Name: generating primary fibroblast cultures from mouse ear and tail tissues
Uploaded: 2016-01-10T13:00:00.000+00:00
Duration: 6 min 35 s
Description: Watch this Scientific Journal Video about Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues at JoVE.com

This work was supported by the NRF grant HUJ-CREATE - Cellular and Molecular Mechanisms of Inflammation.

I topi sono stati alloggiati in condizioni di assenza di patogeni in conformità con le linee guida istituzionali fino euthanization (La cura istituzionale degli animali e del Comitato uso (IACUC) le linee guida presso l&#39;Università Nazionale di Singapore e del Comitato consultivo nazionale per Research Laboratory Animal (NACLAR) le linee guida). 1. Topi <ol><li> Ordinare un mouse del background genetico appropriato. Questo protocollo è basato sul tessuto derivato da uno C57BL / 6 del mouse. </li></ol> 2. Preparazione del terreno completo <ol><li> Preparare terreno completo aggiungendo le seguenti componenti al Roswell Park Memorial Institute (RPMI) 1640: siero fetale bovino al 10% (FCS), 50 mM 2-mercaptoetanolo, 100 micron asparagina, glutammina 2 mM, soluzione di penicillina-streptomicina 1%. </li></ol> 3. Preparazione di enzima Solutions <ol><li> Preparare la soluzione collagenasi D in un tubo da 15 ml fondo conico. <ol><li> Pesare 10 mg di collagenasi D. Dissolve collagenasi D in 4 ml di terreno completo. </li></ol></li><li> Preparare la soluzione pronasi in una provetta 1,7 ml microcentrifuga. <ol><li> Pesare 10 mg di Pronasi. </li><li> Aggiungere 5 ml di 1 M tampone Tris (pH 8,0). Aggiungere 1 ml di 0,5 M EDTA (pH 8,0). </li><li> Rabboccare con 494 ml di acqua sterile. </li><li> Incubare la soluzione pronase a 37 ° C a bagnomaria per 30 minuti. </li></ol></li></ol> 4. Preparazione di Collagenasi D-pronasi Mix (≤2 Tails) Nota: Eseguire le fasi successive in una cappa sterile coltura cellulare. <ol><li> Aggiungere 250 microlitri di soluzione pronase a 4 ml di soluzione di collagenasi D. </li><li> Passare il composto collagenasi D-pronasi attraverso un filtro siringa da 0,2 micron in una sterile 15 ml tubo fondo conico. </li></ol> 5. Estrazione di fibroblasti da tessuti dell&#39;orecchio e coda <ol><li> Euthanize topi secondo le linee guida istituzionali appropriate.</li><li> Mettere in autoclave chirurgici strumenti (forbici e pinze) nel cofano coltura cellulare. </li><li> Aggiungere 10 ml di terreno completo in due 10 cm piatti di coltura di cellule ciascuno. </li><li> Tagliare orecchie (~ 1 cm raggio) e 5 cm di coda (dalla punta della coda) di un topo con le forbici e incubare per 5 minuti in 40 ml di etanolo 70% in una provetta sterile da 50 ml a fondo conico. </li><li> Orecchie con aria secca e la coda di collocarli in un 10 centimetri piatto di coltura cellulare aperta nel cappuccio per 5 min. Una volta essiccati, trasferimento orecchio e pezzi di coda a due piatti della cultura etichettati &quot;orecchie&quot; e &quot;coda&quot; che contiene 10 ml di terreno completo come descritto al punto 5.3. </li><li> Eliminare i peli dalle orecchie e forbici coda utilizzando. </li><li> Tagliare orecchie e la coda in pezzi più piccoli di 3 mm di dimensioni con le forbici. </li><li> Trasferire i tessuti tagliati in 1,8 ml fiale cryotube etichettate &quot;orecchie&quot; e &quot;coda&quot; e aggiungere collagenasi sufficiente soluzione D-pronasi per il volume di raggiungere i 1,8 ml sul flacone. </li><li> Pallacciare le fiale cryotube orizzontalmente su un ed agitare i campioni a 200 rpm per 90 min a 37 ° C. </li><li> Dopo 90 minuti di incubazione, rimuovere le fiale cryotube dal shaker e posizionare le fiale nella cappa. </li><li> Aggiungere 10 ml di terreno completo in due 10 cm di colture cellulari piatti, con l&#39;etichetta &quot;orecchie&quot; e &quot;coda&quot; ciascuno, e mettere un filtro 70 micron cella in ogni piatto. </li><li> Posizionare le orecchie e coda tessuti digeriti nel colino 70 micron cella nei piatti di conseguenza etichettati previste al punto 5.11 e con forza macinare i tessuti con una siringa da 10 ml stantuffo per&gt; 5 min. Agitare il filtro cellule occasionalmente nel mezzo per lavare le cellule fuori del filtro cella. </li><li> Pipetta la sospensione cellulare da ogni piatto in provette fondo due 15 ml coniche etichettati &quot;orecchie&quot; e &quot;coda&quot;. Lavare il piatto e il filtro con l&#39;aggiunta di 10 ml di terreno completo e aggiungere il supporto per le appropriate da 15 ml provette fondo conico. </li><li> Spin giù la cellasospensione per 7 minuti a 580 xg ~ e 4 ° C utilizzando una centrifuga cella frigorifera. </li><li> Rimuovere il surnatante, aggiungere 10 ml di mezzo completo per il pellet cellulare in 15 ml tubo fondo conico e risospendere le cellule. </li><li> Ripetere il passaggio 5.14 e 5.15. </li></ol> 6. Coltura di miscela cellulare <ol><li> Rimuovere il surnatante. Assicurarsi che il pellet di cellule rimane indisturbato. </li><li> Risospendere le cellule in 10 ml di terreno completo e aggiungere il relativo composto per due 10 cm piatti di coltura di cellule etichettate &quot;orecchie&quot; e &quot;coda&quot;. </li><li> Aggiungere 10 ml soluzione amfotericina B (soluzione madre: 250 mg / ml) per la cultura. </li><li> Incubare le cellule a 37 ° C in un umidificata al 5% CO 2 incubatore. </li><li> Al terzo giorno, sostituire il mezzo con terreno completo contenente 10 ml fresco 10 ml di amfotericina B per rimuovere detriti (figure 1 e 2). </li></ol> 7. Sotto-cultura della fibroblasti <ol><li> Whecultura n raggiunge circa il 70% di confluenza (giorno 3-4 della cultura) rimuovere il supporto e lavare le cellule con 5 ml sterile 1x tampone fosfato salino (PBS). </li><li> Rimuovere PBS e aggiungere 2 ml di soluzione sterile 1x tripsina-EDTA per le cellule. </li><li> Incubare le cellule per 5 min a 37 ° C in un umidificata al 5% CO 2 incubatore. </li><li> Dopo 5 minuti, delicatamente toccare il piatto cultura e aggiungere 6 ml di terreno completo alle cellule. </li><li> Trasferire la sospensione cellulare in una provetta da 15 ml conica fondo e girare il tubo per 5 minuti a 450 xg ~ e 4 ° C utilizzando una centrifuga cella frigorifera. </li><li> Rimuovere il surnatante, e delicatamente risospendere il pellet cellulare in 5 ml di mezzo completo. </li><li> Seed 2 x 10 5 cellule in un piatto di coltura cellulare 10 cm e incubare le cellule a 37 ° C in un umidificata al 5% CO 2 incubatore per 3-4 giorni prima di ripetere passaggi da 7.1 a 7.6. </li></ol> 8. Preparazione di orecchie per la spedizione Nota: eseguire tegli fasi successive in una cappa sterile coltura cellulare. <ol><li> Euthanize topi secondo le linee guida istituzionali appropriate. </li><li> Posizionare forbici e pinze in autoclave nel cofano coltura cellulare. </li><li> Aggiungere 50 ml di terreno completo in un tubo da 50 ml fondo conico. </li><li> Tagliare le orecchie (~ 1 cm di raggio) di un mouse con le forbici. </li><li> Trasferire le orecchie nel tubo da 50 ml a fondo conico (come preparato al punto 3) con pinze. </li><li> Sigillare il tubo da 50 ml fondo conico con parafilm prima della spedizione a temperatura ambiente in una scatola appropriata. </li></ol>

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The authors declare no conflict of financial interests.

Qui forniamo una procedura semplice, economico e veloce sperimentale di stabilire colture di fibroblasti primari da orecchie e le code di topi. L&#39;estrazione dovrebbe risultare in fibroblasti divisorie aderenti e rapidamente entro 3 giorni post-isolamento del tessuto. Un importante limite di pile è senescenza, una crescita arresto permanente 15. Utilizzando il protocollo, colture di fibroblasti possono essere diversi passaggi da 5 a 6 volte prima fibroblasti diventano senescente, indicato dalla appiattime...

Cellule primarie sono derivati ​​da tessuti e coltivate che vivono in condizioni in vitro. Si ritiene generalmente che le cellule primarie avvicinano maggiormente lo stato fisiologico e background genetico del tessuto da cui sono derivati ​​di linee cellulari immortalizzate o tumorali 1. Per questo motivo, cellule primarie rappresentano un modello utile per studiare le domande biologica 2,3. Tuttavia, a differenza di linee cellulari stabilizzate che crescono indefinitamente, pile infine subiscono senescenza nella cultura e devono essere frequentemente ristabilita. Pile comunemente usati includono fibroblasti, cellule epiteliali, cellule endoteliali, cellule T, cellule B, i macrofagi del midollo osseo (BMDM) e le cellule dendritiche derivate dal midollo osseo (BMDC). I fibroblasti sono spesso utilizzati come cellula primaria modello di coltura. Essi offrono vantaggi chiave rispetto ad altre cellule primarie. Le colture cellulari sono facilmente stabiliti, prontamente mantenuto e non richiedono purificazione di cellule precedenti alla cultura. Hanno rapida proliferazione iniziale e nessun requisito per i protocolli di media o di attivazione specializzate. I fibroblasti possono essere efficacemente trasfettate utilizzando protocolli fisici 4,5 biologici, chimici, e. C&#39;è la possibilità di memorizzare le orecchie fino a 10 giorni a temperatura ambiente, prima di stabilire colture cellulari. Colture di fibroblasti sono favorevoli alla visualizzazione dei processi citoplasmatici e adatto per la riprogrammazione in staminali pluripotenti indotte (iPS), cellule 6. I fibroblasti sono importanti cellule del tessuto connettivo che secernono proteine ​​collagene e della matrice extracellulare 7. Essi costituiscono il quadro strutturale in molti tessuti 8 e svolgono un ruolo fondamentale nella guarigione delle ferite e la riparazione dei tessuti 9,10. Qui, descriviamo un protocollo (4 ore &lt;) semplice e veloce per creare colture di fibroblasti da orecchie e le code di topo 11. Il protocollo richiede una minima del mouseesperienza per raccogliere i tessuti (a differenza di altri protocolli 12,13) ​​e può essere utilizzato per stabilire culture di orecchie memorizzati nel mezzo a temperatura ambiente per 10 giorni.

<table><tbody><tr><td>RPMI-1640</td><td>HyClone</td><td>SH30027.01</td><td></td></tr><tr><td>Fetal Calf Serum</td><td>HyClone</td><td>SV30160.03</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Sigma-Aldrich</td><td>M3148</td><td></td></tr><tr><td>Asparagine</td><td>Sigma-Aldrich</td><td>A4159</td><td></td></tr><tr><td>Glutamine</td><td>Sigma-Aldrich</td><td>G8540</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>HyClone</td><td>SV30010</td><td></td></tr><tr><td>Ethanol</td><td>Merck Millipore</td><td>107017</td><td>Absolute for analysis</td></tr><tr><td>Collagenase D</td><td>Roche Diagnostics</td><td>11088866001</td><td>From &lt;em&gt;Clostridium histolyticum&lt;/em&gt;, lyophilized, non-sterile</td></tr><tr><td>Pronase protease</td><td>Merck Millipore</td><td>53702</td><td>From &lt;em&gt;Streptomyces griseus&amp;nbsp;&lt;/em&gt;</td></tr><tr><td>Tris buffer (pH 8)</td><td>1st BASE</td><td>1415</td><td>Ultra pure grade</td></tr><tr><td>0.5M EDTA (pH 8)</td><td>1st BASE</td><td>BUF-1053</td><td>Biotechnology grade</td></tr><tr><td>10X Phosphate Buffered Saline (PBS)</td><td>1st BASE</td><td>BUF-2040-10X4L</td><td>Ultra pure grade</td></tr><tr><td>Trypsin-EDTA solution 10X</td><td>Sigma-Aldrich</td><td>59418C-100ML</td><td>0.5% trypsin, 0.2% EDTA, trypsin gamma irradiated by SER-TAIN process, without phenol red, in saline</td></tr><tr><td>Amphotericin B</td><td>Sigma-Aldrich</td><td>A2492-20ml</td><td>250 &amp;mu;g/ml in deionized water, sterile-filtered</td></tr><tr><td>Scissors</td><td>Aesculap</td><td></td><td></td></tr><tr><td>Forcep</td><td>Aesculap</td><td>AE-BD312R</td><td></td></tr><tr><td>0.2 &amp;mu;M syringe filter</td><td>Sartorius Stedim</td><td>16534</td><td></td></tr><tr><td>70 &amp;mu;M cell strainer</td><td>SPL</td><td>93070</td><td></td></tr><tr><td>Syringe plunger</td><td>Terumo</td><td>SS+10L</td><td></td></tr><tr><td>Cryovial tube</td><td>NUNC</td><td>368362</td><td></td></tr><tr><td>1.7 ml microcentrifuge tube</td><td>Axygen</td><td>MCT-175-C</td><td></td></tr><tr><td>10 cm cell culture dish</td><td>Greiner</td><td>664160</td><td>Cell culture treated dish&amp;nbsp;</td></tr><tr><td>15 ml conical bottom tube</td><td>Greiner</td><td>188271</td><td></td></tr><tr><td>50 ml conical bottom tube</td><td>Greiner</td><td>227261</td><td></td></tr><tr><td>Water bath</td><td>GFL</td><td>1002</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810R</td><td></td></tr><tr><td>Incubation shaker</td><td>Sartorius Stedim</td><td></td><td>Certomat-BS1</td></tr><tr><td>Zeiss Axiovert 25 light microscope</td><td>Carl Zeiss AG</td><td></td><td></td></tr></tbody></table>

generating primary fibroblast cultures from mouse ear and tail tissues

Cellule primarie derivano direttamente dal tessuto e si pensa di essere più rappresentativo dello stato fisiologico delle cellule in vivo di linee cellulari stabilizzate. Tuttavia, colture cellulari primarie di solito hanno una durata di vita limitata e devono essere frequentemente ristabilita. I fibroblasti sono una fonte facilmente accessibile di cellule primarie. Qui, si discute una procedura semplice e rapida sperimentale di stabilire colture di fibroblasti primari da orecchie e le code di topi. Il protocollo può essere utilizzato per stabilire colture primarie di fibroblasti da spighe conservati a temperatura ambiente per 10 giorni. Quando il protocollo è attentamente seguita, contaminazioni sono improbabili nonostante l&#39;uso di tessuti non sterile immagazzinato per tempo prolungato in alcuni casi. I fibroblasti proliferano rapidamente in coltura e possono essere espanse per un numero considerevole prima di subire la senescenza replicativa.

Estrazione di fibroblasti da risultati tessuto in una notevole quantità di frammenti di tessuto (Figura 1). In contrasto con i residui di tessuto, fibroblasti aderire a superfici di plastica coltura dei tessuti tra il giorno 1 e 3 della cultura. Il mezzo di colture di fibroblasti può essere modificato in modo sicuro il giorno 3 di coltura, che dovrebbe ridurre significativamente i livelli di detriti presenti nella cultura (Figura 2). I fibroblasti mostrano una morfologia allungata e u...

Watch this Scientific Journal Video about Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues at JoVE.com

Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

Descriviamo una procedura sperimentale semplice e rapido per la generazione di fibroblasti primari dalle orecchie e le code di topi. La procedura non richiede addestramento speciale animali e può essere utilizzato per la generazione di colture di fibroblasti da spighe conservati a temperatura ambiente per 10 giorni.

Generazione di primaria fibroblasti culture da orecchio e coda tessuti di topo

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell ...

generating-primary-fibroblast-cultures-from-mouse-ear-and-tail-tissues

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

33.8K Views.  Yong Loo Lin School of Medicine, National University of Singapore.  Descriviamo una procedura sperimentale semplice e rapido per la generazione di fibroblasti primari dalle orecchie e le code di topi. La procedura non richiede addestramento speciale animali e può essere utilizzato per la generazione di colture di fibroblasti da spighe conservati a temperatura ambiente per 10 giorni. 

Video: Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative strategies for treatment of heart disease. This conversion has been achieved by forced expression of defined factors such as Gata4 (G), Mef2c (M) and Tbx5 (T). Traditionally, iCMs are generated by a cocktail of viruses expressing these individual factors. However, reprogramming efficiency is relatively low and most of the in vitro G,M,T-transduced fibroblasts do not become fully reprogrammed, making it difficult to study the reprogramming mechanisms. We recently have shown that the stoichiometry of G,M,T is crucial for efficient iCM reprogramming. An optimal stoichiometry of G,M,T with relative high level of M and low levels of G and T achieved by using our polycistronic MGT vector (hereafter referred to as MGT) significantly increased reprogramming efficiency and improved iCM quality in vitro. Here we provide a detailed description of the methodology used to generate iCMs with MGT construct from cardiac fibroblasts. Isolation of cardiac fibroblasts, generation of virus for reprogramming and evaluation of the reprogramming process are also included to provide a platform for efficient and reproducible generation of iCMs.

We describe here a protocol for the generation of iCMs using retrovirus-mediated delivery of Gata4, Tbx5 and Mef2c in a polycistronic construct. This protocol yields a relatively homogeneous population of reprogrammed cells with improved efficiency and quality and is valuable for future studies of iCM reprogramming.

Migliorata la generazione di cardiomiociti indotte usando un costrutto policistronico Esprimendo rapporto ottimale di Gata4, MEF2C e Tbx5

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative ...

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Biologia dello sviluppo

Rapid Generation of Primary Murine Melanocyte and Fibroblast Cultures

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation defects and cancer. Vital to the understanding and amelioration of these diseases are experiments in primary fibroblast and melanocyte cultures. Nevertheless, current protocols for melanocyte isolation require that the epidermal and dermal layers of the skin are trypsinized and manually disassociated. This process is time consuming, technically challenging and contributes to inconsistent yields. Furthermore, methods to simultaneously generate pure fibroblast cultures from the same tissue sample are not readily available. Here, we describe an improved protocol for isolating melanocytes and fibroblasts from the skin of mice on postnatal days 0-4. In this protocol, whole skin is mechanically homogenized using a tissue chopper and then briefly digested with collagenase and trypsin. Cell populations are then isolated through selective plating followed by G418 treatment. This procedure results in consistent melanocyte and fibroblast yields from a single mouse in less than 90 min. This protocol is also easily scalable, allowing researchers to process large cohorts of animals without a significant increase in hands-on time. We show through flow cytometric assessments that cultures established using this protocol are highly enriched for melanocytes or fibroblasts.

This protocol outlines a rapid method to simultaneously generate melanocyte and fibroblast cultures from the skin of 0-4 day old mice. These primary cultures can be maintained and manipulated in vitro to study a variety of physiologically relevant processes, including skin cell biology, pigmentation, wound healing and melanoma.

Generazione rapida delle culture primarie di Murine Melanocite e Fibroblast

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation ...

Ricerca sul cancro

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

Generazione e innesto delle navi tissutale in un modello di topo

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Bioingegneria

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

Diretto lignaggio riprogrammazione di fibroblasti di topo adulto ai progenitori eritroidi

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

Generazione di cellule di topo Airway epiteliali ESC-derivati ​​dall&#39;uso di decellularized Lung Ponteggi

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

Stabilire colture primarie di fibroblasti adulti dai roditori

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Biologia

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

Preparazione di cellule di fibroblasti embrionali di topo adatto per colture di cellule pluripotenti umane embrionali e staminali indotta

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generazione di topi derivati ​​da cellule staminali pluripotenti indotte

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

Tessuto connettivo ingegnerizzato umano derivato da fibroblasti per applicazioni di screening

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

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 ...

Generazione di primaria fibroblasti culture da orecchio e coda tessuti di topo

Riepilogo

Esplora Altri Video

Capitoli in questo video

Migliorata la generazione di cardiomiociti indotte usando un costrutto policistronico Esprimendo rapporto ottimale di Gata4, MEF2C e Tbx5

Generazione rapida delle culture primarie di Murine Melanocite e Fibroblast

Generazione e innesto delle navi tissutale in un modello di topo

Diretto lignaggio riprogrammazione di fibroblasti di topo adulto ai progenitori eritroidi

Generazione di cellule di topo Airway epiteliali ESC-derivati dall'uso di decellularized Lung Ponteggi

Stabilire colture primarie di fibroblasti adulti dai roditori

Preparazione di cellule di fibroblasti embrionali di topo adatto per colture di cellule pluripotenti umane embrionali e staminali indotta

Generazione di topi derivati da cellule staminali pluripotenti indotte

Tessuto connettivo ingegnerizzato umano derivato da fibroblasti per applicazioni di screening

Isolamento fibroblasto adulto primario aumentata ad ultrasuoni

Generazione di primaria fibroblasti culture da orecchio e coda tessuti di topo

Riepilogo

Esplora Altri Video

Capitoli in questo video

Migliorata la generazione di cardiomiociti indotte usando un costrutto policistronico Esprimendo rapporto ottimale di Gata4, MEF2C e Tbx5

Generazione rapida delle culture primarie di Murine Melanocite e Fibroblast

Generazione e innesto delle navi tissutale in un modello di topo

Diretto lignaggio riprogrammazione di fibroblasti di topo adulto ai progenitori eritroidi

Generazione di cellule di topo Airway epiteliali ESC-derivati ​​dall'uso di decellularized Lung Ponteggi

Stabilire colture primarie di fibroblasti adulti dai roditori

Preparazione di cellule di fibroblasti embrionali di topo adatto per colture di cellule pluripotenti umane embrionali e staminali indotta

Generazione di topi derivati ​​da cellule staminali pluripotenti indotte

Tessuto connettivo ingegnerizzato umano derivato da fibroblasti per applicazioni di screening

Isolamento fibroblasto adulto primario aumentata ad ultrasuoni

Generazione di cellule di topo Airway epiteliali ESC-derivati dall'uso di decellularized Lung Ponteggi

Generazione di topi derivati da cellule staminali pluripotenti indotte