo-typic özellikleri böbrek epitel ile şifa kapasiteleri15, kPSCs böbrek hastalıklarında hücre tedavisi yara ve nakli ilginç bir gelecek uygulama olurdu. Bu amaçla, hücre/ürün teslimat birkaç stratejileri vardır. İlk strateji kPSCs, IV infüzyon şu anda bmMSC klinik çalışmalarda kullanılan gibidir. Başka bir ilginç uygulama kPSCs veya makine perfüzyon nakli önce kPSC atılır faktörler kullanımıdır. Bu şekilde, hangi sonra nakli için geliştirilmiş Böbrek fonksiyonlarının neden olabilir explanted böbrek kalitesini artırmak. Hem stratejileri için kPSCs daha fazla klinik amaçlar için keşfetmek için ilginç bir yeni hücre kaynağı vardır.

Mezenkimal Stromal hücre (MSCs) kemik iliği aslında izole edildi immün düzenleyici hücrelerdir. Onlar onların iğ şeklinde Morfoloji, yağ, kemik ve kıkırdak ve plastik bağlılık ayırt etmek için yeteneği ile karakterizedir. MSCs stromal işaretleri CD73, CD90, işaretçileri CD31 ve CD451,2,3için negatif olurken CD105 hızlı. MSCs doku homeostatik ve immunomodulatory kapasiteleri nedeniyle hücre tedavisi için umut verici bir aday olduğunu. bmMSCs şu anda böbrek hastalıkları ve başka bir yerde4gözden olarak böbrek nakli dahil olmak üzere çeşitli hastalıklar için klinik çalışmalarda incelendiği.
Daha önce bu Perivasküler yağ dokusu da dahil olmak üzere birkaç farklı solid organ, hücreleri, plasenta ve iskelet kası özellikleri MSCs9ile paylaşan gösterilmiştir. Ancak, bu hücreler aynı zamanda organotypic onarım10neden olabilir doku özgü işlevleri sergi. İnsan miyokardiyal Perivasküler hücreleri, örneğin, hipoksi sonra anjiogenik yanıt teşvik ve diğer dokulardan izole Perivasküler hücreleri bu potansiyeller11belli etmedi ise cardiomyocytes farklı.
Perivasküler stromal hücreler de fare12,13,14 ve insan böbrek15,16ayrılmış olabilir. Yaygın olarak kPSCs ile karakterize olan ve bunları bmMSCs karşılaştırıldığında. KPSCs, bmMSCs için benzer immünsüpresif kapasitesine sahip ve vasküler pleksus oluşumu destekleyebilir bulduk. Ancak, doku hücre tipleri, HoxD10 ve HoxD11 nephrogenic transkripsiyon faktörleri de dahil olmak üzere bir organotypic transkripsiyon ifadesi imzasını gösterdin kPSCs olarak arasında belirli farklar vardır. kPSCs, bmMSCs, aksine myofibroblast stimülasyon ile TGF-β sonra dönüşüme değil ve adiposit ayırt koyamadık. Ayrıca, kPSCs epitel bütünlük içinde bir böbrek borulu epitel yara karalama yöntemi, bmMSCs ile gözlenen değil bir fenomen hız. Bu gelişmiş yara onarım hepatosit büyüme faktörü yayın aracılı. Ayrıca, kPSCs böbrek yaralanma Akut böbrek yaralanma15, bir fare modeli iyileştirmiştir. Bu nedenle, kPSCs üstün böbrek onarım kapasitesine sahip görünmektedir ve böbrek hastalıklarında hücre tedavisi için ilginç bir yeni kaynak vardır.
Hücre tedavisi amacıyla kPSCs kullanabilmek için kPSCs klinik sınıf enzimler ve protokolleri ile klinik sınıf şekilde izole. Ayrıca, birkaç kPSCs 1 donör tedavisinde edebilmek için yeterli hücre sayıları alınmalıdır. İşte ayrıntılı, kPSCs gelen tüm organ nakli sınıf böbrekler, klinik sınıf izolasyon prosedürü yeterli sayıda klinik hücresel tedavi için kullanılmak üzere hücre verimli göstermektedir.

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			<td style="width:117px;">Sigma-Aldrich</td>
			<td style="width:119px;">10043-52-4</td>
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			<td>SERVA</td>
			<td style="width:119px;">17455.03</td>
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			<td>custom made</td>
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			<td>Masterflex</td>
			<td>HV-96410-25</td>
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			<td height="21" style="height:21px;width:216px;">accessory spike</td>
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			<td style="width:119px;">6038020</td>
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			<td style="width:119px;">690175</td>
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			<td height="21" style="height:21px;">culture flask 75 cm2</td>
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			<td style="width:119px;">658170</td>
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			<td height="21" style="height:21px;width:216px;">culture flask 175 cm2</td>
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			<td style="width:119px;">661160</td>
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			<td>sigma</td>
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			<td>miltenyi</td>
			<td>130-090-452</td>
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			<td>Corning</td>
			<td>352350</td>
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			<td>130-042-401</td>
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			<td style="width:117px;">miltenyi</td>
			<td>130-090-976</td>
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			<td height="21" style="height:21px;width:216px;">CD34 FITC</td>
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			<td>555821</td>
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			<td>555485</td>
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			<td style="width:117px;">BD&#160;</td>
			<td>550315</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">NG2 APC</td>
			<td style="width:117px;">R&#38;D</td>
			<td>FAB2585A</td>
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			<td height="21" style="height:21px;width:216px;">CD90 PE</td>
			<td style="width:117px;">BD&#160;</td>
			<td>555596</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">HLA ABC APC</td>
			<td style="width:117px;">BD&#160;</td>
			<td>555555</td>
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			<td height="21" style="height:21px;width:216px;">CD105 FITC</td>
			<td>Ancell</td>
			<td>326-040</td>
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			<td height="21" style="height:21px;width:216px;">HLA DR APC</td>
			<td style="width:117px;">BD&#160;</td>
			<td>559866</td>
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			<td height="21" style="height:21px;width:216px;">CD56 PE</td>
			<td>BD&#160;</td>
			<td>555516</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">CD73 PE</td>
			<td style="width:117px;">BD&#160;</td>
			<td>550257</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD31 FITC</td>
			<td style="width:117px;">BD&#160;</td>
			<td>555445</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">CD133 PE</td>
			<td style="width:117px;">miltenyi</td>
			<td>130-090-853</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PDGF-r&#160;</td>
			<td style="width:117px;">R&#38;D</td>
			<td>mab 1263</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mouse IgG1 FITC</td>
			<td style="width:117px;">BD&#160;</td>
			<td>345815</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mouse IgG1 PE</td>
			<td style="width:117px;">BD&#160;</td>
			<td>345816</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mouse IgG1 APC</td>
			<td style="width:117px;">BD&#160;</td>
			<td>345818</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IgG2b PE</td>
			<td style="width:117px;">BD&#160;</td>
			<td>555743</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">goat anti mouse PE</td>
			<td style="width:117px;">Dako</td>
			<td style="width:119px;">R0480</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sodium azide</td>
			<td style="width:117px;">Merck</td>
			<td>822335</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">DMEM Ham&#8217;s F12</td>
			<td>Gibco</td>
			<td>31331-028</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">ITS (insul, transferrin, selenium)</td>
			<td style="width:117px;">Sigma</td>
			<td>I1884</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">hydrocortisone</td>
			<td>Sigma</td>
			<td>H0135</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">triiodothyrinine</td>
			<td style="width:117px;">Sigma</td>
			<td>T5516</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">epidermal growth factor</td>
			<td>sigma</td>
			<td>E9644</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immortalized human renal PTEC (HK2)</td>
			<td style="width:119px;"></td>
			<td></td>
			<td>courtesey of M. Ryan, university college Dublin</td>
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	</tbody>
</table>

a novel clinical grade isolation method for human kidney perivascular stromal cells

Mezenkimal Stromal hücreler (MSCs) yararlı etkileri böbrek hastalıkları ve nakli göstermiştir doku homeostatik ve bağışıklık düzenleyici hücrelerdir. Perivasküler Stromal hücreler (PSC) kemik iliği MSCs (bmMSCs) ile ortak özelliklere sahiptir. Ancak, onlar da, büyük olasılıkla yerel sürecinden, doku özgü özellikleri ve yerel doku homeostazı bir rol oynar nedeniyle sahip. Bu doku özgüllük doku belirli tamir, insan böbreğe içinde de neden olabilir. Biz daha önce insan böbrek PSC'ler (kPSCs) bmMSCs bu potansiyelin yoktu Oysa şifa böbrek epitel yara gelişmiş gösterdi. Ayrıca, kPSCs böbrek yaralanma içinde vivoiyileştirmek. Bu nedenle, kPSCs hücre tedavisi, özellikle böbrek hastalıkları ve böbrek nakli için ilginç bir kaynak oluşturmaktadır. İşte bütün organ perfüzyon renal arter ve zenginleştirme Perivasküler işaretçi NG2 için üzerinden sindirim enzimleri, temel insan böbrek nakli-sınıftan kPSCs için detaylı yalıtım ve kültür yöntemi göstermektedir. Bu şekilde, büyük hücreli miktarları hücresel tedavi için uygun elde edilebilir.

Klinik sınıf kPSCs yalıtım yöntemini şekil 1' de özetlenmiştir. Ham böbrek hücreleri insan nakli sınıf böbrekler collagenase perfüzyon tarafından izole edilmiştir. Elde edilen hücre süspansiyon %5 trombosit lysates birleşmesi kadar kültürlü. O zaman Perivasküler stromal hücre kesir NG2 ifadeye dayalı izole edilmiştir.
kPSCs plastik yapisan seklinde hücreler (Şekil 2A) ve negatif CD31, CD34, CD45 ve HLA-DR (Şekil 2B) için ise CD73, CD90, CD105 ve Perivasküler stromal işaretleri işaretleri NG2, PDGFR-B ve CD146, olumludur. Genellikle, kPSCs homojen bir nüfusa geçiş 4 ulaşılabilir ve kPSCs yaşlanma etrafında geçit 9-10 (Şekil 2C) ulaşır. Bu yüzden deneyler geçit 5-8 arasında gerçekleştirmek için tavsiye ederiz.
Biz daha önce kPSCs şartına orta bu karalama tahlil15dakika içinde epitel yara iyileşmesi hızlandırabilir gösterdiğim gibi izole kPSCs fonksiyonel kapasitesini değerlendirmek için bir vitro böbrek epitel yara karalama tahlil kPSCs, her yeni toplu gerçekleştiriyoruz. KPSC şartına orta kPSCs alphaMEM % 5 trombosit lysates 48 h için kültür ve süpernatant toplama yapılır. Ardından, ölümsüzleştirdi insan böbrek proksimal tübül epitel hücreleri (HK-2)17 birleşmesi kadar kültürlü ve sıfırdan bir yara daha sonra yapılır. O zaman ya kPSCs veya denetim orta şartına orta wells için eklenir ve yara iyileşme hızı ölçülür. KPSCs klimalı ortam eklendiğinde, yara önemli ölçüde daha hızlı (Şekil 2D) kapatır.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55841/55841fig1.jpg"> 
Resim 1 : Klinik sınıf izolasyon yöntemi, insan kPSCs. Organ nakli sınıf böbrek cannulated ve collagenase yolu ile renal arter (A - G) ile derin ve elde edilen hücre süspansiyon yıkanmış ve ya cryopreserved ya da kültür (H - K) koymak. Hücreleri confluency (L) ulaştıktan sonra hücre zenginleştirme olduğunu NG2 (M) gerçekleştirilen. Hücrelerin trypsinized ne zaman onlar are ikisinden biri Konfluent, Proliferasyona veya 3D yapılar (N - P) görüntülenirken durduruldu. KPSCs sürüm ölçütlerini kısırlık, işaretleyici ifade ve (Q) borulu epitel yara iyileşmesi geliştirmek için yeteneği vardır. Oku: renal arter. Ölçek çubuğu 200 µm. = <a href="http://ecsource.jove.com/files/ftp_upload/55841/55841fig1large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55841/55841fig2.jpg"> 
Resim 2: karakterizasyonu insan kPSCs. Bir) kPSCs seklinde, plastik yapışık hücreleri vardır. B) kPSCs Mezenkimal işaretleri CD73, CD90, CD105, Perivasküler işaretleri NG2, PDGFR-β ve CD146, negatif CD31, CD34 ve CD45 için süre için olumludur. kPSCs express MHC sınıf ı (HLA-ABC) ama değil sınıf II (HLA-DR). C) Akış Sitometresi üç farklı kPSC bağışçılardan büyüme özellikleri (Bölüm 4), homojen NG2 olumlu nüfus doğruladı. kPSCs yaşlanma etrafında geçit 9-10 ulaşmak. D) kPSCs böbrek epitel onarım bir yara karalama tahlil geliştirmek edebiliyoruz. Temsilcisi görüntüleri kontrol orta ve şartına kPSC orta t = 0, 4, 8 ve 12 h gösterilir. A ölçek çubuğu) = d 200 µm) 100 µm. = <a href="http://ecsource.jove.com/files/ftp_upload/55841/55841fig2large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>

Watch this Scientific Journal Video about A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells at JoVE.com

A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells

Burada böbrek Perivasküler Stromal bütün organ perfüzyon sindirim enzimleri ve NG2 hücreli zenginleştirme dayalı hücreleri (kPSCs) için bir roman klinik sınıf izolasyon ve kültür yöntem mevcut. Bu yöntemle, hücresel tedavi için yeterli hücre sayıları elde etmek mümkündür.

İnsan böbrek Perivasküler Stromal hücreler için bir roman klinik sınıf izolasyon yöntemi

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and ...

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JoVE Journal

Medicine

6.8K Görüntüleme.  Leiden University Medical Centre. Burada böbrek Perivasküler Stromal bütün organ perfüzyon sindirim enzimleri ve NG2 hücreli zenginleştirme dayalı hücreleri (kPSCs) için bir roman klinik sınıf izolasyon ve kültür yöntem mevcut. Bu yöntemle, hücresel tedavi için yeterli hücre sayıları elde etmek mümkündür.

Video: A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

Murin Islet İzolasyon ve subkapsüler Böbrek Nakli Bir Yöntem

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

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Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

İnsan Meme Fibroblastlar Karsinom İlişkili Fibroblastlar Deneysel Nesil (CAFs)

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

İnsan Diş Pulp Kök Hücre İzolasyonu, Karakterizasyonu ve Karşılaştırmalı Farklılaşma İki Farklı Yöntemler Kullanılarak Daimi Dişler türetilen

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

Histerektomi Örneklerden İlköğretim İnsan endometrial stroma hücrelerinde Kurulması İçin İki Yöntem

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

İzolasyon, Kriyoprezervasyon ve Kültür Klinik Uygulamaları İnsan Amniyon Epitel Hücreleri

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

İnsan Prostat Kanseri klinik öncesi Ortotopik murin modeli

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...

A Detailed Protocol for Perspiration Monitoring Using a Novel, Small, Wireless Device

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the patients are unaware of such disorders or are having difficulty expressing their symptoms. Until now, several devices for perspiration monitoring have been developed; however, such devices tend to have a relatively large exterior, considerable power consumption, and/or less sensitivity.
Recently, we developed a small, wireless device for perspiration monitoring. The device consists of a temperature/relative humidity (T/RH) sensor, battery-driven small data logger, and silica gel as a desiccant in a small cylindrical exterior. The T/RH sensor is placed between the detection windows (through which the water vapor from the skin enters) and the silica gel. The underlying principle of the perspiration monitoring device is based on Fick&#39;s law of diffusion, which means that water vapor flux from the skin to the silica gel (i.e.&#160;transepidermal water loss and perspiration) can be captured by change in humidity at the T/RH sensor. In addition, a baseline subtraction method was adopted to distinguish perspiration and transepidermal water loss.
As shown in the previous report, the developed device can monitor the perspiration at any sites of the body in an easy, wireless manner. However, detailed methods of how to use the device have not been disclosed yet. In this article, therefore, we would like to show the point-by-point tutorials of how to use the device for perspiration monitoring, by showing the sympathetic activity test with the sympathetic skin response monitoring as an example.

Recently, we developed a small wireless device for perspiration monitoring. In this article, we present detailed protocols on how to use the device for perspiration monitoring with an example of the sympathetic activity test.

Bir Roman kullanarak Terleme İzleme Detaylı Protokol, Küçük Kablosuz Aygıt

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the ...

Clinical Protocol of Producing Adipose Tissue-Derived Stromal Vascular Fraction for Potential Cartilage Regeneration

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the knees by using different forms of mesenchymal stem cells (MSCs). In Korea, bone marrow concentrates and cord blood-derived stem cells have been approved by the Korean Food and Drug Administration (KFDA) for cartilage regeneration. In addition, an adipose tissue-derived stromal vascular fraction (SVF) has been allowed by the KFDA for joint injections in human patients. Autologous adipose tissue-derived SVF contains extracellular matrix (ECM) in addition to mesenchymal stem cells. ECM excretes various cytokines that, along with hyaluronic acid (HA) and platelet-rich plasma (PRP) activated by calcium chloride, may help MSCs to regenerate cartilage and improve knee functions. In this article, we presented a protocol to improve knee functions by regenerating cartilage-like tissue in human patients with OA. The result of the protocol was first reported in 2011 followed by a few additional publications. The protocol involves liposuction to obtain autologous lipoaspirates that are mixed with collagenase. This lipoaspirates-collagenase mixture is then cut and homogenized to remove large fibrous tissue that may clog up the needle during the injection. Afterwards, the mixture is incubated to obtain adipose tissue-derived SVF. The resulting adipose tissue-derived SVF, containing both adipose tissue-derived MSCs and remnants of ECM, is injected into knees of patients, combined with HA and calcium chloride activated PRP. Included are three cases of patients who were treated with our protocol resulting in improvement of knee pain, swelling, and range of motion along with MRI evidence of hyaline cartilage-like tissue.

Here, we present a protocol to produce an adipose tissue-derived stromal vascular fraction and its application to improve knee functions by regenerating cartilage-like tissue in human patients with osteoarthritis.

Yağ dokusundan elde edilen Stromal vasküler kesir için potansiyel kıkırdak rejenerasyon üreten klinik protokol

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the ...

Isolation of Adipose Derived Regenerative Cells for the Treatment of Erectile Dysfunction Following Radical Prostatectomy

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem cells can be obtained from different tissues and several methods for isolation have been described. The presented method for the isolation of adipose-derived regenerative cells (ADRCs) can be used within many therapeutic areas because the method is a general procedure and, therefore, not limited to erectile dysfunction (ED) therapy. ED is a common and serious side effect to radical prostatectomy (RP) since ED often is not well treated with conventional therapy. Using ADRC&#8217;s as treatment for ED has attracted great interest due to the initial positive results after a single injection of cells into the corpora cavernosum. The method used for the isolation of ADRC&#8217;s is a simple, automated process, that is reproducible and ensures a uniform product. Furthermore, the sterility of the isolated product is ensured because the entire process takes place in a closed system. It is important to minimize the risk of contamination and infection since the stem cells are used for injection in humans. The whole procedure can be done within 2.5-3.5 hours and does not require a classified laboratory which eliminates the need for shipping tissue to an off-site. However, the procedure has some limitations since the minimum amount of drained lipoaspirate for the isolation device to function is 100 g.

Precise disclosure of methods and protocols is crucial for large scale uptake of stem cell therapies. Here, we present a protocol to isolate adipose-derived regenerative cells, used for a single intracavernous injection as treatment of erectile dysfunction (ED) following radical prostatectomy (RP).

Radikal Prostatektomi Sonrası Erektil Disfonksiyon Tedavisinde Yağ Kaynaklı Rejeneratif Hücrelerin İzolasyonu

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem ...

Orthotopic Rat Kidney Transplantation: A Novel and Simplified Surgical Approach

Kidney transplantation offers increased survival rates and improved quality of life for patients with end-stage renal disease, as compared to any type of renal replacement therapy. Over the past few decades, the rat kidney transplantation model has been used to study the immunological phenomena of rejection and tolerance. This model has become an indispensable tool to test new immunomodulatory pharmaceuticals and regimens prior to proceeding with expensive preclinical large animal studies.
This protocol provides a detailed overview of how to reliably perform orthotopic kidney transplantation in rats. This protocol includes three distinctive steps that increase the probability of success: perfusion of the donor kidney by flushing through the portal vein and the use of a cuff system to anastomose the renal veins and ureters, thereby decreasing cold and warm ischemia times. Using this technique, we have achieved survival rates beyond 6 months with normal serum creatinine in animals with syngeneic or tolerant kidney transplants. Depending on the aim of the study, this model can be modified by pre- or posttransplant treatments to study the acute, chronic, cellular, or antibody-mediated rejection. It is a reproducible, reliable, and cost-effective animal model to study different aspects of kidney transplantation.

The purpose of this manuscript and protocol is to explain and demonstrate in detail the surgical procedure of orthotopic kidney transplantation in rats. This method is simplified to achieve the correct perfusion of the donor kidney and shorten the reperfusion time by using the venous and ureteral cuff anastomosis technique.

Ortootopik Rat böbrek nakli: bir roman ve Basitleştirilmiş cerrahi yaklaşım

Kidney transplantation offers increased survival rates and improved quality of life for patients with end-stage renal disease, as compared to any type of ...