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>
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			<td>Sanquin</td>
			<td></td>
			<td>hospital surplus material expired for less than 2 days is used</td>
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			<td>custom made</td>
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			<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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		<tr height="21">
			<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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		<tr height="21">
			<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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			<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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			<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>
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			<td height="21" style="height:21px;width:216px;">MACS magnet</td>
			<td style="width:117px;">miltenyi</td>
			<td>130-090-976</td>
			<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

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

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JoVE Journal

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6.8K Views.  Leiden University Medical Centre. 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.

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

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

Un metodo per isolamento Islet murini e trapianto renale subcapsulare

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

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Medicina

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

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

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

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

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

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

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 ...

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

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 culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

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

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

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

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

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 ...

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

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the patients are unaware of such disorders or are having difficulty expressing their symptoms. Until now, several devices for perspiration monitoring have been developed; however, such devices tend to have a relatively large exterior, considerable power consumption, and/or less sensitivity.
Recently, we developed a small, wireless device for perspiration monitoring. The device consists of a temperature/relative humidity (T/RH) sensor, battery-driven small data logger, and silica gel as a desiccant in a small cylindrical exterior. The T/RH sensor is placed between the detection windows (through which the water vapor from the skin enters) and the silica gel. The underlying principle of the perspiration monitoring device is based on Fick&#39;s law of diffusion, which means that water vapor flux from the skin to the silica gel (i.e.&#160;transepidermal water loss and perspiration) can be captured by change in humidity at the T/RH sensor. In addition, a baseline subtraction method was adopted to distinguish perspiration and transepidermal water loss.
As shown in the previous report, the developed device can monitor the perspiration at any sites of the body in an easy, wireless manner. However, detailed methods of how to use the device have not been disclosed yet. In this article, therefore, we would like to show the point-by-point tutorials of how to use the device for perspiration monitoring, by showing the sympathetic activity test with the sympathetic skin response monitoring as an example.

Recently, we developed a small wireless device for perspiration monitoring. In this article, we present detailed protocols on how to use the device for perspiration monitoring with an example of the sympathetic activity test.

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

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the knees by using different forms of mesenchymal stem cells (MSCs). In Korea, bone marrow concentrates and cord blood-derived stem cells have been approved by the Korean Food and Drug Administration (KFDA) for cartilage regeneration. In addition, an adipose tissue-derived stromal vascular fraction (SVF) has been allowed by the KFDA for joint injections in human patients. Autologous adipose tissue-derived SVF contains extracellular matrix (ECM) in addition to mesenchymal stem cells. ECM excretes various cytokines that, along with hyaluronic acid (HA) and platelet-rich plasma (PRP) activated by calcium chloride, may help MSCs to regenerate cartilage and improve knee functions. In this article, we presented a protocol to improve knee functions by regenerating cartilage-like tissue in human patients with OA. The result of the protocol was first reported in 2011 followed by a few additional publications. The protocol involves liposuction to obtain autologous lipoaspirates that are mixed with collagenase. This lipoaspirates-collagenase mixture is then cut and homogenized to remove large fibrous tissue that may clog up the needle during the injection. Afterwards, the mixture is incubated to obtain adipose tissue-derived SVF. The resulting adipose tissue-derived SVF, containing both adipose tissue-derived MSCs and remnants of ECM, is injected into knees of patients, combined with HA and calcium chloride activated PRP. Included are three cases of patients who were treated with our protocol resulting in improvement of knee pain, swelling, and range of motion along with MRI evidence of hyaline cartilage-like tissue.

Here, we present a protocol to produce an adipose tissue-derived stromal vascular fraction and its application to improve knee functions by regenerating cartilage-like tissue in human patients with osteoarthritis.

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 ...

Isolation of Adipose Derived Regenerative Cells for the Treatment of Erectile Dysfunction Following Radical Prostatectomy

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem cells can be obtained from different tissues and several methods for isolation have been described. The presented method for the isolation of adipose-derived regenerative cells (ADRCs) can be used within many therapeutic areas because the method is a general procedure and, therefore, not limited to erectile dysfunction (ED) therapy. ED is a common and serious side effect to radical prostatectomy (RP) since ED often is not well treated with conventional therapy. Using ADRC&#8217;s as treatment for ED has attracted great interest due to the initial positive results after a single injection of cells into the corpora cavernosum. The method used for the isolation of ADRC&#8217;s is a simple, automated process, that is reproducible and ensures a uniform product. Furthermore, the sterility of the isolated product is ensured because the entire process takes place in a closed system. It is important to minimize the risk of contamination and infection since the stem cells are used for injection in humans. The whole procedure can be done within 2.5-3.5 hours and does not require a classified laboratory which eliminates the need for shipping tissue to an off-site. However, the procedure has some limitations since the minimum amount of drained lipoaspirate for the isolation device to function is 100 g.

Precise disclosure of methods and protocols is crucial for large scale uptake of stem cell therapies. Here, we present a protocol to isolate adipose-derived regenerative cells, used for a single intracavernous injection as treatment of erectile dysfunction (ED) following radical prostatectomy (RP).

Isolamento di cellule rigenerative di derivazione adiposa per il trattamento della disfunzione erettile a seguito di prostatectomia radicale

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem ...

Orthotopic Rat Kidney Transplantation: A Novel and Simplified Surgical Approach

Kidney transplantation offers increased survival rates and improved quality of life for patients with end-stage renal disease, as compared to any type of renal replacement therapy. Over the past few decades, the rat kidney transplantation model has been used to study the immunological phenomena of rejection and tolerance. This model has become an indispensable tool to test new immunomodulatory pharmaceuticals and regimens prior to proceeding with expensive preclinical large animal studies.
This protocol provides a detailed overview of how to reliably perform orthotopic kidney transplantation in rats. This protocol includes three distinctive steps that increase the probability of success: perfusion of the donor kidney by flushing through the portal vein and the use of a cuff system to anastomose the renal veins and ureters, thereby decreasing cold and warm ischemia times. Using this technique, we have achieved survival rates beyond 6 months with normal serum creatinine in animals with syngeneic or tolerant kidney transplants. Depending on the aim of the study, this model can be modified by pre- or posttransplant treatments to study the acute, chronic, cellular, or antibody-mediated rejection. It is a reproducible, reliable, and cost-effective animal model to study different aspects of kidney transplantation.

The purpose of this manuscript and protocol is to explain and demonstrate in detail the surgical procedure of orthotopic kidney transplantation in rats. This method is simplified to achieve the correct perfusion of the donor kidney and shorten the reperfusion time by using the venous and ureteral cuff anastomosis technique.

Trapianto di rene ortotopico ratto: un nuovo approccio chirurgico semplificato

Kidney transplantation offers increased survival rates and improved quality of life for patients with end-stage renal disease, as compared to any type of ...