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

Esta pesquisa recebeu financiamento do sétimo programa-quadro da Comunidade Europeia (FP7/2007-2013) sob o número 305436 do contrato de concessão estelar.

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. estão associados com um pedido de patente em relação esta pesquisa. Os outros autores não têm nada a divulgar.

Perivasculares células têm sido isoladas de muitos diferentes sólidos órgãos, incluindo o rim9,15,16, cartilagem, gordura e pâncreas humanos. A maioria dos métodos, no entanto, são baseados em pequenas amostras de tecido, que são dissecados e posteriormente tratadas com enzimas digestivas. Além disso, isso geralmente não é executado com produtos grau clínico. Isto faz estas estratégias menos adequados para tradução clínica direta onde grandes quantidades de células de grau clínico são necessárias.
Aqui nós mostramos um método de isolamento de novela de kPSCs humana para órgãos inteiros, com base na perfusão com grau clínico enzimas e materiais. O protocolo é uma adaptação do protocolo de isolamento clínico ilhotas de Langerhans atualmente em uso para aplicação clínica em nosso centro de18.
Este é o primeiro método de grau clínico onde grandes quantidades de kPSCs podem ser alcançadas. A variabilidade no rendimento de célula é largamente dependente de doador. No entanto, quando o rendimento de célula que atualmente obtemos de uma fração da suspensão celular bruto de três diferentes doadores é extrapolado, em teoria, poderia ser alcançado um rendimento médio de 2,7 x 1012 kPSCs por doador. Como terapia MSC normalmente consiste de 2 infusões de célula com 1-2 x 106 células/kg corpo peso4, esses números de celular são suficientes para tratamento alogênico de vários pacientes.
Um passo crítico no processo de isolamento é a duração da digestão de colagenase. Quando o período de digestão é muito curto, grandes aglomerações de tecido continuará a ser, que será mais difícil para a cultura. Quando o período de perfusão é muito longo, pode ser observada morte celular aumentada. Portanto, quando o rim começa a se tornar macio e os fluidos menos transparentes, o rim deve ser massageado suavemente e o tratamento de colagenase deve ser interrompido.
Outro passo crítico é a cultura das células após enriquecimento de célula NG2. Às vezes após o enriquecimento de célula NG2, as células de perivascular não começam a proliferar, ou começar a crescer em estruturas 3D. Neste caso, quando as células são trypsinized e propagado novamente, as células geralmente vão começar a crescer na cultura de monocamada.
Como material de partida, rins de grau de transplante humano descartados principalmente por razões cirúrgicos foram usados. Estes são órgãos funcionais sem fibrose principal. Reconhecemos que essa poderia ser uma limitação, pois esta é uma doença relativamente rara e difícil de obter a fonte do órgão. Rins explantados podem ser outra fonte; no entanto, dependendo a razão da explantação rim, estes rins podem conter mais fibrose e, portanto, miofibroblastos e, portanto, os cuidados devem ser tomados desde myofibroblasts podem ser isoladas e cultivadas em vez de células perivasculares.
Como o kPSCs isolado com esse show de protocolo Propriedades organo-quar com rim epitelial ferida cura capacidades15, terapia celular, com kPSCs em doenças renais e transplante seria uma interessante aplicação futura. Para este efeito, existem várias estratégias de entrega de produtos de célula/célula. A primeira estratégia é a infusão IV de kPSCs, como usado atualmente nos estudos clínicos bmMSC. Outra aplicação interessante é o uso de kPSCs ou fatores kPSC-excretado na perfusão da máquina antes do transplante. Desta forma, a qualidade do rim explantado pode melhorar o que pode levar a função renal melhorou após transplante. Para ambas as estratégias, kPSCs são uma interessante fonte de novas células para explorar para fins clínicos.

Células estromais mesenquimais (MSCs) são células do sistema imunológico moduladora que foram originalmente isoladas a partir da medula óssea. Eles são caracterizados por seu eixo em forma de morfologia, capacidade de se diferenciarem em gordura, osso e cartilagem e aderência de plástico. MSCs expressam os marcadores do estroma CD73, CD90, CD105 sendo negativo para os marcadores CD31 e CD451,2,3. MSCs são um candidato promissor para terapia celular, devido ao seu tecido homeostático e capacidades de imunomoduladores. bmMSCs, atualmente, estão sendo estudados em ensaios clínicos para várias doenças, incluindo doenças renais e transplante de rim, como comentado em outro lugar4.
Anteriormente foi mostrado que perivasculares células de vários órgãos sólidos diferentes, incluindo o tecido adiposo, placenta e músculo esquelético compartilham características com MSCs9. No entanto, estas células também apresentam funções específicas do tecido, que podem resultar em reparação de organotypic10. Células humanas perivascular miocárdica, por exemplo, estimularam angiogênico respostas após hipóxia e diferenciados em cardiomyocytes, enquanto perivascular de células isoladas de outros tecidos não mostrou esses potenciais11.
Perivascular de células do estroma também podem ser isoladas do rato12,13,14 e15,de rim humano16. Nós extensivamente caracterizada kPSCs e comparei estas para bmMSCs. Descobrimos que kPSCs, semelhantes ao bmMSCs, têm capacidades de imunossupressores e pode apoiar a formação do plexo vascular. Entretanto, há tecido específicas diferenças entre os tipos de célula, como kPSCs mostrou uma assinatura de expressão transcricional de organotypic, incluindo os fatores de transcrição nefrogênico, HoxD10 e HoxD11. kPSCs, em contraste com o bmMSCs, não sofrem transformação Miofibroblasto após estimulação com TGF-β e não foram capazes de se diferenciar em adipócitos. Além disso, o kPSCs acelerado integridade epitelial em um ensaio zero ferimento epitelial tubular renal, um fenômeno que não foi observado com bmMSCs. Esta ferida reforçada reparação foi mediada através da liberação de fator de crescimento do hepatócito. Além disso, kPSCs amenizada lesão renal em um modelo do rato do15. lesão renal aguda. Portanto, kPSCs parecem ter capacidades de reparação renal superior e são uma interessante fonte nova para a terapia celular em doenças renais.
A fim de ser capaz de usar kPSCs para fins de terapia celular, kPSCs deve ser isolado de forma clínica grau com grau clínico enzimas e protocolos. Além disso, para ser capaz de tratar vários pacientes com kPSCs de 1 doador, número suficiente de células deve ser obtido. Aqui nós mostramos em detalhes, o procedimento de isolamento clínico da classe de kPSCs de rins de transplante de todo ano, produzindo um número suficiente de células a ser usado para terapia celular clínica.

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			<td>hospital surplus material expired for less than 2 days is used</td>
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			<td style="width:117px;">Lonza</td>
			<td style="width:119px;">BE17-613E</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>
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			<td height="21" style="height:21px;">UW</td>
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			<td style="width:119px;">32911</td>
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			<td height="21" style="height:21px;">Heparin</td>
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			<td>SERVA</td>
			<td style="width:119px;">17455.03</td>
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			<td style="width:119px;">DH999969404</td>
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			<td height="21" style="height:21px;">perfusion tray</td>
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			<td>custom made</td>
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			<td>Masterflex</td>
			<td>HV-96410-25</td>
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			<td height="21" style="height:21px;width:216px;">accessory spike</td>
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			<td style="width:119px;">6038020</td>
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			<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>
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			<td height="21" style="height:21px;">culture flask 75 cm2</td>
			<td style="width:117px;">greiner</td>
			<td style="width:119px;">658170</td>
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			<td height="21" style="height:21px;width:216px;">culture flask 175 cm2</td>
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			<td style="width:119px;">661160</td>
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			<td>miltenyi</td>
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			<td>555485</td>
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			<td style="width:117px;">BD&#160;</td>
			<td>550315</td>
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			<td height="21" style="height:21px;width:216px;">NG2 APC</td>
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			<td height="21" style="height:21px;width:216px;">CD90 PE</td>
			<td style="width:117px;">BD&#160;</td>
			<td>555596</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">HLA ABC APC</td>
			<td style="width:117px;">BD&#160;</td>
			<td>555555</td>
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			<td height="21" style="height:21px;width:216px;">CD105 FITC</td>
			<td>Ancell</td>
			<td>326-040</td>
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			<td height="21" style="height:21px;width:216px;">HLA DR APC</td>
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			<td>BD&#160;</td>
			<td>555516</td>
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			<td height="21" style="height:21px;width:216px;">CD73 PE</td>
			<td style="width:117px;">BD&#160;</td>
			<td>550257</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD31 FITC</td>
			<td style="width:117px;">BD&#160;</td>
			<td>555445</td>
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			<td height="21" style="height:21px;width:216px;">CD133 PE</td>
			<td style="width:117px;">miltenyi</td>
			<td>130-090-853</td>
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			<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

Células estromais mesenquimais (MSCs) são tecidos homeostático e imune moduladora células que têm demonstrado efeitos benéficos em transplante e doenças renais. Células do estroma perivascular (EPS) compartilham características com medula óssea MSCs (bmMSCs). No entanto, eles também possuem, provavelmente devido a impressão local, propriedades de tecido-específica e desempenhar um papel na homeostase do tecido local. Essa especificidade de tecido pode resultar em reparação de tecidos específicos, também dentro do rim humano. Mostramos anteriormente que PSCs rim humano (kPSCs) têm melhorado o ferimento epitelial renal, Considerando que bmMSCs não tem esse potencial de cura. Além disso, kPSCs amenizar de lesão renal em vivo. Portanto, kPSCs constituem uma fonte interessante para terapia celular, especialmente para doenças renais e transplante renal. Aqui nós mostramos o método de isolamento e cultura detalhado para kPSCs de transplante-grau rins humanos com base na perfusão de todo órgão de enzimas digestivas, através da artéria renal e enriquecimento para o marcador perivascular NG2. Desta forma, as quantidades de células grandes podem ser obtidas que são apropriados para terapia celular.

O método de isolamento de kPSCs de grau clínico está resumido na Figura 1. Células de rim bruto são isoladas humano transplante de rins de grau por perfusão de colagenase. A suspensão resultante é cultivada até confluente em 5% plaquetas lisados. Em seguida, a fração de células estromais perivascular é isolada com base na expressão de NG2.
kPSCs são plásticas aderentes células fusiformes(Figura 2)e são positivos para os marcadores de marcadores do estroma CD73, CD90, CD105 e perivascular NG2, PDGFR-B e CD146, enquanto negativo para CD31 e CD34, CD45, HLA-DR (Figura 2B). Normalmente, uma população homogénea de kPSCs pode ser alcangada na passagem 4 e kPSCs chegar a senescência em torno de passagem 9-10 (Figura 2C). Portanto, aconselhamos a realizar experimentos entre passagem 5-8.
Para avaliar a capacidade funcional da kPSCs isolada, realizamos um em vitro rim epiteliais ferido zero ensaio sobre cada novo lote de kPSCs, como mostramos anteriormente que o meio condicionado de kPSCs pode acelerar epiteliais cicatrização neste ensaio zero15. O meio condicionado kPSC feito pelo cultivo kPSCs por 48 h em alphaMEM 5% plaquetas lysates e recolher o sobrenadante. Em seguida, o rim humano imortalizado túbulo proximal células epiteliais (HK-2)17 são cultivadas até confluente e então feita uma ferida zero. Então ou o meio condicionado de médio kPSCs ou controle é adicionado aos poços e mede-se a velocidade de cicatrização de feridas. Quando o meio condicionado de kPSCs é adicionado, a ferida fecha significativamente mais rápido (Figura 2D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55841/55841fig1.jpg"> 
Figura 1 : Clínica grau isolamento método de humano kPSCs. Transplante de rins de grau são canulados e pintados com colagenase através da artéria renal (A - G) e a suspensão de células resultante é lavada e também criopreservadas ou colocado em cultura (H - K). Depois que as células alcançar confluência (L), NG2 célula enriquecimento é executada (M). As células são trypsinized quando eles também são confluentes, pararam de se proliferando ou quando estruturas 3D aparecem (N - P). Os critérios de liberação de kPSCs são a esterilidade, expressão do marcador e a capacidade de melhorar a cicatrização da ferida epitelial tubular (Q). Seta: artéria renal. Barra de escala = 200 µm. <a href="http://ecsource.jove.com/files/ftp_upload/55841/55841fig1large.jpg" target="_blank">clique aqui para ver uma versão maior desta figura.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55841/55841fig2.jpg"> 
Figura 2: kPSCs caracterização de humano. Um) kPSCs são células aderentes fusiformes, plásticas. B) kPSCs são positivos para marcadores mesenquimais CD73, CD90, CD105, perivascular marcadores NG2, PDGFR-β e CD146, enquanto negativo para CD31 e CD34 CD45. kPSCs expressar o MHC-classe eu (HLA-ABC), mas não a classe II (HLA-DR). C) características de crescimento de três diferentes kPSC doadores de citometria de fluxo confirmaram homogênea NG2 populações positivas (na passagem 4). kPSCs chegar a senescência em torno de 9-10 de passagem. D) kPSCs são capazes de aumentar a reparação epitelial de rim em um ensaio de risco de ferimento. Imagens representativas de controle médio e médio kPSC condicionado em t = 0, 4, 8 e 12 h são mostrados. Barra de escala na) = 200 µm, em D) = 100 µm. <a href="http://ecsource.jove.com/files/ftp_upload/55841/55841fig2large.jpg" target="_blank">clique aqui para ver uma versão maior desta 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

Aqui nós apresentamos um método de isolamento e cultura de romance grau clínico para rim perivasculares células do estroma (kPSCs) com base na perfusão de órgão inteiro com enzimas digestivas e enriquecimento de NG2-célula. Com este método, é possível adquirir o número suficiente de células para terapia celular.

Um método de isolamento do romance grau clínico para rim humano Perivascular de células do estroma

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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

Isolamento, caracterização e comparativa Diferenciação de células-tronco da polpa dentária Derivado de dentes permanentes usando dois métodos diferentes

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

Um método de estabilização Novel Vertebral para a Produção de contusão Lesão Medular

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

Dois métodos para estabelecer células endometriais Humano estromais primários de espécimes de histerectomia

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, criopreservação e cultivo de células epiteliais humanas Amnion para aplicações clínicas

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

Pré-clínica Ortotópico Modelo murino de cancro da próstata humano

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 cultura de células endoteliais primárias de Artérias e Veias canina

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

Um protocolo detalhado para Monitoramento transpiração Usando um Novel, Pequeno, dispositivo sem fios

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

Protocolo clínico de produzir tecido adiposo-derivado do estroma Vascular fração para a regeneração da cartilagem potenciais

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