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

Denne forskning har modtaget støtte fra Det Europæiske Fællesskabs syvende ramme-Program (FP7/2007-2013) under grant aftalenummer 305436 STELLAR.

The local medical ethical committee and ethical advisory board of the European consortium (STELLAR) approved the research and collection of human transplant grade kidneys discarded mainly for surgical reasons. Research consent was given for all kidneys.
1. Preparations for Cell Culture
<ol>
	<li>Prepare a pool of platelet lysates.
	<ol>
		<li>Store the platelets that have expired for less than 2 d in -80 &#176;C until use (for a maximum of 1 year). 
		NOTE: The platelets were originally sourced from a commercial vendor. Hospital surplus material distributed by the local blood bank were obtained.</li>
		<li>Thaw the platelets of at least 5 donors (preferably 10) O/N at 4 &#176;C.</li>
		<li>Pool the platelets in a large sterile bottle, transfer to conical centrifuge tubes and spin down for 10 min at 1,960 x g at 4 &#176;C. 
		NOTE: The volume of platelets depends on the number of donors and the amount of hospital surplus material per donor.</li>
		<li>Pipette the supernatant to blood bags (50 mL/bag) through the barrel of a 50 mL syringe. Store at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Preparation of cell culture medium.
	<ol>
		<li>Defrost one bag of platelet lysates in a water bath at 37 &#176;C.</li>
		<li>Add 1 L (i.e. 2 bottles) of Minimum Essential Medium - alpha modification (alphaMEM), 5 mL 200 mM glutamine, and 20 mL of penicillin (5,000 U/mL)/streptomycin (5,000 &#181;g/mL) (pen/strep) to the bag.</li>
		<li>Incubate for 3 h at 37 &#176;C.</li>
		<li>Shake the bag for degelling by gently tapping on the bag when the bag is resting on a flat surface.</li>
		<li>Filter the 5% platelet lysates medium by pulling the lysates through the filter of the transfusion system with a sterile 50 mL syringe.</li>
		<li>Aliquot the medium in 50 mL tubes and store in -80 &#176;C until use. 
		NOTE: Every new batch of platelet lysates is tested in cell culture and compared to the previous batch by growing cells of interest (bmMSCs or kPSCs) to confluency in both batches. In cell numbers and viability, the kPSCs or bmMCs grown in the new batch should not differ more than 10%, and the marker expression of CD73, CD90, CD105 and CD31, CD34 and CD45 as analyzed by flow cytometry should not differ. The methods of cell culture are described in section 6 of the protocol (Culture of kPSC).</li>
	</ol>
	</li>
</ol>
2. Preparations for Cell Harvest
<ol>
	<li>Preparation of solutions.
	<ol>
		<li>Prepare the plain medium by adding 10 mL of pen/strep to 500 mL (1 bottle) of Dulbecco&#39;s Minimum Essential Medium (DMEM)-F12.</li>
		<li>Prepare the washing medium by adding 50 mL of Normal Human Serum (NHS) and 10 mL of pen/strep to 1 bottle of DMEM-F12.</li>
		<li>Prepare the enzyme-stock buffer by adding 2.9 mL 1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1.2 mL NaHCO3 2.5% [w/v] and 160 &#181;L CaCl2 (1 M) to 160 mL University of Wisconsin solution.</li>
		<li>Prepare the collagenase solution by slowly adding 20 mL of enzyme stock buffer to the bottle of GMP-grade collagenase containing &#62;2,000 U/bottle collagenase. Dissolve at 4 &#176;C for approximately 40 min. Gently shake several times during the dissolving period.</li>
		<li>Prepare the freeze medium by adding 5 mL dimethyl sulfoxide (DMSO) to 45 mL NHS.</li>
	</ol>
	</li>
	<li>Preparation of perfusion tray (Figure 1A).
	<ol>
		<li>Clean the flow cabin and cover with surgical drapes. Heat a water bath to approximately 40 - 45 &#176;C to heat the perfusion tray to 37 &#176;C. Put on surgical gowns and surgical gloves.</li>
		<li>Connect a sterilized perfusion tray to the water bath to preheat the perfusion tray with warm water. Make sure that there is no air in the perfusion tray by starting with the perfusion tray in the vertical position. If necessary add more water to the water bath to prevent air infusion into the perfusion tray.</li>
		<li>Place the LS25 tube in the pump. Leave both sterile ends of the tube in the flow cabinet. Place a Kocher&#39;s forceps on one end of the tube and allow to rest on the bottom of the perfusion tray. Place a sterile gauze on top of the tube to prevent obstruction. Attach a Luer connector on the other tube end.</li>
		<li>Add approximately 100 mL of plain medium into the perfusion tray and start flushing the tube on a pump speed of 100 mL/min.</li>
	</ol>
	</li>
</ol>
3. Kidney Cell Isolation
<ol>
	<li>Preparation of the kidney.
	<ol>
		<li>Remove the kidney from the double sterile bags and place on the sterile perfusion tray (Figure 1B). Remove the perirenal adipose tissue and kidney capsule with scissors and gentle tearing (Figure 1C) and identify the renal artery on the aortic patch as shown in Figure 1D.</li>
		<li>Cannulate the renal artery with the accessory spike, fix with a sterilized tie-rip, and attach to the pump tube end with the Luer connector while the pump is on. If the tubes are not completely filled, first add more medium to the tray and flush tubes by starting the pump (Figure 1E - F). Flush the kidney with plain medium via the pump with a flow of 100 mL/min.</li>
	</ol>
	</li>
	<li>Collagenase treatment and kidney cell isolation.
	<ol>
		<li>Add collagenase (20 mL, &#62;2,000 U) and DNase (2.5 mL, 1 mg/mL) to the perfusion tray (Figure 1G). Turn the kidney from time to time gently by hand. Wait until the kidney becomes soft and the perfusion liquid becomes less transparent (approximately 30 - 40 min).</li>
		<li>Gently massage the kidney (Figure 1H) until a cell suspension is obtained (Figure 1I). Remove non-digested material and the spike in the renal artery.</li>
	</ol>
	</li>
	<li>Washing and collection of kidney cells.
	<ol>
		<li>Add 50 mL of the NHS to the cell suspension. Drain the cell suspension from the perfusion tray by putting the pump at low flow and placing the tube first connected to the kidney above the 50 mL tubes (Figure 1J).</li>
		<li>Centrifuge at 300 x g for 7 min at 4 &#730;C and remove the supernatant. Wash the pellets with washing medium containing 10% NHS and again centrifuge at 300 x g for 7 min. Repeat washing once more. Measure the volume of the cell pellets and add an equal volume of cell culture medium.</li>
		<li>Either cryopreserve the cells from here by adding freezing medium 1:1 to the cell suspension and store in cryogenic vials according to standard protocols, or continue to culture the cells (section 4). 
		NOTE: The cell suspension contains cell clumps and requires extra steps to obtain single cells, which results in an increase in cell death. Therefore, the cells are kept in suspension and are not counted at this stage.</li>
	</ol>
	</li>
</ol>
4. Cell Culture of Crude Kidney Cells
<ol>
	<li>Add approximately 1 mL of the cell suspension to 24 mL of alphaMEM 5% platelet lysates (cell culture medium) in a T175 cell culture flask (Figure 1K) and culture at 37 &#176;C, 5% CO2. Remove the medium and cell debris after 2 d&#160;from the adherent cells and refresh the medium with 25 mL of cell culture medium.</li>
	<li>Refresh the culture medium twice a week by removing half of the medium (12.5 mL) and adding fresh medium (12.5 mL) using aseptic techniques.</li>
	<li>Culture until confluent (usually 5 - 7 d) (Figure 1L). Remove the medium, wash twice with PBS and trypsinize the cells by adding 5 mL trypsin to each T175 flask for 5 min at 37 &#176;C. Wash in culture medium and centrifuge at 490 x g for 5 min and remove the supernatant.</li>
</ol>
5. Cell Enrichment for NG2 by Magnetic Cell Separation (Figure 1M)
<ol>
	<li>Prepare the cell separation buffer according to manufacturer&#39;s protocol.</li>
	<li>Resuspend the trypsinized kPSCs in 10 mL cell separation buffer. Centrifuge at 490 x g for 5 min, discard the supernatant and resuspend the cells in 300 &#181;L cell separation buffer.</li>
	<li>Add 100 &#181;L FcR blocking reagen/5 x 107 cells, add 100 &#181;L anti-melanoma (NG2) beads per 5 x 107 cells, and incubate for 30 min at 4 &#176;C. Separate the NG2-positive fraction according to manufacturer&#39;s protocol. Add 10 mL of medium and centrifuge cells for 5 min at 490 x g.</li>
	<li>Count the cells using a B&#252;rker counting chamber according to manufacturer&#39;s protocol. Seed the cells in a culture flask (approximately 4 x 103 cells/cm2) and incubate. 
	NOTE: Seed the cells in the following concentrations: &#60;250,000 in a T25 flask, 250,000 - 500,000 in a T75 flask, and 700,000 - 800,000 in a T175 flask. After magnetic cell enrichment, a positive fraction of approximately 1 - 2% is isolated. However, as this step is cell enrichment and not cell sorting, other cell types will be present in the culture (Figure 1N). After several passages (usually around 4 - 5), only a homogenous kPSC-population is present (Figure 1P).</li>
</ol>
6. Culture of kPSCs
<ol>
	<li>Refresh the medium twice a week by removing half of the medium and replace it with freshly thawed culture medium (Figure 1N). 
	NOTE: The kPSCs tend to be more viable and proliferative when only half of the medium is refreshed.</li>
	<li>Passaging of kPSCs. 
	NOTE: Passage the kPSCs at either 90 - 100% confluency, or when 3D structures appear (see Figure 1O), or when there hasn&#39;t been cell growth for more than a week. The latter two particularly might occur at early passages after cell enrichment. In this case, after passaging, the cells will usually start to grow again in monolayer culture (Figure 1P).
	<ol>
		<li>Remove the medium from the 90 - 100% confluent cells (keep the medium for later use). Wash the flask twice with PBS (1 mL in T25, 5 mL in T75, 10 mL in T175). Add trypsin (0.5 mL in T25, 2 mL in T75, 5 mL in T175) and incubate for 5 min at 37 &#176;C. Add the old medium to the flask and resuspend gently.</li>
		<li>Transfer to a 15&#160;or 50 mL tube (depending on the volume) and centrifuge at 490 x g for 5 min. Count the viable cells and plate &#60;250,000 in a T25, 250,000 - 500,000 in a T75, or 700,000 - 800,000 in a T175 (approximately 4 x 103 cells/cm2).</li>
	</ol>
	</li>
	<li>Test the kPSCs at passage 5 or 6 (e.g., scratch assay, section 7). 
	NOTE: Characterize the propagated cells by flow cytometry using standard protocols. Marker expression of NG2, PDGFR-&#946;, CD146, CD73, CD90, CD105, HLA-ABC should all be positive and CD31, CD34, CD45 and HLA-DR should be negative. Test for mycoplasma, bacteria and fungi in the culture medium according to standard protocols of the clinical microbiology lab. Test the kPSCs functionally in a kidney epithelial wound scratch assay. Experiments are usually performed with sterile, flow cytometry-confirmed homogeneous kPSC populations with wound healing capacity between passage 6-8.</li>
	<li>Cryopreserve the kPSCs by adding 0.5 mL cryopreservation medium to 0.5 mL cell suspension (2 million cells per mL of culture medium) using standard protocols. 
	NOTE: After thawing, seed the kPSCs at a higher density (106&#160;cells in a T175).</li>
</ol>
7. Functional Test of kPSCs: Kidney Epithelial Wound Scratch Assay
<ol>
	<li>Culture the kPSCs in a 6-well plate at a density of 200,000 cells/well in 2 mL culture medium. After 48 h of culture at 37 &#176;C, 5% CO2, collect the supernatant. This is the conditioned medium.</li>
	<li>Seed the HK-2 (proximal tubular epithelial cells)17 in 2 mL of Proximal Tubular Epithelial Cell (PTEC) medium consisting of a 1:1 ratio of DMEM-F12 supplemented with insulin (5 mg/mL), transferrin (5 mg/mL), selenium (5 ng/mL), hydrocortisone (36 ng/mL), triiodothyrinine (40 pg/mL), epidermal growth factor (10 ng/mL) and pen/strep, in a density of 500,000 cells per well in a 6-well cell culture plate.</li>
	<li>Culture for 48 h at 37 &#176;C, 5% CO2. Remove the cell culture medium of the HK-2s and create a scratch wound in the monolayer of HK-2s by making a scratch with the tip of a yellow pipette point from the top to the bottom of the well. Wash the HK-2s with PBS and add the conditioned medium of the kPSCs or control medium. Mark on the bottom of the plate the area to be imaged. 
	NOTE: The HK-2s should be confluent. Image two areas per well.</li>
	<li>Image the scratch at 4, 8, 12 and 24 h at the same marked position with an inverted bright-field microscope.</li>
	<li>Measure the scratch area in Image J using the polygon selection tool to determine the percentage of closure.</li>
</ol>

<ol>
	<li>Friedenstein, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precursors+for+fibroblasts+in+different+populations+of+hematopoietic+cells+as+detected+by+the+in+vitro+colony+assay+method.">Precursors for fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method.</a> Exp Hematol. 2 (2), 83-92 (1974).</li><li>Dominici, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+criteria+for+defining+multipotent+mesenchymal+stromal+cells.+The+International+Society+for+Cellular+Therapy+position+statement.">Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.</a> Cytotherapy. 8 (4), 315-317 (2006).</li><li>Pittenger, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilineage+potential+of+adult+human+mesenchymal+stem+cells.">Multilineage potential of adult human mesenchymal stem cells.</a> Science. 284 (5411), 143-147 (1999).</li><li>Leuning, D. G., Reinders, M. E., de Fijter, J. W., Rabelink, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+translation+of+multipotent+mesenchymal+stromal+cells+in+transplantation.">Clinical translation of multipotent mesenchymal stromal cells in transplantation.</a> Semin Nephrol. 34 (4), 351-364 (2014).</li><li>Reinders, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+bone+marrow-derived+mesenchymal+stromal+cells+for+the+treatment+of+allograft+rejection+after+renal+transplantation:+results+of+a+phase+I+study.">Autologous bone marrow-derived mesenchymal stromal cells for the treatment of allograft rejection after renal transplantation: results of a phase I study.</a> Stem Cells Transl Med. 2 (2), 107-111 (2013).</li><li>Perico, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+mesenchymal+stromal+cells+and+kidney+transplantation:+a+pilot+study+of+safety+and+clinical+feasibility.">Autologous mesenchymal stromal cells and kidney transplantation: a pilot study of safety and clinical feasibility.</a> Clin J Am Soc Nephrol. 6 (2), 412-422 (2011).</li><li>Perico, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stromal+cells+and+kidney+transplantation:+pretransplant+infusion+protects+from+graft+dysfunction+while+fostering+immunoregulation.">Mesenchymal stromal cells and kidney transplantation: pretransplant infusion protects from graft dysfunction while fostering immunoregulation.</a> Transpl Int. 26 (9), 867-878 (2013).</li><li>Tan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+therapy+with+autologous+mesenchymal+stem+cells+in+living-related+kidney+transplants:+a+randomized+controlled+trial.">Induction therapy with autologous mesenchymal stem cells in living-related kidney transplants: a randomized controlled trial.</a> JAMA. 307 (11), 1169-1177 (2012).</li><li>Crisan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+perivascular+origin+for+mesenchymal+stem+cells+in+multiple+human+organs.">A perivascular origin for mesenchymal stem cells in multiple human organs.</a> Cell Stem Cell. 3 (3), 301-313 (2008).</li><li>Sacchetti, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No+Identical+"Mesenchymal+Stem+Cells"+at+Different+Times+and+Sites:+Human+Committed+Progenitors+of+Distinct+Origin+and+Differentiation+Potential+Are+Incorporated+as+Adventitial+Cells+in+Microvessels.">No Identical "Mesenchymal Stem Cells" at Different Times and Sites: Human Committed Progenitors of Distinct Origin and Differentiation Potential Are Incorporated as Adventitial Cells in Microvessels.</a> Stem Cell Reports. 6 (6), 897-913 (2016).</li><li>Chen, W. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+myocardial+pericytes:+multipotent+mesodermal+precursors+exhibiting+cardiac+specificity.">Human myocardial pericytes: multipotent mesodermal precursors exhibiting cardiac specificity.</a> Stem Cells. 33 (2), 557-573 (2015).</li><li>Dekel, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+nontubular+sca-1+lin-+multipotent+stem/progenitor+cells+from+adult+mouse+kidney.">Isolation and characterization of nontubular sca-1+lin- multipotent stem/progenitor cells from adult mouse kidney.</a> J Am Soc Nephrol. 17 (12), 3300-3314 (2006).</li><li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collecting+duct-derived+cells+display+mesenchymal+stem+cell+properties+and+retain+selective+in+vitro+and+in+vivo+epithelial+capacity.">Collecting duct-derived cells display mesenchymal stem cell properties and retain selective in vitro and in vivo epithelial capacity.</a> J Am Soc Nephrol. 26 (1), 81-94 (2015).</li><li>Pelekanos, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+transcriptome+and+immunophenotype+analysis+of+renal+and+cardiac+MSC-like+populations+supports+strong+congruence+with+bone+marrow+MSC+despite+maintenance+of+distinct+identities.">Comprehensive transcriptome and immunophenotype analysis of renal and cardiac MSC-like populations supports strong congruence with bone marrow MSC despite maintenance of distinct identities.</a> Stem Cell Res. 8 (1), 58-73 (2012).</li><li>Leuning, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical-Grade+Isolated+Human+Kidney+Perivascular+Stromal+Cells+as+an+Organotypic+Cell+Source+for+Kidney+Regenerative+Medicine.">Clinical-Grade Isolated Human Kidney Perivascular Stromal Cells as an Organotypic Cell Source for Kidney Regenerative Medicine.</a> Stem Cells Transl Med. , (2016).</li><li>Bruno, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+resident+mesenchymal+stem+cells+in+human+glomeruli.">Isolation and characterization of resident mesenchymal stem cells in human glomeruli.</a> Stem Cells Dev. 18 (6), 867-880 (2009).</li><li>Ryan, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HK-2:+an+immortalized+proximal+tubule+epithelial+cell+line+from+normal+adult+human+kidney.">HK-2: an immortalized proximal tubule epithelial cell line from normal adult human kidney.</a> Kidney Int. 45 (1), 48-57 (1994).</li><li>Nijhoff, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycemic+Stability+Through+Islet-After-Kidney+Transplantation+Using+an+Alemtuzumab-Based+Induction+Regimen+and+Long-Term+Triple-Maintenance+Immunosuppression.">Glycemic Stability Through Islet-After-Kidney Transplantation Using an Alemtuzumab-Based Induction Regimen and Long-Term Triple-Maintenance Immunosuppression.</a> Am J Transplant. , (2015).</li></ol>

D.G.L., M.A.E., og T.J.R. er forbundet med en patent ansøgning vedrørende denne forskning. Andre forfattere har intet at videregive.

Perivascular celler er blevet isoleret fra mange forskellige menneskelige fast organer, herunder bugspytkirtlen, fedt, brusk og nyre9,15,16. De fleste metoder, men er baseret på små prøver af væv, som er dissekeret og bagefter behandlet med fordøjelsesenzymer. Det er desuden normalt ikke udføres med kliniske grade produkter. Dette gør disse strategier mindre egnet til direkte kliniske oversættelse hvor store mængder af klinisk-grade celler er nødvendige.
Her viser vi en roman isolation metode af menneskelige kPSCs for hele organer baseret på perfusion med kliniske grade enzymer og materialer. Protokollen er tilpasset fra den kliniske Holme af Langerhanske isolation protokol i øjeblikket i brug for klinisk anvendelse i vores center18.
Dette er den første kliniske grade metode, hvor store mængder af kPSCs kan opnås. Variation i celle udbytte er stort set donor afhængige. Når cellen udbytte vi i øjeblikket få fra en brøkdel af rå cellesuspension af tre forskellige donorer er ekstrapoleres, i teorien, opnås imidlertid en gennemsnitlige afkast på 2,7 x 1012 kPSCs per donor. Som MSC terapi består typisk af 2 celle infusioner med 1-2 x 106 celler/kg krop vægt4, er disse celle numre tilstrækkelig for allogen behandling af flere patienter.
Et kritisk trin i proceduren isolation er varigheden af collagenase fordøjelsen. Når perioden fordøjelsen er for kort, forbliver store klumper af væv, som vil være sværere at kultur. Når perfusion er alt for lang, kan øget celledød observeres. Derfor, så snart nyren begynder at blive bløde og mindre gennemsigtig væske, nyrerne bør være masseres forsigtigt og collagenase behandlingen bør stoppes.
En anden kritisk trin er kulturen i cellerne efter NG2 celle berigelse. Nogle gange efter den NG2 celle berigelse begynder perivascular celler ikke at formere eller begynder at vokse i 3D strukturer. I dette tilfælde, når cellerne er trypsinized og tilsås, vil cellerne normalt begynder at vokse i éncellelag kultur.
Som udgangspunkt materiale, blev menneskelig transplantation grade nyrerne kasseret hovedsageligt for kirurgisk grunde brugt. Disse er funktionelle organer uden større fibrose. Vi anerkender, at dette kunne være en begrænsning, da dette er en forholdsvis sjælden og svært at få orgel kilde. Eksplanterede nyrerne kan være en anden kilde; men afhængigt af grunden af nyre explantation, disse nyrer kan indeholde flere fibrose og dermed myofibroblasts, og derfor omhu bør tages, da myofibroblasts kan være isoleret og kulturperler i stedet for perivascular celler.
Som kPSCs isoleret med denne protokol Vis organo-mest egenskaber med nyre epitelial sår healing kapacitet15, celleterapi med kPSCs i nyresygdomme og transplantation vil være en interessant fremtidig anvendelse. Til dette formål er der flere strategier i cellen/cellerne produkt levering. Den første strategi er IV infusion af kPSCs, som anvendes i øjeblikket i de kliniske undersøgelser, bmMSC. En anden interessant program er brugen af kPSCs eller kPSC-udskilles faktorer i maskinen perfusion før transplantation. På denne måde, kan kvaliteten af det eksplanterede nyre forbedre, som kunne føre til forbedret nyrefunktion efter transplantation. For begge strategier er kPSCs en interessant ny celle kilde til yderligere at udforske til kliniske formål.

Mesenchymale stromale celler (MSCs) er modulerende immunceller, der blev oprindeligt isoleret fra knoglemarven. De er kendetegnet ved deres spindel formet morfologi, evnen til at skelne i fedt, knogle og brusk og overholdelse af plast. MSC'er express stromale markører CD73, CD90, CD105 samtidig med at være negativ for markører CD31 og CD451,2,3. MSC'er er en lovende kandidat til celleterapi på grund af deres væv homeostatiske og immunmodulerende kapacitet. bmMSCs er i øjeblikket undersøgt i kliniske forsøg for flere sygdomme, herunder nyresygdomme og nyre transplantation som gennemgik andetsteds4.
Tidligere var det vist at perivascular celler fra flere forskellige fast organer, herunder fedtvæv, moderkagen og skeletmuskulatur deler egenskaber med MSCs9. Men, disse celler også udviser væv-specifikke funktioner, som kan resultere i organotypic reparation10. Menneskelige myokardiets perivascular celler, for eksempel, stimuleret angiogene svar efter hypoxi og opdeles i cardiomyocytes, mens perivascular celler isoleret fra andre væv ikke vise disse potentialer11.
Perivascular stromale celler kan også isoleres fra mus12,13,14 og menneskelige nyre15,16. Vi grundigt karakteriseret kPSCs og sammenlignet disse til bmMSCs. Vi fandt, at kPSCs, svarende til bmMSCs, har immunosuppressive kapacitet og kan støtte vaskulær plexus dannelse. Der er dog væv specifikke forskelle mellem celletyper, som kPSCs viste en organotypic transcriptional udtryk signatur, herunder nefrogen transskriptionsfaktorer HoxD10 og HoxD11. kPSCs, i modsætning til bmMSCs, undergår ikke myofibroblast transformation efter stimulation med TGF-β og var ude af stand til at differentiere i adipocytter. Derudover accelererede kPSCs epitelial integritet i en nyre rørformede epitelial sår scratch assay, et fænomen, der ikke blev observeret med bmMSCs. Denne forbedrede sår reparation var medieret gennem hepatocyt vækstfaktor frigivelse. Desuden forbedret kPSCs nyre skade i en musemodel af akutte nyre skade15. Derfor kPSCs synes at have superior renal reparation kapacitet og er en interessant ny kilde til celleterapi i nyresygdomme.
For at kunne bruge kPSCs til celle terapi formål, bør kPSCs være isoleret i en kliniske grade måde med kliniske grade enzymer og protokoller. Desuden, for at være i stand til at behandle flere patienter med kPSCs fra 1 donor, tilstrækkelig celle numre bør opnås. Her viser vi i detaljer, proceduren kliniske grade isolation af kPSCs fra hele transplantation grade nyrer, giver tilstrækkeligt antal celler skal bruges til klinisk cellulære terapi.

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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>SERVA</td>
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			<td>metrohm</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>sigma</td>
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			<td height="21" style="height:21px;width:216px;">CD45 APC</td>
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			<td>555485</td>
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			<td height="21" style="height:21px;width:216px;">CD146 PE</td>
			<td style="width:117px;">BD&#160;</td>
			<td>550315</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NG2 APC</td>
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			<td>FAB2585A</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>
			<td style="width:117px;">BD&#160;</td>
			<td>559866</td>
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			<td height="21" style="height:21px;width:216px;">CD56 PE</td>
			<td>BD&#160;</td>
			<td>555516</td>
			<td></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>
			<td></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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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PDGF-r&#160;</td>
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			<td>mab 1263</td>
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		<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

Mesenchymale stromale celler (MSCs) er væv homeostatiske og immun modulerende celler, der har vist positive effekter i nyresygdomme og transplantation. Perivascular stromale celler (PSC) deler egenskaber med knoglemarv MSCs (bmMSCs). Men de også besidder, sandsynligvis på grund af lokale prægning, væv-specifikke egenskaber og spille en rolle i lokale væv homøostase. Dette væv specificitet kan resultere i væv specifikke reparation, også inden for den menneskelige nyrerne. Tidligere viste vi, at menneskelige nyre PSC'er (kPSCs) har udvidet nyre epitelial sårheling der henviser til, at bmMSCs ikke har dette potentiale. KPSCs kan desuden forbedre nyre skade in vivo. Derfor, kPSCs er en interessant informationskilde for Celleterapi, især for nyresygdomme og renal transplantation. Her viser vi de detaljerede isolation og kultur metode for kPSCs fra transplantation-grade menneskelige nyrer baseret på hele-orgel perfusion af fordøjelsesenzymer via nyrearteriestenose og berigelse for perivascular markøren NG2. På denne måde, kan være fremstillet store celle mængder, der er egnet til cellulære terapi.

Kliniske grade kPSCs isolation metode er sammenfattet i figur 1. Rå nyreceller er isoleret fra menneskelig transplantation grade nyrerne af collagenase perfusion. Den resulterende cellesuspension er kulturperler indtil sammenflydende i 5% trombocyttal lysates. Derefter er perivascular stromale celle brøkdel isoleret baseret på NG2 udtryk.
kPSCs er plast vedhængende spindel-formet celler (fig. 2A) og er positive for de stromale markører perivascular, CD105, CD73 og CD90 markører NG2, PDGFR-B og CD146, mens negative for CD31, CD34, CD45 og HLA-DR (figur 2B). Typisk, en homogen population af kPSCs kan nås på passage 4 og kPSCs nå gulne omkring passage 9-10 (figur 2C). Vi anbefaler derfor for at udføre eksperimenter mellem passage 5-8.
For at evaluere den funktionelle kapacitet af de isolerede kPSCs, udfører vi en in vitro- nyre epitelial sår scratch assay på hver ny batch af kPSCs, som vi tidligere viste at konditioneret medium af kPSCs kan fremskynde epitelial sårheling i denne scratch assay15. KPSC konditioneret medium er lavet af dyrkning kPSCs for 48 h i alphaMEM 5% trombocyttal lysates og indsamle supernatanten. Næste, udødeliggjort menneskelige nyre proksimale tubulus epitelceller (HK-2)17 er kulturperler indtil sammenflydende og derefter et skrab sår er lavet. Derefter enten kPSCs eller kontrol medium konditioneret medium føjes til brøndene og hastigheden af sårheling måles. Såret lukkes når konditioneret medium af kPSCs er tilføjet, betydeligt hurtigere (figur 2D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55841/55841fig1.jpg"> 
Figur 1 : Kliniske Grade Isolation metode af menneskelige kPSCs. Transplantation grade nyrerne er kanylerede og perfunderet med collagenase via nyrearteriestenose (A - G) og den resulterende cellesuspension er vasket og enten befrugtede eller sætte i kultur (H - K). Efter cellerne nå confluency (L), udført NG2 celle berigelse er (M). Celler er trypsinized når de enten er sammenflydende, har stoppet prolifererende eller Hvornår 3D strukturer vises (N - P). Release kriterierne for kPSCs er sterilitet, markør udtryk og evnen til at øge rørformede epitelial sårheling (Q). Pil: nyrearteriestenose. Skalalinjen = 200 µm. <a href="http://ecsource.jove.com/files/ftp_upload/55841/55841fig1large.jpg" target="_blank">venligst klik her for at se en større version af dette tal.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55841/55841fig2.jpg"> 
Figur 2: karakterisering af menneskelige kPSCs. En) kPSCs er spindel-formet, plast vedhængende celler. B) kPSCs er positive for mesenchymale markører CD73, CD90, CD105, perivascular markører NG2, PDGFR-β og CD146, mens negative for CD31, CD34 og CD45. kPSCs express MHC-klasse jeg (HLA-ABC) men ikke klasse II (HLA-DR). C) vækst egenskaber af tre forskellige kPSC donorer fra flowcytometri bekræftet homogene NG2 positive populationer (på passage 4). kPSCs nå gulne omkring passage 9-10. D) kPSCs er købedygtig forøge nyre epitelial reparation i et sår scratch assay. Repræsentative billeder af kontrol medium og kPSC aircondition medium til t = 0, 4, 8 og 12 h er vist. Skalalinjen i A) = 200 µm i D) = 100 µm. <a href="http://ecsource.jove.com/files/ftp_upload/55841/55841fig2large.jpg" target="_blank">venligst klik her for at se en større version af dette tal.</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

Her præsenterer vi en roman kliniske grade isolation og kultur metode for nyre Perivascular stromale celler (kPSCs) baseret på hele orglet perfusion med fordøjelsesenzymer og NG2-celle berigelse. Med denne metode er det muligt at erhverve tilstrækkeligt celle numre for cellulære terapi.

En roman kliniske Grade Isolation metode for menneskelige nyre Perivascular stromale celler

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

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A Novel Vertebral Stabilization fremgangsmåde til fremstilling Contusive Rygmarvsskader

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

To metoder til fastsættelse primære humane Endometriecancer stromacellers fra Hysterektomi Prøver

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

Isolation, Kryopræservering og dyrkning af humane amnion epitelceller ved kliniske anvendelser

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

Prækliniske ortotopisk Murine Model of Human Prostata Cancer

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

Isolering og dyrkning af Primære endotelceller fra Canine arterier og vener

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

En detaljeret protokol for Sved Overvågning Ved hjælp af en roman, Small, trådløs enhed

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

Klinisk protokol producere fedtvæv-afledte stromale vaskulære brøkdel for potentielle brusk regenerering

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