Gli autori ringraziano per la fornitura di Katharina Schallmoser pHPL, Eva Rohde e Nicole Hofmann per gli utili commenti al manoscritto, Daniela Thaler e Margaretha P. hwirth di assistenza tecnica, Monica Farrell per la modifica linguistica, Uwe Lang, Margit Holzapfel e Sandra Eppich al Dipartimento di Ostetricia per fornire le corde. Questo lavoro è stato sostenuto dalla Fondazione per la ricerca austriaco (FWF, concedere N211-NAN per DS), l&#39;Agenzia austriaca per la promozione della ricerca (FFG, concedere N200 per DS) e da The Adult Stem Cell Research Foundation (AR). AR è un collega della Medicina Molecolare del programma di dottorato dell&#39;Università Medica di Graz.

Parte 1: Installazione 1. Preparazione del terreno di coltura Prima di iniziare la preparazione media di raccogliere tutti i materiali e gli strumenti di cui avrete bisogno. Scongelare 2 x 56 ml di lisato pool delle piastrine umane (pHPL, self-made: riferimento 1 &amp; JOVE # 1523), 10 ml di una penicillina 100x / soluzione di streptomicina, 2 x 5 mL di L-Glutammina (entrambi Sigma). Scongelare la citochina e aliquote fattore di crescita (VEGF, bFGF, EGF, IGF, idrocortisone, acido ascorbico, eparina, Amfotericina forniti come &#39;quots singolo&#39;), rettificato per integrare 1 bottiglia (500 ml) di EGM-2 medio (Lonza). Dal frigo fare un flacone da 500 ml ciascuna (i) alfa-modificato minima quantità di terreno essenziale (a-MEM) e (ii) media endoteliale basale (EBM). Tutte le seguenti operazioni vengono eseguite in una cappa flusso laminare cultura dei tessuti in condizioni sterili. Preparare eparina senza conservanti (ad esempio, Biochrom), sciogliendo la polvere ad una concentrazione finale di 1000 UI / mL con acqua sterile. MSC-media: Utilizzare 500 ml di a-MEM, aggiungere 56 ml di scongelato pHPL (vedi anche riferimento 1 per ulteriori dettagli) e 2 IU / mL (= ml di soluzione madre 224) senza conservanti di eparina (evita la coagulazione del mezzo attraverso aggregazione di il fibrinogeno nel plasma) per raggiungere una concentrazione finale del 10% pHPL. Inoltre aggiungere penicillina (100U/mL) / streptomicina (100μg/mL) soluzione e 2mM di L-Glutamin (entrambi Sigma). Filtrare il medium attraverso un poro 20 micron filtro dimensioni del vuoto (Millipore). Etichetta della bottiglia appropriato (contenuto, data). ECFC-media: Utilizzare una bottiglia (500 ml) di EBM, aggiungere il citochine aliquote, 56 ml di pHPL, 10 UI / ml (= 1120μl di soluzione madre) di conservanti eparina, penicillina (100U/mL) / Streptomicina (100 g / mL) e 2mM soluzione di L-Glutamin al mezzo basale e il filtro con 20 l poro vuoto dimensione del filtro (Millipore). Etichetta della bottiglia appropriato (contenuto, data). 2. Sterilizzazione degli strumenti chirurgici Pinze, paio di forbici affilate e titolare bisturi chirurgico deve essere caldo e getta minerale sterilizzati sterile. 3. Preparazione di tubi per la raccolta del midollo Aggiungere 500 UI di conservante - eparina libera a 50 tubi in polipropilene ml (Falcon) e regolare di un volume finale di 20 ml con PBS. Trasferire i tubi in sala parto. 4. Il consenso informato (confermate dal comitato etico locale o IRB). Chiedere ai genitori prima della consegna se volesse donare un pezzo di corda e adeguatamente li informa delle finalità servirà. 5. Donazione del midollo Dopo la consegna e l&#39;ispezione di placenta e cordone tagliare un pezzo di 10 cm e immediatamente trasferire al tubo preriempita con eparina per evitare la coagulazione del sangue all&#39;interno dei vasi rimanenti cavo. Continuare con isolamento delle cellule il più presto possibile. Parte 2: isolamento delle cellule <ol><li> Preparare cappa a flusso laminare tessuti cultura pulizia delle superfici con Incidin o 70% EtOH. Luogo sterile 150 centimetri 2 piastre di coltura cellulare, 75 cm fiasche di coltura cellulare (entrambi Corning), PBS, strumenti chirurgici sterilizzati, raschietti cellulare, titolare del tubo, 25 stripettes ml e 5 ml siringhe dotate di aghi senza punta giustamente in vostro cappuccio pulito. </li><li> Preriscaldare il vostro preparato α-MEM e EGM-2 terreno di coltura cellulare a 37 ° C in un bagno d&#39;acqua. </li><li> Portare il cavo dalla sala parto al laboratorio dotato di cappuccio colture cellulari. Dopo aver trasferito il cavo nel flusso laminare lavarlo due volte in PBS (attraverso il trasferimento di una nuova piastra) per sbarazzarsi di contaminazione cellule del sangue. Sciacquare la vena ombelicale dopo canulating su un lato con una siringa da 5 ml sterili con ago senza punta con PBS fino a quando il liquido che esce l&#39;altra estremità del cavo diventa completamente trasparente (rossastro indica la contaminazione del sangue). </li></ol> ECFCs: <ol start="4"><li> Preparare un centimetro 75 2 pallone (Corning), si etichetta e compilare 15-20 ml di pre-riscaldato EGM-2 di media. </li><li> Posizionare il cavo in una nuova piastra piena di PBS sterile. Prendete le forbici chirurgiche e tagliare il cordone ombelicale in due pezzi di circa 5 cm di lunghezza (brevi pezzi sono più facili da lavorare, perché i vasi ombelicali sono intrecciati lungo il loro asse). </li><li> Prova a trovare la vena ombelicale (recipiente di grandi dimensioni), inserire un filo d&#39;tuo forbici chirurgiche e tagliare la vena longitudinalmente. A questo punto potrebbe essere utile se un assistente può contenere la vena già tagliato con due pinze, mentre si taglia ulteriormente. </li><li> Posizionare il cavo di tortace con la vena tagliata in un piatto nuovo senza PBS, prendere il raschietto cellulare e di raschiare l&#39;interno (endotelio) della superficie della vena ombelicale da sfregamenti da un capo all&#39;altro. Trasferire le cellule al fiasco preparato da pulire la punta del raschietto nel mezzo. Ripetere questa procedura almeno 10 volte. </li><li> Chiudere il pallone e metterlo in un incubatore (37 ° C, 5% CO 2, 95% di umidità). </li></ol> MSC: <ol start="4"><li> Preparare un centimetro 150 2 piastra di coltura (Corning) ed etichettarlo. </li><li> Prendete il pezzo di corda (dopo raschiando via lo strato endoteliale) e tagliare il cordone tutto in pezzi molto piccoli, utilizzando forbici sterili e bisturi sterile. Dimensione massima dei pezzi cavo devono essere 1-2 mm. </li><li> Trasferite i vostri pezzi di cavo con la pinza sterile nel pre-etichettati piastra di coltura. Lasciando aperta la piastra per 5 minuti prima riempiendo di media, l&#39;attaccamento dei pezzi per la superficie in plastica si rafforza. </li><li> Una volta che i pezzi di cavo sono collegati opportunamente alla plastica, aggiungere delicatamente in 30-35 ml di pre-riscaldato alfa modificato MEM. Cercate di usare la velocità minima per essere sicuri che tutti i pezzi rimangono attaccati. </li><li> Chiudere la piastra e metterla in un incubatore (37 ° C, 5% CO 2, 95% di umidità). </li></ol> Parte 3: l&#39;espansione cellulare e la conferma di immuno-fenotipo mediante citometria di flusso Cellula di espansione EFCF: <ol><li> Controllare il fissaggio delle cellule endoteliali del giorno successivo su un microscopio invertito e lo scambio di tutto EGM-2 di media per sbarazzarsi di tutti i non iscritti cellule e particelle. </li><li> Espandere le cellule fino a confluenza e di scambio un terzo della media due volte alla settimana. Quando confluenza viene raggiunto, staccare le cellule con 0,05% Trypsin/0.7 mM EDTA e trasferirli alle navi nuova cultura. A seconda del numero di cellule all&#39;interno del vostro T75 fiasco, si consiglia di scegliere le dimensioni e il numero dei tuoi fiaschi nuovo per garantire bassa densità di semina per un&#39;ulteriore espansione. </li><li> Scambio un terzo della media due volte alla settimana durante l&#39;espansione delle cellule. </li></ol> MSC: <ol><li> La conseguenza di cellule staminali mesenchimali dal midollo ci vorrà più tempo, in modo da poter attendere almeno 3-4 giorni fino a che controllare su un microscopio invertito per la comparsa di cellule fusiformi nei dintorni dei pezzi di tessuto attaccato. Quando c&#39;è di cellule sufficiente out-crescita, i pezzi tendono a staccarsi, perché perdere il contatto con la plastica. </li><li> Dopo 10-12 giorni rimuovere i pezzi di tessuto, staccare le cellule staminali mesenchimali dalla plastica con 0,05% Trypsin/0.7 mM EDTA e trasferirli alle navi nuova cultura. A seconda della quantità di cellule all&#39;interno del vostro piatto si dovrebbe decidere le dimensioni e il numero dei tuoi fiaschi nuovo per garantire bassa densità di semina per un&#39;ulteriore espansione. </li><li> Scambio un terzo della media due volte alla settimana durante l&#39;espansione delle cellule. </li></ol> Citometria a flusso Attrezzatura necessaria: Citometro a flusso (BD FACSCalibur), micronici tubi (Corning), titolare del tubo, pecore siero (il blocco dei reagenti), Eppifuge (Eppendorf), gli anticorpi monoclonali MSC: CD45, CD14, CD19, HLA-DR, CD31, CD73, CD90, CD105, controlli isotipo ECFCs: CD45, CD14, CD19, HLA-DR, CD31, CD34, CD90, CD105, CD144, CD146, controlli isotipo <ol><li> Dopo aver raggiunto confluenza, staccare il cellulare con il 0,05% Trypsin/0.7 mM EDTA e centrifugare a 300g per 7 minuti a 4 ° C. </li><li> Risospendere le cellule ad una concentrazione di 1x10 6 cellule / ml in PBS e modificato legame degli anticorpi aspecifici blocco con il 10% v / v pecore siero per 20 minuti a 4 ° C. </li><li> Etichettare le provette micronici e aggiungere la giusta concentrazione di anticorpi marcati fluorescenti. </li><li> Aggiungere 50 ml di sospensione cellulare (bloccato) a ciascuna provetta ed incubare per 30 minuti a 4 ° C. </li><li> Lavare le cellule modificate con l&#39;aggiunta di PBS per rimuovere gli anticorpi non vincolante. </li><li> Vai con il tuo cellulare etichettati al citofluorimetro ed eseguire l&#39;analisi. </li></ol> Parte 4: ECFC crescita colonia, fissazione, colorazione e analisi gerarchia <ol><li> Il seme ECFCs praticamente puro raccolte a densità molto bassa placcatura di 3 o 10 cellule per centimetro quadrato in piastre di coltura cellulare per consentire la crescita singola colonia. </li><li> Scambio un terzo della media due volte alla settimana. </li><li> Dopo 14 giorni (fine della cultura) rimuovere media, lavare due volte con PBS e fissare le colonie con una soluzione di fissaggio ghiacciata (Acetone: Metanolo = 3: 2) per 15 minuti in frigorifero. </li><li> Scartare la soluzione di fissaggio e lasciare le piastre aperto ad asciugare per 10 minuti. </li><li> Reidratare le cellule con acqua distillata per 10 minuti. </li><li> Aggiungi Harris soluzione ematossilina e macchia le colonie fisso per 12 minuti. </li><li> Rimuovere H matoxylin soluzione e sciacquare i piatti con acqua di rubinetto per eliminare la soluzione colorante rimanente. </li><li> Scatta foto di ogni colonia diutilizzando uno stereomicroscopio e importare *. jpg o *. tif file in formato sul computer. </li><li> Aprire la foto con il software ImageJ ed analizzare il numero preciso delle cellule di ogni colonia, come descritto nella animazione. </li><li> Aprire il software ImageJ e importare un&#39;immagine colonia utilizzando il &#39;File \ Apri&#39; la funzione nel menu a discesa. </li><li> Procedere utilizzando il &#39;Processo \ Background Sottrarre&#39; funzione </li><li> Utilizzare la funzione &#39;selezione ellittica * * o pennello&#39; nel menu ImageJ per segnare il margine di colonia. </li><li> Chiaro che circonda l&#39;immagine utilizzando il &#39;Modifica \ Fuori Clear&#39; funzione e ritagliare l&#39;immagine, selezionando &#39;Immagine Crop \&#39;. </li><li> Regolare la soglia manualmente con &#39;Image \ Regolare la soglia \&#39; funzione, in modo che tutte le cellule sono completamente colorati in rosso. </li><li> Contare il numero di cellule della colonia, selezionando &#39;Analizza \ analizzare particelle&#39;. Scegliere le impostazioni appropriate (ottimizzate per ogni tipo di cellula) e selezionare &#39;OK&#39;. La casella di risultati, tra cui numero di celle e una nuova immagine contenente i contorni corrispondente delle cellule contate apparirà. <img src="/files/ftp_upload/1525/1525op2.jpg" alt="Figura 1" /></li></ol>

<ol>
	<li>Schallmoser, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+platelet+lysate+can+replace+fetal+bovine+serum+for+clinical-scale+expansion+of+functional+mesenchymal+stromal+cells.">Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells.</a> Transfusion. 47, 1436-1446 (2007).</li><li>Reinisch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+system+to+propagate+cord+blood-derived+multipotent+mesenchymal+stromal+cells+for+clinical+application.">Humanized system to propagate cord blood-derived multipotent mesenchymal stromal cells for clinical application.</a> Regen Med. 2, 371-382 (2007).</li><li>Reinisch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+large-scale+expanded+endothelial+colony-forming+cells+function+in+vitro+and+in+vivo.">Humanized large-scale expanded endothelial colony-forming cells function in vitro and in vivo.</a> Blood. 113, 6716-6725 (2009).</li><li>Strunk, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+characterization+and+preclinical+production+of+human+lineage-negative+cells+for+regenerative+stem+cell+therapy.">Phenotypic characterization and preclinical production of human lineage-negative cells for regenerative stem cell therapy.</a> Transfusion. 45, 315-326 (2005).</li></ol>

Gli autori non hanno alcun conflitto di interessi da dichiarare.

C&#39;è una varietà di fonti da cui ottenere cellule staminali mesenchimali o ECFCs compreso il midollo osseo, tessuto adiposo, sangue del cordone ombelicale, ecc E &#39;necessario per ottenere entrambi i tipi di progenitori dalla stessa fonte di confrontare direttamente il coinvolgimento dei diversi tipi di cellule in processi come tessuti e la rigenerazione nave o contributo alla progressione del tumore. La facilità con cui isolare e successivamente studiare...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>alpha modified MEM</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>L-GLutamin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>EBM</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Single cytokine quots</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>75 cm2 flasks</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>150 cm2 plates</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cell scraper</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin/EDTA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Monoclonal antibodies</td>
<td>&nbsp;</td>
<td>Becton Dickenson</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>50 ml tubes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>500 mL filter 20 &mu;m pore size </td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Preservative free heparin</td>
<td>&nbsp;</td>
<td>Biochrom</td>
<td>&nbsp;</td>
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isolation and animal serum free expansion of human umbilical cord derived mesenchymal stromal cells (mscs) and endothelial colony forming progenitor cells (ecfcs)

Il cordone ombelicale è una ricca fonte di cellule progenitrici con alto potenziale proliferativo delle cellule stromali mesenchimali compreso (designato anche le cellule staminali mesenchimali, MSC) e cellule progenitrici endoteliali formanti colonia (ECFCs). Entrambi i tipi di cellule hanno un ruolo chiave nel mantenere l&#39;integrità dei tessuti e sono probabilmente coinvolti nei processi di rigenerazione e formazione di tumori. Per studiare la loro biologia e la funzione in termini comparativi, è importante avere entrambi i tipi di cellule a disposizione dallo stesso donatore. Può anche essere utile per scopi rigenerativi per derivare cellule staminali mesenchimali e ECFCs dal tessuto stesso. Poiché la terapia cellulare dovrebbe finalmente trovare la loro strada dal laboratorio al letto abbiamo stabilito un nuovo metodo per isolare ed espandere ulteriormente le cellule progenitrici senza l&#39;uso di proteine ​​animali. Pool di lisato piastrinico umano (pHPL) sostituito siero fetale bovino in tutte le fasi del nostro protocollo al fine di evitare completamente il contatto delle cellule alle proteine ​​xenogene. Questo video dimostra una metodologia per l&#39;isolamento e l&#39;espansione delle cellule progenitrici da un cordone ombelicale. Tutti i materiali e le procedure saranno descritte.

Watch this Scientific Journal Video about Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

Questo protocollo descrive l&#39;isolamento e la successiva espansione delle cellule stromali mesenchimali e le cellule endoteliali che formano colonie senza l&#39;uso di siero animale per generare le coppie a fini di trapianto autologo sperimentali.

Isolamento e di espansione degli animali siero gratuito di cordone ombelicale umano derivato cellule stromali mesenchimali (MSC) e cellule endoteliali progenitrici formanti colonia (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

isolation-animal-serum-free-expansion-human-umbilical-cord-derived

Research

JoVE Journal

Biology

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

15.4K Views.  Medical University of Graz, Austria. Questo protocollo descrive l'isolamento e la successiva espansione delle cellule stromali mesenchimali e le cellule endoteliali che formano colonie senza l'uso di siero animale per generare le coppie a fini di trapianto autologo sperimentali.

Video: Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab.

Isolamento di cellule umane endoteliali della vena ombelicale (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps ...

Ricerca

Biologia

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

Isolamento e di espansione su vasta scala di umano adulto cellule progenitrici endoteliali che formano colonie

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and can be easily separated from the hematopoietic stem cells (HSCs) due to their plastic adherence. This protocol describes how to isolate MSCs from rat femurs and tibias. The isolated cells were further enriched against two MSCs surface markers CD54 and CD90 by magnetic cell sorting. Expression of surface markers CD54 and CD90 were then confirmed by flow cytometry analysis. HSC marker CD45 was also included to check if the sorted MSCs were depleted of HSCs. MSCs are naturally quite heterogeneous. There are subpopulations of cells that have different shapes, proliferation and differentiation abilities. These subpopulations all express the known MSCs markers and no unique marker has yet been identified for the different subpopulations. Therefore, an alternative approach to separate out the different subpopulations is using cloning cylinders to separate out single-colony derived cells. The cells derived from the single-colonies can then be cultured and evaluated separately.

Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs

Rat MSCs were isolated from femurs and tibias and then enriched by magnetic cell sorting. Sorted cells were confirmed for the expression of surface markers by flow cytometry. These cells were also cultured at clonal density to form single colonies and then these colonies were separated by cloning cylinders.

L&#39;isolamento e l&#39;arricchimento delle cellule staminali mesenchimali di ratto (MSC) e la separazione dei Single-colonia di cellule staminali mesenchimali derivate

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and ...

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded and their collection does not imply any added risk to the newborn or mother s health. Moreover no ethical concerns are raised. The UC is composed by one vein and two arteries from which both endothelial cells (ECs) 1 and smooth muscle cells (SMCs) 2, two of the main cellular components of blood vessels, can be isolated. In this project the SMCs were obtained after enzymatic treatment of the UC arteries accordingly the experimental procedure previously described by Jaffe et al 3. After cell isolation they were kept in t-flash with DMEM-F12 supplemented with 5% of fetal bovine serum and were cultured for several passages. Cells maintained their morphological and other phenotypic characteristics in the different generations. The aim of this study was to isolate smooth muscle cells in order to use them as models for future assays with constrictor drugs, isolate and structurally characterize L-type calcium channels, to study cellular and molecular aspects of the vascular function 4 and to use them in tissue engineering.

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

The umbilical cords are used to isolate smooth muscle cells by different ways. In this work we used the enzymatic treatment to isolated smooth muscle cells.

Isolamento di cellule umane ombelicale muscolatura liscia arteriosa (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of circulating endothelial progenitor cells (EPCs) were first identified in adult human peripheral blood by Asahara et al. in 1997 2 bringing an infusion of new hypotheses and strategies for vascular regeneration and repair. EPCs are rare but normal components of circulating blood that home to sites of blood vessel formation or vascular remodeling, and facilitate either postnatal vasculogenesis, angiogenesis, or arteriogenesis largely via paracrine stimulation of existing vessel wall derived cells3. No specific marker to identify an EPC has been identified, and at present the state of the field is to understand that numerous cell types including proangiogenic hematopoietic stem and progenitor cells, circulating angiogenic cells, Tie2+ monocytes, myeloid progenitor cells, tumor associated macrophages, and M2 activated macrophages participate in stimulating the angiogenic process in a variety of preclinical animal model systems and in human subjects in numerous disease states4, 5. Endothelial colony forming cells (ECFCs) are rare circulating viable endothelial cells characterized by robust clonal proliferative potential, secondary and tertiary colony forming ability upon replating, and ability to form intrinsic in vivo vessels upon transplantation into immunodeficient mice6-8. While ECFCs have been successfully isolated from the peripheral blood of healthy adult subjects, umbilical cord blood (CB) of healthy newborn infants, and vessel wall of numerous human arterial and venous vessels 6-9, CB possesses the highest frequency of ECFCs7 that display the most robust clonal proliferative potential and form durable and functional blood vessels in vivo8, 10-13. While the derivation of ECFC from adult peripheral blood has been presented14, 15, here we describe the methodologies for the derivation, cloning, expansion, and in vitro as well as in vivo characterization of ECFCs from the human umbilical CB.

Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

Caratterizzazione fenotipica e funzionale delle cellule endoteliali Colony Forming derivato dal sangue del cordone ombelicale umano

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

Brown adipocytes have the ability to uncouple the respiratory chain in mitochondria and dissipate chemical energy as heat. Development of UCP1-positive brown adipocytes in white adipose tissues (so called beige or brite cells) is highly induced by a variety of environmental cues such as chronic cold exposure or by PPAR&gamma; agonists, therefore, this cell type has potential as a therapeutic target for obesity treatment. Although most immortalized adipocyte lines cannot recapitulate the process of "browning" of white fat in culture, primary adipocytes isolated from stromal vascular fraction in subcutaneous white adipose tissue (WAT) provide a reliable cellular system to study the molecular control of beige/brite cell development. Here we describe a protocol for effective isolation of primary preadipocytes and for inducing differentiation to beige/brite cells in culture. The browning effect can be assessed by the expression of brown fat-selective markers such as UCP1.

Primary white preadipocytes isolated from white adipose tissues in mice can be differentiated into beige/brite cells. Presented here is a reliable cellular model system to study the molecular regulation of "browning" of white fat.

Isolamento e la differenziazione di cellule stromali vascolari alle celle Beige / Brite

Brown adipocytes have the ability to uncouple the  respiratory chain in mitochondria and dissipate chemical energy as heat.  Development of UCP1-positive ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Induzione e Analisi dei epiteliale a Mesenchymal Transizione

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Isolamento di cellule murine endoteliali Valve

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

Isolamento di CD146<sup&gt; +</sup&gt; cellule residenti del polmone mesenchimali stromali da Rat Polmoni

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

Isolamento e arricchimento di cellule progenitrici epiteliali polmonari umane per la coltura di organoidi

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...

Isolamento e di espansione degli animali siero gratuito di cordone ombelicale umano derivato cellule stromali mesenchimali (MSC) e cellule endoteliali progenitrici formanti colonia (ECFCs)

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