M.M. and C.A.R. acknowledge the financial support of the Ministero della Salute, Ricerca Finalizzata/giovani ricercatori anno 2009 of the Fondazione IRCCS Ospedale San Matteo, Pavia (I). M.M and C.A.R. wish to thank Marianna Longo for helping with the images preparation.

Questo protocollo è condotto in conformità con i principi ispiratori del Europea (UE 63/2010) e Italiano (26/2014) leggi che proteggono gli animali utilizzati per la ricerca scientifica. Cervicale dislocazione l&#39;eutanasia viene utilizzato per l&#39;isolamento ovaie ed è eseguita da esperti e soggetti elencati nel protocollo approvato che copre questo studio addestrato. 1. Gli animali <ol><li> Mantenere adulti da 3 a 6 settimane di età topi femmina in una stanza a temperatura e umidità controllate (con fasi di 12L: 12D e alimentati ad libitum). </li><li> Iniettare topi per via intraperitoneale con 2.5 UI di gonadotropina del siero di cavalla incinta (PMSG) per stimolare la crescita follicolare imitando gli effetti dell&#39;ormone follicolo-stimolante (FSH). Isolare le ovaie di ovociti antrali di raccolta 48 ore successive. Euthanize il mouse femminile precedentemente iniettato con PMSG, in base alle proprie linee guida istituzionali. </li><li> Rimuovere rapidamente le ovaie dalla cavità del corpo e metterli nel piatto cont Petriaining M2 mezzo per successivo isolamento ovociti (vedi sezione 3). <ol><li> Effettuare un&#39;incisione sulla pelle che copre la parete addominale seguita da un&#39;incisione nel peritoneo utilizzando forbici dissezione sterili. Aprire l&#39;incisione per esporre l&#39;addome, spostare le viscere da una parte con una pinzetta sterile e localizzare i tubi uterine, formando una forma a V a partire dalla vescica. </li><li> Osservare le ovaie si trovano al di sotto dei reni. Tenere ogni tubo con pinzetta sterile e fare una piccola incisione nella parte inferiore delle ovaie a rilasciarli. Trasferire entrambe le ovaie nella parte inferiore di una piastra di Petri contenenti terreno pre-equilibrata. </li></ol></li></ol> 2. Ormone Preparazione <ol><li> Diluire PMSG (disponibile in diverse concentrazioni archivi) con soluzione salina isotonica sterile per ottenere una concentrazione di lavoro di 2,5 UI di ormone per 0,1 ml di un volume di iniezione appropriata. NOTA: Le soluzioni di ormoni devono essere aliquotati in provette da 0,5 ml e conservati a -20 ° C fino al giorno di utilizzo. Non tenere aliquote diluito superiori a tre mesi a -20 ° C. Una volta scongelato, se si eseguono iniezioni seriali, memorizzare le aliquote delle soluzioni ormonali a +4 ° C e utilizzare entro 4 giorni. </li></ol> Isolamento 3. Antral Ovociti Per un corretto isolamento ovociti: <ol><li> Equilibrare M2 medio 13 (M2) al 5% di CO 2 e 37 ° C per almeno 1 ora prima di avviare la procedura di isolamento oociti. Preparare due piastre di Petri (35 mm x 10 mm), una con 1 ml di m2 per punzonamento ovaie e la seconda con piccole gocce (20 ml) di M2 coperto con olio minerale (~ 1,5 ml) per il trasferimento ovociti dopo isolamento dal ovaia (vedi 3,4-3,6). Tenere entrambe le capsule di Petri in incubatore al 5% di CO 2 e 37 ° C fino al momento di essere utilizzati. </li><li> Utilizzare uno stereomicroscopio durante le procedure di isolamento ovociti. </li><li> Preparare sottili pipette di vetro bocca per raccogliere gli ovociti stetchhing pipette Pasteur in vetro tenute orizzontalmente (tenendo le estremità della pipetta con entrambe le mani), a fiamma verticale proveniente dal bruciatore Bunsen (Figura 1). <ol><li> Una volta che il vetro inizia a fondere, prendere la pipetta della fiamma ed eseguire un movimento di trazione rapido avere la pipetta desiderata. Saldamente tagliare il vetro in eccesso vicino al punto stretto della pipetta con unghie per regolare la lunghezza e il diametro interno del capillare. Assicurarsi che il sottile capillare ha un diametro interno di ~ 100 micron, leggermente maggiore del diametro del ovocita (~ 80 micron). </li><li> Per bocca pipetta, collegare un&#39;estremità del tubo di aspirazione alla pipetta tirato utilizzando una punta appropriata e posizionare l&#39;altra estremità in bocca, fissandolo con i denti. </li></ol></li><li> Raccogliere ovaie usando pinzette sterili e depositarli sul fondo di un piatto di coltura cellulare di Petri (35 mm x 10 mm). Coprire con 1 ml di M2 precedentemente equilibrata a 37 ° Ce 5% CO 2 (Figura 2). </li><li> Forare delicatamente i follicoli antrali delle ovaie con un ago sterile siringa da insulina per liberare oociti antrali a M2 (Figura 3). </li><li> Delicatamente bocca pipettare gli ovociti attraverso uno stretto pipetta Pasteur per rimuovere le cellule follicolari e qualsiasi altri detriti tessuto possibile (Figura 4A) e poi depositarsi sul fondo di piccole gocce (20 mL) di M2 coperti con olio minerale adatto a cultura dell&#39;embrione (precedentemente preparati; Figura 4B). NOTA: E &#39;importante rimuovere tutte le cellule del cumulo collegati alla zona pellucida degli ovociti pipettando continuo attraverso diversi e diverse gocce di M2. Eseguire questo passaggio il più velocemente possibile per evitare alterazioni del pH del mezzo. </li></ol> 4. Antral ovociti colorazione con Hoechst33342 NOTA: ovociti Antral può essere distinta in SN (Circondato Nucleolo), NSN (Non Circondato Nucleolo) oin qualsiasi altra forma intermedia, in base alla loro organizzazione della cromatina dopo colorazione con concentrazione sopravitale di Hoechst33342 (o qualsiasi altro marker specifici per le basi AT) 14. In effetti, gli ovociti SN mostrano un anello Hoechst-positivo attorno al nucleolo mentre la NSN non lo fanno, da qui il suo nome. Per una corretta colorazione ovociti: <ol><li> Trasferire gli ovociti usando una pipetta bocca in gocce di Hoechst33342 (20 ml) diluito in M2 alla concentrazione sopravitale di 50 ng / ml per 10 min in scuro. </li><li> Dopo l&#39;incubazione con Hoechst33342, trasferire ogni singolo ovocita con una pipetta a bocca in fondo piccole gocce (5 mL) di M2 coperti con olio minerale, per l&#39;ordinamento basato su organizzazione della cromatina (SN o NSN). Utilizzare qualsiasi microscopio a fluorescenza dotato di filtro Hoechst33342 (lunghezza d&#39;onda di emissione 486 nm) per visualizzare la configurazione della cromatina degli ovociti. NOTA: A seconda del tipo di microscopio a fluorescenza utilizzato, it può diventare indispensabile utilizzare vetro Petri fondo per l&#39;acquisizione delle immagini. La microscopia confocale Olympus Fluoview Fv10i quando dotata di supporto del campione per 35 millimetri piatto richiede solo vetro bottom-piatti per l&#39;acquisizione di immagini, con spessore standard 0,17 millimetri e un range da 0,13 a 0,21 millimetri accettabili. </li><li> Eccitare gli ovociti per non più di 5-10 sec, probabilmente abbastanza tempo per visualizzare la presenza o l&#39;assenza di un anello attorno al nucleolo (Figura 5). </li><li> Dopo la cernita, la cultura SN antrale e ovociti NSN in M2 al 5% di CO 2 e 37 ° C per indurre la maturazione in vitro o tenerli in buffer appropriate per analisi molecolari e cellulari. </li></ol>

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O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+recombinant+human+gonadotropins+on+human,+bovine+and+murine+oocyte+meiosis,+fertilization+and+embryonic+development+in+vitro.">Effect of recombinant human gonadotropins on human, bovine and murine oocyte meiosis, fertilization and embryonic development in vitro.</a> Human Reprod. 15 (5), 1140-1148 (2000).</li><li>Mattson, B. A., Albertini, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oogenesis:+Chromatin+and+microtubule+dynamics+during+meiotic+prophase.">Oogenesis: Chromatin and microtubule dynamics during meiotic prophase.</a> Mol. Reprod. Dev. 25 (4), 374-383 (1990).</li><li>Debey, P., Szollosi, M. S., Szollosi, D., Vautier, D., Girousse, A., Besomber, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Competent+mouse+oocytes+isolated+from+antral+follicles+exhibit+different+chromatin+organization+and+follow+different+maturation+dynamics.">Competent mouse oocytes isolated from antral follicles exhibit different chromatin organization and follow different maturation dynamics.</a> Mol. Reprod. Dev. 36 (1), 59-74 (1993).</li><li>Zuccotti, M., Piccinelli, A., Giorgi Rossi, P., Garagna, S., Redi, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+organisation+during+mouse+oocyte+growth.">Chromatin organisation during mouse oocyte growth.</a> Mol. Reprod. Dev. 41 (1-2), 479-485 (1995).</li><li>Wu, Y. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+oocytes+for+in+vitro+maturation+by+brilliant+cresyl+blue+staining:+a+study+using+the+mouse+model.">Selection of oocytes for in vitro maturation by brilliant cresyl blue staining: a study using the mouse model.</a> Cell Res. 17, 722-731 (2007).</li><li>Opiela, Y., K&#261;tska-Ksi&#261;&#380;kiewicz, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+utility+of+Brilliant+Cresyl+Blue+(BCB)+staining+of+mammalian+oocytes+used+for+in+vitro+embryo+production+(IVP).">The utility of Brilliant Cresyl Blue (BCB) staining of mammalian oocytes used for in vitro embryo production (IVP).</a> Reproductive Biol. 13, 177-183 (2013).</li><li>Labrecque, R., Sirard, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+study+of+mammalian+oocyte+competence+by+transcriptome+analysis:+progress+and+challenges.">The study of mammalian oocyte competence by transcriptome analysis: progress and challenges.</a> Mol. Human Reprod. 20 (2), 103-116 (2014).</li><li>Monti, M., Zanoni, M., Calligaro, A., Ko, M. S., Mauri, P. L., Redi, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+arrest+and+mouse+antral+Not-Surrouded+Nucleolus+oocyte.">Developmental arrest and mouse antral Not-Surrouded Nucleolus oocyte.</a> Biol. Reprod. 88 (1), 1-7 (2013).</li><li>Yurttas, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+for+PADI6+and+the+cytoplasmic+lattices+in+ribosomal+storage+in+oocytes+and+translational+control+in+the+early+mouse+embryo.">Role for PADI6 and the cytoplasmic lattices in ribosomal storage in oocytes and translational control in the early mouse embryo.</a> Development. 135 (15), 2627-2636 (2008).</li><li>Li, L., Baibakov, B., Dean, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+subcortical+maternal+complex+essential+for+preimplantation+mouse+embryogenesis.">A subcortical maternal complex essential for preimplantation mouse embryogenesis.</a> Dev. Cell. 15 (3), 416-425 (2008).</li><li>Ami, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FT-IR+spectra+signatures+of+mouse+antral+oocytes:+molecular+markers+of+oocyte+maturation+and+developmental+competence.">FT-IR spectra signatures of mouse antral oocytes: molecular markers of oocyte maturation and developmental competence.</a> Biochim. Biophys. Acta. 1813 (6), 1220-1222 (2011).</li><li>Fulton, B. P., Whittingam, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+mammalian+oocytes+by+intercellular+injection+of+calcium.">Activation of mammalian oocytes by intercellular injection of calcium.</a> Nature. 273 (5658), 149-151 (1978).</li><li>Luttmer, S. J., Longo, F. 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The authors have nothing to disclose

Questo protocollo descrive il metodo utilizzato per isolare e classificare il mouse antrali GV ovociti. Non vi sono particolari passaggi critici per sottolineare, con poche eccezioni. Primo: questa procedura deve essere eseguita il più rapidamente possibile: a) mantenere la vitalità degli ovociti e b) evitare variazioni di pH del mezzo utilizzato. Secondo: gli ovociti devono essere brevemente eccitato (5-10 sec) per Hoechst33342 per evitare danni cromatina e la successiva morte degli ovociti, soprattutto se IVM e IVF ...

Antrale ovociti completamente sviluppati (GV, vescicola germinale) isolati dal vano antrale dell&#39;ovario sono abitualmente utilizzati per scopi diversi, come, per esempio, lo studio dei meccanismi cellulari e molecolari alla base maturazione in vitro fino metafase II. Nonostante le loro bella apparenza oociti antrali più, ma non tutti, sono in grado di completare la meiosi e raggiungere lo stadio di blastocisti; infatti, in molte specie di mammiferi, compreso l&#39;uomo 1,2, circa il 30% del loro arresto loro sviluppo nella fase 2 cellule, se avviene la fecondazione 3-5. Ad oggi, alcuni parametri sono utilizzati per distinguere tra gli ovociti in grado di sostenere lo sviluppo embrionale da quelli in grado di farlo. Ad esempio, cresolo colorazione blu (BCB) è un valido metodo per ordinare gli ovociti basati sull&#39;attività di glucosio-6-fosfato deidrogenasi, sebbene l&#39;utilità di questo metodo di ordinamento correlate a BCB + o colorazione è BCB-ancora dipendente 6-8 laboratorio. Un altro metodo consolidato risiede nella loro organizzazione della cromatina: se macchiata di eventuali coloranti intercalanti del DNA (come Hoechst33342), il 70% degli ovociti antrali mostrano un anello colorante positivo attorno al nucleolo e sono chiamati Circondato Nucleolo (SN), mentre il restante 30% mostrare un segnali positivi più maculati e sono chiamate Non Circondato Nucleolo (NSN) 3-5. Recentemente abbiamo dimostrato che l&#39;assenza di reticoli citoplasmatici 9 (CPL, siti di stoccaggio importanti per mRNA di origine materna e ribosomi sia in ovociti di mammiferi ed embrioni) 10 e la down-regolazione di MATER (essenziale per CPL formazione 11), unitamente alla presenza di gocce lipidiche nei loro cytoplasms 9,12, possono essere altre cause legate alla ovociti incapacità NSN a procedere oltre la fase 2 cellule. Purtroppo, poche tecniche possono essere applicate per ordinare SN antrale e oociti NSN e,per questa ragione, lo sviluppo di un metodo meno invasivo (senza colorazione nucleare, le procedure di fissaggio o di manipolazione con pipette bocca) potrebbe diventare molto importante aumentare i tassi di entrambi sviluppo embrionale umano e animale. In questo lavoro, abbiamo dettaglio il protocollo semplice e veloce utilizzato per isolare annuncio sorta SN e NSN oociti antrali di topi di sesso femminile e si rivolge a tutti gli scienziati interessati allo studio della gametogenesi femminile, la maturazione degli ovociti e sviluppo embrionale.

<table>
	<tbody>
		<tr>
			<td>PMSG</td>
			<td>Sigma</td>
			<td>G4527</td>
			<td></td>
		</tr>
		<tr>
			<td>EmbrioMax M2</td>
			<td>Millipore</td>
			<td>MR-015-PD</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst33342</td>
			<td>Thermo Scientific</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral Oil</td>
			<td>Sigma</td>
			<td>M8410</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Dish 35mmx10mm</td>
			<td>Corning</td>
			<td>430165</td>
			<td></td>
		</tr>
		<tr>
			<td>&#181;-Dish 35mm, high glass bottom</td>
			<td>Ibidi</td>
			<td>81158</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Pasteur Borosilicate</td>
			<td>Corning</td>
			<td>7095D-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Aspirator tube assemblies for calibrated micropillary pipettes</td>
			<td>Sigma</td>
			<td>A5177</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and characterization of mouse antral oocytes based on nucleolar chromatin organization

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded Nucleolus) or unable (NSN, Not Surrounded Nucleolus) to develop to the blastocyst stage after in vitro maturation (IVM) and in vitro fertilization (IVF). It makes use of Hoeschst33342 (or any other DNA intercalating dye) able to bind to the heterochromatin of the nucleolus showing a ring in the SN oocytes or not, like in the NSN oocytes. This represents the easiest and quickest way to sort both antral oocytes that can be eventually used for IVM or IVF procedures. 
Briefly, the protocol consists of the following steps: hormone injection to stimulate follicular growth; isolation of the oocytes at the GV stage from the antral compartment by puncturing the ovary with a sterile needle; preparation of thin glass pipettes for mouth pipetting of the oocytes; sorting of the oocytes with Hoechst33342 prepared at a supravital concentration; IVM, IVF or any other molecular/cellular analysis.
Unfortunately there are still few evidences to sort SN and NSN oocytes using less invasive techniques. If and once they will be identified, they could be potentially applied to human assisted reproductive technologies, although with several aspects that should be modified. To date, this technique has potential implications to dramatically increase IVM and IVF successful procedures in both endangered and species with economic interest.

Le seguenti immagini mostrano il metodo utilizzato per isolare oociti antrali da ovaie di topo, a partire dalle pipette preparazione alla selezione degli ovociti &#39;per mezzo di Hoechst33342. Questo metodo è molto semplice e può essere eseguita in qualsiasi laboratorio dotato di CO 2 incubatore, uno stereomicroscopio e un microscopio a fluorescenza equipaggiato con il filtro corretto per l&#39;osservazione del Hoechst33342 Figura 1 mostra la fase iniziale ...

Watch this Scientific Journal Video about Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization at JoVE.com

Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization

Qui vi presentiamo un protocollo per la caratterizzazione di oociti antrali di topo sulla base di organizzazione nucleolare.

Isolamento e caratterizzazione di mouse Antral ovociti Sulla base Nucleolare cromatina Organizzazione

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded ...

isolation-characterization-mouse-antral-oocytes-based-on-nucleolar

Research

JoVE Journal

Developmental Biology

13.2K Views.  Fondazione IRCCS Policlinico San Matteo.  Qui vi presentiamo un protocollo per la caratterizzazione di oociti antrali di topo sulla base di organizzazione nucleolare. 

Video: Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization

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

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

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

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

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

Ricerca

Biologia dello sviluppo

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.

This protocol describes the isolation of satellite cells from branchiomeric head muscles of a 9 week-old rat. The muscles originate from different branchial arches. Subsequently, the satellite cells are cultured on a spot coating of millimeter size to study their differentiation. This approach avoids the expansion and passaging of satellite cells.

Isolamento e caratterizzazione di cellule satelliti da muscoli Rat Capo Branchiomeric

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

Isolamento e caratterizzazione di singole cellule da embrioni di zebrafish

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

Isolamento e caratterizzazione di cellule mesenchimali stromali dal Cordone ombelicale e placenta fetale

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

Un uso novello di tridimensionale ad alta frequenza l'ecografia per la caratterizzazione di gravidanza precoce nel topo

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

A Neural Network-Based Identification of Developmentally Competent or Incompetent Mouse Fully-Grown Oocytes

Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus improving the overall pregnancy outcome. We recently developed a classification method based on microscopic live observations of mouse oocytes during their in vitro maturation from the germinal vesicle (GV) to the metaphase II stage, followed by the analysis of the cytoplasmic movements occurring during this time-lapse period. Here, we present detailed protocols of this procedure. Oocytes are isolated from fully-grown antral follicles and cultured for 15 h inside a microscope equipped for time-lapse analysis at 37 &#176;C and 5% CO2. Pictures are taken at 8 min intervals. The images are analyzed using the Particle Image Velocimetry (PIV) method that calculates, for each oocyte, the profile of Cytoplasmic Movement Velocities (CMVs) occurring throughout the culture period. Finally, the CMVs of each single oocyte are fed through a mathematical classification tool (Feed-forward Artificial Neural Network, FANN), which predicts the probability of a gamete to be developmentally competent or incompetent with an accuracy of 91.03%. This protocol, set up for the mouse, could now be tested on oocytes of other species, including humans.

Here, we present a protocol for non-invasive assessment of oocyte developmental competence performed during their in vitro maturation from the germinal vesicle to the metaphase II stage. This method combines time-lapse imaging with particle image velocimetry (PIV) and neural network analyses.

Un'identificazione basato su rete neurale di ovociti adulto Mouse inerente allo sviluppo competente o incompetente

Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus ...

Isolation and Characterization of Human Umbilical Cord-derived Mesenchymal Stem Cells from Preterm and Term Infants

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally isolated and characterized from bone marrow (BM), then acquired from tissues including adipose tissue, synovium, skin, dental pulp, and fetal appendages such as placenta, umbilical cord blood (UCB), and umbilical cord (UC). MSCs are a heterogeneous cell population with the capacity for (1) adherence to plastic in standard culture conditions, (2) surface marker expression of CD73+/CD90+/CD105+/CD45-/CD34-/CD14-/CD19-/HLA-DR- phenotypes, and (3) trilineage differentiation into adipocytes, osteocytes, and chondrocytes, as currently defined by the International Society for Cellular Therapy (ISCT). Although BM is the most widely used source of MSCs, the invasive nature of BM aspiration ethically limits its accessibility. Proliferation and differentiation capacity of MSCs obtained from BM generally decline with the age of the donor. In contrast, fetal MSCs obtained from UC have advantages such as vigorous proliferation and differentiation capacity. There is no ethical concern for UC sampling, as it is typically regarded as medical waste. Human UC starts to develop with continuing growth of the amniotic cavity at 4-8 weeks of gestation and keeps growing until reaching 50-60 cm in length, and it can be isolated during the whole newborn delivery period. To gain insight into the pathophysiology of intractable diseases, we have used UC-derived MSCs (UC-MSCs) from infants delivered at various gestational ages. In this protocol, we describe the isolation and characterization of UC-MSCs from fetuses/infants at 19-40 weeks of gestation.

Human umbilical cord (UC) can be obtained during the perinatal period as a result of preterm, term, and postterm delivery. In this protocol, we describe the isolation and characterization of UC-derived mesenchymal stem cells (UC-MSCs) from fetuses/infants at 19-40 weeks of gestation.

Isolamento e caratterizzazione di umani cellule staminali mesenchimali del cordone ombelicale-derivato da prematuri e neonati a termine

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally ...

Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays to characterize the differentiated cells. The E14 mouse embryonic stem cells were used to form embryoid bodies through the hanging drop method, and then induced to differentiate into neural progenitor cells by retinoic acid, and finally differentiated into neurons. Quantitative&#160;reverse transcription&#160;polymerase chain reaction (RT-qPCR) and immunofluorescence experiments revealed that the neural progenitors and neurons exhibit corresponding markers (nestin for neural progenitors and neurofilament for neurons) at day 8 and 12 post-differentiation, respectively. Flow cytometry experiments on an E14 line expressing a Sox1 promoter-driven GFP reporter showed that about 60% of cells at day 8 are GFP positive, indicating the successful differentiation of neural progenitor cells at this stage. Finally, RNA-seq analysis was used to profile the global transcriptomic changes. These methods are useful for analyzing the involvement of specific genes and pathways in regulating the cell identity transition during neuronal differentiation.

We describe the procedure for the in vitro differentiation of mouse embryonic stem cells into neuronal cells using the hanging drop method. Furthermore, we perform a comprehensive phenotypic analysis through RT-qPCR, immunofluorescence, RNA-seq, and flow cytometry.

Differenziazione e caratterizzazione di progenitori neurali e neuroni da cellule staminali embrionali di topo

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays ...

Minimum Volume Vitrification of Immature Feline Oocytes

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to promote biodiversity preservation. In felids, most species are threatened with extinction, and domestic breeds are used as a model to increase the efficiency of protocols for germplasm banking. Among oocyte cryopreservation techniques, vitrification is more and more popular in human and veterinary assisted reproduction. Cryotop vitrification, which was at first developed for human oocytes and embryos, has demonstrated to be well-suited for cat oocytes. This method offers several advantages, such as the feasibility in field conditions and the speed of the procedure, which can be helpful when several samples need to be processed. However, the efficiency is strongly dependent on the operator&#8217;s skills, and intra- and inter-laboratory standardization are needed, as well as personnel training. This protocol describes minimum volume vitrification of immature feline oocytes on a commercial support in a step by step field-friendly protocol, from oocyte collection to warming. Following the protocol, preservation of oocyte integrity and viability at warming (as high as 90%) can be expected, although there is still room for improvement in post-warming maturation and embryonic development outcomes.

This manuscript describes a protocol for the minimum volume vitrification of immature cat oocytes with laboratory-made media on commercial supports. It covers every step from oocyte isolation from ex vivo gonads to vitrification and warming.

Vitrificazione del volume minimo degli ovociti felini immaturi

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to ...

In Vitro Culture Strategy for Oocytes from Early Antral Follicle in Cattle

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that during the reproductive life span only about 1% of the oocytes in an ovary mature and ovulate, several techniques have been developed to increase the exploitation of the ovarian reserve to the growing population of non-ovulatory follicles. Such technologies have allowed interventions of fertility preservation, selection programs in livestock, and conservation of endangered species. However, the vast potential of the ovarian reserve is still largely unexploited. In cows, for instance, some attempts have been made to support in vitro culture of oocytes at specific developmental stages, but efficient and reliable protocols have not yet been developed. Here we describe a culture system that reproduce the physiological conditions of the corresponding follicular stage, defined to develop in vitro growing oocytes collected from bovine early antral follicles to the fully-grown stage, corresponding to the medium antral follicle in vivo. A combination of hormones and a phosphodiesterase 3 inhibitor was used to prevent untimely meiotic resumption and to guide oocyte&#39;s differentiation.

We describe the procedures for isolation of growing oocytes from ovarian follicles at early stages of development, as well as the setup of an in vitro culture system which can support the growth and differentiation up to the fully-grown stage. &#160;

Strategia di coltura in vitro per oociti dal follicolo antralico precoce nel bestiame

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that ...