Name: a novel clinical grade isolation method for human kidney perivascular stromal cells
Uploaded: 2017-08-07T13:00:00
Duration: 9 min 5 s
Description: Watch this Scientific Journal Video about A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells at JoVE.com

この研究資金を受けている欧州共同体の第 7 フレームワーク プログラム (FP7/2007-2013) から付与契約番号 305436 の下恒星。

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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機械知能系、D.G.L.、T.J.R. は、この研究に関する特許出願に関連付けられます。他の作家が何を開示するあります。

血管周囲細胞は多くの異なった人間固体器官、膵臓、脂肪、軟骨、腎臓9,,1516などから分離されています。ただし、ほとんどのメソッドは、組織を切除し、その後消化酵素で処理の小さなサンプルに基づいています。また、これは通常行われません臨床グレード製品。これにより、臨床グレード細胞の大量が必要これらの戦略を直接臨床翻訳には適して。
ここで臨床グレード酵素および物質による血液灌流に基づく全臓器の人間 kPSCs の新規分離法を示す.プロトコルは、現在臨床応用センター18使用中臨床ランゲルハンス島の隔離のプロトコルから適応。
これは、大量の kPSCs を実現できます最初の臨床グレード方法です。細胞収率の変動は主としてドナー依存です。しかし、我々 は現在 3 つの異なるドナー原油細胞懸濁液の一部から得る細胞収量を推定すると、理論的には、ドナーごと 1012 kPSCs x 2.7 の平均利回りが達成できます。MSC 療法は、通常、1 - 2 × 106セル/kg 本体重量42 細胞注入で構成されますとこれらの細胞の数はいくつかの患者の同種の治療のため十分です。
分離のプロシージャの 1 つの重要なステップは、コラゲナーゼの消化力の期間です。消化期間が短すぎると、文化が困難となる、組織の大きな塊保たれます。潅流期間が長すぎると、細胞死が増加を観察することがあります。したがって、腎臓ソフトになり、流体透過性が低下し始めると、すぐに腎臓を優しくマッサージする必要があります、コラゲナーゼ処理を停止する必要があります。
別の重要なステップは、NG2 細胞濃縮後、細胞の培養です。NG2 細胞濃縮後時々 血管周囲細胞は増殖や立体構造で成長を開始する開始されません。この場合ときに、細胞がトリプシンおよび再シード、細胞が単層培養による成長を開始通常。
出発材料として人間の移植グレード腎臓手術の理由主に破棄されたが使用されました。これらの主要な線維化なし機能器官です。我々 は、これは比較的まれな制限をする可能性がありますこれを認めるとオルガンのソースを取得するは難しい。Explanted 腎臓可能性があります別のソース;しかし、腎植の理由に応じてより多くの線維化およびこうして芽、これらの腎臓があります、したがってケアしなければならないので芽を分離、培養血管細胞ではなく可能性があります。
KPSCs が分離されたこのプロトコルを見ると腎臓上皮とオルガノ typic プロパティ創傷治癒能力15、腎臓疾患における kPSCs を用いた細胞治療、移植は今後の興味深いアプリケーションになります。このため、セル/セル製品出荷のいくつかの戦略があります。BmMSC の臨床試験で現在使われている、最初の戦略は、kPSCs の点滴です。別の興味深いアプリケーションは、kPSCs, 移植前に機械灌流 kPSC 排泄要因の使用です。このように、移植後改善された腎機能につながる可能性があるが explanted 腎臓の質を改善するかもしれない。両方の戦略 kPSCs 臨床目的のためさらに探索する興味深い新しいセル源であります。

間葉系間質細胞 (MSCs) は、もともと骨の髄から分離された免疫の調節細胞です。彼らは彼らの紡錘の形態、脂肪、骨、軟骨、およびプラスチックの付着に分化する能力を形によって特徴付けられます。MSCs は、間質マーカー CD73、CD90、マーカー CD31、CD451,2,3負ながら CD105 を表現します。MSCs は、彼らの組織の恒常性維持と免疫調節能力のための細胞療法の有望な候補です。bmMSCs は、腎臓病と腎移植4として他の場所を含むいくつかの疾患の臨床試験で現在検討中です。
以前血管周囲脂肪組織を含むいくつかの異なった固体器官から細胞、胎盤および骨格筋は MSCs9と特性を共有ことが示されました。しかし、これらの細胞はまた脊髄修復10になることができます組織固有の関数を表わします。ひと心筋血管周囲細胞低酸素後の血管新生反応の刺激など、一方、他の組織から分離した血管周囲細胞はこれらの電位11を表示されませんでした、心筋細胞に分化します。
血管周囲間質細胞は、マウス12,13,14ひと腎臓15,16から分離することができます。我々 の広範囲 kPSCs を特徴し、bmMSCs にこれらを比較します。KPSCs、bmMSCs と同様、免疫能力および血管叢形成をサポートすることができますがわかった。ただし、特定セル型では、腎の転写因子 HoxD10 および HoxD11 を含めて、脊髄転写式署名を示した kPSCs と違い組織があります。kPSCs、bmMSCs と対照をなして TGF-β 刺激後の myofibroblast 変換を受けていないと、脂肪細胞に分化することができませんでした。さらに、kPSCs は、腎尿細管上皮創傷スクラッチ アッセイ、bmMSCs と見られなかった現象で上皮の完全性を加速しました。この強化された傷修理だった肝細胞成長因子の遊離を介する。さらに、kPSCs は、急性腎傷害15マウス モデルにおける腎障害を改善しました。したがって、kPSCs は優れた腎修復能力を持っているようだ、腎臓病の細胞療法のための興味深い新しいソースです。
細胞療法目的で kPSCs を使用するために、kPSCs は臨床グレード酵素とプロトコル臨床グレードの方法で分離する必要があります。また、1 ドナーから kPSCs と数人の患者を治療することには、十分な細胞数を取得必要があります。ここで我々 は臨床細胞療法に使用する細胞の十分な数を降伏詳細、全体移植グレード腎臓から kPSCs の臨床グレードの隔離プロシージャに表示します。

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			<td style="width:119px;">09-761-11</td>
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			<td height="21" style="height:21px;">500ml PP centrifuge tubes</td>
			<td style="width:117px;">Corning</td>
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			<td height="21" style="height:21px;">a-MEM medium</td>
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			<td>BE12-169F</td>
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			<td height="21" style="height:21px;">glutamax</td>
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			<td height="21" style="height:21px;">pen/strep&#160;</td>
			<td>Invitrogen</td>
			<td>15070063</td>
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			<td height="21" style="height:21px;">transfusion system</td>
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			<td>455609</td>
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			<td height="21" style="height:21px;">DMEM-F12</td>
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			<td>11320-074</td>
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			<td height="21" style="height:21px;">normal human serum</td>
			<td style="width:117px;">Sanquin</td>
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			<td height="21" style="height:21px;">Hepes 1M</td>
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			<td style="width:119px;">BE-17-737E</td>
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			<td height="21" style="height:21px;">NaHCO3 7.5%</td>
			<td style="width:117px;">Lonza</td>
			<td style="width:119px;">BE17-613E</td>
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			<td height="21" style="height:21px;">CaCl2 1M</td>
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			<td height="21" style="height:21px;">UW</td>
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			<td height="21" style="height:21px;">Heparin</td>
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			<td></td>
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			<td height="21" style="height:21px;">collagenase NB1 GMP grade</td>
			<td>SERVA</td>
			<td style="width:119px;">17455.03</td>
			<td></td>
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			<td height="21" style="height:21px;">DMSO</td>
			<td>Sigma Aldrich</td>
			<td>D2650-100ml</td>
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			<td style="width:117px;">3M Nederland</td>
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			<td>metrohm</td>
			<td>x007528300</td>
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			<td>metrohm</td>
			<td>x077202600</td>
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			<td style="width:119px;"></td>
			<td>custom made</td>
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			<td height="21" style="height:21px;">LS25 masterflex tubes</td>
			<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 height="21" style="height:21px;">pulmozyme</td>
			<td style="width:117px;">Roche</td>
			<td style="width:119px;"></td>
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			<td height="24" style="height:24px;width:216px;">culture flasks 25 cm2</td>
			<td style="width:117px;">greiner</td>
			<td style="width:119px;">690175</td>
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		<tr height="21">
			<td height="21" style="height:21px;">culture flask 75 cm2</td>
			<td style="width:117px;">greiner</td>
			<td style="width:119px;">658170</td>
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			<td height="21" style="height:21px;width:216px;">culture flask 175 cm2</td>
			<td style="width:117px;">greiner</td>
			<td style="width:119px;">661160</td>
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			<td height="21" style="height:21px;width:216px;">Trypsin</td>
			<td>sigma</td>
			<td>t4174</td>
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			<td height="21" style="height:21px;width:216px;">BSA</td>
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			<td style="width:119px;">A2153</td>
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			<td height="21" style="height:21px;width:216px;">EDTA</td>
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			<td>E5134-500g</td>
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			<td height="21" style="height:21px;width:216px;">FcR blocking reagent</td>
			<td>miltenyi</td>
			<td>130-059-901</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 height="21" style="height:21px;width:216px;">LS columns</td>
			<td>Miltenyi</td>
			<td>130-042-401</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">MACS magnet</td>
			<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 height="21" style="height:21px;width:216px;">CD45 APC</td>
			<td style="width:117px;">BD&#160;</td>
			<td>555485</td>
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			<td height="21" style="height:21px;width:216px;">CD146 PE</td>
			<td style="width:117px;">BD&#160;</td>
			<td>550315</td>
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		<tr height="21">
			<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>
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		<tr height="21">
			<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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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD105 FITC</td>
			<td>Ancell</td>
			<td>326-040</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HLA DR APC</td>
			<td style="width:117px;">BD&#160;</td>
			<td>559866</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD56 PE</td>
			<td>BD&#160;</td>
			<td>555516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD73 PE</td>
			<td style="width:117px;">BD&#160;</td>
			<td>550257</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD133 PE</td>
			<td style="width:117px;">miltenyi</td>
			<td>130-090-853</td>
			<td></td>
		</tr>
		<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>
			<td></td>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM Ham&#8217;s F12</td>
			<td>Gibco</td>
			<td>31331-028</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">hydrocortisone</td>
			<td>Sigma</td>
			<td>H0135</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">triiodothyrinine</td>
			<td style="width:117px;">Sigma</td>
			<td>T5516</td>
			<td></td>
		</tr>
		<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>
		</tr>
	</tbody>
</table>

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

間葉系間質細胞 (MSCs) が恒常性や免疫調節細胞移植や腎臓疾患の有益な効果を示すです。血管周囲間質細胞 (Psc) は、骨髄 MSCs (bmMSCs) の特性を共有します。しかし、彼らも所有している、最も可能性の高いローカル刷り込み、組織固有のプロパティおよびローカル組織の恒常性の役割を果たすのため。ティッシュ特定修理、また、ヒトの腎臓内でこの組織の特異性があります。以前、ひと腎臓 Psc (kPSCs) が腎臓上皮創傷治癒の bmMSCs は、この可能性を持っていなかった一方を強化しました。さらに、kPSCs は、腎臓損傷の生体を改善できます。したがって、kPSCs は、細胞治療、特に腎臓病と腎移植のための興味深いソースを構成します。ここで腎動脈と血管マーカー NG2 の濃縮による消化酵素の全体臓器灌流に基づく人間の腎臓移植グレードから kPSCs の詳細な分離と培養法を示す.この方法では、細胞療法に適している大規模な細胞量を取得できます。

臨床グレード kPSCs 分離法は、図 1のとおりです。原油の腎臓の細胞は、コラゲナーゼ灌流によって人間の移植グレード腎臓から分離されます。結果として得られる細胞懸濁液は 5% 血小板 lysates の合流まで培養しました。その後、血管周囲間質細胞画分は、NG2 式に基づいて分離されます。
kPSCs はプラスチック付着性紡錘形細胞 (図 2A)、CD31 CD34、CD45、HLA-DR (図 2B) 負ながら CD73、CD90, CD105、血管周囲間質マーカー マーカー NG2、試験 B、CD146、陽性です。通常、kPSCs の同質な人口は通路 4 に到達することができ、kPSCs に達する通路 9-10 (図 2C) 周りの老化。したがって、通路 5-8 間実験を実行する勧めします。
分離 kPSCs の機能を評価するため、以前示した kPSCs の馴化培地がこのスクラッチ法15の上皮の創傷治癒過程を加速することが、私たちに kPSCs のすべての新しいバッチに体外腎上皮創傷スクラッチ アッセイを実行します。KPSC 馴化培地 kPSCs alphaMEM 5% 血小板溶解液で 48 時間の培養、培養上清を収集させた。次に、不死化ヒト腎近位尿細管上皮細胞 (HK-2)17合流まで培養し、スクラッチ傷が行われます。どちらか kPSCs またはコントロール中の馴化培地は井戸に追加されます、創傷治癒の速度を測定します。KPSCs の馴化培地を追加すると、傷は格段に速くなります (図 2D) を閉じます。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55841/55841fig1.jpg"> 
図 1: 臨床等級分離法の人間 kPSCs.移植グレード腎臓を cannulated、腎動脈 (A G) を介してコラゲナーゼと灌流し結果細胞懸濁液を洗浄およびどちらかが凍結したまたは文化 (H ・ K) に入れます。セルに達する confluency (L) と、細胞濃縮は NG2 は (M) を実行しました。どちらかが合流、細胞がトリプシンが増殖または 3 D 構造が (N - P) を表示するときに停止しています。KPSCs のリリース条件は、不妊、マーカー式管状の上皮創傷治癒 (Q) を強化する機能を。矢印: 腎動脈。スケール バー = 200 μ m.<a href="http://ecsource.jove.com/files/ftp_upload/55841/55841fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55841/55841fig2.jpg"> 
図 2:特性の人間 kPSCs。) KPSCs、紡錘形、プラスチックの付着性のセル。B) kPSCs は、間葉系マーカー CD73、CD90、CD105、血管マーカー、NG2、試験 β および CD146、CD31 CD34、CD45 陰性間の肯定的です。kPSCs エクスプレス MHC クラス (HLA-ABC) けどクラス II (HLA) ではありません。C) フローサイトメトリーから 3 つの異なる kPSC ドナーの生育特性 (通路 4) で同種の NG2 肯定的な集団を確認します。kPSCs に達する通路 9 10 周り老化。D) kPSCs が傷傷アッセイの腎臓上皮修復を強化することができます。制御媒体と t でエアコン kPSC 媒体の代表的な画像 = 0、4、8、12 h が表示されます。A スケール バー) = 200 μ m、D) = 100 μ m.<a href="http://ecsource.jove.com/files/ftp_upload/55841/55841fig2large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</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

腎臓血管周囲間質細胞 (kPSCs) の消化酵素と NG2 細胞濃縮全臓器灌流に基づく新規臨床等級分離と培養法をご紹介します。この方法では、細胞療法の十分なセル番号を取得することが可能です。

ひと腎臓血管周囲間質細胞の新規臨床等級分離法

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汗は小説、小型、ワイヤレスデバイスを使用した監視するための詳細なプロトコル

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