Questo lavoro è stato sostenuto dal National Institutes of Health di Grant 5KO8DK085136-02 a JY.

1. Ottenere Colon Tissue Mouse Un protocollo per eseguire esperimenti su animali, specificando le motivazioni e gli obiettivi della ricerca, è stato presentato e approvato dal nostro ufficio istituzionale di Animal Research Vigilanza. <ol><li> Euthanize il mouse con isoflurano inalato seguita da dislocazione cervicale. </li><li> Effettuare una incisione mediana ventrale, ritrarre il piccolo intestino di distanza e identificare il colon. Ritrarre la vescica e dell&#39;utero (se presente) lateralmente e identificare l&#39;osso pubico e tagliare con forbici pregiati per esporre il retto. </li><li> Sezionare il mesentere dorsale del retto off del colon e mobilizzare il colon dall&#39;ano al cieco, e la transezione per scollegare il colon dal piccolo intestino. </li><li> Porre il campione in tampone fosfato ghiacciata (PBS). </li><li> Riempire una siringa da 10 ml con PBS freddo e lavare il colon lume diverse volte per cancellare il contenuto, quindi tagliare l&#39;ope colonn senso della lunghezza con le forbici. </li><li> Posizionare il colon in una provetta conica da 50 ml contenente 20 ml di PBS ghiacciato. I due punti non deve essere collocato in qualsiasi orientamento particolare. Lavare il colon sostituendo il PBS fino a quando il liquido è trasparente senza particolato. </li></ol> Umano Un protocollo di ottenere tessuti umani da pazienti chirurgici, specificando l&#39;importanza di ottenere questo tessuto, nonché una valutazione dei potenziali rischi e benefici, è stato presentato e approvato dal nostro Ufficio istituzionale del programma di protezione Human Research. <ol><li> Collaborazione di ricerca con un chirurgo colorettale è necessario essere in grado di ottenere tessuti umani per la ricerca. Un protocollo di ottenere tessuto del colon umano dovrebbe essere presentato e approvato dal comitato istituzionale di revisione. </li><li> Il consenso informato scritto è ottenuto dal paziente prima della procedura chirurgica. </li><li> Il chirurgo esegue una resezione del colon in standla moda ard. Una volta che il colon è stato rimosso dal paziente, il campione viene immediatamente portata patologia. 0,5 nella sezione a tutto spessore della parete del colon non necessario per la valutazione patologica è previsto ed è subito posizionato sulla PBS freddo. Questo esemplare non dovrebbe includere il mesentere colon, e appendici premistoppa deve essere rimosso e, dal momento che la presenza di grasso influenzerà il processo di digestione. </li></ol> 2. Denudare Cellule epiteliali <ol><li> Incubare tutto il colon topo in 25 ml di EDTA 5 mM in HBSS (temperatura ambiente) in una provetta conica da 50 ml. Posizionare il tubo conico in C agitazione bagno aria 37 ° (250 rpm) per 15 minuti. Dopo 15 min, versare con cura il liquido e riempire il tubo con 25 ml di EDTA 5 mM, ripetere per un totale di 5 lavaggi. Per colon umano, dopo aver ottenuto una striscia ½ centimetro di parete del colon dal patologo tagliare il colon con le forbici in quattro pezzi uguali dimensioni. Posizionare due dei pezzi in una provetta conica da 50 ml, ecollocare i restanti due pezzi di tessuto del colon in una provetta conica da 50 ml separata. Poi incubare con 25 ml di EDTA 5 mM in HBSS e effettuare lavaggi, come descritto sopra. </li><li> Sciacquare il colon due volte in 20 ml di PBS freddo. </li><li> Posizionare il colon tessuto topo in una nuova provetta da 50 ml contenente 20 ml di RPMI-5, 10 U di dispasi, e 2.000 U di collagenasi D (temperatura ambiente). Per il tessuto colon umano, usare il doppio della quantità di dispasi e collagenasi (20 U dispasi, 4.000 U collagenasi D). </li><li> Porre il tubo in un bagno di aria 37 ° C agitando orientato verticalmente (se posto orizzontalmente, c&#39;è troppa aerazione e danno cellulare). Agitare a 250 rpm per un massimo di 60 min. Dopo l&#39;esposizione al dispasi e collagenasi D, il tessuto colon inizierà a digerire e il tessuto colon inizierà a guardare filante. Questo richiede di solito circa 30 min. Quando il colon ha questo aspetto, rimuovere il colon dagli enzimi. </li><li> Agitare ogni provetta su e giù 2-4 volte a rompere il tessuto. PEllet tessuto a 200 xg in una centrifuga da tavolo (4 ° C) per 5 min. Versare con cautela il surnatante e gettarlo. </li></ol> 3. Isolamento miofibroblasti e cultura <ol><li> Risospendere ogni pellet aggiungendo 10 ml di tampone di lisi ACK (47 ° C). Centrifugare nuovamente a 200 xg per 5 min (4 ° C), versare fuori e scartare il surnatante. </li><li> Risospendere ogni pellet aggiungendo 10 ml RPMI-5 con una pipetta. </li><li> Passare metà della sospensione cellulare (5 ml) attraverso un colino a maglie 70 micron con una pipetta 10 ml in un piatto TC-trattati 100 mm. Quindi aggiungere altri 15 ml di RPMI-5. Ripetere con i rimanenti 5 ml di sospensione cellulare in da 100 mm piatto TC-trattati differente. Un colon mouse quindi produrrà due 100 millimetri piatti TC-trattati. Una striscia di ½ pollice di tessuto del colon umano produrrà un totale di quattro piatti TC-trattata da 100 mm. </li><li> Porre la cultura piatti di tessuto in un incubatore CO 2 del 10% a 37 ° C. Le condizioni di coltura sono le stesse fo il mouse e cellule primarie umane. </li><li> Dopo 3 ore, lavare delicatamente le cellule non aderenti, con due cambi di HBSS. Il galleggiamento detriti dovrebbe essere lavato delicatamente. Cellule aderenti sono composti da cellule epiteliali, macrofagi e miofibroblasti. Una settimana dopo la semina, miofibroblasti solo dividere, macrofagi e cellule epiteliali senesce dopo il primo passaggio. Aggiungere fresco RPMI-5 e crescere le cellule in coltura cellulare. </li><li> Le cellule sono diversi passaggi per tripsinizzazione per 5 minuti e sono divisi 2:1. </li></ol> Abbreviazioni HBSS: soluzione salina bilanciata di Hank; RPMI-5: Roswell Park Memorial Institute media; ACK: ammonio-cloruro-potassio; FCS: siero fetale di vitello; TC-trattati: la cultura tessuto trattato Soluzioni Tampone di lisi ACK - (4.15 g di NH 4 Cl, 0,5 g KHCO 3, 18,6 mg Na 2 EDTA, 400 ml H 2 O, aggiustare il pH a 7,2-7,4 con 1 N HCl). Filtro sterilizzabilee attraverso un filtro 0,2 micron e conservare a 4 ° C. RPMI-5 - (per fare 500 ml: 454,5 ml RPMI, 25 ml FCS, 5 ml di 200 mM L-glutammina, 5 ml di 1 M HEPES, pH 7,4, 5 ml 100 mM sodio piruvato, 5 ml 100x Pen-Strep, 500 ml di 50 mM B-ME in PBS).

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	<li>Armaka, 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=Mesenchymal+cell+targeting+by+TNF+as+a+common+pathogenic+principle+in+chronic+inflammatory+joint+and+intestinal+diseases.">Mesenchymal cell targeting by TNF as a common pathogenic principle in chronic inflammatory joint and intestinal diseases.</a> J. Exp. Med. 205, 331-337 (2008).</li><li>Powell, D. W., 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=Paracrine+cells+important+in+health+and+disease.">Paracrine cells important in health and disease.</a> Am. J. Physiol. 277, C1-C9 (1999).</li><li>Yoo, J., Rodriguez Perez, C. E., Nie, W., Sinnett-Smith, J., Rozengurt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+D1+mediates+synergistic+MMP-3+expression+induced+by+TNF-alpha+and+bradykinin+in+human+colonic+myofibroblasts.">Protein kinase D1 mediates synergistic MMP-3 expression induced by TNF-alpha and bradykinin in human colonic myofibroblasts.</a> Biochem. Biophys. Res. Commun. 413, 30-35 (2011).</li><li>Yoo, J., Chung, C., Slice, L., Sinnett-Smith, J., Rozengurt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+D+mediates+synergistic+expression+of+COX-2+induced+by+TNF-{alpha}+and+bradykinin+in+human+colonic+myofibroblasts.">Protein kinase D mediates synergistic expression of COX-2 induced by TNF-{alpha} and bradykinin in human colonic myofibroblasts.</a> Am. J. Physiol. Cell Physiol. 297, C1576-C1587 (2009).</li><li>Valentich, J. D., Popov, V., Saada, J. I., Powell, D. W. <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+of+an+intestinal+subepithelial+myofibroblast+cell+line.">Phenotypic characterization of an intestinal subepithelial myofibroblast cell line.</a> Am. J. Physiol. 272, C1513-C1524 (1997).</li><li>Rodriguez Perez, C. E., Nie, W., Sinnett-Smith, J., Rozengurt, E., Yoo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF-alpha+potentiates+lysophosphatidic+acid-induced+COX-2+expression+via+PKD+in+human+colonic+myofibroblasts.">TNF-alpha potentiates lysophosphatidic acid-induced COX-2 expression via PKD in human colonic myofibroblasts.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 300, G637-G646 (2011).</li><li>Yoo, 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=TNF-alpha+induces+upregulation+of+EGFR+expression+and+signaling+in+human+colonic+myofibroblasts.">TNF-alpha induces upregulation of EGFR expression and signaling in human colonic myofibroblasts.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 302, G805-G814 (2012).</li><li>Mahida, Y. R., 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=Adult+human+colonic+subepithelial+myofibroblasts+express+extracellular+matrix+proteins+and+cyclooxygenase-1+and+-2.">Adult human colonic subepithelial myofibroblasts express extracellular matrix proteins and cyclooxygenase-1 and -2.</a> Am. J. Physiol. 273, G1341-G1348 (1997).</li><li>De Wever, O., 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=Priming+and+potentiation+of+DNA+damage+response+by+fibronectin+in+human+colon+cancer+cells+and+tumor-derived+myofibroblasts.">Priming and potentiation of DNA damage response by fibronectin in human colon cancer cells and tumor-derived myofibroblasts.</a> Int. J. Oncol. 39, 393-400 (2011).</li><li>Edwards, R. A., Smock, A. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+arachidonate+release+and+PGE2+production+in+Gi+alpha2-deficient+intestinal+and+colonic+subepithelial+myofibroblasts.">Defective arachidonate release and PGE2 production in Gi alpha2-deficient intestinal and colonic subepithelial myofibroblasts.</a> Inflamm. Bowel Dis. 12, 153-165 (2006).</li><li>Hoang, B., Trinh, A., Birnbaumer, L., Edwards, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+MAPK-+and+PGE2-dependent+IL-11+production+in+Gialpha2-/-+colonic+myofibroblasts.">Decreased MAPK- and PGE2-dependent IL-11 production in Gialpha2-/- colonic myofibroblasts.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 292, G1511-G1519 (2007).</li><li>Li, Z. B., Kollias, H. D., Wagner, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myostatin+directly+regulates+skeletal+muscle+fibrosis.">Myostatin directly regulates skeletal muscle fibrosis.</a> J. Biol. Chem. 283, 19371-19378 (2008).</li><li>Saada, J. 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La tecnica generale è simile usando il mouse o colon umano. Questa tecnica può essere utilizzata per isolare miofibroblasti dal piccolo intestino. I dettagli specifici per l&#39;isolamento dei miofibroblasti da 1. mouse e 2. colon umano sono discussi di seguito. 1. Topo Colon Irrigazione del colon mouse viene facilitata collegando un 4, 16 G Popper ago smussato-end a un&#39;estremità del colon. Questo aiuta anche durante il taglio del colon longitudinalmente. È ...

La regolazione della funzione gastrointestinale del tratto (GI) comporta complesse, interazioni dinamiche tra lo strato mucoso e il suo mesenchima sottostante. Queste interazioni normalmente servono a mantenere l&#39;omeostasi ma possono anche portare allo sviluppo di stati di malattia in condizioni patologiche 1. Miofibroblasti sono una sottopopolazione di cellule stromali situati soggiacente allo strato epiteliale che comunicano in modo paracrino con circostante sottopopolazioni di cellule per regolare un numero di processi tratto GI critici che includono rigenerazione della mucosa, riparazione e fibrosi 2. La linea cellulare 18Co è un esempio di una linea umana colon miofibroblasti cella che è stato ampiamente utilizzato per studiare la funzione miofibroblasti perché condivide molte delle caratteristiche di primaria in miofibroblasti situ. Questi includono l&#39;espressione della proteina di a-actina muscolare liscia (α-SMA) e vimentina, nonché l&#39;espressione di un numerodei recettori di superficie cellulare, come il recettore del fattore di crescita epidermico, o recettori per 3,4 acido lysophosphatidic. A causa di queste caratteristiche ultrastrutturali condivisi, nonché similitudini nella funzione biologica 5, la linea cellulare 18Co è stato ampiamente utilizzato per studiare la funzione miofibroblasti nel contesto di molti stati patologici quali la malattia infiammatoria intestinale o colorettale 3,6 cancro. Tuttavia, vi sono limitazioni intrinseche e preoccupazioni quando si utilizza una linea cellulare. Questi includono instabilità genotipica nel tempo che differenzia linee cellulari di cellule primarie, alterando il fenotipo e funzione biologica della cellula, compresi tassi di crescita e le interazioni con altre popolazioni cellulari. Linee cellulari mancano anche i normali componenti del microambiente GI (epiteliale, stromale e componenti vascolari), che è un importante limitazione al suo uso. Pertanto, le conclusioni tratte dalla ricerca sperimentale linea cellulare richiede un&#39;ulteriore conferma per ridurre il risk di errori di interpretazione. Miofibroblasti primari possono essere ottenuti da colon tessuto umano e il mouse per confermare i risultati sperimentali individuati in una linea cellulare 7. Il metodo è stato originariamente descritto da Mahida, et al. 8, e il protocollo è una delle molte tecniche ben definite che possono essere utilizzati per isolare miofibroblasti primari di topo e del colon umano 9. Per ogni modello murino della malattia del colon, questa tecnica può essere utilizzata per isolare myofibroblasts primarie per studiare come interagiscono con le cellule vicine o contribuire sia normale o patologico GI elabora 10,11. Questa tecnica può essere utilizzata per studiare come miofibroblasti contribuiscono alla guarigione della mucosa, fibrosi, e lo sviluppo di adenomi e carcinomi colorettali. Miofibroblasti base possono anche essere ottenuti da tessuti del colon umano che è stato asportato al momento della operazione per benigna (malattia infiammatoria intestinale, stenosi, diverticolite) o maligni condizioni. Cellule primarie isolate da tessuti umani colon e mouse possono essere coltivate in coltura cellulare ed utilizzati per un numero limitato di passaggi.

<table><tbody><tr><td>Name of the reagent</td><td>Company</td><td>Catalogue number</td><td>Comments (optional)</td></tr><tr><td>Dispase I</td><td>Invitrogen/GIBCO</td><td>#17105-041</td><td></td></tr><tr><td>Collagenase D</td><td>Roche</td><td>#1 088 858</td><td></td></tr><tr><td>4 in, 16 G Popper blunt-end needle</td><td>Fisher</td><td>#14-825-16K</td><td></td></tr></tbody></table>

isolation of primary myofibroblasts from mouse and human colon tissue

Il miofibroblasti è una cellula stromale del tratto che sta guadagnando una notevole attenzione per il suo ruolo fondamentale in molte funzioni GI gastrointestinale (GI). Mentre diverse linee cellulari miofibroblasti sono disponibili in commercio per studiare queste cellule in vitro, i risultati della ricerca di una linea di cellule esposte a condizioni di coltura cellulare sperimentali hanno delle limitazioni intrinseche dovute al carattere eccessivamente riduttiva del lavoro. L&#39;uso di miofibroblasti primarie offre un grande vantaggio in termini di conferma risultati sperimentali individuati in una linea cellulare. Isolamento di miofibroblasti primarie da un modello animale consente lo studio dei miofibroblasti in condizioni che imitare maggiormente lo stato di malattia in fase di studio. Isolamento di miofibroblasti primari dal tessuto del colon umano fornisce senza dubbio i dati sperimentali più rilevanti, dal momento che le cellule provengono direttamente dai pazienti con la malattia di base. Descriviamo una tecnica ben consolidata che può essere utilized isolare myofibroblasts primarie sia da mouse e tessuti del colon umano. Queste cellule isolate sono stati caratterizzati per essere alfa-actina del muscolo liscio e vimentina-positive, e desmina-negativo, in linea con miofibroblasti intestinali subepiteliali. Cellule myofibroblast primarie possono essere coltivate in coltura cellulare e utilizzati per scopi sperimentali su un numero limitato di passaggi.

Una volta isolato, miofibroblasti umani primari possono essere coltivate in coltura cellulare ed utilizzati per un numero limitato di passaggi (fino a passaggio 4). Queste cellule sono caratterizzate come un liscio-actina muscolare e vimentina positivo, e desmin-negativi (Figura 1A), in linea con intestinali myofibroblasts subepiteliali 5,7. Essi hanno anche una caratteristica stellato morfologia (Figura 1B). Miofibroblasti primari possono poi essere utilizzati per confermare...

Watch this Scientific Journal Video about Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue at JoVE.com

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

Il miofibroblasti è una cellula stromale influente del tratto gastrointestinale che regola importanti processi fisiologici in stati normali e patologici. Descriviamo una tecnica che consente l&#39;isolamento dei miofibroblasti primarie sia da mouse e tessuti del colon umano, che può essere utilizzato per la sperimentazione in vitro.

Isolamento di primaria miofibroblasti da mouse e Colon Tessuto umano

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

isolation-of-primary-myofibroblasts-from-mouse-and-human-colon-tissue

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

19.6K Views.  UCLA.  Il miofibroblasti è una cellula stromale influente del tratto gastrointestinale che regola importanti processi fisiologici in stati normali e patologici. Descriviamo una tecnica che consente l'isolamento dei miofibroblasti primarie sia da mouse e tessuti del colon umano, che può essere utilizzato per la sperimentazione in vitro. 

Video: Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

Isolamento e quantitativa immunocitochimica caratterizzazione di cellule miogeniche primarie e fibroblasti da muscolo scheletrico umano

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Ricerca

Biologia dello sviluppo

Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens

Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and promotion of the tumor microenvironment. While there are currently many techniques for the isolation of MFs from gastrointestinal (GI) tissues, this protocol introduces a novel element of isolation of these stromal cells from frozen tissue. Freezing GI tissue specimens not only allows the researcher to acquire samples from worldwide collaborators, biobanks, and commercial vendors, it also permits the delayed processing of fresh samples. The described protocol will consistently yield characteristic spindle-shaped cells with the MF phenotype that express the markers CD90, &#945;-SMA and vimentin. As these cells are derived from patient samples, the use of primary cells also confers the benefit of closely mimicking MFs from disease states-namely cancer and inflammatory bowel diseases. This technique has been validated in gastric, small bowel, and colonic MF primary culture generation. Primary MF cultures can be used in a vast array of experiments over a number of passage and their purity assessed by both immunocytochemistry and flow cytometry analysis.

Here, a protocol to isolate and establish primary fibroblast/myofibroblast (MF) cultures from frozen gastric, small intestinal, and colonic tissue-yielding cells with a MF phenotype-is presented. These cells express CD90, &#945;-SMA and vimentin. MFs can be used for a variety of functional assays including enzymatic activity and cytokine production.

Isolamento di CD 90+ fibroblasti / miofibroblasti da umani congelati gastrointestinali campioni


Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and ...

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

Isolamento di primarie cellule del colon tumorali umane dai tessuti chirurgici e Coltura direttamente su più elastica e flessibile substrati per trazione citometria

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

Bioingegneria

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

Isolamento e differenziazione dei mioblasti primari dagli esopanti muscolari scheletrici del topo

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

Isolamento delle cellule mononucleari Di Lamina propria dal colon Murine utilizzando Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...

Immunologia e infezioni

Ultrasonic-augmented Primary Adult Fibroblast Isolation

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary fibroblasts cannot divide indefinitely due to myofibroblast differentiation or senescence induction, new cultures must be established regularly. However, there are several obstacles to overcome during the processes of developing a reliable isolation protocol and primary fibroblast isolation itself: the method&#8217;s degree of difficulty (especially for beginners), the risk of bacterial contamination, the required time until primary fibroblasts can be used for experiments, and subsequent cell quality and viability. In this study, a fast, reliable and easy-to-learn protocol to isolate and culture primary adult fibroblasts from mouse heart, lung, liver and kidney combining enzymatic digestion and ultrasonic agitation is provided.

We present a protocol to isolate primary adult fibroblasts in an easy, fast and reliable way, performable by beginners (e.g., students). The procedure combines enzymatic tissue digestion and mechanical agitation with ultrasonic waves to obtain primary fibroblasts. The protocol can easily be adapted to specific experimental requirements (e.g., human tissue).

Isolamento fibroblasto adulto primario aumentata ad ultrasuoni

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary ...

Medicina

Isolation of Myofibroblasts from Mouse and Human Esophagus

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, and subjected to enzymatic digestion with collagenase and dispase for 25 min. Human esophageal resection specimens are stripped of muscularis propria and adventitia and the remaining mucosa is minced, and subjected to enzymatic digestion with collagenase and dispase for up to 6 hr. Cultured cells express &#945;-SMA and vimentin and express desmin weakly or not at all. Culture conditions are not conducive to growth of epithelial, hematopoietic, or endothelial cells. Culture purity is further confirmed by flow cytometric evaluation of cell surface marker expression of potential contaminating hematopoietic and endothelial cells. The described technique is straightforward and results in consistent generation of non-hematopoieitc, non-endothelial stromal cells. Limitations of this technique are inherent to the use of primary cultures in molecular biology studies, i.e., the unavoidable variability encountered among cultures established across different mice or humans. Primary cultures however are a more representative reflection of the in vivo state compared to cell lines. These methods also provide investigators the ability to isolate and culture stromal cells from different clinical and experimental conditions, allowing comparisons between groups. Characterized esophageal stromal cells can also be used in functional studies investigating epithelial-stromal interactions in esophageal disorders.

We present a protocol to generate primary cultures of murine and human esophageal stromal cells with a myofibroblast phenotype. Cultured cells have spindle shaped morphology, express &#945;-SMA and vimentin, and lack epithelial, hematopoietic and endothelial cell surface markers. Characterized stromal cells can be used in functional studies of epithelial-stromal interactions.

Isolamento di miofibroblasti da mouse e Esofago umano

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, ...

Biologia

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

Metodo criosezionamento per Microdissezione di Murine colon mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...

Isolation of Murine Intestinal Mesenchyme Resulting in a High Yield of Telocytes

The murine small intestine, or colon mesenchyme, is highly heterogenous, containing distinct cell types including blood and lymphatic endothelium, nerves, fibroblasts, myofibroblasts, smooth muscle cells, immune cells, and the recently identified cell type, telocytes. Telocytes are unique mesenchymal cells with long cytoplasmic processes, reaching a distance of tens to hundreds of microns from the cell body. Telocytes have recently emerged as an important intestinal stem cell niche component, providing Wnt proteins that are essential for stem and progenitor cell proliferation.
Although protocols on how to isolate mesenchyme from the mouse intestine are available, it is not clear whether these procedures allow the efficient isolation of telocytes. Isolating telocytes efficiently requires special protocol adjustments that would allow dissociation of the strong cell-cell contact between telocytes and neighboring cells without affecting their viability. Here, available intestinal mesenchyme isolation protocols were adjusted to support the successful isolation and culture of mesenchyme containing a relatively high yield of viable single-cell telocytes.
The obtained single-cell suspension can be analyzed by several techniques, such as immunostaining, cell sorting, imaging, and mRNA experiments. This protocol yields mesenchyme with sufficiently conserved antigenic and functional properties of telocytes, and can be used for several applications. For example, they can be used for co-culture with mouse- or human-derived organoids to support organoid growth with no growth factor supplementation, to better reflect the situation in the original tissue.

Here, we present a protocol to isolate murine intestinal mesenchyme, including telocytes. These can be used for several applications, such as co-culture with mouse or human-derived organoids, to support growth and better reflect the situation in the original tissue.

Isolamento del mesenchima intestinale murino con conseguente alta resa di telociti

The murine small intestine, or colon mesenchyme, is highly heterogenous, containing distinct cell types including blood and lymphatic endothelium, nerves, ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

Isolamento e caratterizzazione di fibroblasti cardiaci adulti e miofibroblasti

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...