Ringraziamo il Dott. Steven Austad per averci fornito la prima versione di questo protocollo. Questo lavoro è stato supportato anche da finanziamenti del NIH e la Ellison Medical Foundation di VG e AS

1. Prima di partire <ol><li> Sterilizzare forbici e pinze con il 70% di etanolo. </li><li> Piccolo agitatore magnetico all&#39;interno di un bicchiere da 30 ml, coprire con due strati di stagnola, e autoclave. </li><li> Preparare un 28 unità Wunsch / mL di soluzione stock Liberase Blendzyme 3 in acqua sterile. Fai aliquote 0,5 ml e congelare a -20 ° C. Scongelare un&#39;aliquota di nuovo prima di ogni utilizzo. La soluzione potrebbe apparire torbida dopo lo scongelamento. Vortex la soluzione fino a quando diventa chiaro. </li><li> Scaldare i media di coltura cellulare. </li></ol> 2. Animal Preparazione del campione Attenzione: gli animali selvatici possono contenere agenti patogeni come virus della rabbia. Siate sempre consapevoli di aperta taglienti. <ol><li> Eutanasia dell&#39;animale e il luogo della carcassa a 4 ° C. E &#39;meglio usare la carcassa subito, tuttavia, se questo non è pratico, come se gli animali sono raccolti sul campo, le carcasse possono essere utilizzati entro 24 ore. Dopo 24 ore il calo di rendimento delle cellule. </li><li> Sezionare l&#39;animale in sala operatoria o in cappa chimica (non nel cofano tessuto cultura). </li><li> Ascellare zona è un luogo comodo per raccogliere campioni di pelle come la pelle ascellare è più sottile, contiene meno grasso, e la pelliccia è meno densa. Pulire il sito di incisione con il 70% di etanolo. Assicurarsi che la pelliccia è imbevuto di etanolo. </li><li> Accorciare il pelo intorno al sito di incisione con un bisturi affilato. Shave una zona più ampia rispetto al sito di incisione desiderata. Cercare di minimizzare i tagli alla pelle. Spruzzare la zona con il 70% di etanolo e lasciare asciugare. </li><li> Per raccogliere un campione di pelle accise un frammento di circa 1 cm 2. Pizzicare la pelle con una pinza dei tessuti, e tagliare con le forbici. Cerca di non tagliare lo strato di grasso con la pelle, sotto forma di grasso interferisce con la digestione collagenasi. Per raccogliere i campioni di polmone, lavare la zona del petto con il 70% di etanolo, e aprire la pelle sul petto con le forbici facendo una forma a T incisione e tirando fuori i lembi della pelle. Lavare l&#39;area muscolare aperto con il 70% di etanolo e lasciare asciugare. Tagliare la gabbia toracica con pinze e forbici sterili (usare frese ossea per animali più grandi). Utilizzare tecniche sterili per evitare di contaminare gli organi interni. Non toccare gli organi interni con strumenti che ha toccato il pelo degli animali. Taglio frammenti polmone di circa 1 cm 2 utilizzando pinze sterili e forbici. </li><li> Frammenti di tessuto posto in 50 mL con PBS sterile per evitare l&#39;essiccamento. </li><li> Lavare la parte esterna dei 50 mL con frammenti di tessuto con etanolo, e portarli alla cappa coltura dei tessuti. </li><li> Smaltire carcassa animale. </li></ol> 3. L&#39;estrazione di cellule <ol><li> Trasferire i frammenti di tessuto in un piatto di coltura di tessuti 10 cm con bisturi sterile. Non trasferire troppo PBS con il campione. </li><li> Tagliare il tessuto in ~ 1 pezzi millimetri utilizzando due bisturi. Utilizza due lame con un&#39;azione a forbice partendo dal centro e tirando a parte. Mantenere il tessuto appallottolato, non tagliare pezzo per pezzo. Quando il taglio è sufficiente la pelle assomiglia a stucco, non verrà separato in pezzi, ma sarà tratto sottile. Il tessuto polmonare è più facile da tagliare, e sarà separato in piccoli pezzi. </li><li> Usando un bisturi, trasferire il tessuto tagliato in sterile da 30 ml bicchiere con ancoretta sterile. Lavare la piastra utilizzata per il taglio dei tessuti con 10 ml di DMEM/F12 media con 0,14 unità Wunsch / mL Liberase Blendzyme 3, e 1X antibiotica / antimicotica, e aggiungere la soluzione nel becher ml 30 con frammenti di tessuto. </li><li> Coprire il becher con un foglio di sterile e incubare a 37 ° C, mescolando lentamente, per 30 a 90 minuti. La lunghezza del incubazione dipende dal tipo di specie e dei tessuti. Fare attenzione a non digerire troppo il tessuto. Migliori rendimenti si ottengono frammenti di tessuto sono ancora presenti alla fine della digestione. La pelle richiede più tempo per digerire quello del polmone. La pelle di animali di grandi dimensioni richiede più tempo per digerire. Controllare la digestione dopo 30 minuti, e poi ogni 10 min. Quando la digestione pelle è completa, i media diventa torbida e frammenti di pelle separati gli uni dagli altri, ed i bordi dei pezzi diventano &quot;fuzzy&quot;. Digestione del polmone dovrebbe essere interrotta quando al polmone cambia frammenti di colore dal rosso al bianco e iniziare a formare le fibre appiccicoso. </li><li> Preparare la soluzione contenente frammenti di tessuto su e giù per rompere i grumi. Trasferire la soluzione sterile tubo 50 ml. Se i frammenti di muoversi facilmente attraverso il taglio pipetta 10 ml e la digestione è stato fatto bene. Lavare il becher 3 volte con 10 ml di caldo DMEM/F12 media con 15% FBS, antibiotico / antimicotico 1X e aggiungere il supporto per il tubo 50 ml con frammenti di tessuto. Chiudere il tubo da 50 ml e mescolare per inversione un paio di volte. Il FBS nei media si ferma la digestione Liberase. </li><li> Girano a 524 g in un oscillante centrifuga cultura secchiello tessuto per 5 min. Rimuovere il surnatante. Risospendere il pellet in 10 ml di caldo DMEM/F12 media con 15% FBS, 1X antibiotico / antimicotico. Preparare la sospensione conmassima forza di rompere i pezzi di tessuto. </li><li> Aggiungere un altro 30 ml di DMEM/F12 media con 15% FBS, 1X antibiotico / antimicotico, mescolare e centrifugare a 524 g in un oscillante centrifuga cultura secchiello tessuto per 5 min. Ripetere ancora una volta per rimuovere le tracce di Liberase. </li><li> Risospendere il pellet in 10 ml di DMEM/F12 media con 15% FBS, 1X antibiotica / antimicotica e trasferirli in un piatto di coltura tissutale 10 cm e posto in un tessuto culturale incubatore a 37 ° C, 5% CO 2, il 3% O 2 . </li><li> Controllare le piastre ogni giorno per i fibroblasti e il colore dei media. Se l&#39;isolamento ha avuto successo, fibroblasti strisciare fuori dei frammenti di tessuto e allegare alla piastra (Figura 1). Il fibroblasti iniziano a uscire frammenti di tessuto entro 2-5 giorni. </li><li> Se il colore dei media diventa giallo, significa potenziale contaminazione o sovraffollamento delle celle. Esaminare le piastre sotto il microscopio ad alto ingrandimento. Se i batteri, funghi, o vermi sono presenti scartare le piastre (questo è raramente un problema con gli animali da laboratorio, tuttavia può verificarsi quando i campioni vengano prelevati dal loro ambiente naturale). In assenza di contaminazione è presente, ma i media cambiato colore, questo è causato da troppe cellule o frammenti di tessuto troppi posti nello stesso piatto. Se oltre il 60% del piatto è coperto da fibroblasti attaccato, cambiare il supporto sul piatto e trasferire i frammenti di tessuto per una nuova piastra con i nuovi media. Se non fibroblasti molti attaccato alla piastra, cambiare i media e dividere i pezzi di tessuto per un 2-4 piatti. </li><li> Dopo 7 giorni, se i media non era stato sostituito in precedenza, il cambiamento dei media e trasferire i frammenti di tessuto per una nuova piastra con i nuovi media. </li><li> Incubare le cellule e frammenti di tessuto per altri 7 giorni. Entro il giorno 14 tutti i fibroblasti praticabile usciti i frammenti di tessuto. </li><li> Dopo 14 giorni dall&#39;inizio della cella di isolamento, scartare i media vecchi e frammenti di tessuto, raccogliere le cellule e la piastra in un piatto nuovo a 5x10 5 cellule / piastra Emem con il 15% FBS, 1X penicillina / streptomicina, non amminoacidi essenziali e sodio piruvato. I media Emem sosterrà la crescita dei fibroblasti tipi di cellule solo e gli altri moriranno o fermare la proliferazione. </li><li> Dopo che le cellule raggiungono 80-90% confluenza, congelare una aliquota di cellule per uso futuro. </li><li> Continuare a coltura le cellule dividerli a 5x10 5 cellule / piastra, quando le cellule raggiungono 80-90% confluenza. </li></ol> 4. Rappresentante Risultati Fibroblasti normali sono cellule di grandi dimensioni con sporgenze prominente (lamellipodia) (Figura 2). I fibroblasti crescere in monostrato. Una cultura sana crescita contiene cellule 1-10% in fase M, riconosciuto come arrotondato cellule elevata sulla superficie della piastra, ma non distaccata dalla piastra (Figura 3). Tipicamente, un piatto di 10 centimetri con la testa di serie 5x10 5 cellule diventa confluenti in 3-4 giorni. Il tempo di raddoppio varia enormemente tra le specie, e può essere maggiore per alcune delle longeve specie di roditori 1. Quando le cellule riempire un piatto che l&#39;arresto della proliferazione in fase G1. Tipico piatto confluenti dei fibroblasti contiene un fitto strato di cellule (Figura 4). Quando le cellule raggiungono il 90% confluenza sono pronti per la scissione. Se lo si desidera, le cellule possono essere mantenute su un piatto arrestato confluenti per lunghi periodi di tempo con le normali modifiche media (una o due volte alla settimana). <img src="/files/ftp_upload/2033/2033fig1.jpg" alt="Figura 1" /> Figura 1. Isolamento da fibroblasti della pelle del mouse (A) e polmone (B). I media è stata modificata per rimuovere le cellule distaccati e detriti prima di prendere le immagini il giorno 7. <img src="/files/ftp_upload/2033/2033fig2.jpg" alt="Figura 2" /> Figura 2. Topo polmone fibroblasti. <img src="/files/ftp_upload/2033/2033fig3.jpg" alt="Figura 3" /> Figura 3. Un campo di fibroblasti di topo celle a M-stadio, fotografato con ingrandimento 10X. <img src="/files/ftp_upload/2033/2033fig4.jpg" alt="Figura 4" /> Figura 4. Un piatto confluenti di fibroblasti di topo, fotografato con ingrandimento 10X.

<ol>
	<li>Seluanov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+tumor+suppressor+mechanisms+evolve+in+rodent+species+that+differ+in+size+and+lifespan.+Aging+.">Distinct tumor suppressor mechanisms evolve in rodent species that differ in size and lifespan. Aging .</a> Cell. 7, 813-823 (2008).</li><li>Parrinello, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+sensitivity+severely+limits+the+replicative+lifespan+of+murine+fibroblasts.">Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts.</a> Nat Cell Biol. 5, 741-747 (2003).</li></ol>

Nessun conflitto di interessi dichiarati.

Normali fibroblasti primari fornire un&#39;ottima alternativa alle linee di cellule tumorali stabilito nella ricerca biologica. Un vantaggio importante dei fibroblasti è che non siano portatori di mutazioni in oncogeni e soppressori tumorali e mantenere intatti i punti di controllo del ciclo cellulare. Questo rende fibroblasti normale un sistema preferito per gli studi di regolazione del ciclo cellulare, la riparazione del DNA e l&#39;apoptosi. Il protocollo qui descritto fornisce una ricetta semplice per l&#39;isolame...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DMEM/F12 media</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11330-032</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum (FBS), Qualified</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>01437-036</td>
<td>The Qualified serum had been pre-tested to provide good growth support for primary fibroblasts.</td>
</tr>
<tr>
<td>Antibiotic/Antimycotic</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15420-096</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Liberase TM Research Grade</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>05401127001</td>
<td>Replacement enzyme.</td>
<tr>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>A note from the authors:
Since Roche discontinued Liberase Blendzyme 3 (11814184001), they recommend using Liberase TM Research Grade medium Thermolysin (Cat. no. 05401119001 - 10 mg, Cat. no. 05401127001 - 100mg) instead. We have switched to this enzyme successfully with no issues.</td>
<tr>
<td>EMEM media</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>30-203</td>
<td>The EMEM media from ATCC already contains nonessential amino acids and sodium pyruvate.</td>
</tr>
<tr>
<td>Feathered #21 disposable, sterile scalpel</td>
<td>&nbsp;</td>
<td>Multiple suppliers</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Three Gas Control incubator</td>
<td>&nbsp;</td>
<td>Forma or Heraeus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

establishing primary adult fibroblast cultures from rodents

L&#39;importanza di utilizzare cellule primarie, piuttosto che linee cellulari tumorali, per ricerche biologiche sta diventando ampiamente riconosciuto. Cellule primarie sono preferite in studi di controllo del ciclo cellulare, apoptosi, e la riparazione del DNA, le cellule tumorali sono portatori di mutazioni in geni coinvolti in questi processi. Cellule primarie non può essere coltivato a tempo indeterminato a causa della comparsa di senescenza replicativa o aneuploidization. Quindi, nuove culture devono essere stabiliti regolarmente. La procedura per l&#39;isolamento di fibroblasti embrionali dei roditori è ben consolidata, ma isolando colture di fibroblasti adulti spesso è una sfida. Fibroblasti roditori adulti isolati da modelli murini di patologie umane può essere un controllo preferito quando li confronto a fibroblasti da pazienti umani. Inoltre, i fibroblasti adulti sono l&#39;unico materiale a disposizione quando si lavora con i roditori selvatici in cui donne in gravidanza non possono essere facilmente ottenuto. Qui forniamo un protocollo per l&#39;isolamento e la cultura dei fibroblasti adulti dalla pelle dei roditori e dei polmoni. Abbiamo usato questa procedura con successo per isolare fibroblasti da oltre venti specie di roditori da laboratorio, topi e ratti di roditori selvatici come il castoro, l&#39;istrice e lo scoiattolo.

Watch this Scientific Journal Video about Establishing Primary Adult Fibroblast Cultures From Rodents at JoVE.com

Establishing Primary Adult Fibroblast Cultures From Rodents

Questo articolo descrive un protocollo per l&#39;isolamento e il mantenimento delle colture primarie di fibroblasti della pelle e tessuto polmonare dei roditori selvatici.

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 ...

establishing-primary-adult-fibroblast-cultures-from-rodents

Research

JoVE Journal

Biology

49.2K Views.  University of Rochester. Questo articolo descrive un protocollo per l'isolamento e il mantenimento delle colture primarie di fibroblasti della pelle e tessuto polmonare dei roditori selvatici.

Video: Establishing Primary Adult Fibroblast Cultures From Rodents

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

Culture primarie neuronale dal cervello di tardo fase Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

Ricerca

Biologia

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

Primaria dissociato mesencefalo colture cellulari dopamina da roditori neonati

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

La preparazione di colture primarie di cellule emopoietiche dal midollo osseo murino per elettroporazione

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

Cultura primario di miociti adulti Cuore Rat

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Colture cellulari primarie da Drosophila Gastrula embrioni

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

Fibroblast rimozione veloce da alta densità Culture staminali embrionali umane

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Naïve Adult Stem Cells isolamento da colture primarie di fibroblasti umani

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.&#160;teleost fish) and full regeneration following appendage loss (i.e.&#160;urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.

In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.

Preparazione di primaria Miogenica precursori cellulari / Culture mioblasti dal basale vertebrati Lignaggi

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell numbers. Therefore, the most widely used cellular system is the osteocyte-like MLO-Y4 cell line.
The method here described refers to the use of retinoic acid to generate a homogeneous population of ramified cells with morphological and molecular osteocyte features.
After isolation of osteoblasts from mouse calvaria, all-trans retinoic acid (ATRA) is added to cell medium, and cell monitoring is conducted daily under an inverted microscope. First morphological changes are detectable after 2 days of treatment and differentiation is generally complete in 5 days, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers and up-regulation of osteocyte-specific molecules.
Daily cell monitoring is needed because of the inherent variability of primary cells, and the protocol can be adapted with minimal variation to cells obtained from different mouse strains and applied to transgenic models. 
The method is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications.

Treatment of primary mouse osteoblasts with retinoic acid produces a homogeneous population of ramified cells bearing morphological and molecular features of osteocytes. The method overcomes the difficulty of obtaining and maintaining primary osteocytes in culture, and can be advantageous to study cells derived from transgenic models.&#160;

Applicazione di acido retinoico per ottenere colture osteocytes da principale del mouse osteoblasti

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Isolamento e cultura dei cheratinociti primari del mouse dalla pelle dei neonati e adulti

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...