1. Istituzione di colture di fibroblasti da espianti di pelle <ol><li> 4 biopsie millimetri punzone acquisiti da avambraccio umano sono posti in MEM addizionato 100 unità / ml di penicillina, 100 ug / ml streptomicina, e 0,25 mcg / ml Fungizone. </li><li> Tagliare il grasso sottocutaneo e tritare finemente la biopsia in piccoli pezzi da dondolo 24 lama di bisturi numero contro il fondo della capsula di Petri. </li><li> Tessuto slurry posto in un centimetro 25 2 pallone di coltura tissutale con mezzi primari di crescita dei fibroblasti (MEM supplementato con 10% FCS, 100 unità / ml di penicillina, 100 ug / ml streptomicina, e 25 ug / ml Fungizone) aggiunto finché la superficie del pallone è coperto ma la profondità del liquido è insufficiente per il tessuto a stare a galla. </li><li> Seguendo tre giorni di incubazione a 37 ° C in atmosfera umidificata al 5% di CO 2, aggiungere 3 ml di mezzo di crescita dei fibroblasti primari. </li><li> Dopo un altro giorno 3 Media cambiamento di incubazione con i media fibroblasti primari di crescita fresche, o se cells sono quasi confluenti possono essere suddivisi 01:04 in mezzi freschi. (Fungizone può essere omesso da questo punto in poi). </li></ol> 2. Fase I - Preparazione del collagene I di code di ratto Nota: protocollo per circa 12-14 code di ratto adolescente (freschi o surgelati) <ol><li> Preparare coda di ratto mediante lavaggio in 70% etanolo e rimuovere tendini come segue: </li><li> Rimuovere la pelle dal coda di ratto mediante tranciatura nel mezzo della coda da cima a fondo con un bisturi e pealing lungo la lunghezza della coda. </li><li> Tendine staccare dal nucleo della regione prossimale della coda. </li><li> Rimuovere tendine verso la regione distale della coda evitando la guaina con pinze dentate. </li><li> 1 g di estratto tendine / 250 ml di 0,5 M di acido acetico agitazione a 4 ° C per 48 h. </li><li> Centrifugare l&#39;estratto (7.500 xg) per 30 minuti e scartare il pellet. </li><li> Aggiungere un volume uguale del 10% (w / v) NaCl al supernatante, e agitare per 30-60 min. </li><lCentrifugare i&gt; (10.000 xg) per 30 minuti, scartare il surnatante, e ri-sciogliere il precipitato in 0,25 M di acido acetico in rapporto 1:1 ~ da agitazione per 24 ore a 4 ° C. </li><li> Dializza la soluzione di collagene contro 6-8 cambi di 6L ~ 17,5 mM di acido acetico (1 ml di acido acetico glaciale per litro di acqua fredda, modifica 2 x al giorno). </li><li> Centrifugare collagene dializzato a 30.000 xg per 1,5 ore. </li><li> Rimuovere il surnatante e mettere in beuta sterile. </li><li> Regolare la concentrazione di collagene I ~ 2 mg / ml usando acido acetico 0,5 mM a 4 ° C. </li></ol> 3. Fase II - Creare la matrice 3D con fibroblasti incorporati consentono di contrarre (per 8 giorni). <ol><li> Assemblare il mix utilizzando reagenti freddi a 4 ° C in un pre-refrigerati bottiglia. I collagen mantenere in ghiaccio. </li><li> Per una T75 pallone confluenti fibroblasti primari (~ cella numero 1 x 10 6) uso: <ul><li> 25 ml di collagene coda di ratto (ca. conc 2mg/ml) </li><li> 3 ml 10xMEM </li><li> 0,22 M di NaOH: in primo luogo, aggiungere 2 ml, poi goccia a goccia sotto agitazione, fino a quando il collagene diventa arancione, </li><li> ma non rosa (solitamente fino a 3 ml). Approssimativo p H = 7.2. </li><li> Nota: è importante garantire che il mezzo / gel rimane neutro o leggermente acido come fibroblasti non contrarre il gel se esposto a condizioni anche leggermente alcaline. </li></ul></li><li> Trypsinise i fibroblasti, centrifugare a 400 xg per 5 minuti e rimuovere il surnatante. </li><li> Durante la centrifuga ad oltre 5 minuti: Preparare 12 x 35 piatti di plastica mm - consegue normalmente 12 piatti da un pallone di fibroblasti / mix collagene. </li><li> Risospendere fibroblasti in 3 ml di FCS e aggiungere immediatamente alla miscela di collagene e mescolare. </li><li> Piastra di circa 2,5 ml di collagene / fibroblasti a piatto più rapidamente possibile. Cercate di evitare le bolle e le impostazioni di collagene. </li><li> Lasciare che il collagene impostato a 37 ° C in atmosfera umidificata al 5% di CO 2 in aria per 10 minuti. </li><li> Aggiungere 1 ml di fibroblasti crescere° media (DMEM + 10% FCS). </li><li> Staccare il collagene / fibroblasti matrice dai lati del piatto con una pipetta. </li><li> Il giorno dopo: Aggiungere 1 ml di terreni di crescita dei fibroblasti (DMEM + 10% FCS). </li><li> Cambiare i media ogni due giorni. Consenti collagene / fibroblasti matrice a contrarre per circa 8 giorni, fino a quando si inseriscono in un piatto da 24 pozzetti (Contrazione da ~ 3,5 cm a 1,5 cm di diametro, si veda la Figura 1). </li></ol> Nota: La concentrazione di collagene deve essere regolata secondo l&#39;applicazione. Il più diluire la soluzione di collagene, più veloce sarà contratto dai fibroblasti. Leggere differenze di velocità di contrazione sarà sperimentato con differenti lotti di collagene alla stessa concentrazione. Allo stesso modo, diverse colture di fibroblasti di contrarre il gel di collagene a ritmi diversi, i fibroblasti e più presenti, più veloce è il tasso di contrazione. Concentrazione di collagene e densità dei fibroblasti possono quindi, regolato per modificare la velocità di contrazione,e la densità finale del gel di collagene. 4. Fase II - cellule Placcatura di interesse sulla parte superiore della matrice Nota: Sterilizzare tutti pinze e le attrezzature con etanolo prima dell&#39;uso. <ol><li> Utilizzando pinze smussato, spostare delicatamente matrice contratto a 24-ben piatto. Assicurarsi che non si piega. </li><li> Preparare la sospensione di cellule di interesse a circa 4 x 10 1 ml 4 / ml e la piastra (dopo che le cellule trypsinising, di selezione per sbarazzarsi di tripsina) sulla parte superiore della matrice. Numero effettivo di cellule necessarie varierà a seconda del tipo cellulare utilizzato. Medie cellulare dovrebbe essere medio normale crescita per le cellule di interesse. </li><li> Permettere alle cellule di crescere fino a confluenza in cima alla matrice, circa 3-5 giorni. </li></ol> 5. Fase III - Trasferire la matrice di una griglia per l&#39;invasione (circa 0-21 giorni) <ol><li> Tagliare griglie in acciaio inox per creare un treppiede e sterilizzare in autoclave prima dell&#39;uso (vedere la figura 2). </li><li> Mettere in rete sterilePiatto 6 cm, aggiungere mezzi di crescita ad un livello sopra la griglia (circa 10.5ml). Matrix posto sulla griglia e delicatamente aspirare supporti modo che il fondo della matrice è in contatto con i media ma non sommersa. Questo è indicato come l&#39;aria / interfaccia liquido, che crea un gradiente che promuove invasione. Sostituire media ogni due giorni (vedi figura 2). </li><li> Vivo delle cellule, l&#39;imaging contestuale utilizzando cellule fluorescenti possono essere esposte in questa fase o precedenti. Il collagene contratto o fibrillare che è possibile creare immagini utilizzando generazione di seconda armonica (SHG, vedi figura 3). Utilizzo multi-fotone di eccitazione combinata con ampio campo di rilevamento si trova che SHG possono essere esposte ad almeno 100 micron nella matrice, mentre le cellule che esprimono GFP citoplasmatica è possibile creare immagini di almeno 200 micron di profondità. </li></ol> Nota: Per quanto riguarda la quantificazione di invasione, il giorno in cui sono posti da matrici sulle griglie definisce Giorno 0. Posizionamento su griglie genera un gradiente di terreni di coltura delle cellule che promuove int invasione<html> o la matrice. I campioni possono essere ripreso nei prossimi 1-21 giorni (o più) la valutazione dei processi biologici come l&#39;invasione, la proliferazione, la sopravvivenza o la differenziazione (Vedi tabella). 6. Fase IV - Fissazione <ol><li> Aggiungere 5 ml del 4% PFA in un tubo Falcon. </li><li> Trasferire la matrice su una superficie piana. Tagliare la matrice a metà con un fresco pulito bisturi (come si sarà colorazione la sezione trasversale), sollevare la matrice con bisturi e trasferimento al 4% PFA, fissare per tutta la notte. </li><li> Matrici organotipiche sono ora pronti a macchiare con l&#39;anticorpo / macchia di scelta (vedi Figura 3). </li></ol> 7. Rappresentante dei risultati: <img alt="Figura 1" src="/files/ftp_upload/3089/3089fig1.jpg" /> Figura 1 Esempio di fibroblasti-contratto Miscela collagene I. fibroblasti collagene e della coda di ratto staccato dalla capsula di Petri e ha permesso di contrarre più di 6-8 giorni. Figura 2 &quot;src =&quot; / files/ftp_upload/3089/3089fig2.jpg &quot;/&gt; Figura 2 progressione organotipica dosaggio. A, schematica di set up organotipica e progressione. B, Esempio di collagene / fibroblasti matrice sopra di acciaio inossidabile, griglia sterile per creare interfaccia aria / liquido. <img alt="Figura 3" src="/files/ftp_upload/3089/3089fig3.jpg" /> Figura 3 Applicazioni di test organotipica. A, B non invasive e invasive pancreatiche duttali adenocarcinoma (PDAC) invade le cellule nel tempo con matrice organotipica. C, vita equivalente della pelle che mostra una epidermide stratificata su un fibroblasto-contratto componente collagene dermico. D, C8161 cellule di melanoma che invadono per un fibroblasto -contratto collagene dermico equivalente. E, invasive cellule PDAC (verde) che interagiscono e invadere con fibroblasti (rosso) all&#39;interno della matrice organotipica. F </strong&gt;, le cellule PDAC invasive (verde) che interagiscono con un degradante circostante matrice extracellulare (viola) visualizzato per mezzo di multifotone basata generazione di seconda armonica.

<ol>
	<li>Yamada, K. M., Cukierman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+tissue+morphogenesis+and+cancer+in+3D.">Modeling tissue morphogenesis and cancer in 3D.</a> Cell. 130, 601-610 (2007).</li><li>Edward, M., Gillan, C., Micha, D., Tammi, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour+regulation+of+fibroblast+hyaluronan+expression:+a+mechanism+to+facilitate+tumour+growth+and+invasion.">Tumour regulation of fibroblast hyaluronan expression: a mechanism to facilitate tumour growth and invasion.</a> Carcinogenesis. 26, 1215-1223 (2005).</li><li>Patsialou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+human+breast+cancer+cells+in+vivo+requires+both+paracrine+and+autocrine+loops+involving+the+colony-stimulating+factor-1+receptor.">Invasion of human breast cancer cells in vivo requires both paracrine and autocrine loops involving the colony-stimulating factor-1 receptor.</a> Cancer Res. 69, 9498-9506 (2009).</li><li>Serrels, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+study+of+E-cadherin+and+membrane+dynamics+in+living+animals:+implications+for+disease+modeling+and+drug+development.">Real-time study of E-cadherin and membrane dynamics in living animals: implications for disease modeling and drug development.</a> Cancer Res. 69, 2714-2719 (2009).</li><li>Timpson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+Regulation+of+RhoA+Activity+during+Pancreatic+Cancer+Cell+Invasion+Driven+by+Mutant+p53.">Spatial Regulation of RhoA Activity during Pancreatic Cancer Cell Invasion Driven by Mutant p53.</a> Cancer Res. 71, 747-757 (2011).</li><li>Edward, M., Quinn, J. A., Pasonen-Seppanen, S. M., McCann, B. A., Tammi, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=4-Methylumbelliferone+inhibits+tumour+cell+growth+and+the+activation+of+stromal+hyaluronan+synthesis+by+melanoma+cell-derived+factors.">4-Methylumbelliferone inhibits tumour cell growth and the activation of stromal hyaluronan synthesis by melanoma cell-derived factors.</a> Br. J. Dermatol. 162, 1224-1232 (2010).</li><li>Fusenig, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+differentiation+characteristics+of+transformed+keratinocytes+from+mouse+and+human+skin+in+vitro+and+in+vivo.">Growth and differentiation characteristics of transformed keratinocytes from mouse and human skin in vitro and in vivo.</a> J. Invest. Dermatol. 81, 168s-175s (1983).</li><li>Nystrom, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+quantitative+method+to+analyse+tumour+cell+invasion+in+organotypic+culture.">Development of a quantitative method to analyse tumour cell invasion in organotypic culture.</a> J. Pathol. 205, 468-475 (2005).</li><li>Sabeh, F., Shimizu-Hirota, R., Weiss, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protease-dependent+versus+-independent+cancer+cell+invasion+programs:+three-dimensional+amoeboid+movement+revisited.">Protease-dependent versus -independent cancer cell invasion programs: three-dimensional amoeboid movement revisited.</a> J. Cell. Biol. 185, 11-19 (2009).</li><li>Amjad, S. B., Carachi, R., Edward, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratinocyte+regulation+of+TGF-beta+and+connective+tissue+growth+factor+expression:+a+role+in+suppression+of+scar+tissue+formation.">Keratinocyte regulation of TGF-beta and connective tissue growth factor expression: a role in suppression of scar tissue formation.</a> Wound Repair Regen. 15, 748-755 (2007).</li><li>Gaggioli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast-led+collective+invasion+of+carcinoma+cells+with+differing+roles+for+RhoGTPases+in+leading+and+following+cells.">Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells.</a> Nat. Cell. Biol. 9, 1392-1400 (2007).</li></ol>

Non abbiamo nulla da rivelare.

Presentiamo qui un metodo per la produzione di una matrice 3-dimensionale adatto per studi di migrazione cellulare invasiva 6-8. La matrice è costituita da collagene di tipo 1 fibrille in subappalto per un periodo di diversi giorni da fibroblasti umani primari epidermiche. Il collagene è preparato mediante estrazione acida non, digestione enzimatica, che conserva reattività alle poli-peptide estremità e promuove reticolazione delle fibrille di collagene in grandi aggregati upon neutralizzazione di condizioni del tampone 9. Ciò comporta una ricostituito gel che più strettamente simula le funzioni di collagene in vivo e ha conseguenze importanti per le proprietà invasive delle cellule coltivate in gel. Non importa se fresco o congelato materiale di partenza viene utilizzato, ma l&#39;uso di code di ratto adolescenti è importante perché il collagene cross-linking è più labile in animali più giovani. Collagene I da animali più giovani è quindi più facile da estrarre, e ri-costituisce with alta fedeltà. La matrice di collagene può essere fissate e colorate con anticorpi diretti contro sia cellule tumorali invasive o fibroblasti stromali 2,10 e sono molto utili per testare l&#39;efficacia dei farmaci che possono inibire l&#39;invasione 5,6. Sebbene questo protocollo suggerisce l&#39;uso di fibroblasti umani primari, è possibile utilizzare fibroblasti primari da altri tipi di tessuto, a seconda del tipo di tessuto in esame. È anche possibile stabilire culture utilizzando fibroblasti immortalizzate, comprese le cellule che sono state stabilmente trasfettate con coniugati proteina fluorescente. In questo modo le cellule stromali e sia tumore può essere etichettato per visualizzare interazioni cellulari durante invasione 11.

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti (opzionale) </th></tr><tr><td> MEM 10x </td><td> Gibco </td><td> 21430 </td><td> - </td></tr><tr><td> NaOH </td><td> Sigma </td><td> 367176-500G </td><td> Preparare magazzino 0,22 M in acqua </td></tr><tr><td> FCS </td><td> PAA Laboratories </td><td> A15-101 </td><td> - </td></tr><tr><td> Piastre da 35 mm </td><td> Falco </td><td> 353001 </td><td> Per il punto 3.4 </td></tr><tr><td> 60 piatti millimetri </td><td> Falco </td><td> 353004 </td><td> Per il punto 5.2 </td></tr><tr><td> Primavera pinza smussato </td><td> Samco </td><td> E003/02 </td><td> Dentata, non è liscia </td></tr><tr><td> 16% paraformaldeide </td><td> Servizi di Microscopia Elettronica </td><td> 15710 </td><td> Diluire al 4% in PBS prima dell&#39;uso </td></tr><tr><td> Schermi per cd-1, taglia 40 maglie </td><td> Sigma </td><td> S07707-5EA </td><td> Griglie in acciaio inox, 5 per confezione </td></tr><tr><td> Dialisi tubo </td><td> Medicell internazionale </td><td> 7607 2295 </td><td> 12-14 kD </td></tr><tr><td> PBS </td><td> Oxoid </td><td> BR0014G </td><td> - </td></tr><tr><td> Acido acetico </td><td> Sigma </td><td> 242853 </td><td> - </td></tr><tr><td> 24 piatto ben </td><td> Falco </td><td> 353047 </td><td> Per Passaggio 4.1 </td></tr><tr><td> Fungizone </td><td> In vitrogen </td><td> 15290018 </td><td> - </td></tr></tbody></table>

organotypic collagen i assay: a malleable platform to assess cell behaviour in a 3-dimensional context

Migrazione cellulare è fondamentale per molti aspetti della biologia, compreso lo sviluppo, la guarigione delle ferite, le risposte cellulari del sistema immunitario, e metastasi delle cellule tumorali. La migrazione è stata studiata su vetrini in modo da rendere dinamiche cellulari suscettibili di indagine al microscopio ottico. Tuttavia, è diventato chiaro che molti aspetti della migrazione cellulare dipendono caratteristiche dell&#39;ambiente locale compresa la sua elasticità, composizione proteica, e la dimensione dei pori, che non sono fedelmente rappresentati da due substrati rigidi dimensionali quali vetro e plastica 1. Inoltre, l&#39;interazione con altri tipi di cellule, inclusi i fibroblasti stromali e cellule immunitarie 2, 3 ha dimostrato di giocare un ruolo critico nel promuovere l&#39;invasione delle cellule tumorali. Indagine a livello molecolare ha sempre dimostrato che dinamica molecolare, compresa la risposta al trattamento farmacologico, di cellule identiche sono significativamente differenti rispetto <em&gt; In vitro e in vivo 4. Idealmente, sarebbe meglio studiare la migrazione cellulare nel suo contesto naturale in organismi viventi, tuttavia questo non è sempre possibile. Intermedi sistemi di coltura di tessuti, come matrice cellulare derivata, Matrigel, coltura organotipica (descritto qui) espianti tissutali, organoidi, xenotrapianti, e sono quindi importanti intermedi sperimentali. Questi aspetti approssimativi sistemi certi di un ambiente in vivo, ma sono più suscettibili di manipolazione sperimentale come l&#39;uso di linee cellulari stabilmente trasfettate, regimi di trattamento farmacologico, a lungo termine e ad alta risoluzione delle immagini. Tali sistemi intermedi sono particolarmente utili come terreni di prova per convalidare le sonde e stabilire i parametri necessari per l&#39;immagine della risposta dinamica di cellule fluorescenti e giornalisti, prima di effettuare l&#39;imaging in vivo 5. Come tali, essi possono svolgere un ruolo importante nel ridurre la necessità di esperimenti su living animali.

Watch this Scientific Journal Video about Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context at JoVE.com

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Un metodo è descritto per la preparazione di un 3-dimensionale matrice costituita da collagene di tipo I e di fibroblasti umani primari. Questo gel organotipica serve come substrato utile per valutare la migrazione delle cellule invasive perché imita caratteristiche di base di stroma tissutale ed è suscettibile di molte forme di microscopia.

Organotipiche Collagene I test: una piattaforma malleabile per valutare Comportamento cella in un 3-dimensionale Contesto

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

organotypic-collagen-i-assay-malleable-platform-to-assess-cell

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Bioengineering

22.3K Views.  University of Glasgow. Un metodo è descritto per la preparazione di un 3-dimensionale matrice costituita da collagene di tipo I e di fibroblasti umani primari. Questo gel organotipica serve come substrato utile per valutare la migrazione delle cellule invasive perché imita caratteristiche di base di stroma tissutale ed è suscettibile di molte forme di microscopia.

Video: Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

Un metodo per l&#39;incapsulamento follicolo ovarico e la cultura in un proteoliticamente degradabile 3 Sistema dimensionale

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

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Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

La creazione di substrati Patterned bidimensionali per confinamento di proteine ​​e cellule

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

La coltivazione di cellule progenitrici neurali umane in un 3-dimensionale di auto-assemblaggio Peptide Hydrogel

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Ponteggi self-reporting per coltura cellulare 3-Dimensional

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

Un metodo migliore per la preparazione di tipo I collagene della pelle

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

Analisi della migrazione delle cellule all&#39;interno di una tridimensionale di collagene Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

Planar gradiente di diffusione del sistema per Indagare Chemiotassi in un 3D collagene Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

Tissue tritata in Compressed Collagene: A Biotransplant cellulare contenente per la riparazione singolo-messa in scena Ricostruttiva

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

Ricombinante collagene Peptide Microcarriers per l'espansione delle cellule e loro potenziale utilizzo come sistema di consegna di cella in un bioreattore modello

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.