This research has received funding from the European Community's Seventh Framework Program (FP7/2007-2013) under grant agreement number 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>

<ol>
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D.G.L., M.A.E., and T.J.R. are associated with a patent application regarding this research. The other authors have nothing to disclose.

Perivascular cells have been isolated from many different human solid organs, including pancreas, fat, cartilage and the kidney9,15,16. Most methods, however, are based on small samples of tissue, which are dissected and afterwards treated with digestive enzymes. Moreover, this is usually not performed with clinical grade products. This makes these strategies less suitable for direct clinical translation where large quantities of clinical-grade cells are necessary.
Here we show a novel isolation method of human kPSCs for whole organs based on perfusion with clinical grade enzymes and materials. The protocol is adapted from the clinical islets of Langerhans isolation protocol currently in use for clinical application in our center18.
This is the first clinical grade method where large quantities of kPSCs can be achieved. The variability in cell yield is largely donor dependent. However, when the cell yield we currently obtain from a fraction of the crude cell suspension of three different donors is extrapolated, in theory, an average yield of 2.7 x 1012 kPSCs per donor could be achieved. As MSC therapy typically consists of 2 cell infusions with 1 - 2 x 106 cells/kg body weight4, these cell numbers are sufficient for allogenic treatment of several patients.
One critical step in the isolation procedure is the duration of collagenase digestion. When the digestion period is too short, large clumps of tissue will remain, which will be harder to culture. When the perfusion period is too long, increased cell death may be observed. Therefore, as soon as the kidney starts to become soft and the fluids less transparent, the kidney should be massaged gently and the collagenase treatment should be stopped.
Another critical step is the culture of the cells after NG2 cell enrichment. Sometimes after the NG2 cell enrichment, the perivascular cells do not start to proliferate or start to grow in 3D structures. In this case, when the cells are trypsinized and reseeded, the cells will usually start to grow in monolayer culture.
As starting material, human transplant grade kidneys discarded mainly for surgical reasons were used. These are functional organs without major fibrosis. We acknowledge that this might be a limitation as this is a relatively rare and hard to obtain organ source. Explanted kidneys might be another source; however, depending on the reason of kidney explantation, these kidneys might contain more fibrosis and thus myofibroblasts, and therefore care should be taken since myofibroblasts might be isolated and cultured instead of perivascular cells.
As the kPSCs isolated with this protocol show organo-typic properties with kidney epithelial wound healing capacities15, cell therapy with kPSCs in kidney diseases and transplantation would be an interesting future application. For this purpose, there are several strategies of cell/cell product delivery. The first strategy is IV infusion of kPSCs, as used currently in the bmMSC clinical studies. Another interesting application is the use of kPSCs or kPSC-excreted factors in machine perfusion before transplantation. In this way, the quality of the explanted kidney might improve which could lead to improved kidney function after transplantation. For both strategies, kPSCs are an interesting new cell source to further explore for clinical purposes.

Mesenchymal Stromal Cells (MSCs) are immune modulatory cells that were originally isolated from the bone marrow. They are characterized by their spindle shaped morphology, ability to differentiate into fat, bone and cartilage, and plastic adherence. MSCs express the stromal markers CD73, CD90, CD105 while being negative for the markers CD31 and CD451,2,3. MSCs are a promising candidate for cell therapy due to their tissue homeostatic and immunomodulatory capacities. bmMSCs are currently being studied in clinical trials for several diseases, including kidney diseases and kidney transplantation as reviewed elsewhere4.
Previously it was shown that perivascular cells from several different solid organs, including adipose tissue, placenta and skeletal muscle share characteristics with MSCs9. However, these cells also exhibit tissue-specific functions which can result in organotypic repair10. Human myocardial perivascular cells, for example, stimulated angiogenic responses after hypoxia and differentiated into cardiomyocytes, while perivascular cells isolated from other tissues did not show these potentials11.
Perivascular stromal cells can also be isolated from the mouse12,13,14 and human kidney15,16. We extensively characterized kPSCs and compared these to bmMSCs. We found that kPSCs, similar to bmMSCs, have immunosuppressive capacities and can support vascular plexus formation. However, there are tissue specific differences between the cell types, as kPSCs showed an organotypic transcriptional expression signature, including the nephrogenic transcription factors HoxD10 and HoxD11. kPSCs, in contrast to bmMSCs, did not undergo myofibroblast transformation after stimulation with TGF-&#946; and were unable to differentiate into adipocytes. Furthermore, kPSCs accelerated epithelial integrity in a kidney tubular epithelial wound scratch assay, a phenomenon which was not observed with bmMSCs. This enhanced wound repair was mediated through hepatocyte growth factor release. Moreover, kPSCs ameliorated kidney injury in a mouse model of acute kidney injury15. Therefore, kPSCs seem to have superior renal repair capacities and are an interesting new source for cell therapy in kidney diseases.
In order to be able to use kPSCs for cell therapy purposes, kPSCs should be isolated in a clinical grade manner with clinical grade enzymes and protocols. Moreover, to be able to treat several patients with kPSCs from 1 donor, sufficient cell numbers should be obtained. Here we show in detail, the clinical grade isolation procedure of kPSCs from whole transplant grade kidneys, yielding sufficient numbers of cells to be used for clinical cellular therapy.

<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 Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and transplantation. Perivascular Stromal Cells (PSCs) share characteristics with bone marrow MSCs (bmMSCs). However, they also possess, most likely due to local imprinting, tissue-specific properties and play a role in local tissue homeostasis. This tissue specificity may result in tissue specific repair, also within the human kidney. We previously showed that human kidney PSCs (kPSCs) have enhanced kidney epithelial wound healing whereas bmMSCs did not have this potential. Moreover, kPSCs can ameliorate kidney injury in vivo. Therefore, kPSCs constitute an interesting source for cell therapy, particularly for kidney diseases and renal transplantation. Here we show the detailed isolation and culture method for kPSCs from transplant-grade human kidneys based on whole-organ perfusion of digestive enzymes via the renal artery and enrichment for the perivascular marker NG2. In this way, large cell quantities can be obtained that are suitable for cellular therapy.

The clinical grade kPSCs isolation method is summarized in Figure 1. Crude kidney cells are isolated from human transplant grade kidneys by collagenase perfusion. The resulting cell suspension is cultured until confluent in 5% platelet lysates. Then the perivascular stromal cell fraction is isolated based on NG2 expression.
kPSCs are plastic adherent spindle-shaped cells (Figure 2A) and are positive for the stromal markers CD73, CD90, CD105 and perivascular markers NG2, PDGFR-B and CD146, while negative for CD31, CD34, CD45 and HLA-DR (Figure 2B). Typically, a homogeneous population of kPSCs can be reached at passage 4 and kPSCs reach senescence around passage 9 - 10 (Figure 2C). We therefore advise to perform experiments between passage 5 - 8.
To evaluate the functional capacity of the isolated kPSCs, we perform an in vitro kidney epithelial wound scratch assay on every new batch of kPSCs, as we previously showed that the conditioned medium of kPSCs can accelerate epithelial wound healing in this scratch assay15. The kPSC conditioned medium is made by culturing kPSCs for 48 h in alphaMEM 5% platelet lysates and collecting the supernatant. Next, the immortalized human kidney proximal tubule epithelial cells (HK-2)17 are cultured until confluent and then a scratch wound is made. Then either the conditioned medium of kPSCs or control medium is added to the wells and the speed of wound healing is measured. When the conditioned medium of kPSCs is added, the wound closes significantly faster (Figure 2D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55841/55841fig1.jpg" /> 
Figure 1: Clinical Grade Isolation Method of Human kPSCs. Transplant grade kidneys are cannulated and perfused with collagenase via the renal artery (A - G) and the resulting cell suspension is washed and either cryopreserved or put into culture (H - K). After the cells reach confluency (L), NG2 cell enrichment is performed (M). Cells are trypsinized when they either are confluent, have stopped proliferating or when 3D structures appear (N - P). The release criteria for kPSCs are sterility, marker expression and the capability to enhance tubular epithelial wound healing (Q). Arrow: renal artery. Scale bar = 200 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55841/55841fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55841/55841fig2.jpg" /> 
Figure 2: Characterization of Human kPSCs. A) kPSCs are spindle-shaped, plastic adherent cells. B) kPSCs are positive for mesenchymal markers CD73, CD90, CD105, perivascular markers NG2, PDGFR-&#946; and CD146, while negative for CD31, CD34 and CD45. kPSCs express MHC-class I (HLA-ABC) but not class II (HLA-DR). C) Growth characteristics of three different kPSC donors from flow cytometry confirmed homogeneous NG2 positive populations (at passage 4). kPSCs reach senescence around passage 9 - 10. D) kPSCs are able to enhance kidney epithelial repair in a wound scratch assay. Representative images of control medium and kPSC conditioned medium at t=0, 4, 8 and 12 h are shown. Scale bar in A) = 200 &#181;m, in D) = 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55841/55841fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</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

Here we present a novel clinical grade isolation and culture method for kidney Perivascular Stromal Cells (kPSCs) based on whole organ perfusion with digestive enzymes and NG2-cell enrichment. With this method, it is possible to acquire sufficient cell numbers for cellular therapy.

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

Nanyang Technological University

Research

JoVE Journal

Medicine

6.8K Views.  Leiden University Medical Centre. Hier presenteren we een nieuwe klinische rang isolatie en cultuur methode voor nieren gerelateerde stromale cellen (kPSCs) op basis van hele orgel perfusie met spijsverteringsenzymen en NG2-cel verrijking. Met deze methode is het mogelijk om het verwerven van voldoende cel nummers voor cellulaire therapie.

Video: 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.

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

Biochemistry

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.

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

Immunology and Infection

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.

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.

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

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.

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

Biology

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.

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.

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.

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.remotexs.ntu.edu.sg/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.

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

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

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