Questo lavoro è stato sostenuto da un assegno di ricerca dal National Institutes of Health (HL068915 di DBC), un nuovo Researcher Award del Fondo di ricerca Thrasher (al PAC), un Grant-in-Aid dalla American Heart Association (12GRNT11910008 a DBC) , un assegno di ricerca da Heart Foundation dei Bambini (da DBC), e le donazioni a Hospital di conduzione cardiaca Fondo dei Bambini di Boston, la Ryan Famiglia Endowment, e da David Pullman.

Qui ci dimostrare l&#39;isolamento di COL1 dalla pelle di agnello. Come scritto, questo protocollo può anche essere utilizzata per isolare con successo COL1 da coniglio e pelle umana. 1. Preparare Dermal campione <ol><li> Equilibrare tutti i reagenti a 4 ° C prima dell&#39;uso. </li><li> Risciacquare campione cutanea (25-50 g) in DH ghiacciata 2 O e rimuovere eventuali lana, pelliccia o capelli con crema depilatoria. </li><li> Utilizzare una lama di rasoio singolo bordo per raschiare il pulito campione di tessuto connettivo e grasso. </li><li> Lavare il campione in ghiaccio-freddo dH 2 O. </li><li> Tagliate il campione di pelle in 1 cm x 1 centimetri pezzi con una lama di rasoio singolo bordo. </li></ol> 2. Rimuovere noncollagenous solubilizzata Materiale <ol><li> Pesare 5 g di campione per tubo da 50 ml e aggiungere 30 ml di ghiacciata 0.5 M acetato di sodio. </li><li> Mescolare provette per 1 min alla regolazione di 6 m / sec utilizzando un adattatore tubo da 50 ml in omogeneizzatore da banco. </li><li> Scartare supernatant e ripetere per un totale di 7 sodio acetato cicli di lavaggio. </li><li> Lavare il campione in DH ghiacciata 2 O e mescolare una volta per rimuovere acetato di sodio residuo. </li><li> Utilizzare una spatola per comprimere il campione contro il lato del tubo per rimuovere il liquido in eccesso e poi trasferire in una provetta conica da 50 ml fresco. </li></ol> 3. Collagene di tipo I Estrazione <ol><li> Lavare il campione due volte in 2 ml / g 0,075 M tampone sodio citrato per 1 min a 6 m / sec nella omogeneizzatore banco comprimendo il campione e scartando il supernatante dopo ogni lavaggio. </li><li> Aggiungere un fresco 2 ml / g aliquota di 0,075 M tampone sodio citrato al campione. </li><li> Eseguire 6 sequenziale 1 min, 6 cicli banco mix omogeneizzatore m / sec di agitazione senza rimuovere il cuscinetto tra ogni ciclo. </li><li> Trasferire la spessa, chiara surnatante in un tubo di raccolta. </li><li> Aggiungere un ulteriore 1 ml / g 0,075 M di sodio tampone citrato al campione ed effettuare un ultimo banco omogeneizzatore agitazioneCiclo zione. </li><li> Aggiungere questo surnatante finale a un tubo di raccolta. </li><li> Centrifugare la provetta di raccolta a 3.200 xg per 10 min a 4 ° C. </li><li> Trasferire il surnatante al vano superiore di un peso molecolare 100.000 tagliare dispositivo di filtro centrifugo. </li><li> Centrifugare a 3200 xg per 30 min a 4 ° C. </li><li> Trasferire il purificato COL1 dal vano superiore in una provetta pulita e conservare a 4 ° C. </li></ol>

<ol>
	<li>Nageotte, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coagulation+fibrillaire+in+vitro+du+collag&#232;ne+dissous+dans+un+acide+dilu&#233;.">Coagulation fibrillaire in vitro du collag&#232;ne dissous dans un acide dilu&#233;.</a> CR hebd. s&#233;ances Acad. Sei. 184, 115 (1927).</li><li>Schmitt, F. O., Hall, C. E., Jakus, 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=Electron+microscope+investigation+of+the+structure+of+collagen.">Electron microscope investigation of the structure of collagen.</a> J. Cell Comp. Physiol. 20, 11-33 (1942).</li><li>Choi, Y. H., 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=Cardiac+conduction+through+engineered+tissue.">Cardiac conduction through engineered tissue.</a> Am. J. Pathol. 169, 72-85 (2006).</li><li>Pacak, C. A., Cowan, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+myogenic+engineered+tissue+constructs.">Fabrication of myogenic engineered tissue constructs.</a> J. Vis. Exp. (27), (2009).</li><li>Cliche, S., Amiot, J., Avezard, C., Gariepy, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+and+characterization+of+collagen+with+or+without+telopeptides+from+chicken+skin.">Extraction and characterization of collagen with or without telopeptides from chicken skin.</a> Poultry Sci. 82, 503-509 (2003).</li><li>Rajan, N., Habermehl, J., Cote, M. F., Doillon, C. J., Mantovani, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+ready-to-use,+storable+and+reconstituted+type+I+collagen+from+rat+tail+tendon+for+tissue+engineering+applications.">Preparation of ready-to-use, storable and reconstituted type I collagen from rat tail tendon for tissue engineering applications.</a> Nat. Protoc. 1, 2753-2758 (2006).</li><li>Seifter, S., Gallop, P., Colowick, S., Kaplan, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+properties+of+soluble+collagens.">Preparation and properties of soluble collagens.</a> Methods in Enzymology. , (1963).</li><li>Pacak, C. A., Powers, J. M., Cowan, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrarapid+purification+of+collagen+type+I+for+tissue+engineering+applications.">Ultrarapid purification of collagen type I for tissue engineering applications.</a> Tissue Eng. Part C Methods. 17, 879-885 (2011).</li><li>Ohan, M. P., Dunn, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+stabilizes+collagen+sterilized+with+gamma+irradiation.">Glucose stabilizes collagen sterilized with gamma irradiation.</a> J. Biomed. Mater. Res. A. 67, 1188-1195 (2003).</li><li>Tyan, Y. C., Liao, J. D., Lin, S. P., Chen, C. C. <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+the+sterilization+effect+of+gamma+ray+irradiation+of+immobilized+collagen+polypropylene+nonwoven+fabric+surfaces.">The study of the sterilization effect of gamma ray irradiation of immobilized collagen polypropylene nonwoven fabric surfaces.</a> J. Biomed. Mater. Res. A. 67, 1033-1043 (2003).</li><li>Ber, S., Torun Kose, G., Hasirci, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+tissue+engineering+on+patterned+collagen+films:+an+in+vitro+study.">Bone tissue engineering on patterned collagen films: an in vitro study.</a> Biomaterials. 26, 1977-1986 (2005).</li><li>Liu, Y., Sun, S., Singha, S., Cho, M. R., Gordon, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3-D+femtosecond+laser+patterning+of+collagen+for+directed+cell+attachment.">3-D femtosecond laser patterning of collagen for directed cell attachment.</a> Biomaterials. 26, 4597-4605 (2005).</li><li>Thakar, R. G., Ho, F., Huang, N. F., Liepmann, D., Li, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+vascular+smooth+muscle+cells+by+micropatterning.">Regulation of vascular smooth muscle cells by micropatterning.</a> Biochem. Biophys. Res. Comm. 307, 883-890 (2003).</li><li>Leighton, J., Justh, G., Esper, M., Kronenthal, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen-coated+cellulose+sponge:+three+dimensional+matrix+for+tissue+culture+of+Walker+tumor+256.">Collagen-coated cellulose sponge: three dimensional matrix for tissue culture of Walker tumor 256.</a> Science. 155, 1259-1261 (1967).</li></ol>

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Un componente fondamentale di questo metodo è la compressione efficiente ripetuta del campione dermica per rimuovere il liquido in eccesso. E &#39;fondamentale per rimuovere acetato di sodio quanto possibile al punto 2.5 comprimendo il campione. Usiamo una spatola per compattare la pelle contro il lato del tubo, tuttavia, garza potrebbe anche essere utilizzato, anche se ciò richiederebbe la rimozione del campione dalla provetta e aumentare il tempo necessario per eseguire questi passaggi. Allo stesso modo, è importan...

Per decenni, i ricercatori e venditori commerciali hanno isolato solubilizzato COL1 da un assortimento di fonti tissutali incluse pelle e tendine con qualche variazione di un protocollo di estrazione acida semplice seguito da neutralizzazione, che si traduce in una risospensione di una matrice di fibrille col1 organizzati utilizzabile per una moltitudine di applicazioni biomediche 1-4. Mentre ci sono molti esempi di applicazioni cliniche per COL1, alcuni di questi impiegano COL1 autologously-derivato per la preparazione di questa proteina richiede estrazioni lunghi prendere giorni o settimane per effettuare 5-7. Come risultato, gli investigatori ricerca e medici usano generalmente costosi, preparazioni commerciali di COL1 che vengono preparati utilizzando tessuti non umani come ratto, bovino, o derma suino o utilizzando pelle rimosso da cadaveri o dopo la circoncisione maschile. Per applicazioni rigenerative, molti di questi prodotti mostrano variabilità rispetto alla loro capacità di formare solidimatrici o costrutti di tessuto in grado di supportare attaccamento e la crescita cellulare. Di conseguenza, vi è la necessità distinto per un nuovo metodo standardizzato progettato per estrarre rapidamente COL1 da fonti tissutali autologhi accessibili e abbondanti. Tale metodo permetterebbe di risparmiare tempo e denaro nel contesto di ricerca in cui COL1 potrebbe essere estratto dal modello animale di interesse e utilizzato per la sperimentazione preclinica dei tessuti ingegnerizzati montati su scaffold a base di collagene. Allo stesso tempo, avendo la possibilità di utilizzare una rapida isolato, COL1 autologously derivata nella clinica aumenterebbe la sicurezza complessiva per i pazienti che altrimenti ricevere allogeniche o xenogeniche preparazioni. Per i pazienti che ricevono una preparazione autogeneic, questo metodo semplificato potrebbe ridurre notevolmente l&#39;intervallo tra raccolta biopsia e l&#39;applicazione di collagene. Per queste ragioni, abbiamo cercato di migliorare un isolamento e la purificazione metodo COL1 lunga tradizione utilizzando ad alta velocità agitazione e le dimensioni esclusione centrifugazione. Questo metodo è semplice da eseguire utilizzando tamponi standard e le attrezzature presenti in molti laboratori. Con l&#39;ausilio di agitazione ad alta velocità, il nostro protocollo riduce il tempo necessario per isolare solubile, dermica COL1 da circa 10 giorni a meno di 3 ore 8. È importante sottolineare che questo metodo può essere facilmente eseguito in ambito clinico per preparare COL1 autologously-derivato per l&#39;uso in pazienti durante un&#39;unica serie di procedure.

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			<td height="43" style="height:43px;width:123px;">FastPrep-24 System</td>
			<td style="width:136px;">MP Biomedicals</td>
			<td style="width:147px;">116004500</td>
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			<td height="190" style="height:190px;width:123px;">Centrifuge</td>
			<td style="width:136px;">Beckman</td>
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			<td style="width:139px;">Swing bucket rotor to accommodate 50 mL conical tubes at 3200 g.</td>
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an improved method for the preparation of type i collagen from skin

Tipo solubile 1 del collagene (COL1) è ampiamente utilizzato come substrato adesivo per colture cellulari e come impalcatura cellulare per applicazioni rigenerative. Clinicamente, questa proteina è ampiamente utilizzato per la chirurgia estetica, iniezioni dermiche, innesto osseo, e chirurgia ricostruttiva. Le fonti di COL1 per queste procedure sono comunemente non umano, che aumenta il potenziale di infiammazione e rigetto nonché trasmissione di malattie xenobiotico. In considerazione di ciò, un metodo per purificare in modo efficiente e rapido da COL1 quantità limitate di tessuti autologously derivati ​​elusione molti di questi problemi, tuttavia, protocolli di isolamento standard sono lunghe e spesso richiedono grandi quantità di tessuti di collagene. Qui, dimostriamo un metodo di estrazione COL1 efficiente che riduce il tempo necessario per isolare e purificare questa proteina da circa 10 giorni a meno di 3 ore. Abbiamo scelto il derma come fonte tessuto a causa della sua disponibilità durante molte procedure chirurgiche. Til suo metodo utilizza i buffer di estrazione tradizionali combinati con agitazione vigorosa e filtrazione centrifuga per ottenere altamente puro, COL1 solubile da piccole quantità di corium. In breve, biopsie dermici sono lavati accuratamente in DH ghiacciata 2 O dopo la rimozione di grasso, tessuto connettivo, e capelli. I campioni di pelle sono spogliati di proteine ​​non collagene e polisaccaridi con 0,5 M di acetato di sodio e un banco omogeneizzatore ad alta velocità. Collagene da residui solidi viene successivamente estratta con un tampone sodio citrato 0,075 M usando l&#39;omogeneizzatore. Questi estratti sono purificati con 100.000 MW di cut-off filtri centrifughi che producono preparazioni col1 di qualità comparabile o superiore ai prodotti commerciali o di quelli ottenuti utilizzando le procedure tradizionali. Prevediamo che questo metodo faciliterà l&#39;utilizzazione di autologously derivato COL1 per una moltitudine di ricerca e applicazioni cliniche.

Questo protocollo isolamento COL1 richiede circa 2 ore e 15 minuti per completare. La Figura 1 mostra un diagramma schematico che illustra le fasi principali di questa procedura. Preparazione del campione ed eseguendo i 7 cicli di agitazioni alta velocità e risciacqui con acetato di sodio richiede circa 35 min (Figure 1A-C). L&#39;esecuzione di una agitazione e risciacquo con dH 2 O richiede circa 5 min (Figure 1D e 1E). Aggiunta di citrato di sodio e sottop...

Watch this Scientific Journal Video about An Improved Method for the Preparation of Type I Collagen From Skin at JoVE.com

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

Procedure tradizionali per l&#39;isolamento di tipo solubile 1 del collagene (COL1) richiedono circa 10 giorni dall&#39;inizio alla fine a causa di incubazione tampone lunghe e laboriose resuspensions di fibrille. Qui, descriviamo un mezzo per purificare COL1 da piccole biopsie dermici in meno di 3 ore.

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. ...

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Bioengineering

22.7K Views.  Boston Children's Hospital.  Procedure tradizionali per l'isolamento di tipo solubile 1 del collagene (COL1) richiedono circa 10 giorni dall'inizio alla fine a causa di incubazione tampone lunghe e laboriose resuspensions di fibrille. Qui, descriviamo un mezzo per purificare COL1 da piccole biopsie dermici in meno di 3 ore. 

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

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

Tridimensionale pelle umana Ricostruire Modello: uno strumento per studiare la pelle normale e progressione Melanoma

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

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Bioingegneria

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

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

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 ...

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules4-5. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold6. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.

A major hurdle in current stem cell therapies is determining the most effective method to deliver these cells to host tissues. Here, we describe a chitosan-based delivery method that is efficient and simple in approach, while allowing adipose-derived stem cells to maintain their multipotency.

Costruire un idrogel collagene per la fornitura di Stem Cell-loaded Chitosan Microsfere

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions3, monitor drug delivery4,5, and encapsulation4. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle.6 Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs)7. In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking8. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters &lt;100 - 400 nm to study their behavior on cell functions3. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest9. Drug encapsulation can be achieved as reported during liposome formation9. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and (2) liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles.
Nonetheless, the current liposome preparation technologies (sonication10, freeze-and-thaw10, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter &lt;100 nm) with high consistency and efficiency10,5, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes.
Manual extrusion using gas-tight syringes and polycarbonate membranes10,5 is a common practice but heterogeneity is often observed when using pore sizes &lt;100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS)10, electron microscopy11 and nanoparticle tracking analysis (NTA)12 were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below.

This protocol describes an extrusion method for preparing lipid vesicles of sub-micron sizes with a high degree of homogeneity. This method uses a pressure-controlled system with controlled nitrogen flow rates for liposome preparation. The lipid preparation1,2, liposome extrusion, and size characterization will be presented herein.

Costante pressione controllata metodo di estrusione per la preparazione del Nano di dimensioni vescicole lipidiche

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform ...

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

Migliorata metodo per la preparazione di un cell-based, Contact modello umano della barriera sanguigna del cervello

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

Imaging denaturate collagene fili<em&gt; In vivo</em&gt; E<em&gt; Ex vivo</em&gt; Via Photo-triggered ibridazione di Caged collagene Mimetic peptidi

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

An Improved Mechanical Testing Method to Assess Bone-implant Anchorage

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a nano-scale. As such, traditional methods of describing implant surfaces -&#160;namely numerical determinants of surface roughness -&#160;are inadequate for predicting in vivo performance. Biomechanical testing provides an accurate and comparative platform to analyze the performance of biomaterial surfaces. An improved mechanical testing method to test the anchorage of bone to candidate implant surfaces is presented. The method is applicable to both early and later stages of healing and can be employed for any range of chemically or mechanically modified surfaces -&#160;but not smooth surfaces. Custom rectangular implants are placed bilaterally in the distal femora of male Wistar rats and collected with the surrounding bone. Test specimens are prepared and potted using a novel breakaway mold and the disruption test is conducted using a mechanical testing machine. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate and reproducible means for isolating an exact peri-implant region for testing.

An improved method to mechanically test bone anchorage to candidate implant surfaces is presented. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate means to direct the disruption forces to an exact peri-implant region.

Una migliorata meccanica metodo di prova per valutare osso-impianto Anchorage

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Ingegneria 3D Cellularized collagene Gel per Vascolare Rigenerazione Tissutale

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

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 ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Preparazione del 3D collagene Gel e microcanali per lo studio delle interazioni 3D<em&gt; In Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

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

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