Diese Forschung wird finanziell von der Europäischen Gemeinschaft siebten Rahmenprogramm (RP7/2007-2013) unter Grant Vertragsnummer 305436 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., und T.J.R. mit einer Patentanmeldung bezüglich dieser Forschung verbunden sind. Die anderen Autoren haben nichts preisgeben.

Perivaskuläre Zellen wurden von vielen verschiedenen menschlichen solide Organe, einschließlich Fett, Knorpel, Bauchspeicheldrüse und Niere9,15,16isoliert. Die meisten Methoden basieren jedoch auf kleine Proben von Gewebe, die seziert und danach mit Verdauungsenzymen behandelt. Darüber hinaus erfolgt dies in der Regel nicht mit klinischen Grade Produkte. Dies macht diese Strategien weniger geeignet für direkte klinische Übersetzung befinden sich große Mengen an klinischen Grade Zellen notwendig.
Hier zeigen wir eine neuartige Isolationsmethode des menschlichen kPSCs für ganze Organe anhand der Perfusion mit klinischen Grade Enzyme und Materialien. Das Protokoll ist vom klinischen Langerhans-Inseln Isolierung Protokoll derzeit im Einsatz für die klinische Anwendung in unserem Zentrum18angepasst.
Dies ist die erste klinische Grade-Methode, wo große Mengen von kPSCs erreicht werden können. Die Variabilität der Zellausbeute ist weitgehend Geber angewiesen. Wenn die Zellausbeute erhalten wir derzeit von einem Bruchteil der rohen Zellsuspension von drei verschiedenen Gebern, in der Theorie extrapoliert ist konnte eine durchschnittliche Rendite von 2,7 x 1012 kPSCs pro Spender erreicht werden. MSC-Therapie besteht in der Regel aus 2 Zelle Infusionen mit 1-2 x 106 Zellen/kg Körper Gewicht4, genügen diese Zellzahlen allogene Behandlung von mehreren Patienten.
Ein entscheidender Schritt in der Isolierung-Prozedur ist die Dauer der Kollagenase Verdauung. Wenn die Verdauung Periode zu kurz ist, bleiben große Klumpen des Gewebes, die schwerer zu Kultur werden. Wenn die Perfusion Periode zu lang ist, kann erhöhte Zelltod beobachtet werden. Daher, sobald die Niere weich und die Flüssigkeiten weniger transparent zu werden beginnt, die Niere sollte sanft massiert und die Kollagenase-Behandlung abgebrochen werden sollte.
Ein weiterer wichtiger Schritt ist die Kultur der Zellen nach der NG2 Zelle Bereicherung. Manchmal nach der NG2 Zelle Bereicherung starten die perivaskuläre Zellen nicht zu vermehren oder in 3D Strukturen zu wachsen beginnen. In diesem Fall wenn die Zellen trypsiniert und eingesät, startet die Zellen in der Regel in Monolayer Kultur wachsen.
Als Ausgangsmaterial, dienten menschliche Transplantation Grade Nieren hauptsächlich aus chirurgischen Gründen verworfen. Dies sind funktionelle Organe ohne großen Fibrose. Wir anerkennen, dass dies möglicherweise eine Einschränkung, da dies eine relativ seltene und schwer zu Orgel Quelle zu erhalten. Explantierten Nieren möglicherweise eine andere Quelle; jedoch, je nach dem Grund der Niere Explantation diese Nieren enthalten mehr Fibrose und somit Myofibroblasts, und daher vorsichtig vorzugehen, da Myofibroblasts könnten isoliert und statt perivaskuläre Zellen kultiviert.
Wie die kPSCs isoliert mit diesem Protokoll Show Organo-typisches Eigenschaften mit Niere epithelialen Wunde heilende Fähigkeiten15, Zell-Therapie mit kPSCs bei Nierenerkrankungen und Transplantation wäre eine interessante zukünftige Anwendung. Zu diesem Zweck gibt es mehrere Strategien der Zelle/Zelle Produktlieferung. Die erste Strategie ist IV-Infusion von kPSCs, wie derzeit in klinischen Studien BmMSC verwendet. Eine weitere interessante Anwendung ist die Verwendung von kPSCs oder kPSC ausgeschieden Faktoren bei der Perfusion der Maschine vor der Transplantation. Auf diese Weise könnte die Qualität der explantierten Niere verbessern die nach der Transplantation zu verbesserten Nierenfunktion führen könnten. Für beide Strategien sind kPSCs eine interessante neue Zelle Quelle für klinische Zwecke weiter zu erkunden.

Mesenchymaler Stromazellen Zellen (MSCs) sind modulierende Immunzellen, die ursprünglich aus dem Knochenmark isoliert wurden. Sie zeichnen sich durch ihre Spindel förmigen Morphologie, Fähigkeit, in Fett, Knochen und Knorpel und Kunststoff Termintreue zu unterscheiden. MSCs express die stromale Marker CD73, CD90, CD105 und negativ für die Marker CD31 und CD451,2,3. MSCs sind ein viel versprechender Kandidat für die Zelltherapie aufgrund ihrer Gewebe homöostatische und immunmodulatorische Kapazitäten. BmMSCs werden derzeit in klinischen Studien für verschiedene Krankheiten, einschließlich Nierenerkrankungen und Nierentransplantation wie an anderer Stelle überprüft4geprüft.
Bisher zeigte sich, dass perivaskuläre Zellen aus mehreren verschiedenen solide Organe, einschließlich Fettgewebe, Plazenta und Skelettmuskulatur mit MSCs9Merkmale teilen. Allerdings zeigen diese Zellen auch Gewebe-spezifische Funktionen die organotypischen Reparatur10führen kann. Menschlichen myokardialen perivaskuläre Zellen, z. B. angeregt angiogenen Antworten nach Hypoxie und differenziert in Kardiomyozyten, während perivaskuläre Zellen isoliert von anderen Geweben nicht diese Potenziale11zeigte.
Perivaskuläre Stromazellen Zellen können auch von der Maus12,13,14 und menschliche Niere15,16isoliert werden. Wir haben ausgiebig gekennzeichnet kPSCs und verglich diese mit BmMSCs. Wir festgestellt, dass kPSCs, ähnlich wie BmMSCs, immunsuppressive Kapazitäten und vaskulären Plexus Bildung unterstützen können. Allerdings gibt es Unterschiede zwischen der Zelltypen als kPSCs zeigte eine organotypischen transkriptionelle Ausdruck Signatur, einschließlich der nephrogenen Transkriptionsfaktoren, HoxD10 und HoxD11 Gewebe. kPSCs, im Gegensatz zu BmMSCs, nicht unterziehen, Myofibroblast Transformation nach Stimulation mit TGF-β und waren nicht in der Lage, sich in Adipozyten differenzieren. Darüber hinaus beschleunigt kPSCs epitheliale Integrität in einem Nieren röhrenförmige epithelialen Wunde kratzen Assay, ein Phänomen, das nicht mit BmMSCs beobachtet wurde. Diese erweiterten Wunde Reparatur wurde durch Hepatozyten-Wachstumsfaktor Freisetzung vermittelt. Darüber hinaus verbessert kPSCs Niere Verletzungen in einem Mausmodell der akuten Nierenversagens Verletzungen15. Daher kPSCs überlegene renal Reparatur Kapazitäten zu haben scheinen und sind eine interessante neue Quelle für die Zelltherapie bei Nierenerkrankungen.
Um kPSCs Zelle Therapie Zwecken nutzen zu können, sollte kPSCs in gewissem Sinne klinische Grade mit klinischen Grade Enzyme und Protokollen isoliert werden. Um mehrere Patienten mit kPSCs von 1 Spender behandeln zu können, sollte darüber hinaus ausreichend Zellzahlen erzielt werden. Hier zeigen wir im Detail, das klinische Grade isoliert Verfahren der kPSCs von ganzen Transplantation Grade Nieren, nachgiebig ausreichende Zahl von Zellen für Klinische Zelltherapie verwendet werden.

<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

Mesenchymaler Stromazellen Zellen (MSCs) sind homöostatische und Immunsystem modulierende Gewebezellen, die wohltuende Wirkung bei Nierenerkrankungen und Transplantation gezeigt haben. Perivaskuläre Stromazellen Zellen (EAP) teilen Eigenschaften mit Knochenmark MSCs (BmMSCs). Jedoch besitzen sie auch, wahrscheinlich aufgrund lokaler Prägung, Gewebe-spezifische Eigenschaften und spielen eine Rolle bei der lokalen Gewebe Homöostase. Diese Gewebespezifität führen bestimmte Gewebereparatur, auch innerhalb der menschlichen Niere. Wir zeigten bisher, dass menschliche Niere EAP (kPSCs) Niere epitheliale Wundheilung während BmMSCs nicht dieses Potenzial hatte verbessert haben. Darüber hinaus können kPSCs Niere Verletzungen in Vivolindern. Daher bilden kPSCs eine interessante Quelle für Zelltherapie, insbesondere für Nierenerkrankungen und Nierentransplantation. Hier zeigen wir die detaillierte Isolation und Kultur-Methode für kPSCs von Transplantation-Grade menschliche Nieren basierend auf ganze Organ Perfusion von Verdauungs-Enzymen über die Nierenarterie und Bereicherung für die perivaskuläre Marker NG2. Auf diese Weise großzellige Mengen erhalten, die für die zelluläre Therapie eignen.

Die klinische Grade kPSCs Isolationsmethode ist in Abbildung 1zusammengefasst. Grobe Nierenzellen sind von menschlichen Transplantation Grade Nieren von Kollagenase Perfusion isoliert. Die daraus resultierende Zellsuspension ist bis zum Zusammenfluss in 5 % Thrombozyten Lysates kultiviert. Dann ist die perivaskuläre Stromazellen Zelle Bruchteil isoliert basierend auf NG2 Ausdruck.
kPSCs sind aus Kunststoff anhaftende spindelförmige Zellen (Abb. 2A) und positiv für die stromale Marker CD73, CD90, CD105 und perivaskuläre Marker NG2, PDGFR-B und CD146, während negativ für CD31, CD34, CD45 und HLA-DR (Abbildung 2B). In der Regel eine homogene Bevölkerung von kPSCs erreichen Sie in der Passage 4 und kPSCs erreichen Seneszenz um Durchgang 9-10 (Abbildung 2C). Wir empfehlen daher Experimente zwischen 5-8 Durchgang durchführen.
Um die Funktionsfähigkeit der isolierten kPSCs zu bewerten, führen wir einen in-vitro- Niere epithelialen Wunde kratzen Assay auf jede neue Charge von kPSCs, wie zuvor gezeigt, dass die konditionierte Medium der kPSCs beschleunigen kann epitheliale Wundheilung in dieser Kratzer Assay-15. Das kPSC konditionierten Medium erfolgt durch Kultivierung kPSCs für 48 h in AlphaMEM 5 % Thrombozyten Lysates und den Überstand zu sammeln. Als nächstes verewigt menschliche Niere proximale Röhrchen Epithelzellen (HK-2)17 sind bis zum Zusammenfluss kultiviert und dann Kratzen Wunde gemacht. Dann entweder die konditionierte Medium der kPSCs oder Kontrolle Medium wird hinzugefügt, um die Brunnen und die Geschwindigkeit der Wundheilung wird gemessen. Wenn die konditionierte Medium der kPSCs hinzugefügt wird, schließt die Wunde deutlich schneller (Abbildung 2D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55841/55841fig1.jpg"> 
Abbildung 1 : Klinische Grade Isolation Methode des menschlichen kPSCs. Transplantation Grade Nieren sind kanülierte durchströmt mit Kollagenase über die Nierenarterie (A - G) und die daraus resultierende Zellsuspension wird gewaschen und entweder kryokonserviert oder in Kultur (H - K). Nachdem die Zellen Konfluenz (L) erreicht haben, durchgeführt NG2 Zelle Bereicherung ist (M). Zellen sind trypsiniert wenn sie entweder konfluierende, sind nicht mehr vermehren oder wenn 3D-Strukturen (N - P) angezeigt. Die Freigabekriterien für kPSCs sind Sterilität, Marker Ausdruck und die Fähigkeit, röhrenförmigen epitheliale Wundheilung (Q) zu verbessern. Pfeil: Nierenarterie. Maßstabsleiste = 200 µm. <a href="http://ecsource.jove.com/files/ftp_upload/55841/55841fig1large.jpg" target="_blank">Bitte klicken Sie hier für eine größere Version dieser Figur.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55841/55841fig2.jpg"> 
Abbildung 2: Charakterisierung des menschlichen kPSCs. Ein) kPSCs sind spindelförmig, Kunststoff adhärente Zellen. ( B) kPSCs sind positiv für mesenchymalen Marker CD73, CD90, CD105, perivaskuläre Marker NG2, PDGFR-β und CD146, während negativ für CD31, CD34 und CD45. kPSCs express MHC-Klasse ich (HLA-ABC) jedoch keine Klasse II (HLA-DR). ( C) Wuchsmerkmalen von drei verschiedenen kPSC Spender aus Durchflusszytometrie bestätigt homogene NG2 positive Populationen (Abschnitt 4). kPSCs erreichen Seneszenz um Durchgang 9-10. D) kPSCs sind in der Lage, Niere epithelialen Reparatur in einer Wunde kratzen Assay zu verbessern. Repräsentative Bilder von Kontrolle und kPSC bedingt Medium bei t = 0, 4, 8 und 12 h gezeigt. Maßstabsleiste a) = 200 µm, in D) = 100 µm. <a href="http://ecsource.jove.com/files/ftp_upload/55841/55841fig2large.jpg" target="_blank">Bitte klicken Sie hier für eine größere Version dieser Figur.</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

Hier präsentieren wir eine neue klinische Grade Isolation und Kultur Methode zur Niere perivaskuläre Stromazellen Zellen (kPSCs) anhand der gesamten Organ Perfusion mit Verdauungsenzymen und NG2-Zelle Bereicherung. Mit dieser Methode ist es möglich, ausreichende Zellzahlen für Zelltherapie erwerben.

Eine neue klinische Grade Isolationsmethode für menschliche Niere perivaskuläre Stromazellen Zellen

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

a-novel-clinical-grade-isolation-method-for-human-kidney-perivascular

Research

JoVE Journal

Medicine

6.8K Aufrufe.  Leiden University Medical Centre. Hier präsentieren wir eine neue klinische Grade Isolation und Kultur Methode zur Niere perivaskuläre Stromazellen Zellen (kPSCs) anhand der gesamten Organ Perfusion mit Verdauungsenzymen und NG2-Zelle Bereicherung. Mit dieser Methode ist es möglich, ausreichende Zellzahlen für Zelltherapie erwerben.

Serie: 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.

Isolierung der Glomeruli und In Vivo Kennzeichnung der glomerulären Zelle Oberflächenproteine

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

Forschung

Biochemie

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.

Isolierung von Double Negative αβ T-Zellen aus der Niere

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

Immunologie und Infektion

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.

Zuverlässig und High Efficiency Extraktion von Nieren-Immunzellen

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.

Eine effiziente Methode der Absiebung, intakten Glomeruli von Erwachsenen Ratte Niere zu isolieren

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

Medizin

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.

Einkanal-Analyse und Calcium Imaging in den Podozyten der frisch isolierten Glomeruli

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

Biologie

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.

Isolierung, Charakterisierung und hohem Durchsatz extrazelluläre Flux-Analyse der Maus primäre renale tubuläre Epithelzellen

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.

Isolierung von bedingt immortalisierten glomerulären Maus-Endothelzellen mit fluoreszierenden Mitochondrien

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.

Isolierung primärer humaner proximaler Tubula-Epithelzellen und ihre Verwendung bei der Erstellung eines mikrophysiologischen Modells des proximalen Tubulus der Niere

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.

Optimierung der Isolierung und Reinigung von murinen glomerulären Mesangialzellen

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.

Isolierung von Nierenzellen an der Maus: Eine Technik zur Isolierung und Erzeugung einer Primärkultur von tubulären Nierenepithelzellen

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