이 연구는 부여 계약 번호 305436에서 유럽 공동체의 일곱 번째 기구 프로그램 (FP7 2007-2013 년)에서 자금 지원을 받은 스텔라.

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., 및 T.J.R.는이 연구에 대 한 특허 출원과 함께 연결 되어 있습니다. 다른 저자는 공개 없다.

혈관 주위 세포는 많은 다른 인간 단단한 장기, 췌 장, 지방, 연골, 신장9,,1516등에서 분리 되었습니다. 그러나 대부분의 방법은,, 기반으로 조직, 해 부 되 고 이후에 소화 효소 치료의 작은 샘플 합니다. 또한, 이것은 일반적으로 수행 되지 않습니다 임상 학년 제품. 이것은 이러한 전략 직접 임상 번역을 위해 보다 적게 적당 한 임상 등급 셀의 대량은 필요 합니다.
여기 우리는 관류 임상 등급 효소와 재료에 따라 전체 기관에 대 한 인간 kPSCs의 소설 격리 방법을 보여줍니다. 프로토콜은 현재 우리의 센터18에 임상 응용 프로그램에 대 한 사용에에서 임상 독도 Langerhans 격리 프로토콜에서 적응.
이것은 첫 번째 임상 등급 방법 kPSCs의 대량을 달성 될 수 있다입니다. 셀 수익률의 변동성은 크게 기증자 의존. 그러나, 이론적으로, 우리는 현재 3 명의 다른 기증자의 조 세포 현 탁 액의 일부분에서 얻을 셀 생산량 추정 때 기증자 당 1012 kPSCs x 2.7의 평균 수익률 달성 될 수 있습니다. MSC 치료는 일반적으로 1-2 106 셀/kg 몸 무게4x 2 셀 혼합 이루어져 있다로이 핸드폰 번호 allogenic 치료의 여러 환자에 대 한 충분 한 있습니다.
분리 절차의 하나의 중요 한 단계는 콜라 소화의 기간입니다. 소화 기간이 너무 짧은 때 문화에 더 있을 것 이다 조직의 큰 덩어리 유지 됩니다. 관류 기간이 너무 긴 경우 증가 세포 죽음을 관찰할 수 있습니다. 따라서, 최대한 빨리 신장 부드럽고 덜 투명 한 체액 되기 시작, 신장 부드럽게 마사지 한다 고 콜라 치료를 중지 합니다.
또 다른 중요 한 단계 NG2 셀 농축 후 셀의 문화입니다. 가끔 NG2 셀 농축 후 혈관 주위 세포 증식 또는 3 차원 구조에 성장 하기 시작 시작 되지 않습니다. 이 경우 셀 trypsinized을 다시 시드해야 하는 경우 셀 것 이다 일반적으로 단층 문화에서 성장 시작 됩니다.
자료를 시작으로 인간 이식 학년 신장 수술 이유로 주로 삭제 사용 되었다. 이러한 주요 섬유 증 없이 기능적인 기관 이다입니다. 우리는 이것이 비교적 드문이 제한 있을 인정 및 기관 소스를 얻기 어렵다. Explanted 신장 있을 다른 소스; 그러나, 신장 explantation의 이유에 따라 이러한 신장에 더 많은 섬유 증 및 따라서 myofibroblasts, 포함 될 수 있습니다 그리고 그러므로 주의 해야 myofibroblasts 수 있습니다 격리 하 고 혈관 주위 세포 대신 교양.
kPSCs 절연이 프로토콜 표시와 함께 신장 상피와 유기 전형적 속성 상처 치유 용량15, 신장 질병에서 kPSCs와 세포 치료 하 고 이식 미래 응용 될 것 이다. 이 목적을 위해 셀/제품 배달의 여러 전략 있습니다. 첫 번째 전략은 bmMSC 임상 연구에서 현재 사용 되는 kPSCs의 IV 주입 이다. 또 다른 흥미로운 응용 프로그램은 kPSCs 또는 kPSC 배설 요소 이식 하기 전에 기계 관류에의 사용 이다. 이 방법으로 이식 후 향상 된 신장 기능을 이어질 수 explanted 신장의 품질 향상 됩니다. 두 전략에 대 한 kPSCs는 추가 임상 목적을 위해 탐구 하는 재미 있는 새로운 셀 소스입니다.

Mesenchymal Stromal 세포 (MSCs)는 면역 modulatory 세포는 골 수에서 원래 고립 되었다. 그들은 그들의 스핀 들 모양의 형태, 지방, 뼈와 연골, 그리고 플라스틱 부착으로 차별 하는 능력에 의해 특징. MSCs stromal 마커 CD73, CD90, CD105 동안 마커 CD31, CD451,2,3에 대 한 부정적인 표현 한다. MSCs는 그들의 조직의 항상성 및 immunomodulatory 용량 세포 치료에 대 한 유망한 후보. bmMSCs는 현재 신장 질환과 신장 이식으로4다른 검토를 포함 하 여 여러 가지 질병에 대 한 임상 실험에서 공부 되 고 있습니다.
이전 그것은 혈관 주위 지방 조직을 포함 하 여 여러 다른 단단한 장기에서 세포, 태 반 및 골격 근육 MSCs9특성을 공유 표시 했다. 그러나,이 세포는 또한 조직 관련 기능 organotypic 수리10귀 착될 수 있는 전시. 예를 들어 인간의 심근 혈관 주위 세포 hypoxia 후 신생 응답을 자극 하 고 혈관 주위 세포가 다른 조직에서 고립이 후보11보여주지 않았다 하는 동안, cardiomyocytes에 분화.
혈관 주위 stromal 세포 마우스12,,1314 및 인간의 신장15,16으로부터 격리 될 수 있습니다. 우리는 광범위 하 게 kPSCs를 특징 하 고 bmMSCs에 비해. 우리는 kPSCs, bmMSCs, 유사한 면역 능력 있고, 혈관 신경 얼 기 형성을 지원할 수 있습니다 발견. 그러나, 조직 세포 유형 HoxD10 및 HoxD11 nephrogenic 전사 인자를 포함 하 여 organotypic transcriptional 식 서명 했다 kPSCs로 구체적인 차이가 있습니다. kPSCs, bmMSCs, 달리 myofibroblast 변환 후 TGF-β와 자극을 받아야 하지 않았다 고 adipocytes에 차별화 하지 못했습니다. 또한, kPSCs는 신장 관 상피 상처 스크래치 분석 결과, bmMSCs와 관찰 하지 현상은 상피 무결성 가속. 이 향상 된 상처 복구 hepatocyte 성장 인자 자료를 통해 중재 했다. 또한, kPSCs ameliorated 급성 신장 상해15의 쥐 모델에서 신장 상해. 따라서, kPSCs 우수한 신장 수리 능력을가지고 것과 신장 질환의 세포 치료에 대 한 흥미로운 새로운 소스.
세포 치료 목적을 위해 kPSCs를 사용할 수 있어야 kPSCs 임상 등급 효소와 프로토콜 임상 등급 방식으로 격리 한다. 또한, 1 기증자에 게 서 kPSCs와 여러 환자를 치료 수 있도록, 충분 한 셀 숫자를 얻은 합니다. 여기 우리가 임상 세포 치료에 사용 되는 셀의 충분 한 숫자 저조한 전체 이식 학년 신장에서 kPSCs의 임상 등급 격리 절차 자세히 보여줍니다.

<table><tbody><tr><td>Baxter bags</td><td>Fenwal</td><td>R4R-7004</td><td></td></tr><tr><td>Human platelets</td><td>Sanquin</td><td></td><td>hospital surplus material expired for less than 2 d is used</td></tr><tr><td>platelet lysate</td><td></td><td></td><td>custom made</td></tr><tr><td>Disposable sterile bottles</td><td>Corning</td><td>09-761-11</td><td></td></tr><tr><td>500 mL PP centrifuge tubes</td><td>Corning</td><td>431123</td><td></td></tr><tr><td>a-MEM medium</td><td>Lonza</td><td>BE12-169F</td><td></td></tr><tr><td>glutamax</td><td>thermo fisher</td><td>35050038</td><td></td></tr><tr><td>pen/strep&amp;nbsp;</td><td>Invitrogen</td><td>15070063</td><td></td></tr><tr><td>transfusion system</td><td>Codan</td><td>455609</td><td></td></tr><tr><td>DMEM-F12</td><td>life technologies</td><td>11320-074</td><td></td></tr><tr><td>Normal Human Serum (NHS)</td><td>Sanquin</td><td></td><td></td></tr><tr><td>Hepes 1 M</td><td>Lonza</td><td>BE-17-737E</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt; 7.5%</td><td>Lonza</td><td>BE17-613E</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; 1 M</td><td>Sigma-Aldrich</td><td>10043-52-4</td><td></td></tr><tr><td>UW</td><td>Bridge to life</td><td>32911</td><td></td></tr><tr><td>Heparin</td><td>Leo Pharma</td><td></td><td></td></tr><tr><td>collagenase NB1 GMP grade</td><td>SERVA</td><td>17455.03</td><td></td></tr><tr><td>DMSO</td><td>Sigma Aldrich</td><td>D2650-100ml</td><td></td></tr><tr><td>surgical drapes</td><td>3M Nederland</td><td>DH999969404</td><td></td></tr><tr><td>perfusion pump</td><td>metrohm</td><td>x007528300</td><td></td></tr><tr><td>pump head</td><td>metrohm</td><td>x077202600</td><td></td></tr><tr><td>perfusion tray</td><td></td><td></td><td>custom made</td></tr><tr><td>LS25 masterflex tubes</td><td>Masterflex</td><td>HV-96410-25</td><td></td></tr><tr><td>accessory spike</td><td>Gambro DASCO</td><td>6038020</td><td></td></tr><tr><td>pulmozyme</td><td>Roche</td><td></td><td></td></tr><tr><td>culture flasks 25 cm&lt;sup&gt;2&lt;/sup&gt;</td><td>greiner</td><td>690175</td><td></td></tr><tr><td>culture flask 75 cm&lt;sup&gt;2&lt;/sup&gt;</td><td>greiner</td><td>658170</td><td></td></tr><tr><td>culture flask 175 cm&lt;sup&gt;2&lt;/sup&gt;</td><td>greiner</td><td>661160</td><td></td></tr><tr><td>Trypsin</td><td>sigma</td><td>t4174</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma</td><td>A2153</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E5134-500g</td><td></td></tr><tr><td>FcR blocking reagent</td><td>miltenyi</td><td>130-059-901</td><td></td></tr><tr><td>anti melanoma beads (NG2)</td><td>miltenyi</td><td>130-090-452</td><td></td></tr><tr><td>cell strainer 70 &amp;micro;m</td><td>Corning</td><td>352350</td><td></td></tr><tr><td>LS columns</td><td>Miltenyi</td><td>130-042-401</td><td></td></tr><tr><td>MACS magnet</td><td>miltenyi</td><td>130-090-976</td><td></td></tr><tr><td>CD34 FITC</td><td>BD&amp;nbsp;</td><td>555821</td><td></td></tr><tr><td>CD45 APC</td><td>BD&amp;nbsp;</td><td>555485</td><td></td></tr><tr><td>CD146 PE</td><td>BD&amp;nbsp;</td><td>550315</td><td></td></tr><tr><td>NG2 APC</td><td>R&amp;amp;D</td><td>FAB2585A</td><td></td></tr><tr><td>CD90 PE</td><td>BD&amp;nbsp;</td><td>555596</td><td></td></tr><tr><td>HLA ABC APC</td><td>BD&amp;nbsp;</td><td>555555</td><td></td></tr><tr><td>CD105 FITC</td><td>Ancell</td><td>326-040</td><td></td></tr><tr><td>HLA DR APC</td><td>BD&amp;nbsp;</td><td>559866</td><td></td></tr><tr><td>CD56 PE</td><td>BD&amp;nbsp;</td><td>555516</td><td></td></tr><tr><td>CD73 PE</td><td>BD&amp;nbsp;</td><td>550257</td><td></td></tr><tr><td>CD31 FITC</td><td>BD&amp;nbsp;</td><td>555445</td><td></td></tr><tr><td>CD133 PE</td><td>miltenyi</td><td>130-090-853</td><td></td></tr><tr><td>PDGF-r&amp;nbsp;</td><td>R&amp;amp;D</td><td>mab 1263</td><td></td></tr><tr><td>mouse IgG1 FITC</td><td>BD&amp;nbsp;</td><td>345815</td><td></td></tr><tr><td>mouse IgG1 PE</td><td>BD&amp;nbsp;</td><td>345816</td><td></td></tr><tr><td>mouse IgG1 APC</td><td>BD&amp;nbsp;</td><td>345818</td><td></td></tr><tr><td>IgG2b PE</td><td>BD&amp;nbsp;</td><td>555743</td><td></td></tr><tr><td>goat anti mouse PE</td><td>Dako</td><td>R0480</td><td></td></tr><tr><td>sodium azide</td><td>Merck</td><td>822335</td><td></td></tr><tr><td>DMEM Ham&amp;rsquo;s F12</td><td>Gibco</td><td>31331-028</td><td></td></tr><tr><td>ITS (insulin, transferrin, selenium)</td><td>Sigma</td><td>I1884</td><td></td></tr><tr><td>hydrocortisone</td><td>Sigma</td><td>H0135</td><td></td></tr><tr><td>triiodothyrinine</td><td>Sigma</td><td>T5516</td><td></td></tr><tr><td>Epidermal Growth Factor (EGF)</td><td>sigma</td><td>E9644</td><td></td></tr><tr><td>Immortalized human renal PTEC (HK2)</td><td></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

Mesenchymal Stromal 세포 (MSCs)는 조직 항상성 및 면역 modulatory 세포 신장 질환과 이식에 유익한 효과 표시. 혈관 주위 Stromal 세포 (PSCs) 골 MSCs (bmMSCs)와 특성을 공유. 그러나, 그들은 또한 소유, 대부분 지역 각 인, 조직 관련 속성 및 지역 조직의 항상성에서 역할. 이 조직 특이성 또한 인간의 신장 내 조직 특정 복구 될 수 있습니다. 우리는 이전 인간의 신장 Psc (kPSCs)는 신장 상피 상처 치유 bmMSCs이이 잠재력 하지 않은 반면 강화 보였다. 또한, kPSCs는 신장 상해 비보개량 수 있습니다. 따라서, kPSCs는 특히 신장 질환과 신장 이식에 대 한 세포 치료에 대 한 재미 있는 소스를 구성합니다. 여기 우리가 인간의 신장 신장 동맥 및 혈관 주위 마커 NG2 농축을 통해 소화 효소의 전체 장기 관류에 따라 이식-학년에서 kPSCs에 대 한 자세한 고립과 문화 메서드를 보여줍니다. 이 방법에서는, 큰 셀 수량 얻어질 수 있다 세포 치료에 적합 합니다.

임상 등급 kPSCs 절연 메서드는 그림 1에 요약 됩니다. 조 신장 세포 콜라 관류에 의해 인간의 이식 학년 신장 으로부터 격리 됩니다. 결과 셀 서 스 펜 션 5% 혈소판 lysates에 합칠 때까지 양식입니다. 다음 혈관 주위 stromal 세포 분수 NG2 식에 따라 격리 됩니다.
kPSCs 플라스틱 부착 스핀 들 모양의 셀 (그림 2A) 고 CD31, CD34, CD45 그리고 HLA-DR (그림 2B)에 대 한 부정적인 동안 stromal 마커 CD73, CD90, CD105 및 혈관 주위 마커 NG2, PDGFR-B와 CD146에 대 한 긍정적인. 일반적으로, kPSCs의 균질 인구 통로 4에 도달 될 수 있다 그리고 kPSCs 도달 통로 9-10 (그림 2C) 주위 노화. 우리는 통로 5-8 사이 실험을 수행 하 따라서 조언 한다.
격리 된 kPSCs의 기능 능력을 평가, 우리 수행 체 외에 신장 상피 상처 스크래치 분석 결과 kPSCs의 모든 새로운 배치에 우리가 이전 kPSCs의 바른된 매체 상피 상처 치유이 스크래치 시험15가속화할 수 있습니다 보여준. KPSC 조건된 매체 alphaMEM 5% 혈소판 lysates 48 h에 대 한 kPSCs를 경작 하 고는 상쾌한 수집 하 여 이루어집니다. 다음, 불멸 하 게 인간의 신장 근 위 관 상피 세포 (홍콩-2)17 합칠까지 교양 하 고 스크래치 상처를 만든 다음. 다음 중 kPSCs 또는 제어 매체의 바른된 매체 우물에 추가 되 고 상처의 치유의 속도 측정. KPSCs의 조절된 중 추가 될 때 상처는 훨씬 빠른 (그림 2D) 닫습니다.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55841/55841fig1.jpg"> 
그림 1 : 인간의의 임상 등급 격리 방법 kPSCs. 이식 학년 신장 cannulated 신장 동맥 (A-G)을 통해 콜라와 함께 끼얹는다 고 결과 세포 현 탁 액을 씻어 그리고 중 cryopreserved 또는 문화 (H-K)에 넣어. 셀에 도달 confluency (L), 후 셀 농축은 NG2 수행 (M). 셀 때 하나 confluent, trypsinized는 확산 또는 3D 구조 (N-P)를 표시 하는 때 중단 했다. KPSCs에 대 한 릴리스 조건 있으며 불 임, 마커 식 관 상피 상처 치유 (Q)을 강화 하는 기능. 화살표:의 신장 동맥 눈금 막대 = 200 µ m. <a href="http://ecsource.jove.com/files/ftp_upload/55841/55841fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55841/55841fig2.jpg"> 
그림 2: 특성의 인간 kPSCs. ) kPSCs는 스핀 들-모양, 플라스틱 부착 세포. B) kPSCs는 엽 마커 CD73, CD90, CD105, CD31 CD34, CD45 부정 하면서 혈관 주위 마커 NG2, PDGFR-β 및 CD146에 대 한 긍정적 이다. kPSCs 익스프레스 MHC 클래스 I (HLA-ABC) 하지만 하지 클래스 II (HLA-DR). C) cytometry에서 3 명의 다른 kPSC 기증자의 성장 특성 확인 (통로 4)에 균질 NG2 긍정적인 인구. kPSCs 노화 통로 9-10 주위에 도달합니다. D) kPSCs 신장 상피 수리 상처 스크래치 분석 결과에서 향상 시킬 수 있습니다. 제어 매체 및 t kPSC 조절 매체의 대표 이미지 = 0, 4, 8 및 12 h 표시 됩니다. A에서 눈금 막대) = 200 µ m, d에서) = 100 µ m. <a href="http://ecsource.jove.com/files/ftp_upload/55841/55841fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</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

여기 우리는 신장 혈관 주위 Stromal 세포 (kPSCs) 소화 효소와 NG2 셀 농축 전체가 장기 관류에 따라 새로운 임상 등급 고립과 문화 방법 제시. 이 방법으로 세포 치료에 대 한 충분 한 셀 번호 취득 가능 하다.

인간의 신장 혈관 주위 Stromal 세포에 대 한 새로운 임상 등급 격리 방법

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

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6.8K 조회수.  Leiden University Medical Centre. 여기 우리는 신장 혈관 주위 Stromal 세포 (kPSCs) 소화 효소와 NG2 셀 농축 전체가 장기 관류에 따라 새로운 임상 등급 고립과 문화 방법 제시. 이 방법으로 세포 치료에 대 한 충분 한 셀 번호 취득 가능 하다.

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

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and podocytes with their slit diaphragms. The delicate structure of the glomerular filter, especially the slit diaphragm, relies on the interplay of diverse cell surface proteins. Studying these cell surface proteins has so far been limited to in vitro studies or histologic analysis. Here, we present a murine in vivo biotinylation labeling method, which enables the study of glomerular cell surface proteins under physiologic and pathophysiologic conditions. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of a protein of interest. Semi-quantitation of glomerular cell surface abundance is readily available with this novel method, and all proteins accessible to biotin perfusion and immunoprecipitation can be studied. In addition, isolation of glomeruli with or without biotinylation enables further analysis of glomerular RNA and protein as well as primary glomerular cell culture (i.e., primary podocyte cell culture).

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Here we present a protocol for murine in vivo labeling of glomerular cell surface proteins with biotin. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of the protein of interest.

절연 Glomeruli와 비보에 사 세포 표면 단백질의 라벨

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and ...

연구

생화학

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in various immune responses. DN T cells are a unique immune cell type that has been implicated in regulating immune and autoimmune responses and tolerance to allotransplants1-6. DN T cells are, however, rare in peripheral blood and secondary lymphoid organs (spleen and lymph nodes), but are major residents of the normal kidney. Very little is known about their pathophysiologic function7 due to their paucity in the periphery. We recently described a comprehensive phenotypic and functional analysis of this population in the kidney8 in steady state and during ischemia reperfusion injury. Analysis of DN T cell function will be greatly enhanced by developing a protocol for their isolation from the kidney.
Here, we describe a novel protocol that allows isolation of highly pure ab CD4+ CD8+ T cells and DN T cells from the murine kidney. Briefly, we digest kidney tissue using collagenase and isolate kidney mononuclear cells (KMNC) by density gradient. This is followed by two steps to enrich hematopoietic T cells from 3% to 70% from KMNC. The first step consists of a positive selection of hematopoietic cells using a CD45+ isolation kit. In the second step, DN T cells are negatively isolated by removal of non-desired cells using CD4, CD8, and MHC class II monoclonal antibodies and CD1d &#945;-galcer tetramer. This strategy leads to a population of more than 90% pure DN T cells. Surface staining with the above mentioned antibodies followed by FACs analysis is used to confirm purity.

DNabT cells are rare among peripheral T cells; however, they are abundant in certain non-lymphoid tissues. Difficulty of isolating DN T cells from non-lymphoid tissue hinders their functional analysis despite increasing recognized pathophysiologic significance. We describe a novel protocol for isolation of highly purified DN T cells from murine kidney.

신장에서 더블 네거티브 (Double Negative) αβ의 T 세포의 분리

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

면역학 및 감염병학

Reliable and High Efficiency Extraction of Kidney Immune Cells

Immune system activation occurs in multiple kidney diseases and pathophysiological processes. The immune system consists of both adaptive and innate components and multiple cell types. Sometimes, the cell type of interest is present in very low numbers among the large numbers of total cells isolated from the kidney. Hence, reliable and efficient isolation of kidney mononuclear cell populations is important in order to study the immunological problems associated with kidney diseases. Traditionally, tissue isolation of kidney mononuclear cells have been performed via enzymatic digestions using different varieties and strengths of collagenases/DNAses yielding varying numbers of viable immune cells. Recently, with the development of the mechanical tissue disruptors for single cell isolation, the collagenase digestion step is avoided and replaced by a simple mechanical disruption of the kidneys after extraction from the mouse. Herein, we demonstrate a simple yet efficient method for the isolation of kidney mononuclear cells for every day immune cell extractions. We further demonstrate an example of subset analysis of immune cells in the kidney. Importantly, this technique can be adapted to other soft and non-fibrous tissues such as the liver and brain.

Techniques that are reliable and efficient for the isolation of kidney immune cells are needed for downstream applications. This requires surface antibody labeling of a small number of kidney immune cells. Herein, we describe a concise method for isolation of kidney immune cells that seemingly achieves this goal.

신장 면역 세포의 신뢰성 및 고효율 추출

Immune system activation occurs in multiple kidney diseases and pathophysiological processes.  The immune system consists of both adaptive and innate ...

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

그대로 Glomeruli 성인 쥐 신장에서 분리 하는 효율적인 체질 하 방법

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

의학

Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells in the pathogenesis of various renal diseases has been established since podocyte injury leads to proteinuria and foot process effacement. It was previously reported that various endogenous agents may cause a dramatic overload in intracellular Ca2+ concentration in podocytes, presumably leading to albuminuria, and this likely occurs via calcium-conducting ion channels. Therefore, it appeared important to study calcium handling in the podocytes both under normal conditions and in various pathological states. However, available experimental approaches have remained somewhat limited to cultured and transfected cells. Although they represent a good basic model for such studies, they are essentially extracted from the native environment of the glomerulus. Here we describe the methodology of studying podocytes as a part of the freshly isolated whole glomerulus. This preparation retains the functional potential of the podocytes, which are still attached to the capillaries; therefore, podocytes remain in the environment that conserves the major parts of the glomeruli filtration apparatus. The present manuscript elaborates on two experimental approaches that allow 1) real-time detection of calcium concentration changes with the help of ratiometric confocal fluorescence microscopy, and 2) the recording of the single ion channels activity in the podocytes of the freshly isolated glomeruli. These methodologies utilize the advantages of the native environment of the glomerulus that enable researchers to resolve acute changes in the intracellular calcium handling in response to applications of various agents, measure basal concentration of calcium within the cells (for instance, to evaluate disease progression), and assess and manipulate calcium conductance at the level of single ion channels.

Changes in the intracellular calcium levels in the podocytes are one of the most important means to control the filtration function of glomeruli. Here we explain a high-throughput approach that allows detection of real-time calcium handling and single ion channels activity in the podocytes of the freshly isolated glomeruli.

갓 격리 된 사구체 족 세포의 단일 채널 분석 및 칼슘 이미징

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells ...

생물학

Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). Therefore, assessing mitochondrial function in primary TECs may provide valuable insight into the bioenergetic status of the cells, providing insight into the pathophysiology of CKD. While there are a number of complex protocols available for the isolation and purification of proximal tubules in different species, the field lacks a cost-effective method optimized for&#160;tubular cell isolation without the need for purification. Here, we provide an isolation protocol that allows for studies focusing on both primary mouse proximal and distal renal TECs. In addition to cost-effective reagents and minimal animal procedures required in this protocol, the isolated cells maintain high energy levels after isolation and can be sub-cultured up to four&#160;passages, allowing for continuous studies. Furthermore, using a high throughput extracellular flux analyzer, we assess the mitochondrial respiration directly in the isolated TECs in a 96-well plate for which we provide recommendations for the optimization of cell density and compound concentration. These observations suggest that this protocol can be used for renal tubular ex vivo studies with a consistent, well-standardized production of renal TECs. This protocol may have broader future applications to study mitochondrial dysfunction associated with renal disorders for drug discovery or drug characterization purposes.

This protocol provides a cost-effective approach to isolate and characterize mouse primary renal tubular cells that can subsequently be sub-cultured to assess renal biological functions ex vivo, including mitochondrial bioenergetics.

마우스 기본 신장 관 상피 세포의 고립, 특성, 및 높은 처리량 세포 외 유출 분석

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). ...

Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria

Glomerular endothelial cell (GEC) dysfunction can initiate and contribute to glomerular filtration barrier breakdown. Increased mitochondrial oxidative stress has been suggested as a mechanism resulting in GEC dysfunction in the pathogenesis of some glomerular diseases. Historically the isolation of GECs from in vivo models has been notoriously challenging due to difficulties in isolating pure cultures from glomeruli. GECs have complex growth requirements in vitro and a very limited lifespan. Here, we describe the procedure for isolating and culturing conditionally immortalized GECs with fluorescent mitochondria, enabling the tracking of mitochondrial fission and fusion events. GECs were isolated from the kidneys of a double transgenic mouse expressing the thermolabile SV40 TAg (from the Immortomouse), conditionally promoting proliferation and suppressing cell differentiation, and a photo-convertible fluorescent protein (Dendra2) in all mitochondria (from the photo-activatable mitochondria [PhAMexcised] mouse). The stable cell line generated allows for cell differentiation after inactivation of the immortalizing SV40 TAg gene and photo-activation of a subset of mitochondria causing a switch in fluorescence from green to red. The use of mitoDendra2-GECs allows for live imaging of fluorescent mitochondria&#39;s distribution, fusion, and fission events without staining the cells.

The article describes the method for isolating conditionally immortalized glomerular endothelial cells from the kidneys of transgenic mice expressing the thermolabile simian virus 40 and photo-activatable mitochondria, PhAMexcised. We describe the procedure for glomeruli isolation from whole kidneys using beads, digestion steps, seeding, and culturing of GECs-CD31 positive.

형광 미토콘드리아를 이용한 조건부 불멸화 마우스 사구체 내피 세포의 분리

Glomerular endothelial cell (GEC) dysfunction can initiate and contribute to glomerular filtration barrier breakdown. Increased mitochondrial oxidative ...

Isolation of Primary Human Proximal Tubule Epithelial Cells and Their Use in Creating a Microphysiological Model of the Renal Proximal Tubule

Kidney disease affects over 850 million people globally, including 37 million Americans. Risk factors for&#160;chronic kidney disease include environmental influences, genetic predispositions, co-existing medical conditions, and a history of acute kidney injury. These factors often take months or years to develop, complicating longitudinal studies of disease etiology and pathophysiology. Advanced kidney models are needed to improve our understanding of disease mechanisms and enhance nephrotoxicity prediction in drug development. Proximal tubule epithelial cells (PTECs) in the kidney play a critical role in xenobiotic and toxin clearance as well as the reabsorption of essential nutrients. We have previously demonstrated that three-dimensional (3D) microphysiological system (MPS) platforms, populated with isolated primary PTECs, can be used to investigate renal drug interactions, assess nephrotoxicity of compounds, and predict drug clearance. Here, we present protocols for isolating and culturing primary PTECs from whole human kidneys and for seeding them into a 3D MPS platform that mimics in vivo renal physiology. This protocol enables long-term studies supporting PTEC viability, physiological morphology, and functional polarization of key transporter proteins in MPS devices for up to 6 months.

We present an optimized protocol for processing whole human kidneys to isolate and culture primary renal proximal tubule epithelial cells and the application of these cells in a three-dimensional, microfluidic, microphysiological platform to recapitulate the renal proximal tubule.

1차 인간 근위세뇨관 상피 세포의 분리 및 신장 근위세뇨관의 미세생리학적 모델 생성에 사용

Kidney disease affects over 850 million people globally, including 37 million Americans. Risk factors for&#160;chronic kidney disease include ...

Optimizing Isolation and Purification of Murine Glomerular Mesangial Cells

Mesangial cells (MCs) are stromal cells located in the middle space of the glomerulus, with pivotal functions in glomerular homeostasis. Methods for isolating, purifying, and culturing glomerular MCs have been developed and optimized since the 1980s for use in biomedical research, particularly in the field of nephrology. Mice are the most frequently used experimental animal models in research on renal diseases. In this study, we developed an optimized protocol for murine MCs isolation and ex vivo cell culture. These cells can be passaged multiple times, frozen, revived, and cultured without compromising cell growth or protein expression. This optimized approach significantly reduces the study duration for researchers and enables long-term cell preservation. The necessary equipment for the procedures is easily accessible in a basic biomedical laboratory, and the procedural steps are straightforward. The acquisition of target cells requires only 2-3 weeks, a reduction of at least 1 week compared to existing methods.

This study developed an optimized protocol for the isolation of murine mesangial cells (MCs) and their ex vivo cell culture. These cells can be passaged multiple times, frozen, revived, and cultured without compromising cell growth or protein expression.

Mesangial cells (MCs) are stromal cells located in the middle space of the glomerulus, with pivotal functions in glomerular homeostasis. Methods for ...

Murine Renal Cell Isolation: A Technique to Isolate and Generate Primary Culture of Renal Tubular Epithelial Cells

Source: Ding, W. et al. <a href="https://www.jove.com/video/57718" target="_blank">Isolation, Characterization and High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells.</a> J. Vis. Exp. (2018)
In this video, we demonstrate the steps to isolate and purify renal tubular epithelial cells for a wide range of molecular biology and cell culture studies.

쥐 신장 세포 분리: 신장 세뇨관 상피 세포의 1차 배양을 분리하고 생성하는 기술

Source: Ding, W. et al. Isolation, Characterization and High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells. J. ...