This work was supported in part by grants from NHRI (CS-104-PP-06 to B.L.Y.).

Il consenso informato del paziente, come approvato dal comitato istituzionale di revisione deve essere ottenuto per l&#39;uso di cellule umane. 1. Densità Isolamento gradiente di cellule periferiche mononucleari Sangue umano (PBMC) <ol><li> Aggiungere 25 ml di sangue intero eparinizzati in un tubo da 50 ml con una pipetta 25 ml. <ol><li> Diluire le cellule con 25 ml di PBS (PBS). </li></ol></li><li> Aggiungere 15 ml di gradiente di densità Ficoll-Paque in un nuovo tubo 50 ml, e mentre inclinando il tubo, molto lentamente e con attenzione aggiungono in 25 ml di sospensione cellulare diluita su gradiente di densità in modo che non vi è alcuna miscelazione del sangue intero con gradiente di densità, cioè., nessun disturbo dell&#39;interfaccia gradiente di densità del sangue. </li><li> Centrifugare la sospensione dal punto 1.2 a 400 g per 30 e 40 minuti in un rotore oscillante secchiello temperatura controllata senza freno a 20 ° C. Nota: Tre strati distinti dovrebbero essere evidenti dopo la centrifugazione: °e l&#39;essere al plasma strato superiore; lo strato inferiore è chiaro gradiente di densità Ficoll-Paque; e uno strato cellulare sottile, centrale è il PBMCs </li><li> Aspirare e scartare lo strato superiore per aspirazione con una pipetta Pasteur, con attenzione a non disturbare lo strato interfase delle cellule mononucleate (cioè, linfociti, monociti e piastrine). </li><li> Raccogliere accuratamente questo strato di cellule mononucleate con una pipetta 10 ml in un tubo da centrifuga da 50 ml. </li><li> Riempire il tubo con 20 ml di PBS, mescolare bene e centrifugare a 300 xg per 10 min a 20 ° C. Rimuovere il surnatante completamente dopo la centrifugazione. </li><li> Risospendere il pellet cellulare in 20 ml di PBS e centrifugare a 200 xg per 10-15 minuti a 20 ° C. Rimuovere il surnatante completamente dopo la centrifugazione. Nota: Questo passaggio rimuove le piastrine-che sono indesiderate, all&#39;interno delle PBMC; questo passaggio può essere ripetuto per assicurare la rimozione completa delle piastrine. </li><li> Se nessuna selezione di popolazioni specifiche èdesiderato, pellet cellulare risospendere in leucociti terreno completo (10% di siero fetale bovino (FBS), 1% di L-glutammina, e 1% di penicillina / streptomicina in terreno RPMI-1640) per la coltura, dopo l&#39;esecuzione di un numero di cellule 16 di sospendere 1 ml di medio per 10 7 PBMC prima di procedere alla fase successiva. </li></ol> 2. magnetica Etichettatura dei leucociti popolazioni (passare alla Fase 4 se nessuna selezione di popolazioni specifiche è obbligatorio) <ol><li> Sospensione centrifugare PBMC a 300 × g per 10 minuti e aspirare il surnatante completamente. </li><li> Pellet cellulare Risospendere in 80 ml ​​di PBS per 10 7 cellule totali. </li><li> Aggiungere 20 ml di sfere magnetiche specifici (ad esempio, CD14 sfere magnetiche per l&#39;isolamento dei monociti, CD4 sfere magnetiche per l&#39;isolamento dei linfociti T) per 10 7 cellule totali. </li><li> Mescolare bene e incubare per 15 minuti a 2 e 8 ° C in frigorifero. </li><li> Aggiungere 1-2 ml di PBS per 10 7 cellule per lavare e centrifugarea 300 g per 10 min. </li><li> Aspirare il surnatante e risospendere completamente fino a 10 8 cellule in 500 ml di PBS. </li></ol> 3. magnetica Selezione delle sottopopolazioni di leucociti Nota: Una varietà di separatori perline magnetiche e le colonne sono disponibili per l&#39;isolamento della specifica popolazione di leucociti da marcatore di superficie cellulare da un certo numero di produttori. L&#39;isolamento può essere da una selezione positiva sulla base di uno o pochi marcatori positivi o selezione negativa dopo l&#39;esaurimento delle popolazioni indesiderate. Un orientamento generale di eseguire selezione positiva utilizzando un marcatore (ad esempio, per i linfociti T CD4 helper, o CD14 per i monociti) è indicato di seguito. <ol><li> Preparare e separatore magnetico primo compresa colonna di separazione secondo le istruzioni del produttore. </li><li> Applicare provetta contenente il campione di PBMC etichettati. Fornire due 15 ml provette per la raccolta dell &#39;ed unlabel etichettata positivamentefrazioni cellulari ndr, seguendo le istruzioni del produttore. </li><li> Contare il numero di cellule 15 e sospendere nuovamente le leucociti selezionati positivamente (ad esempio, linfociti T CD4 + CD14 + o cellule) in leucociti terreno completo, utilizzando 1 ml di terreno per 10 7 cellule. </li></ol> 4. CFSE colorazione di leucociti per la valutazione di proliferazione Nota: CFSE è stato ampiamente utilizzato in indagini immunologiche, sia in vivo e in vitro. Il nostro protocollo è stato ottimizzato per lo studio in vitro di MSC / PBMC (o altri leucociti) interazioni. Mentre i passaggi generali coinvolti nella conduzione CFSE etichettatura in vitro sono simili, ci possono essere differenze di dosaggio specifica e la tempistica del protocollo 14. Ciò può essere dovuto a una serie di fattori, vale a dire, il tipo di cellula specifico utilizzato, il numero di cellule utilizzate-che possono probabilmente influenzare l&#39;intensità del segnale fluorescente CFSE. <ol&gt; <li> Diluire la soluzione CFSE magazzino (10 mm) in PBS alla concentrazione di lavoro desiderata di 10 micron (soluzione CFSE-lavoro). Preparare 10 7 cellule (ad esempio, PBMC o cellule T) per l&#39;etichettatura con la soluzione CFSE-lavoro. </li><li> Centrifugare a 300 g per 10 minuti per ottenere un pellet di cellule e aspirare il surnatante. </li><li> Risospendere delicatamente le cellule in 1 ml di soluzione di pre-riscaldato (37 ° C) CFSE-lavoro e incubare le cellule per 10 min a 37 ° C. </li><li> Per lavare eccesso CFSE, diluire la sospensione cellulare con 10x (in volume) di preraffreddato (4 ° C) RPMI mezzo contenente 10% FBS. Sedimentare le cellule per centrifugazione a 300 xg per 5 minuti e scartare il surnatante. Lavare il pellet di cellule in questo modo per altre due volte. </li><li> Re-agglomerare le cellule per centrifugazione e contare il numero di cellule 15. Risospendere 10 7 cellule (sia PBMC o cellule T) in 1 ml di fresco leucociti preriscaldata terreno completo. </li></ol> 5. Co-coltura di MSC con leucociti e attivazione dei leucociti <ol><li> Pre-caldo MSC terreno completo (10% FBS (pretestati per una crescita ottimale MSC), 1% L-glutammina, 1% di penicillina e / streptomicina in terreno di glucosio DMEM-basso) a 37 ° C per non più di 30 min. </li><li> MSC Seed a 50.000 cellule in 1 ml di MSC terreno completo in piastre da 24 pozzetti (densità MSC: 25.000 cellule / cm 2) per il fissaggio O / N in un incubatore a 37 °, consentendo alle cellule staminali di raggiungere l&#39;80% di confluenza. </li><li> Media Aspirare, e sulla base di numeri MSC teste di serie, aggiungere PBMC CFSE etichettati (precedentemente etichettata-consultare sopra protocollo punto 4) a MSC coltivati ​​(testa di serie il giorno precedente in piastre da 24 pozzetti) in 1 ml di leucociti terreno completo in un 01:10 (rapporto cellulare) Rapporto co-coltura delle cellule staminali mesenchimali per PBMC. </li><li> Aggiungere il mitogeno fitoemoagglutinina (PHA), un non-specifico attivatore leucociti, ad una concentrazione finale di 10 ug / ml in un volume totale di 1 ml leucociti terreno completo per pozzetto. <ol><li> Alternatively, per stimolare l&#39;attivazione dei linfociti T in particolare: utilizzare alfa-CD3 / 28 microsfere e aggiungere al due cellule co-coltura per ottenere un rapporto di linfociti da tallone a T di 1: 1. </li></ol></li><li> Per il controllo negativo, piatto 500.000 PBMC / bene (o di una specifica popolazione effettore leucocitaria; densità cellulare: 250.000 leucociti / cm 2) in un 24-pozzetti con 1 ml di leucociti solo terreno completo; per il controllo positivo, oltre alla placcatura lo stesso numero di PBMC / pozzetto, aggiungere PHA ad una concentrazione finale di 10 mg / ml. </li><li> Al 3 ° e 5 ° giorno dell&#39;esperimento co-coltura, valutare la proliferazione dei leucociti CFSE marcati (posto in tubi tondi-basso) mediante analisi di citometria di flusso 17 con 488 nm di eccitazione ed emissione di appositi filtri per fluoresceina. Nota: intracellulare colorazione citochine per citometria a flusso può essere eseguita anche a questi leucociti CFSE marcato a questo punto per valutare per i cambiamenti nel profilo di espressione dei leucociti citochine come modulated da MSC. Poiché CFSE viene valutata con un adeguato filtro per fluoresceina, gli anticorpi selezionati per valutare varie citochine devono essere coniugato con fluorocromi diversi fluoresceina o spettro simile (ad esempio, ficoeritrina, peridinina complesso proteico clorofilla (PerCP)). </li></ol> 6. Variazione: Effector Soppressione Assay magnetico Bead-selezionata, MSC-indotta immunomodulante leucociti attivati ​​su CFSE etichettati cellule effettrici CD4 + T <ol><li> MSC Seed a 250.000 cellule in 3 ml di MSC terreno completo a 6 pozzetti (densità MSC: 25.000 cellule / cm 2) per il fissaggio O / N in un incubatore a 37 °, consentendo alle cellule staminali di raggiungere l&#39;80% di confluenza. Almeno 3 6 pozzetti sono necessari per garantire sufficiente di PBMC MSC-messi in coltura per la selezione sottopopolazione. </li><li> Aspirare il terreno, e aggiungere PBMC separati secondo la Fase 1, ma in 6 pozzetti a 2,5 x 10 6 cellule / pozzetto (densità delle cellule: 250.000 leukocytes / cm 2) con 3 ml di terreno completo leucociti. Co-coltura per 48-72 ore in un incubatore a 37 °. </li><li> Magnetic bead-seleziona specifica popolazione di leucociti immunomodulatori MSC-indotta (ad esempio, CD14 + cellule) come da Sezioni 2-3. Note: colorazione intracellulare di citochine per l&#39;analisi citometrica possono essere eseguite a questo punto per valutare variazioni di leucociti citochine profilo di espressione, cioè, espressione di interleuchina-10 come modulati dal MSC. </li><li> Add CFSE marcato allogeniche cellule T CD4 + generati secondo le sezioni 2-4 in piastre da 24 pozzetti in 1 ml di leucociti completa (densità delle cellule T: 250.000 cellule / cm 2) a medio leucociti MSC-indotta tallone-selezionato a vari rapporti , cioè, 1:10, 1: 5, 1: 2 e 1: 1 (cella per cella) rapporti. </li><li> Per stimolare le cellule T CD4 +, aggiungere alfa-CD3 / 28 microsfere coniugati per ottenere un rapporto di tallone-cellula di 1: 1. </li><li> Per un controllo negativo, piatto 500.000 cellule T CD4 + / bene in un (densità cellulare T: 250.000 cellule / cm 2) 24-pozzetti con 1 ml di leucociti solo terreno completo; per il controllo positivo, oltre alla placcatura lo stesso numero di cellule CD4 + T / pozzetto, aggiungere alfa-CD3 / 28 microsfere coniugate ad ottenere un rapporto da tallone a cella di 1: 1. </li><li> Il giorno 3 ° di co-coltura, valutare la proliferazione di CFSE etichettati cellule T CD4 + (posti in tubi tondi fondo) per citometria a flusso di analisi 16 con 488 nm di eccitazione ed emissione di appositi filtri per fluoresceina. </li></ol>

<ol>
	<li>Friedenstein, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precursor+cells+of+mechanocytes.">Precursor cells of mechanocytes.</a> Int Rev Cytol. 47, 327-359 (1976).</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, 143-147 (1999).</li><li>Prockop, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marrow+stromal+cells+as+stem+cells+for+nonhematopoietic+tissues.">Marrow stromal cells as stem cells for nonhematopoietic tissues.</a> Science. 276, 71-74 (1997).</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, 315-317 (2006).</li><li>Engler, A. J., Sen, S., Sweeney, H. L., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+elasticity+directs+stem+cell+lineage+specification.">Matrix elasticity directs stem cell lineage specification.</a> Cell. 126, 677-689 (2006).</li><li>Zuk, P. 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=Multilineage+cells+from+human+adipose+tissue:+implications+for+cell-based+therapies.">Multilineage cells from human adipose tissue: implications for cell-based therapies.</a> Tissue Eng. 7, 211-228 (2001).</li><li>Yen, B. L., 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+of+multipotent+cells+from+human+term+placenta.">Isolation of multipotent cells from human term placenta.</a> Stem Cells. 23, 3-9 (2005).</li><li>Erices, A., Conget, P., Minguell, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+progenitor+cells+in+human+umbilical+cord+blood.">Mesenchymal progenitor cells in human umbilical cord blood.</a> Br J Haematol. 109, 235-242 (2000).</li><li>Bartholomew, 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=Mesenchymal+stem+cells+suppress+lymphocyte+proliferation+in+vitro+and+prolong+skin+graft+survival+in+vivo.">Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo.</a> Exp Hematol. 30, 42-48 (2002).</li><li>Chang, C. 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=Placenta-derived+multipotent+cells+exhibit+immunosuppressive+properties+that+are+enhanced+in+the+presence+of+interferon-gamma.">Placenta-derived multipotent cells exhibit immunosuppressive properties that are enhanced in the presence of interferon-gamma.</a> Stem Cells. 24, 2466-2477 (2006).</li><li>Uccelli, A., Moretta, L., Pistoia, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+in+health+and+disease.">Mesenchymal stem cells in health and disease.</a> Nat Rev Immunol. 8, 726-736 (2008).</li><li>Chen, P. M., Yen, M. L., Liu, K. J., Sytwu, H. K., Yen, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomodulatory+properties+of+human+adult+and+fetal+multipotent+mesenchymal+stem+cells.">Immunomodulatory properties of human adult and fetal multipotent mesenchymal stem cells.</a> J Biomed Sci. 18, 49-59 (2011).</li><li>Muul, L. 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=Measurement+of+proliferative+responses+of+cultured+lymphocytes.">Measurement of proliferative responses of cultured lymphocytes.</a> Curr Protoc Immunol. , (2011).</li><li>Quah, B. J., Warren, H. S., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+lymphocyte+proliferation+in+vitro+and+in+vivo+with+the+intracellular+fluorescent+dye+carboxyfluorescein+diacetate+succinimidyl+ester.">Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester.</a> Nat Protoc. 2, 2049-2056 (2007).</li><li>Chen, P. 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=Induction+of+immunomodulatory+monocytes+by+human+mesenchymal+stem+cell-derived+hepatocyte+growth+factor+through+ERK1/2.">Induction of immunomodulatory monocytes by human mesenchymal stem cell-derived hepatocyte growth factor through ERK1/2.</a> J Leukoc Biol. 96, 295-303 (2014).</li><li>Oughton, J. A., Kerkvliet, N. I., Costa, L. G., Davila, J. C., Lawrence, D. A., Reed, D. J., Will, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UNIT+18.8+Immune+cell+phenotyping+using+flow+cytometry.">UNIT 18.8 Immune cell phenotyping using flow cytometry.</a> Curr Protoc Toxicol. , 18.8.1-18.8.24 (2005).</li><li>Phelan, M. C., Lawler, G., Robinson, J. P., 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=APPENDIX+3A+Cell+counting.">APPENDIX 3A Cell counting.</a> Curr Protoc Cytom. , A.3A.1-A.3A.4 (2001).</li><li>Gebler, A., Zabel, O., Seliger, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immunomodulatory+capacity+of+mesenchymal+stem+cells.">The immunomodulatory capacity of mesenchymal stem cells.</a> Trends Mol Med. 18, 128-134 (2012).</li><li>Le Blanc, K., 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+stem+cells+for+treatment+of+steroid-resistant,+severe,+acute+graft-versus-host+disease:+a+phase+II+study.">Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study.</a> Lancet. 371, 1579-1586 (2008).</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, 1169-1177 (2012).</li><li>Le Blanc, K., 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+stem+cells+inhibit+and+stimulate+mixed+lymphocyte+cultures+and+mitogenic+responses+independently+of+the+major+histocompatibility+complex.">Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex.</a> Scand J Immunol. 57, 11-20 (2003).</li><li>Li, X. Y., 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=Long-term+culture+in+vitro+impairs+the+immunosuppressive+activity+of+mesenchymal+stem+cells+on+T+cells.">Long-term culture in vitro impairs the immunosuppressive activity of mesenchymal stem cells on T cells.</a> Mol Med Rep. 6, 1183-1189 (2012).</li><li>Moll, 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=Do+cryopreserved+mesenchymal+stromal+cells+display+impaired+immunomodulatory+and+therapeutic+properties?.">Do cryopreserved mesenchymal stromal cells display impaired immunomodulatory and therapeutic properties?.</a> Stem Cells. 32, 2430-2442 (2012).</li><li>Ho, P. J., Yen, M. L., Tang, B. C., Chen, C. T., Yen, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H2O2+accumulation+mediates+differentiation+capacity+alteration,+but+not+proliferative+decline,+in+senescent+human+fetal+mesenchymal+stem+cells.">H2O2 accumulation mediates differentiation capacity alteration, but not proliferative decline, in senescent human fetal mesenchymal stem cells.</a> Antioxid Redox Signal. 18, 1895-1905 (2013).</li></ol>

We have nothing to disclose.

Increasingly, the immunomodulatory properties of MSCs are being translated into clinical use more rapidly than the multilineage capacity of these stem cells18-20. Thus, co-culture techniques of MSCs with leukocytes and assays to evaluate immune function are important to further delineate the specific mechanisms involved in these properties for optimizing effective therapeutic application. 
One of the most critical technical aspects for success in these assays is having adequate PBMC...

Le cellule staminali mesenchimali umane (MSC) sono progenitrici somatiche che possono differenziarsi in lignaggi mesodermiche parassiali di ossa, cartilagine e tessuto adiposo 1-4, così come ad alcuni lignaggi extramesodermal 5. In primo luogo isolato dal midollo osseo adulto, questi progenitori multilineage ora sono state trovate in numerosi tessuti 6-8 e, inaspettatamente, dimostrato di avere forti proprietà immunomodulanti che sembrano altamente suscettibili di applicazione clinica 9-12. Meccanismi dettagliati coinvolti negli effetti immunomodulatori sono attivamente indagati dell&#39;effettiva applicazione in entità patologiche specifiche. Uno dei modi più semplici per valutare immunomodulazione è valutando per la soppressione di effettori leucociti proliferazione 13. La maggior parte dei leucociti effettrici quali T linfociti e monociti proliferano prodigiosamente quando stimolato o attivato. Funzione immunomodulante può essere valutata quando la soppressione diproliferazione è evidenziato. Tradizionalmente, effettore leucociti proliferazione è stata valutata mediante rilevamento di [3 H] timidina nel DNA. Tuttavia, questo metodo ha svantaggi significativi a causa delle preoccupazioni di radiazioni e smaltimento post-utilizzo, così come il complesso attrezzature necessarie. Mentre ci sono saggi non radioattivi per valutare la proliferazione cellulare, l&#39;estere carboxyfluorescein succinimidil (CFSE) test ha anche altri vantaggi come ad esempio consentendo l&#39;identificazione delle popolazioni cellulari specifiche, il che è particolarmente utile in esperimenti di co-coltura di cellule che coinvolgono molteplici tipi. CFSE è un colorante cellulare fluorescente che può essere valutata mediante l&#39;analisi di citometria di flusso. Come le cellule si dividono, l&#39;intensità di questa etichetta cellulare diminuisce proporzionalmente; questo permette non solo la determinazione della proliferazione cellulare complessiva ma anche permette di valutare il numero di divisioni cellulari fino a 8 divisioni prima fluorescenza diventa difficile detect contro segnale di fondo. Inoltre, la stabilità della fluorescenza CFSE permette il monitoraggio in vivo di cellule marcate tali che le cellule possono essere visualizzati fino a molti mesi 14. Questo test può anche essere variata per valutare specifici tipi di leucociti effettrici o la funzione immunomodulante di specifiche popolazioni di MSC indotta leucociti-come immunomodulatori l&#39;interleuchina-10 (IL-10), la produzione di monociti CD14 + 15 eseguendo magnetico tallone selezione marcatore superficie popolazioni di cellule di interesse prima o dopo co-coltura a seconda dei casi. Il nostro protocollo descrive il saggio base di valutare gli effetti immunomodulatorie delle MSC sui leucociti effettrici (diagramma di flusso mostrato nella Figura 1) e una variazione su questo test base per la valutazione di MSC indotta immunomodulazione leucociti su allogeniche CD4 + effettrici linfociti T (diagramma di flusso mostrato in figura 4).

<table border="0" cellpadding="0" cellspacing="0" width="1205">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Ficoll-Paque PLUS</td>
			<td style="width:259px;">GE Healthcare</td>
			<td style="width:121px;">71-7167-00 AG</td>
			<td style="width:549px;">Density grandient for isolation of peripheral blood mononuclear cells (PBMCs)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:276px;">Vibrant CFDA-SE Cell Tracer Kit (CFSE)</td>
			<td style="width:259px;">Life Technologies</td>
			<td style="width:121px;">V12883</td>
			<td>Cellular label for detection of cell division</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Phytoagglutinin (PHA)</td>
			<td style="width:259px;">Sigma</td>
			<td>L8902</td>
			<td>Activation of human PBMCs</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:276px;">Dynabeads Human T-Activator CD3/28</td>
			<td style="width:259px;">Life Technologies</td>
			<td style="width:121px;">111.32D</td>
			<td style="width:549px;">Activation of human T lymphocytes, e.g. CD4+ T cells, CD8+ T cells,etc.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">autoMACS&#8482; Separator</td>
			<td>Miltenyi Biotec</td>
			<td style="width:121px;">autoMACS&#8482; Separator</td>
			<td>Magnetic based cell separator</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">autoMACS&#174; Columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-021-101</td>
			<td>separation columns</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:276px;">CD14 microbeads, human&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-050-201</td>
			<td style="width:549px;">For positive selection of CD14+ human monocytes and macrophages from PBMCs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">CD4 microbeads, human&#160;</td>
			<td>Miltenyi Biotec</td>
			<td style="width:121px;">130-045-101</td>
			<td style="width:549px;">For positive selection of CD4+ human T lymphocytes from PBMCs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">RPMI 1640 Medium</td>
			<td style="width:259px;">Life Technologies</td>
			<td style="width:121px;">11875</td>
			<td>Human PBMC/leukocyte culture medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">DMEM, Low glucose, pyruvate</td>
			<td style="width:259px;">Life Technologies</td>
			<td style="width:121px;">11885</td>
			<td style="width:549px;">Human mesenchymal stem cell (MSC) culture medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">L-glutamine</td>
			<td style="width:259px;">Life Technologies</td>
			<td>25030-081</td>
			<td>Supplementation for MSC complete medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Penicillin/Streptomycin</td>
			<td style="width:259px;">Life Technologies</td>
			<td>15070-063</td>
			<td>Supplementation for PBMC/leukocyte and MSC complete medium</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:276px;">Fetal bovine serum (FBS)</td>
			<td style="width:259px;">1) Hyclone, for MSC culture&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 2) Life Technologies, for all other cells (i.e. PBMCs, specific leukocyte populations)</td>
			<td style="width:121px;">1) SH30070.03M 2) 10091-148</td>
			<td>Pre-test lots for support of MSC in vitro culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">24-well cell culture plate</td>
			<td style="width:259px;">Corning</td>
			<td>COR3524</td>
			<td>Co-culture plate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">50 mL centrifuge tube</td>
			<td style="width:259px;">Corning</td>
			<td style="width:121px;">430291</td>
			<td>Isolation PBMCs from whole blood by Ficoll-Paque PLUS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">15 mL centrifuge tube&#160;</td>
			<td style="width:259px;">Corning</td>
			<td style="width:121px;">430766</td>
			<td>Collection of the labeled and unlabeled cell fractions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Round-bottom tubes</td>
			<td style="width:259px;">BD Falcon&#160;</td>
			<td style="width:121px;">352008</td>
			<td>Collection of cells for flow cytometric analysis</td>
		</tr>
	</tbody>
</table>

assessment of the immunomodulatory properties of human mesenchymal stem cells (mscs)

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of immune-related diseases. Mechanisms involved are increasingly being elucidated and in this article, we describe the basic experiment to assess MSC immunomodulation by assaying for suppression of effector leukocyte proliferation. Representing activation, leukocyte proliferation can be assessed by a number of techniques, and we describe in this protocol the use of the fluorescent cellular dye carboxyfluorescein succinimidyl ester (CFSE) to label leukocytes with subsequent flow cytometric analyses. This technique can not only assess proliferation without radioactivity, but also the number of cell divisions that have occurred as well as allowing for identification of the specific population of proliferating cells and intracellular cytokine/factor expression. Moreover, the assay can be tailored to evaluate specific populations of effector leukocytes by magnetic bead surface marker selection of single peripheral blood mononuclear cell populations prior to co-culture with MSCs. The flexibility of this co-culture assay is useful for investigating cellular interactions between MSCs and leukocytes.

Figura 1 denota lo schema generale dell&#39;esperimento, e la figura 2 mostra l&#39;aspetto delle diverse condizioni di coltura cellulare come visualizzato in contrasto di fase invertita microscopia. Le MSC sono cellule aderenti con fibroblastica, morfologia a forma di fuso, mentre PBMC e leucociti sono piccole cellule non aderenti rotonde. Questi due tipi di cellule morfologicamente differenti può essere visto chiaramente nella co-coltura. Al termine del test, quando le PBMC (o leuckotyes) vengono aspirat...

Watch this Scientific Journal Video about Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs at JoVE.com

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

Le proprietà immunomodulanti di cellule umane staminali mesenchimali (MSC) appaiono sempre più rilevante per l&#39;applicazione clinica. Utilizzando un sistema di co-coltura di cellule staminali mesenchimali e leucociti del sangue periferico pre-colorati con il colorante fluorescente carbossifluoresceina succinimidil estere (CFSE), si descrive la valutazione in vitro di MSC immunomodulazione sulla proliferazione dei leucociti effettrici e sottopopolazioni specifiche.

Valutazione della proprietà immunomodulanti di cellule staminali mesenchimali umane (MSC)

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of ...

assessment-immunomodulatory-properties-human-mesenchymal-stem-cells

Research

JoVE Journal

Immunology and Infection

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs)

12.1K Views.  National Health Research Institutes (NHRI).  Le proprietà immunomodulanti di cellule umane staminali mesenchimali (MSC) appaiono sempre più rilevante per l'applicazione clinica. Utilizzando un sistema di co-coltura di cellule staminali mesenchimali e leucociti del sangue periferico pre-colorati con il colorante fluorescente carbossifluoresceina succinimidil estere (CFSE), si descrive la valutazione in vitro di MSC immunomodulazione sulla proliferazione dei leucociti effettrici e sottopopolazioni spec...

Video: Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Espansione, di purificazione, e la valutazione funzionale delle cellule del sangue umano periferico NK

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Ricerca

Immunologia e infezioni

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

La valutazione quantitativa delle cellule immunitarie nel tessuto danneggiato del midollo spinale mediante citometria di flusso: un nuovo uso per un metodo di purificazione cellulare

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, transmission of antibiotic resistant bacteria between animals and humans, and the high cost of treating infections.
In this study, we disclose a new strategy that may be effective in preventing implant-associated infection based on the potential antimicrobial properties of platelet-rich plasma (PRP). Due to its well-studied properties for promoting healing, PRP (a biological product) has been increasingly used for clinical applications including orthopaedic surgeries, periodontal and oral surgeries, maxillofacial surgeries, plastic surgeries, sports medicine, etc.
PRP could be an advanced alternative to conventional antibiotic treatments in preventing implant-associated infections. The use of PRP may be advantageous compared to conventional antibiotic treatments since PRP is less likely to induce antibiotic resistance and PRP's antimicrobial and healing-promoting properties may have a synergistic effect on infection prevention. It is well known that pathogens and human cells are racing for implant surfaces, and PRP's properties of promoting healing could improve human cell attachment thereby reducing the odds for infection. In addition, PRP is inherently biocompatible, and safe and free from the risk of transmissible diseases.
For our study, we have selected several clinical bacterial strains that are commonly found in orthopaedic infections and examined whether PRP has in vitro antimicrobial properties against these bacteria. We have prepared PRP using a twice centrifugation approach which allows the same platelet concentration to be obtained for all samples. We have achieved consistent antimicrobial findings and found that PRP has strong in vitro antimicrobial properties against bacteria like methicillin-sensitive and methicillin-resistant Staphylococcus aureus, Group A Streptococcus, and Neisseria gonorrhoeae. Therefore, the use of PRP may have the potential to prevent infection and to reduce the need for costly post-operative treatment of implant-associated infections.

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is a significant clinical complication. This study describes an approach using platelet-rich plasma (PRP) to prevent implant-associated infections, presents the protocol for preparing PRP with constant platelet concentration, and reports the newly identified antimicrobial properties of PRP and related protocols for examining such antimicrobial properties in vitro.

PRP come un nuovo approccio per prevenire l&#39;infezione: Preparazione e<em&gt; In vitro</em&gt; Proprietà antimicrobiche di PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Lo sviluppo di cellule staminali derivate antigene-specifiche cellule T regolatorie contro autoimmunità

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Destrano migliora l'efficienza di trasduzione lentivirale di cellule NK primarie Murine ed umane

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode larvae of the genus Trichinella. The most important sources of human infections worldwide are game meat and pork or pork products. In many countries, the prevention of human trichinellosis is based on the identification of infected animals by means of the artificial digestion of muscle samples from susceptible animal carcasses.
There are several methods based on the digestion of meat but the magnetic stirrer method is considered the gold standard. This method allows the detection of Trichinella larvae by microscopy after the enzymatic digestion of muscle samples and subsequent filtration and sedimentation steps. Although this method does not require special and expensive equipment, internal controls cannot be used. Therefore, stringent quality management should be applied throughout the test. The aim of the present work is to provide detailed handling instructions and critical control points of the method to analysts, based on the experience of the European Union Reference Laboratory for Parasites and the National Reference Laboratory of Germany for Trichinella.

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

The prevention of human trichinellosis in many countries worldwide is based on the laboratory examination of muscle samples from susceptible animals by methods of digestion, of which the magnetic stirrer method is considered the gold standard.

Metodo dell&#39;agitatore magnetico per la rilevazione di<em&gt; Trichinella</em&gt; Le larve in campioni di muscolo

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

Un'analisi di citotossicità di base di citometria a flusso per la valutazione dell'attività delle cellule NK umane

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

Valutazione dei citotossici e gli effetti immunomodulatori di sostanze in fette di polmone umano di taglio di precisione

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

Valutazione dell'interfaccia sinaptica primaria umana delle cellule di T dal sangue periferico e tessuto linfoide

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Induced Differentiation of M Cell-like Cells in Human Stem Cell-derived Ileal Enteroid Monolayers

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where immune cells reside and therefore contribute to mucosal immunity in the intestine. A complete understanding of how M cells differentiate in the intestine as well as the molecular mechanisms of antigen uptake by M cells is lacking. This is because M cells are a rare population of cells in the intestine and because in vitro models for M cells are not robust. The discovery of a self-renewing stem cell culture system of the intestine, termed enteroids, has provided new possibilities for culturing M cells. Enteroids are advantageous over standard cultured cell lines because they can be differentiated into several major cell types found in the intestine, including goblet cells, Paneth cells, enteroendocrine cells and enterocytes. The cytokine RANKL is essential in M cell development, and addition of RANKL and TNF-&#945; to culture media promotes a subset of cells from ileal enteroids to differentiate into M cells. The following protocol describes a method for the differentiation of M cells in a transwell epithelial polarized monolayer system of the intestine using human ileal enteroids. This method can be applied to the study of M cell development and function.

This protocol describes how to induce the differentiation of M cells in human stem cell-derived ileal monolayers and methods to assess their development.

Differenziazione indotta di cellule M in monostrati Enteroidi Ileal Imbecilli derivati da cellule staminali umane

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where ...