This work was supported by National Institute of Health (1R01DK103150-01A1, T32 DK007639, R01CA175803, K08CA125209) and the Institute for Translational Sciences at the University of Texas Medical Branch, and a Clinical and Translational Science Award (8UL1TR000071).

Il protocollo per l&#39;ottenimento di tessuti umani scartati da pazienti chirurgici e la creazione di colture primarie è stato approvato dalla University of Texas Medical Branch e University of New Mexico revisione istituzionale Boards. I requisiti generali per l&#39;approvvigionamento di campioni di tessuto umano è descritto di seguito. Si noti che, poiché MF può essere isolato da tessuti congelati, le biobanche e fornitori commerciali sono anche opzioni praticabili. 1. Ottenere Human Tissue <ol><li> Invia un protocollo per ottenere dei tessuti del colon umano al comitato istituzionale di revisione per l&#39;approvazione. </li><li> Stabilire collaborazioni con chirurghi generali e / o chirurghi colorettali al fine di ottenere tessuti umani per la ricerca. La collaborazione con il reparto di patologia è obbligatoria anche in quanto saranno determinare il tessuto che non è richiesto per la diagnosi e può essere designato per la ricerca. </li><li> Informare il patologo di fornire il tessuto dalla neoplasia e poi grossly mucosa normale di almeno 5 cm dal tumore. Porre immediatamente i campioni nei media lavaggio gelide e disporli sul ghiaccio. Il patologo determina la quantità di tessuto che può essere dato in modo sicuro per la ricerca. Il tessuto minimo richiesto per l&#39;isolamento MF è di 1,5 mm 2. </li><li> Utilizzare il tessuto ottenuto o immediatamente per stabilire le colture primarie o congelamento e posto a -80 ° C per l&#39;isolamento futuro. <ol><li> Se il congelamento per l&#39;isolamento più tardi, tagliate il tessuto in circa 1-2 mm 2 pezzi, mettere in un tubo criogenico con 1 ml di mezzi di congelamento (vedi sotto), e conservare a -80 ° C. Nota: Utilizzando questo protocollo, è stato osservato con successo l&#39;isolamento di MF da tessuto congelato per oltre 4 anni. </li></ol></li></ol> 2. Preparare Reagenti <ol><li> Per la soluzione Collagenase: Preparare 100 U / ml di collagenasi I, II e IV, in soluzione salina bilanciata di Hank (HBSS) con Ca2 + / Mg 2+ (vedi Tabella Materiali </strong&gt;). ATTENZIONE: collagenasi concentrazione unità attiva e purezza può varia da produttore a produttore, che possono ostacolare l&#39;efficienza della digestione tessuti e / o conservazione dell&#39;epitopo sulla superficie delle cellule della mucosa. Per evitare questi potenziali insidie, utilizzare collagenasi con più alto grado di purezza (≥95%) e preparare la soluzione madre basata sulla concentrazione unità attiva. </li><li> Preparare la soluzione di DNasi magazzino a base di acqua a 10 mg / ml (3.55 UA / mg). </li><li> Preparare lavaggio mezzi: (MEM: 89 ml, antibiotici contro funghi, 100x: 1 ml; FCS inattivato al calore: 10 ml) </li><li> Preparare Fibroblast crescita dei media: MEM: flacone da 500 ml; L-glutammina (200 mM): 5 ml; MEM non essenziali aminoacidi, 100x soluzione: 5 ml; ciprofloxacina HCl (10 mg / ml): 570 ml; Antibiotico-Antimicotici soluzione 100x: 10 ml; piruvato di sodio (100 mM): 5 ml; FCS inattivato al calore: 50 ml </li><li> Preparare supporti Fermo: i media di crescita dei fibroblasti (reagente 2.4) con l&#39;aggiunta del 10% di dimetilsolfossido (DMSO), poi filtrare-sterilize con un filtro di 0,22 micron. </li></ol> Tissue 3. enzimaticamente Digest umana <ol><li> Se il tessuto è stato congelato nei media congelare, posto esageratamente a (~ 37 ° C) acqua calda fino a quando viene scongelato dei tessuti e dei media. </li><li> Per 4-5 di 2 mm 2 pezzi di tessuto, lavare il tessuto due volte con 10 ml di HBSS senza Ca 2 + / Mg 2+. </li><li> Una volta che il tessuto è stabilito per gravità, eliminare accuratamente il surnatante, senza girare campione. </li><li> Aggiungere 10 ml della soluzione di collagenasi al campione e trasferire la miscela di tessuto in una soluzione sterile tubo DNasi / RNasi-free con incorporato rotori per campione dissociazione. Utilizzare collagenasi dissociare la matrice extracellulare che circonda l&#39;aspetto basale delle cellule epiteliali e costituisce la frazione non cellulare della propria mucosa lamina. </li><li> Chiudere bene il tubo e collegarlo a testa in giù sul manicotto del dissociatore (ad esempio, gentleMACS). Se il laboratorio non ha l&#39;accesso a tegli summenzionato attrezzature, aumentare il tempo di ciascuna fase di digestione per ottenere risultati comparabili. </li><li> Eseguire il h_tumor_01 Programma, un profilo preimpostato in dotazione con la macchina (durata complessiva di 36 secondi, con impulsi intermittenti che vanno da 1.000 - 4.000 giri, con 268 colpi per corsa). </li><li> Dopo la cessazione del programma, staccare il tubo dal dissociatore. </li><li> Incubare campione per 45 min a 37 ° C in rotazione continua su agitatore a 140 rpm. Nota: Per le diverse quantità di campioni di tessuto, il tempo di digestione può essere proporzionalmente ridotto o aumentato (ad esempio, grandi quantità di tessuto possono richiedere tempo supplementare). </li><li> Collegare il tubo a testa in giù sul manicotto del dissociatore. </li><li> Scegliere ed eseguire il programma h_tumor_02 (durata complessiva di 37 secondi, con impulsi intermittenti che vanno da 1.000 - 4.000 giri, con 235 colpi per corsa). </li><li> Dopo la cessazione del programma, staccare il tubo dal dissociatore. </li><li> Aggiungere 50 ml di soluzione madre DNasi. Utilizzare DNAse dissociare / rimuovere le cellule morte e la frazione di DNA di detriti cellulari che porta alla cella aggregazione. </li><li> Incubare campione per 30 min a 37 ° C in rotazione continua su agitatore a 60 rpm. </li><li> Collegare il tubo a testa in giù sul manicotto del dissociatore di nuovo. </li><li> Scegliere ed eseguire il programma h_tumor_03 (durata complessiva di 37 secondi, con impulsi intermittenti che vanno da 1.000 - 4.000 giri, con 168 colpi per corsa). Nota: Se il tessuto non è completamente digerito, centrifugare a 250 xg per 10 min a 20 ° C, scartare il surnatante, risospendere in 2 ml di soluzione di dissociazione cellulare e incubare a temperatura ambiente per 10 min. </li><li> Passare la sospensione cellulare attraverso sterile 70 micron colino cella. </li><li> Sospensione cellulare Centrifugare a 250 xg per 10 min a 20 ° C. Aspirare il surnatante completamente. </li><li> Lavare pellet di cellule due volte con 25 ml di HBSS senza Ca 2 + / Mg 2+ e scartare il surnatante. </li><li> Prima dell&#39;ultima erah, contare il numero di cellule utilizzando un sistema di conteggio delle cellule automatizzato disponibile in laboratorio o manualmente utilizzando emocitometro. Per MF generazione coltura primaria proseguire per passo 3.19.1, per l&#39;analisi o l&#39;ordinamento di MF in citometria a flusso proseguire per passo 3.19.2. <ol><li> Per l&#39;isolamento e la crescita della coltura pura MF (Figura 1), scartare il surnatante da ultima centrifugazione, il pellet cellulare risospendere in quantità appropriata di terreni di isolamento fibroblasti necessaria per seminare fino a 4 x 10 6 cellule in 3 ml di media per pozzetto in 6 piatti ben, coltura cellulare trattati. Passare al punto 3.20. </li><li> Per l&#39;analisi o l&#39;ordinamento di MF in citometria a flusso, collocare fino a 2 x 10 6 a 2 ml di terreni di isolamento dei fibroblasti in 24 bene, piatto a basso legame e incubare O / N a 37 ° C con 5% di CO 2. Questo è quello di ripristinare le epitopi di superficie delle cellule che potrebbero essere interessati dalla procedura enzimatica. Poi raccogliere sospensione cellulare e contare le cellule recuperate e procedereper la immunocolorazione e citometria a flusso di analisi 8. </li></ol></li><li> Crescere le cellule a 37 ° C con 5% di CO 2. Modificare i media ogni 2 - 3 giorni fino alla formazione del ~ 80% confluenti MF monostrato (Figura 1D-E). </li><li> Cellule Passage in fusti T25 con da 6 pozzetti in rapporto 1: 1 e crescere per circa 10 - 14 giorni nei media di crescita dei fibroblasti per raggiungere 80-100% di confluenza. </li><li> Espandere cultura, passaging cellule da T25 a cellule T75 in rapporto 1: 2. Una volta che le cellule raggiungono la confluenza, usare un pallone per l&#39;analisi di isolati purezza MF usando da citofluorimetria come descritto in precedenza 5. Congelare o di passaggio un altro pallone T75 della cultura MF in rapporto 1: 3. Nota: Quando analizzate mediante citometria di flusso, si prevede che la mucosa GI stromale MF di origine mesenchimale quando cresce nella cultura avrà seguente fenotipo: EpCAM-, CD31-, CD45-, vimentina +, α-SMA +, CD90 +. </li></ol>

<ol>
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The authors have nothing to disclose.

Mentre questo protocollo è stato sviluppato per l&#39;applicazione solo ricerca, alla luce della crescente importanza critica di cellule stromali come un cancro potenziali biomarker prognostici e obiettivo terapeutico, la capacità di congelare campioni biologici &quot;funzionali&quot; per un uso successivo offre un vantaggio significativo. Questo rappresenta un vantaggio unico per la creazione di biorepository clinica, che può servire come strumenti aggiuntivi che servono per promuovere lo sviluppo della medicina per...

Miofibroblasti / fibroblasti (MFS) rappresentano una popolazione abbondante di cellule gastrointestinali (GI) mucosa. Queste cellule stromali si trovano appena sotto l&#39;epitelio e formano una rete interconnessa all&#39;interno della mucosa lamina propria nell&#39;intestino. MF non sono responsabili solo per la deposizione della matrice extracellulare, ma attraverso la regolamentazione paracrina possono influenzare il trasporto di elettroliti, la restituzione, e la funzione di barriera dell&#39;epitelio adiacente 1, 2. Inoltre, MF hanno dimostrato di svolgere un ruolo chiave nel processo infiammatorio e tessuti rimodellamento 3, 4. miofibroblasti sono una parte critica del microambiente tumorale, dove sono conosciuti anche come i fibroblasti associati al cancro, e contribuire alla crescita delle cellule tumorali e servire come una nicchia per le cellule staminali del cancro 3. Dati emergenti suggeriscono che MF può anche servire le cellule presentanti l&#39;antigene come locali. Inoltre, la funzione MF come importanti regolatori della risposta immunitaria innata e adattativa, prcing una varietà di citochine e fattori di crescita 5. Negli individui sani, le cellule MFS si ritiene da differenziare da cellule staminali mesenchimali ed esprimere sulla loro superficie delle cellule, il marcatore mesenchimale, CD90 3, 5. Queste cellule sono anche positivi per vimentina, ma negativo per epiteliali e marker delle cellule ematopoietiche, EpCAM e CD45 rispettivamente. Miofibroblasti sono suggeriti per essere una forma attiva di fibroblasti e possono essere distinti dai fibroblasti non attivati ​​mediante l&#39;espressione di α-SMA 5. Negli ultimi dieci anni, più approcci per l&#39;isolamento di miofibroblasti dalla mucosa del colon umani sono stati pubblicati per lo più basato sul metodo originariamente descritto da Mahida et al. 5-8. Mentre i singoli studi riportano procedure di isolamento di varie mucosa intestinale, nessun protocollo universale per l&#39;isolamento di MF da più zone del tratto gastrointestinale (ad esempio, gastrico, piccolo e large mucosa intestinale) è stato pubblicato. Il protocollo qui presentata è stato testato ed usato con successo per tutti e tre tipo di tessuto sopra menzionato. . Inoltre, non sono stati segnalati procedure per isolare MFs da mucosa GI congelati. Qui, vi presentiamo un metodo ottimizzato, che si basa sulla digestione enzimatica, e permette contemporaneamente per l&#39;isolamento di mucosa dell&#39;intestino umano MF per la cultura e citometria a flusso analisi appena digerito, singola cella, i preparativi della mucosa. Questa tecnica produce in modo affidabile colture primarie con un fenotipo MF. Inoltre, gli stessi metodi possono essere utilizzati per isolare MFs da campioni congelati di tessuti intestinali gastriche e piccoli pure. Isolamento di miofibroblasti da tessuto fresco GI è stata precedentemente descritta; tuttavia, l&#39;utilizzo di campioni congelati presenta molti vantaggi. Vale a dire, i ricercatori saranno in grado di raccogliere e conservare i tessuti da un qualsiasi numero di collaboratori in tutto il mondo che hanno il capabilità per spedire campioni di tessuto congelato. Inoltre, i ricercatori potrebbero trovare la collezione di tessuti scartati dalla sala operatoria e / o suite endoscopia e immediata elaborazione del tessuto per l&#39;isolamento di conflitto con il loro programma di sperimentazione in corso. Inoltre, a causa della natura imprevedibile di un intervento chirurgico, di approvvigionamento dei tessuti può verificarsi molto tardi nel corso della giornata, che limiterà il tempo rimasto per il tessuto lavorazione. Congelamento dei tessuti per successive elaborazioni si migliorare queste sfide. Infine, questi metodi sono stati utilizzati con successo nell&#39;isolamento di colon, gastrici e piccole miofibroblasti intestinali in stati di malattia come il carcinoma colorettale e malattie infiammatorie intestinali.

<table><tbody><tr><td>MEM, 1x</td><td>Corning</td><td>MT-10-010-CV</td><td></td></tr><tr><td>Hanks&#039; Balanced Salt Solution</td><td>Sigma-Aldrich</td><td>H6648</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>S8636</td><td></td></tr><tr><td>Antibiotic-Antimycotic, 100x</td><td>Gibco</td><td>15240-062</td><td></td></tr><tr><td>Ciprofloxacin HCl</td><td>Corning</td><td>61-277-RF</td><td></td></tr><tr><td>L-Glutamine, 100x</td><td>Corning</td><td>25-005-CI</td><td></td></tr><tr><td>MEM Nonessential Amino Acids</td><td>Corning</td><td>25-025-CI</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO) Hybri-Max</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>DL-Dithiothreitol solution (DTT)</td><td>Sigma-Aldrich</td><td>646563</td><td></td></tr><tr><td>0.5 M EDTA, pH 8.0</td><td>Cellgro</td><td>46-034-CI</td><td></td></tr><tr><td>DNAse</td><td>Worthington</td><td>LS002139</td><td></td></tr><tr><td>Collagenase from&amp;nbsp;Clostridium histolyticum, Type I</td><td>Sigma-Aldrich</td><td>C1639</td><td></td></tr><tr><td>Collagenase from&amp;nbsp;Clostridium histolyticum, Type II</td><td>Sigma-Aldrich</td><td>C1764</td><td></td></tr><tr><td>Collagenase from Clostridium histolyticum, Type IV</td><td>Sigma-Aldrich</td><td>C5138</td><td></td></tr><tr><td>Accumax cell dissociation solution</td><td>Sigma-Aldrich</td><td>A7089</td><td></td></tr><tr><td>gentleMACS C Tubes</td><td>Miltenyi Biotec</td><td>130-093-237</td><td></td></tr><tr><td>gentleMACS Dissociator</td><td>Miltenyi Biotec</td><td>130-093-235</td><td></td></tr><tr><td>Countess Automated Cell Counter</td><td>Invitrogen</td><td>C10227</td><td></td></tr><tr><td>Cell Strainer (70 &amp;micro;m)</td><td>Corning</td><td>352350</td><td></td></tr><tr><td>PE Mouse Anti-Human Vimentin</td><td>BD Biosciences</td><td>562337</td><td>Clone RV202</td></tr><tr><td>FITC Monoclonal Anti-Actin, &amp;alpha;-Smooth Muscle</td><td>Sigma-Aldrich</td><td>F3777</td><td>Clone 1A4</td></tr><tr><td>APC Mouse Anti-Human CD90</td><td>BD Biosciences</td><td>559869</td><td>Clone 5E10</td></tr></tbody></table>

isolation of cd 90+ fibroblast/myofibroblasts from human frozen gastrointestinal specimens

Fibroblasti / miofibroblasti (MFS) stanno guadagnando sempre maggiore attenzione per il loro ruolo nella patogenesi e il loro contributo sia per la guarigione della ferita e la promozione del microambiente tumorale. Mentre ci sono attualmente molte tecniche per l&#39;isolamento di MF da gastrointestinali (GI) tessuti, questo protocollo introduce un elemento nuovo di isolamento di queste cellule stromali di tessuti congelati. Congelamento campioni di tessuto GI non solo permette al ricercatore di acquisire campioni di collaboratori in tutto il mondo, le biobanche e fornitori commerciali, permette anche il trattamento ritardato dei campioni freschi. Il protocollo descritto sarà costantemente produrrà caratteristici cellule fusiformi con il fenotipo MF che esprimono i marcatori CD90, α-SMA e vimentina. Poiché queste cellule sono derivate da campioni di pazienti, l&#39;uso di cellule primarie conferisce anche il vantaggio di MF mimando da stati, cioè il cancro e malattie infiammatorie intestinali malattie. Questa tecnica ha apen convalidato gastrico, piccolo intestino, colon e MF generazione coltura principale. Colture primarie MF possono essere utilizzati in una vasta gamma di esperimenti su un numero di passaggio e la loro purezza valutate sia immunocitochimica e analisi citofluorimetrica.

Usando questo protocollo digestione enzimatica, abbiamo sempre stati in grado di isolare e far crescere CD90 + cellule stromali della mucosa intestinale MF popolazione da GI campioni chirurgici e biopsie (Figura 1). Visibile MF colonia proliferazione potrebbe essere osservato il giorno 2-5 dopo la semina di cellule mononucleate sospensioni in 6 pozzetti (Figura 1A-B). Le colture primarie MF raggiungono ~ 50 - 70% confluenza di giorno 7 - 11 <s...

Watch this Scientific Journal Video about Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens at JoVE.com

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

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-cd-90-fibroblastmyofibroblasts-from-human-frozen

Research

JoVE Journal

Developmental Biology

9.6K Views.  University of Texas Medical Branch. 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, α-SMA and vimentin. MFs can be used for a variety of functional assays including enzymatic activity and cytokine production. 

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

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

Murine Dermal Fibroblast Isolation by FACS

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin-) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.

Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.

Murino Dermal fibroblasti Isolation di FACS

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast ...

Isolation of Papillary and Reticular Fibroblasts from Human Skin by Fluorescence-activated Cell Sorting

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have traditionally been attributed to the superficial papillary or lower reticular dermis according to their histological localization. In mouse dermis, papillary and reticular fibroblasts originate from two different lineages with diverging functions regarding physiological and pathological processes and a distinct cell surface marker expression profile by which they can be distinguished.
Importantly, evidence from explant cultures from superficial and lower dermal layers suggest that at least two functionally distinct dermal fibroblasts lineages exist in human skin dermis as well. However, unlike for mouse skin, cell surface markers enabling the discrimination of different fibroblast subsets have not yet been established for human skin. We developed a novel protocol for the isolation of human papillary and reticular fibroblast populations via fluorescence-activated cell sorting (FACS) using the two cell surface markers Fibroblast Activation Protein (FAP) and Thymocyte antigen 1 (Thy1)/CD90. This method enables the isolation of pure fibroblast subsets without in vitro manipulation, which was shown to affect gene expression, thus permitting accurate functional analysis of human dermal fibroblast subsets in regard to tissue homeostasis or disease pathology.

This manuscript describes a FACS-based protocol for isolation of papillary and reticular fibroblasts from human skin. It circumvents in vitro culture which was inevitable with the commonly used isolation protocol via explant cultures. The emanating fibroblast subsets are functionally distinct and display differential gene expression and localization within the dermis.

Isolamento dei fibroblasti papillari e reticolari dalla pelle umana mediante selezione cellulare attivata a fluorescenza

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have ...

Genetica

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

Isolamento e Immortalizzazione di paziente derivati ​​da linee cellulari provenienti da Muscle Biopsia per la malattia Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Medicina

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

Isolamento di fibroblasti dermici umani adulti dalla pelle addominale e generazione di cellule staminali pluripotenti indotte utilizzando un metodo non integrato

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Bioingegneria

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

Isolamento di fibroblasti normali e tumorali associata da tessuti freschi di fluorescenza activated cell sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

Biologia

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

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 ...

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 ...

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, ...

Isolation, Culture, and Characterization of Primary Dermal Fibroblasts from Human Keloid Tissue

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of primary fibroblasts derived from keloid tissue are the basis for further studies of the biological function and molecular mechanisms of keloids, as well as new therapeutic strategies for treating them. The traditional method of obtaining primary fibroblasts has limitations, such as poor cellular state, mixing with other types of cells, and susceptibility to contamination. This paper describes an optimized and easily reproducible protocol that could reduce the occurrence of possible issues when obtaining fibroblasts. In this protocol, fibroblasts can be observed 5 days after isolation and reach nearly 80% confluency after 10 days of culture. Then, the fibroblasts are passaged and verified using PDGFR&#945; and vimentin antibodies for immunofluorescence assays and CD90 antibodies for flow cytometry. In conclusion, fibroblasts from keloid tissue can be easily acquired through this protocol, which can provide an abundant and stable source of cells in the laboratory for keloid research.

This study describes an optimized protocol for establishing primary fibroblasts from keloid tissues that can effectively and steadily provide pure and viable fibroblasts.

Isolamento, coltura e caratterizzazione di fibroblasti dermici primari da tessuto cheloide umano

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of ...

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

Riepilogo

Esplora Altri Video

Capitoli in questo video

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

Murino Dermal fibroblasti Isolation di FACS

Isolamento dei fibroblasti papillari e reticolari dalla pelle umana mediante selezione cellulare attivata a fluorescenza

Isolamento e Immortalizzazione di paziente derivati da linee cellulari provenienti da Muscle Biopsia per la malattia Modeling

Isolamento di fibroblasti dermici umani adulti dalla pelle addominale e generazione di cellule staminali pluripotenti indotte utilizzando un metodo non integrato

Isolamento di fibroblasti normali e tumorali associata da tessuti freschi di fluorescenza activated cell sorting (FACS)

Isolamento di primaria miofibroblasti da mouse e Colon Tessuto umano

Isolamento fibroblasto adulto primario aumentata ad ultrasuoni

Isolamento di miofibroblasti da mouse e Esofago umano

Isolamento, coltura e caratterizzazione di fibroblasti dermici primari da tessuto cheloide umano

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

Riepilogo

Esplora Altri Video

Capitoli in questo video

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

Murino Dermal fibroblasti Isolation di FACS

Isolamento dei fibroblasti papillari e reticolari dalla pelle umana mediante selezione cellulare attivata a fluorescenza

Isolamento e Immortalizzazione di paziente derivati ​​da linee cellulari provenienti da Muscle Biopsia per la malattia Modeling

Isolamento di fibroblasti dermici umani adulti dalla pelle addominale e generazione di cellule staminali pluripotenti indotte utilizzando un metodo non integrato

Isolamento di fibroblasti normali e tumorali associata da tessuti freschi di fluorescenza activated cell sorting (FACS)

Isolamento di primaria miofibroblasti da mouse e Colon Tessuto umano

Isolamento fibroblasto adulto primario aumentata ad ultrasuoni

Isolamento di miofibroblasti da mouse e Esofago umano

Isolamento, coltura e caratterizzazione di fibroblasti dermici primari da tessuto cheloide umano

Isolamento e Immortalizzazione di paziente derivati da linee cellulari provenienti da Muscle Biopsia per la malattia Modeling