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

Questa ricerca ha ricevuto un finanziamento dalla settimo programma Comunità europea quadro (FP7/2007-2013) sotto la concessione numero di contratto 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., e T.J.R. sono associati con una domanda di brevetto per quanto riguarda questa ricerca. Gli altri autori non hanno nulla a rivelare.

Cellule perivascolari sono state isolate da molti diversi organi solidi umani, tra cui pancreas, grasso, cartilagine e il rene9,15,16. Maggior parte dei metodi, tuttavia, sono basata su piccoli campioni di tessuto, che vengono sezionati e successivamente trattati con gli enzimi digestivi. Inoltre, questo solitamente non viene eseguito con prodotti grado clinico. Questo rende queste strategie meno adatto per traduzione clinica diretta dove sono necessarie grandi quantità di cellule di uso clinico.
Qui vi mostriamo un metodo di isolamento romanzo di kPSCs umana per organi interi basato su aspersione con enzimi di grado clinico e materiali. Il protocollo è adattato dal protocollo di isolamento clinico isolotti di Langerhans attualmente in uso per l'applicazione clinica nel nostro centro18.
Questo è il primo metodo di grado clinico in cui è possibile ottenere grandi quantità di kPSCs. La variabilità nel rendimento delle celle è in gran parte dipendente da donatore. Tuttavia, quando il rendimento delle celle che attualmente otteniamo da una frazione della sospensione delle cellule grezza di tre diversi donatori è estrapolato, in teoria, potrebbe conseguire un rendimento medio di 2,7 x 1012 kPSCs per donatore. Come terapia MSC è in genere costituito da 2 infusioni delle cellule con 1-2 x 106 cellule/kg corpo peso4, questi numeri di cellulare sono sufficienti per il trattamento allogenico di parecchi pazienti.
Un passaggio critico nella procedura di isolamento è la durata della digestione della collagenosi. Quando il periodo di digestione è troppo breve, grandi ciuffi di tessuto rimarrà, che sarà più difficile alla cultura. Quando il periodo di perfusione è troppo lungo, si può osservare aumentata delle cellule morte. Pertanto, non appena il rene inizia a diventare morbida e i fluidi meno trasparenti, il rene deve essere massaggiato delicatamente e il collagenosi trattamento deve essere interrotto.
Un altro punto critico è la cultura delle cellule dopo arricchimento cella NG2. A volte dopo l'arricchimento di cella NG2, le cellule perivascolari non iniziare a proliferare o iniziano a crescere in strutture 3D. In questo caso, quando le cellule vengono tripsinizzate e reinizializzate, le cellule di solito inizia a crescere in coltura dello strato monomolecolare.
Come materiale di partenza, trapianto umano reni di grado scartati principalmente per motivi chirurgici sono stati usati. Questi sono organi funzionali senza fibrosi principale. Riconosciamo che questo potrebbe essere una limitazione come questo è una malattia relativamente rara e difficile da ottenere organo sorgente. Reni espiantati potrebbero essere un'altra fonte; Tuttavia, a seconda del motivo dell'espianto di rene, questi reni potrebbero contenere più fibrosi e così myofibroblasts e pertanto si dovrebbe prestare attenzione poiché myofibroblasts potrebbe essere isolati e coltivati invece di cellule perivascolari.
Come il kPSCs isolato con questo spettacolo di protocollo organo-typic proprietà con rene epiteliale ferita guarigione capacità15, terapia cellulare con kPSCs in malattie del rene e trapianto sarebbe un'interessante applicazione futura. Per questo scopo, ci sono diverse strategie cellula/cellula della consegna del prodotto. La prima strategia è infusione IV di kPSCs, come attualmente utilizzati negli studi clinici bmMSC. Un'altra applicazione interessante è l'uso di kPSCs o fattori kPSC-escreto nell'aspersione di macchina prima di trapianto. In questo modo, la qualità del rene espiantato potrebbe migliorare che potrebbe portare ad una migliore funzionalità renale dopo trapianto. Per entrambe le strategie, i kPSCs sono un'interessante fonte cellulare nuovo di esplorare ulteriormente per scopi clinici.

Cellule stromali mesenchimali (MSC) sono cellule modulatore immuni che sono state originariamente isolate dal midollo osseo. Si distinguono per la loro morfologia, capacità di differenziarsi in grasso, osso e cartilagine e aderenza plastica fusiformi. MSCs esprimono i marcatori stromali CD73, CD90, CD105 pur essendo negativo per i marcatori CD31 e CD451,2,3. MSCs sono un candidato promettente per la terapia cellulare dovuto il loro tessuto omeostatico e capacità immunomodulatoria. bmMSCs sono attualmente allo studio in studi clinici per diverse malattie, tra cui malattie del rene e trapianto di rene come Recensito altrove4.
Precedentemente è stato dimostrato che cellule perivascolari da diversi organi solidi differenti, compreso il tessuto adiposo, placenta e muscolo scheletrico condividono caratteristiche con MSCs9. Tuttavia, queste cellule presentano anche funzioni di tessuto-specifici che possono provocare organotipiche riparazione10. Le cellule perivascolari del miocardio umano, ad esempio, ha stimolato risposte angiogeniche dopo ipossia e differenziano nei cardiomiociti, mentre cellule perivascolari isolate da altri tessuti non hanno mostrato questi potenziali11.
Cellule stromali perivascolare possono anche essere isolate dal mouse12,13,14 e rene umano15,16. Abbiamo ampiamente caratterizzata kPSCs e confrontato questi per bmMSCs. Abbiamo trovato che kPSCs, simile a bmMSCs, hanno capacità immunosoppressive e può supportare la formazione di plesso vascolare. Tuttavia, ci è tessuto specifiche differenze tra i tipi di cellule, come kPSCs ha mostrato una firma di espressione trascrizionale organotipiche, compresi i fattori di trascrizione nephrogenic HoxD10 e HoxD11. kPSCs, in contrasto con bmMSCs, non hanno subito la trasformazione dei miofibroblasti dopo stimolazione con TGF-β e sono stati in grado di differenziarsi in adipociti. Inoltre, kPSCs accelerato l'integrità epiteliale in un'analisi gratta e Vinci della ferita epiteliale tubolare renale, un fenomeno che non è stato osservato con bmMSCs. Questa ferita avanzata riparazione è stata mediata attraverso il rilascio di fattore di crescita dell'epatocita. Inoltre, kPSCs ha migliorato il danno renale in un modello murino di insufficienza renale acuta lesioni15. Pertanto, kPSCs sembrano avere la capacità di riparazione renale superiore e sono una fonte interessante di nuova per la terapia cellulare in patologie renali.
Per poter utilizzare kPSCs per scopi di terapia cellulare, kPSCs dovrebbe essere isolato in un modo di grado clinico con gli enzimi grado clinici e protocolli. Inoltre, per essere in grado di trattare parecchi pazienti con kPSCs da 1 donatore, sufficiente numeri di cell dovrebbero essere ottenuti. Qui vi mostriamo nel dettaglio, la procedura di isolamento clinico grado di kPSCs da reni di grado intero trapianto, producendo un numero sufficiente di cellule da utilizzare per cliniche di terapia cellulare.

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			<td height="21" style="height:21px;width:216px;">Baxter bags</td>
			<td style="width:117px;">Fenwal</td>
			<td style="width:119px;">R4R-7004</td>
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			<td height="21" style="height:21px;">Human platelets</td>
			<td>Sanquin</td>
			<td></td>
			<td>hospital surplus material expired for less than 2 days is used</td>
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			<td style="width:117px;"></td>
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			<td>custom made</td>
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			<td height="21" style="height:21px;">Disposable sterile bottles</td>
			<td style="width:117px;">Corning</td>
			<td style="width:119px;">09-761-11</td>
			<td></td>
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			<td height="21" style="height:21px;">500ml PP centrifuge tubes</td>
			<td style="width:117px;">Corning</td>
			<td style="width:119px;">431123</td>
			<td></td>
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			<td height="21" style="height:21px;">a-MEM medium</td>
			<td>Lonza</td>
			<td>BE12-169F</td>
			<td></td>
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			<td height="21" style="height:21px;">glutamax</td>
			<td style="width:117px;">thermo fisher</td>
			<td>35050038</td>
			<td></td>
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			<td height="21" style="height:21px;">pen/strep&#160;</td>
			<td>Invitrogen</td>
			<td>15070063</td>
			<td></td>
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			<td height="21" style="height:21px;">transfusion system</td>
			<td>Codan</td>
			<td>455609</td>
			<td></td>
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			<td height="21" style="height:21px;">DMEM-F12</td>
			<td>life technologies</td>
			<td>11320-074</td>
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			<td height="21" style="height:21px;">normal human serum</td>
			<td style="width:117px;">Sanquin</td>
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			<td></td>
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			<td height="21" style="height:21px;">Hepes 1M</td>
			<td style="width:117px;">Lonza</td>
			<td style="width:119px;">BE-17-737E</td>
			<td></td>
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			<td height="21" style="height:21px;">NaHCO3 7.5%</td>
			<td style="width:117px;">Lonza</td>
			<td style="width:119px;">BE17-613E</td>
			<td></td>
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			<td height="21" style="height:21px;">CaCl2 1M</td>
			<td style="width:117px;">Sigma-Aldrich</td>
			<td style="width:119px;">10043-52-4</td>
			<td></td>
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			<td height="21" style="height:21px;">UW</td>
			<td style="width:117px;">Bridge to life</td>
			<td style="width:119px;">32911</td>
			<td></td>
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			<td height="21" style="height:21px;">Heparin</td>
			<td style="width:117px;">Leo Pharma</td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;">collagenase NB1 GMP grade</td>
			<td>SERVA</td>
			<td style="width:119px;">17455.03</td>
			<td></td>
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			<td height="21" style="height:21px;">DMSO</td>
			<td>Sigma Aldrich</td>
			<td>D2650-100ml</td>
			<td></td>
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			<td height="21" style="height:21px;">surgical drapes</td>
			<td style="width:117px;">3M Nederland</td>
			<td style="width:119px;">DH999969404</td>
			<td></td>
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			<td height="21" style="height:21px;">perfusion pump</td>
			<td>metrohm</td>
			<td>x007528300</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">pump head</td>
			<td>metrohm</td>
			<td>x077202600</td>
			<td></td>
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			<td height="21" style="height:21px;">perfusion tray</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td>custom made</td>
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			<td height="21" style="height:21px;">LS25 masterflex tubes</td>
			<td>Masterflex</td>
			<td>HV-96410-25</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">accessory spike</td>
			<td style="width:117px;">Gambro DASCO</td>
			<td style="width:119px;">6038020</td>
			<td></td>
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			<td height="21" style="height:21px;">pulmozyme</td>
			<td style="width:117px;">Roche</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="24">
			<td height="24" style="height:24px;width:216px;">culture flasks 25 cm2</td>
			<td style="width:117px;">greiner</td>
			<td style="width:119px;">690175</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">culture flask 75 cm2</td>
			<td style="width:117px;">greiner</td>
			<td style="width:119px;">658170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">culture flask 175 cm2</td>
			<td style="width:117px;">greiner</td>
			<td style="width:119px;">661160</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Trypsin</td>
			<td>sigma</td>
			<td>t4174</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BSA</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">A2153</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E5134-500g</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">FcR blocking reagent</td>
			<td>miltenyi</td>
			<td>130-059-901</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti melanoma beads (NG2)</td>
			<td>miltenyi</td>
			<td>130-090-452</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">cellstrainer 70 &#181;m</td>
			<td>Corning</td>
			<td>352350</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LS columns</td>
			<td>Miltenyi</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MACS magnet</td>
			<td style="width:117px;">miltenyi</td>
			<td>130-090-976</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">CD34 FITC</td>
			<td style="width:117px;">BD&#160;</td>
			<td>555821</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD45 APC</td>
			<td style="width:117px;">BD&#160;</td>
			<td>555485</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD146 PE</td>
			<td style="width:117px;">BD&#160;</td>
			<td>550315</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NG2 APC</td>
			<td style="width:117px;">R&#38;D</td>
			<td>FAB2585A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD90 PE</td>
			<td style="width:117px;">BD&#160;</td>
			<td>555596</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HLA ABC APC</td>
			<td style="width:117px;">BD&#160;</td>
			<td>555555</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD105 FITC</td>
			<td>Ancell</td>
			<td>326-040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HLA DR APC</td>
			<td style="width:117px;">BD&#160;</td>
			<td>559866</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD56 PE</td>
			<td>BD&#160;</td>
			<td>555516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD73 PE</td>
			<td style="width:117px;">BD&#160;</td>
			<td>550257</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD31 FITC</td>
			<td style="width:117px;">BD&#160;</td>
			<td>555445</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD133 PE</td>
			<td style="width:117px;">miltenyi</td>
			<td>130-090-853</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PDGF-r&#160;</td>
			<td style="width:117px;">R&#38;D</td>
			<td>mab 1263</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mouse IgG1 FITC</td>
			<td style="width:117px;">BD&#160;</td>
			<td>345815</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mouse IgG1 PE</td>
			<td style="width:117px;">BD&#160;</td>
			<td>345816</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mouse IgG1 APC</td>
			<td style="width:117px;">BD&#160;</td>
			<td>345818</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IgG2b PE</td>
			<td style="width:117px;">BD&#160;</td>
			<td>555743</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">goat anti mouse PE</td>
			<td style="width:117px;">Dako</td>
			<td style="width:119px;">R0480</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sodium azide</td>
			<td style="width:117px;">Merck</td>
			<td>822335</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM Ham&#8217;s F12</td>
			<td>Gibco</td>
			<td>31331-028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ITS (insul, transferrin, selenium)</td>
			<td style="width:117px;">Sigma</td>
			<td>I1884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">hydrocortisone</td>
			<td>Sigma</td>
			<td>H0135</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">triiodothyrinine</td>
			<td style="width:117px;">Sigma</td>
			<td>T5516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">epidermal growth factor</td>
			<td>sigma</td>
			<td>E9644</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immortalized human renal PTEC (HK2)</td>
			<td style="width:119px;"></td>
			<td></td>
			<td>courtesey of M. Ryan, university college Dublin</td>
		</tr>
	</tbody>
</table>

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

Cellule stromali mesenchimali (MSCs) sono omeostatici e immuni modulatoria cellule del tessuto che hanno dimostrato gli effetti benefici nel trapianto e malattie renali. Le cellule Stromal perivascolari (PSC) condividono caratteristiche con midollo osseo MSCs (bmMSCs). Tuttavia, possiedono anche, molto probabilmente a causa di locali imprinting, proprietà di tessuto-specifici e giocare un ruolo nell'omeostasi del tessuto locale. Questa specificità tissutale può provocare riparazione specifico del tessuto, anche all'interno del rene umano. Precedentemente abbiamo indicato che gli sportelli unici di rene umano (kPSCs) sono migliorati arrotolata epiteliale renale mentre bmMSCs non ha avuto questo potenziale di guarigione. Inoltre, kPSCs può migliorare di lesioni renali in vivo. Pertanto, kPSCs costituiscono una fonte interessante per la terapia cellulare, in particolare per malattie del rene e trapianto renale. Qui vi mostriamo il metodo di isolamento e coltura dettagliato per kPSCs dal grado di trapianto di reni umani basati su tutto-organo aspersione di enzimi digestivi tramite l'arteria renale e un arricchimento per il marcatore perivascolare NG2. In questo modo, può essere ottenuto quantità di grandi cellule che sono adatti per la terapia cellulare.

Il metodo di isolamento kPSCs grado clinico è riassunto nella Figura 1. Cellule di rene di greggio sono isolate dal trapianto umano reni di grado tramite aspersione di collagenasi. La sospensione cellulare risultante è coltivata fino al confluente nel 5% delle piastrine lisati. Quindi la frazione di cellule stromali perivascolare è isolata basato sull'espressione di NG2.
kPSCs sono cellule fusiformi aderenti in plastica (Figura 2A) e sono positivi per gli indicatori di stromal marcatori CD73, CD90, CD105 e perivascolare NG2, PDGFR-B e CD146, mentre negativo per CD31, CD34, CD45 e HLA-DR (Figura 2B). In genere, una popolazione omogenea di kPSCs può essere raggiunto al passaggio 4 e kPSCs raggiungere senescenza intorno passaggio 9-10 (Figura 2C). Consigliamo quindi di eseguire esperimenti tra passaggio 5-8.
Per valutare la capacità funzionale del kPSCs isolato, eseguiamo un'analisi gratta e Vinci in vitro rene epiteliale ferita su ogni nuovo lotto di kPSCs, come abbiamo dimostrato in precedenza che il medium condizionato di kPSCs può accelerare la guarigione arrotolata epiteliale in questo dosaggio zero15. Il medium condizionato kPSC fatta di coltura kPSCs per 48 h in alphaMEM 5% della piastrina lisati e raccogliere il surnatante. Successivamente, il rene umano immortalato tubulo prossimale cellule epiteliali (HK-2)17 sono coltivate fino a confluenza e quindi viene effettuata una ferita gratta e Vinci. Quindi sia il medium condizionato di mezzo kPSCs o controllo viene aggiunto ai pozzetti e viene misurata la velocità di guarigione delle ferite. Quando viene aggiunto il medium condizionato di kPSCs, la ferita si chiude significativamente più veloce (Figura 2D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55841/55841fig1.jpg"> 
Figura 1 : Grado clinico metodo di isolamento di umana kPSCs. Trapianto di reni di grado sono cannulati e irrorati con collagenasi via l'arteria renale (A - G) e la sospensione cellulare risultante viene lavata e sia crioconservato o messi in coltura (H - K). Dopo che le cellule raggiungono la confluenza (L), NG2 arricchimento delle cellule è effettuato (M). Le cellule vengono tripsinizzate quando entrambi sono confluenti, hanno smesso di proliferazione o quando strutture 3D appaiono (N - P). I criteri di rilascio per kPSCs sono sterilità, espressione del marcatore e la capacità di migliorare la guarigione arrotolata epiteliale tubolare (Q). Freccia: dell'arteria renale. Barra della scala = 200 µm. <a href="http://ecsource.jove.com/files/ftp_upload/55841/55841fig1large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55841/55841fig2.jpg"> 
Figura 2: kPSCs caratterizzazione dei diritti dell'uomo. Un) kPSCs sono fusiformi, plastica cellule aderenti. B) kPSCs sono positivi per gli indicatori mesenchymal CD73, CD90, CD105, perivascolare marcatori NG2, PDGFR-β e CD146, mentre negativo per CD31, CD34 e CD45. kPSCs express MHC di classe I (HLA-ABC) ma non di classe II (HLA-DR). C) caratteristiche di crescita di tre differenti kPSC donatori da citometria a flusso ha confermato popolazioni positivi NG2 omogenee (al passaggio 4). kPSCs raggiungere senescenza intorno passaggio 9-10. D) kPSCs sono in grado di esaltare il riparazione epiteliale renale in un'analisi di gratta e Vinci di ferita. Immagini rappresentative di controllo medio e medio kPSC condizionata a t = 0, 4, 8 e 12 h sono mostrati. Barra della scala nel A) = 200 µm, d) = 100 µm. <a href="http://ecsource.jove.com/files/ftp_upload/55841/55841fig2large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</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

Qui presentiamo un metodo di isolamento e coltura di romanzo grado clinico per rene perivascolare di cellule stromali (kPSCs) basato sulla perfusione d'organo intero con gli enzimi digestivi e arricchimento NG2-cella. Con questo metodo, è possibile acquisire il numero di celle sufficiente per una terapia cellulare.

Un metodo di isolamento di romanzo Clinical Grade per rene umano perivascolari cellule stromali

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

Generazione sperimentale di carcinoma-Associated fibroblasti (CAF) da fibroblasti umani mammari

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

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

Differenziazione Isolamento, caratterizzazione e comparata delle cellule umane Dental Pulp staminali derivate da denti permanenti mediante due diversi metodi

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

A Novel Vertebral Stabilization Method for Producing  Contusive Spinal Cord Injury

A Novel vertebrale metodo di stabilizzazione per la produzione di Contusivi Cord Injury spinale

Clinically-relevant animal cervical spinal cord injury (SCI) models are essential for developing and testing potential therapies; however, producing ...

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

Due metodi per stabilire primarie cellule umane endometriale stromali da isterectomia campioni

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

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

Isolamento, Crioconservazione e Cultura umani Cellule epiteliali Amnion per le applicazioni cliniche

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

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

Pre-clinica ortotopico Modello murino di Human cancro alla prostata

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

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Isolamento e coltura di cellule endoteliali primarie dalle arterie e vene Canine

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

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

Un protocollo dettagliato per la sudorazione monitoraggio Utilizzando un romanzo, Piccolo, dispositivo wireless

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

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

Protocollo clinico di produrre tessuto adiposo-derivato Stromal frazione vascolare per potenziale rigenerazione della cartilagine

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