Ce travail a été soutenu par la recherche sur le cancer ovarien Fonds [accorder LT/UIC/01.2011], le Centre de l&#39;UIC pour la science clinique et translationnelle, le Cancer Center UIC, et le National Institutes of Health octroi C06RR15482. Expériences sur les animaux ont été effectuées en conformité avec les directives et les règlements stipulés par AAALAC sous protocoles approuvés par les soins des animaux protection des animaux et du Comité utilisation à l&#39;UIC. Les animaux de ce projet sont logés dans des chambres barrière dans le laboratoire biologique Ressources (BRL) à l&#39;Université de l&#39;Illinois à Chicago. Le BRL dispose d&#39;un personnel totalement vétérinaires qui surveille les animaux au moins deux fois par jour et fournit des conseils sur les soins aux animaux.

1. La dissection des ovaires et de l&#39;isolement des tissus <ol><li> Préparer une solution à 0,5% (p / v) d&#39;alginate de sodium, de préparer à partir d&#39;une version modifiée du protocole décrit précédemment 5, en utilisant PBS stérile et la chaleur à 37 ° C. Mélanger par inversion ou basculant, mais ne vortex. Dessinez l&#39;alginate dans une seringue de 1 ml avec une aiguille 25 G et de maintenir à 37 ° C. </li><li> Préparer 10mM stérile solution de CaCl 2 pour la réticulation de l&#39;alginate sur l&#39;encapsulation de tissu et chaud à 37 ° C. </li><li> Préparer 1 mL de milieu de culture (+ 5 unités aMEM pénicilline et 5 ug / ml de streptomycine) par puits dans une plaque à 24 puits pour chaque expérience. Inclure diverses drogues, les peptides, ou de virus dans le milieu de culture. Pour la transfection avant encapsulation d&#39;organes, de tissus avec incuber Lipofectamine 2000 et l&#39;ADN plasmidique pendant quatre heures à 37 ° C. </li><li> Disséquer les organes de l&#39;animal, enlever tout le tissu adipeux ou résiduelles, retirez doucement la membrane entourant l&#39;ovaire Bursa, et couper l&#39;ovaire ou l&#39;oviducte en quatre morceaux par la bissectrice de l&#39;orgue. Les animaux ont été euthanasiés par asphyxie CO 2 suivie par dislocation cervicale. Pour l&#39;oviducte, doucement dérouler le tissu en tirant en dehors des membranes de raccordement et étendant les extrémités du tube dans des directions opposées l&#39;aide de pinces avant de couper. </li><li> Placez une goutte d&#39;alginate unique (uL ~ 1-3) sur une fibre mailles stérilisé. </li><li> En utilisant une pince stérile, déplacer une pièce d&#39;orgue dans la goutte d&#39;alginate et le centre de la goutte à l&#39;aide de forceps ou d&#39;une aiguille stérile. </li><li> Inverser la gouttelette contenant la pièce pour orgue (organoïde) en saisissant la fibre filet avec des pinces stériles et en le tournant à l&#39;envers pour permettre à la gravité de forcer l&#39;alginate dans une goutte suspendue. </li><li> Appuyez sur le pince sur le bord d&#39;un plat de 10cm de Pétri contenant 10 mM de CaCl2 solution afin que la goutte tombe dans la solution. </li><li> Incuber pendant 2 minutes, retirer avec une cuillère stérile contenant de libérer les pores supplémentaires 10mM CaCl 2, et le transfert dans le milieu de culture. Jusqu&#39;à quatre gels d&#39;alginate peut être transféré dans un seul puits d&#39;une plaque de 24 puits contenant le milieu de culture. Culture pour une période de temps définie. </li></ol> 2. La dégradation de l&#39;alginate et la collecte de cellules <ol><li> Pour la solubilisation du gel d&#39;alginate, incuber les cultures dans l&#39;alginate lyase (3,4 mg / ml) dissous dans Leibovitz L-15 des médias à 37 ° C pendant 30 minutes ou jusqu&#39;à ce gel est complètement dissous. </li><li> Retirez le tissu de la lyase d&#39;alginate. </li><li> Pour la collecte de l&#39;OSE, incuber les organoïde avec 500 unités de collagénase dans Leibovitz L-15 des médias pendant 1 heure 8. Vortex à 2 impulsions seconde (2 secondes et 2 secondes de vortex reste) à puissance moyenne pendant 1 minute, suivie par une seconde impulsions (1 vortex secondes et 1 secondes de repos) pendant 1 minute. Laissez tomber des pièces d&#39;orgue au fond du microtube et le surnageant de pipette dans le tube à centrifuger stérile frais. Centrifuger 3 minutes à 3000 rpm à l&#39;épithélium de surface de l&#39;ovaire granulés. </li><li> Pour la collecte de l&#39;épithélium des trompes, couper le tube de la longueur et les placer sur la culture de tissus en plastique dans du DMEM avec 5 unités de pénicilline et de 5 ug / ml de streptomycine et de 4% (v / v) de FBS. Plus de 3-5 jours, les cellules «ramper» hors du stroma des trompes et poussent sur plastic9 culture de tissus. </li></ol> 3. Fixation des cultures d&#39;alginate organes encapsulés pour des sections de paraffine <ol><li> Recueillir l&#39;alginate encapsulé perle du milieu de culture au point de temps sélectionné. </li><li> Recrosslink l&#39;alginate de rigidifier le gel en laissant tomber la culture dans 10 mM stérile solution de CaCl 2 pendant deux minutes. </li><li> Fixer l&#39;organe d&#39;alginate encapsulé dans du paraformaldéhyde à 2% contenant 50 mM de saccharose, 10 mM de CaCl2 et 50 mM cacodylate de sodium pour s&#39;assurer que le gel reste solide et encapsule totalement les tissus lors de la fixation. </li><li> Après traitement des tissus, intégrer l&#39;ensemble de l&#39;alginate-encapsulée morceau culture d&#39;organe dans la paraffine et de la section avec un microtome à générer 5 sections um. </li><li> L&#39;alginate sera dissoute et retirée de la diapositive si performants immunohistochimie due à la chaleur de récupération d&#39;antigène base. L&#39;alginate restera aux taches et pour l&#39;éosine en date hématoxyline et éosine. </li></ol> 4. Les résultats représentatifs: Un exemple d&#39;une culture d&#39;organe en trois dimensions de l&#39;ovaire et l&#39;oviducte est montré dans la figure 1. Les ovaires peuvent être coupés en morceaux de taille différente, et l&#39;OSE prolifèrent et migrent vers la surface de réparation qui a été blessé par l&#39;incision avec le scalpel. Pendant ce processus, l&#39;orientation correcte de l&#39;OSE par rapport à la stroma sous-jacent est maintenu, avec l&#39;OSE encapsulant l&#39;ovaire. Comme le montre la figure 2, l&#39;OSE des ovaires réduit de moitié d&#39;encapsuler l&#39;ovaire plus complètement par rapport aux ovaires coupés en quartiers ou des huitièmes. La réparation des plaies de l&#39;ovairede surface peuvent être étudiés dans le contexte de l&#39;ovaire entier et peut faire la lumière sur la façon dont la réparation de l&#39;ovulation et de surface induit la transformation de surface de l&#39;ovaire. Le critère de l&#39;expérience dépend de l&#39;information qui est requise. Architecture des tissus, la morphologie cellulaire, et les taux de croissance cellulaire sont facilement contrôlés à l&#39;aide des sections de paraffine. Par exemple, une seule couche de l&#39;épithélium de surface de l&#39;ovaire est observée après culture en milieu sans sérum aMEM pendant 7 jours (figure 3A), alors que plus de 5 ug / ml d&#39;insuline pendant 7 jours induit une hyperprolifération de l&#39;épithélium de surface de l&#39;ovaire et la formation de plusieurs couches de cellules (figure 3B). En ajoutant bromodésoxyuridine aux cultures, à une concentration finale de 10 pM 24 heures avant la fixation, la prolifération des cellules peut être mesurée (en médaillon 3A et 3B). Pour les cultures maintenues dans le sérum sans aMEM, environ 3-5% de l&#39;OSE subissent la prolifération pendant la période de 24 h étiquetage (3A en médaillon), tandis qu&#39;un plus grand pourcentage de cellules de la granulosa prolifèrent. Ajout d&#39;insuline pour les milieux de culture augmente le pourcentage de prolifération OSE à environ 30% du total des OSE (3B en médaillon). Immunofluorescence de la protéine fluorescente verte (GFP) peuvent être visualisées à travers le gel d&#39;alginate avec la microscopie standard après transfection d&#39;ADNc (figure 3C). Alors que la culture d&#39;organes alginate fournit de croissance et de l&#39;analyse de l&#39;épithélium de surface de l&#39;ovaire et des follicules primaires, il convient de noter que la maturité, les follicules secondaires ne parviennent pas à survivre dans ce système de culture (figure 3D). Culture de follicules à maturité au sein d&#39;une matrice d&#39;hydrogel d&#39;alginate a été décrit ailleurs 10. Les altérations des protéines ou d&#39;ARN peuvent être analysées à partir de l&#39;organe entier ou de types cellulaires spécifiques en utilisant la dégradation enzymatique (figure 4). Traitement des organites ovariens cultivés avec de la collagénase quitte le stroma sous-jacent intact (figure 4C), tout en permettant l&#39;enrichissement de l&#39;OSE dans le culot cellulaire obtenu après le traitement, bien que certaines cellules stromales sont également isolés (figure 4D). <img alt="Figure 1" src="/files/ftp_upload/2804/2804fig1.jpg" /> Figure 1. Ovaire de souris culture système en trois dimensions utilisé pour propager l&#39;OSE après avoir blessé. A) Sous le microscope, la bourse et le tampon entourant la graisse ont été enlevés. B) des ovaires intacts (O) avec tube de l&#39;oviducte (T) et graisses de pad (F). C) Ovaire avec Bursa et coussinet adipeux retirés (à gauche) et déroulé l&#39;oviducte (à droite). D) organites ovaire ou l&#39;oviducte ont été placés dans une goutte d&#39;alginate. E) ont été enfermés organites alginate est tombé dans une solution de CaCl 2 réticulation de l&#39;alginate pour former un gel. F) Alginate encapsulé organoïde ovaire. G) Alginate encapsulé organoïde oviducte. H) organites alginate encapsulé incubés dans un milieu de culture. <img alt="Figure 2" src="/files/ftp_upload/2804/2804fig2.jpg" /> Figure 2. Surface régénérée par la surface de l&#39;ovaire mesurée par cytokératine 8. L&#39;encapsulation des organites ovariens par l&#39;OSE, après avoir été coupé en huitièmes, quarts et demis, cultivées dans un milieu de BSA, et mesurée par une analyse immunohistochimique utilisant cytokératine 8 (CK8) anticorps. <img alt="Figure 3" src="/files/ftp_upload/2804/2804fig3.jpg" /> Figure 3. Système de culture permet le passage d&#39;alginate de facteurs de croissance et de transfection des ADNc dans l&#39;OSE, mais ne supporte pas la croissance rapide et maturation des follicules. A) organoïde ovaire cultivées pendant 7 jours dans le milieu de base analysés pour l&#39;expression CK8, qui marque l&#39;OSE. En médaillon, la section de série colorées pour l&#39;incorporation de BrdU. B) Ajout d&#39;insuline pour les milieux de culture induit une hyperplasie de l&#39;OSE. En médaillon, la section de série colorées pour l&#39;incorporation de BrdU. C) organoïde ovaire transfectées avec l&#39;ADNc pour exprimer la GFP. D) système de culture alginate soutient la croissance des follicules primordiaux (rouge pointe de flèche), mais pas la maturité des follicules secondaires (flèche noire). <img alt="Figure 4" src="/files/ftp_upload/2804/2804fig4.jpg" /> Figure 4. OSE peut être isolé pour la culture analyse qui suit. A) organoïde cultivées dans un milieu pour un maximum de 2 semaines. B) organoïde encapsulé est traitée avec lyase d&#39;alginate de quitter organoïde intacte. L&#39;ovaire est tachée de CK8 pour marquer l&#39;OSE. C) organoïde est traitée avec de la collagénase d&#39;OSE séparer de stroma sous-jacent. Pièce de tissu restant était tachée de CK8 de mettre en évidence l&#39;enlèvement de la surface de l&#39;ovaire. D) les cellules recueillies après le traitement sont enrichis pour OSE que colorées pour CK8 (rouge). Les cellules sont contre-colorées avec du DAPI pour marquer les noyaux.

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Il n&#39;ya rien à révéler.

Le développement d&#39;un système en trois dimensions d&#39;orgue de la culture en utilisant des hydrogels d&#39;alginate représente une méthode polyvalente pour analyser de nombreux types de tissus différents à partir d&#39;un large éventail d&#39;organismes. L&#39;utilisation de cultures 3D peut être étendu aux domaines de l&#39;ingénierie tissulaire et la médecine régénérative car il fournit un échafaudage sur lequel les cellules peuvent croître 11. Actuellement, notre laboratoire est en co...

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires (optionnel) </th></tr><tr><td> Leibovitz milieu L-15 </td><td> Gibco </td><td> 11415 </td><td></td></tr><tr><td> aMEM moyennes </td><td> Gibco </td><td> 12000-022 </td><td></td></tr><tr><td> L&#39;alginate de sodium </td><td> Novamatrix </td><td></td><td></td></tr><tr><td> Cacodylate de sodium </td><td> Sigma-Aldrich </td><td> C0250 </td><td></td></tr><tr><td> Treillis en fibre de polypropylène </td><td> McMaster Carr </td><td></td><td> 45 &quot;large rouleau; 0,0041« ouverture </td></tr><tr><td> Lipofectamine 2000 </td><td> Invitrogen </td><td> 11668 </td><td></td></tr><tr><td> Pénicilline / streptomycine </td><td> Gibco </td><td> 15070-063 </td><td></td></tr><tr><td> Lyase d&#39;alginate </td><td> Sigma-Aldrich </td><td> A1603 </td><td></td></tr><tr><td> A1603 </td><td> Sigma-Aldrich </td><td> C9697 </td><td> De Clostridium histolyticum </td></tr><tr><td> L&#39;albumine sérique bovine </td><td> MP Biomédical </td><td> 103700 </td><td></td></tr><tr><td> Sérum fœtal bovin </td><td> Gibco </td><td> Gibco </td><td></td></tr><tr><td> Solution ITS </td><td> Roche </td><td> 11074547001 </td><td> Insuline / transferrine / sélénite de sodium </td></tr><tr><td> Cytokératine 8 anticorps </td><td> Des études de développement de la Banque d&#39;hybridomes </td><td> TROMA-1 </td><td> Utilisez au 1:200 </td></tr><tr><td> DAPI </td><td> Vector Labs </td><td> H1200 </td><td></td></tr><tr><td> Bromodésoxyuridine </td><td> Sigma </td><td> B50002-1G </td><td> La concentration finale 10 uM </td></tr></tbody></table>

alginate hydrogels for three-dimensional organ culture of ovaries and oviducts

Cancer de l&#39;ovaire est la cinquième cause de décès par cancer chez la femme et a un taux de mortalité de 63% dans l&#39;une aux États-Unis. Le type de cellules d&#39;origine pour les cancers de l&#39;ovaire est toujours en question et pourrait être soit l&#39;épithélium de surface de l&#39;ovaire (OSE) ou l&#39;épithélium distale de la trompe de Fallope fimbriae 2,3. La culture des cellules normales comme une culture primaire in vitro, permettra aux scientifiques de modéliser les changements spécifiques qui pourraient conduire au cancer de l&#39;ovaire dans l&#39;épithélium distincts, ainsi définitivement la détermination du type de cellules d&#39;origine. Ceci permettra le développement de biomarqueurs plus précises, des modèles animaux avec des changements tissu-spécifique des gènes, et les stratégies de prévention mieux ciblées à cette maladie. Le maintien des cellules normales dans les hydrogels d&#39;alginate favorise à court terme dans la culture in vitro de cellules dans leurs trois dimensions contexte et permet l&#39;introduction d&#39;ADN plasmidique, siARN, et des petites molécules. Par des organes en culture dans des pièces qui proviennent de coupures stratégiques à l&#39;aide d&#39;un scalpel, plusieurs cultures à partir d&#39;un seul organe peut être généré, en augmentant le nombre d&#39;expériences à partir d&#39;un seul animal. Ces réductions des aspects du modèle de l&#39;ovulation entraînant la prolifération de l&#39;OSE, qui est associée à la formation du cancer de l&#39;ovaire. Les types cellulaires tels que l&#39;OSE qui ne poussent pas bien sur les surfaces en plastique peuvent être cultivés en utilisant cette méthode et faciliter les enquêtes dans les processus cellulaires normaux ou les premiers événements dans la formation du cancer 4. Hydrogels d&#39;alginate peut être utilisé pour soutenir la croissance de nombreux types de tissus 5. Alginate est un polysaccharide linéaire composé d&#39;unités répétitives de β-D-mannuronique acide et α-L-guluronique acides qui peuvent être réticulés avec des ions calcium, résultant en une action gélifiante douce qui n&#39;a pas les tissus des dommages 6,7. Comme d&#39;autres matrices tridimensionnelles telles que la culture de cellules de Matrigel, alginate fournit un support mécanique pour les tissus, mais les protéines ne réagissent pas avec la matrice d&#39;alginate, et donc les fonctions d&#39;alginate comme une matrice extracellulaire qui synthétiques ne lance pas la signalisation cellulaire 5. L&#39;hydrogel d&#39;alginate flotte dans milieu de culture cellulaire standard et prend en charge l&#39;architecture de la croissance des tissus in vitro. Une méthode est présentée pour la préparation, la séparation, et l&#39;incorporation de l&#39;ovaire et de pièces d&#39;orgue dans l&#39;oviducte hydrogels d&#39;alginate, qui peuvent être maintenues en culture pendant jusqu&#39;à deux semaines. La libération enzymatique des cellules pour l&#39;analyse des protéines et des échantillons d&#39;ARN à partir de la culture d&#39;organe est également décrite. Enfin, la croissance des types de cellules primaires n&#39;est possible sans l&#39;immortalisation génétique de souris et permet aux enquêteurs d&#39;utiliser et de souris transgéniques knockout.

Watch this Scientific Journal Video about Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts at JoVE.com

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Culture de cellules normales dans leurs trois dimensions contexte représente une méthode alternative pour étudier les événements précoces nécessaires pour la transformation cellulaire et la tumorigenèse. Cette méthode est utilisée pour cultiver des cellules ovariennes normales et l&#39;oviducte pour étudier les événements précoces de la formation de cancer de l&#39;ovaire.

Les hydrogels d&#39;alginate de culture d&#39;organe en trois dimensions des ovaires et des trompes

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

alginate-hydrogels-for-three-dimensional-organ-culture-ovaries

Research

JoVE Journal

Bioengineering

21.8K vues.  University of Illinois at Chicago. Culture de cellules normales dans leurs trois dimensions contexte représente une méthode alternative pour étudier les événements précoces nécessaires pour la transformation cellulaire et la tumorigenèse. Cette méthode est utilisée pour cultiver des cellules ovariennes normales et l'oviducte pour étudier les événements précoces de la formation de cancer de l'ovaire.

Vidéo: Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Impression Planar et en trois dimensions des encres conductrices

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Recherche

Bio-ingénierie

Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

The intervertebral disc (IVD) is the joint of the spine connecting vertebra to vertebra. It functions to transmit loading of the spine and give flexibility to the spine. It composes of three compartments: the innermost nucleus pulposus (NP) encompassing by the annulus fibrosus (AF), and two cartilaginous endplates connecting the NP and AF to the vertebral body on both sides. Discogenic pain possibly caused by degenerative intervertebral disc disease (DDD) and disc herniations has been identified as a major problem in our modern society. To study possible mechanisms of IVD degeneration, in vitro organ culture systems with live disc cells are highly appealing. The in vitro culture of intact bovine coccygeal IVDs has advanced to a relevant model system, which allows the study of mechano-biological aspects in a well-controlled physiological and mechanical environment. Bovine tail IVDs can be obtained relatively easy in higher numbers and are very similar to the human lumbar IVDs with respect to cell density, cell population and dimensions. However, previous bovine caudal IVD harvesting techniques retaining cartilaginous endplates and bony endplates failed after 1-2 days of culture since the nutrition pathways were obviously blocked by clotted blood. IVDs are the biggest avascular organs, thus, the nutrients to the cells in the NP are solely dependent on diffusion via the capillary buds from the adjacent vertebral body. Presence of bone debris and clotted blood on the endplate surfaces can hinder nutrient diffusion into the center of the disc and compromise cell viability. Our group established a relatively quick protocol to "crack"-out the IVDs from the tail with a low risk for contamination. We are able to permeabilize the freshly-cut bony endplate surfaces by using a surgical jet lavage system, which removes the blood clots and cutting debris and very efficiently reopens the nutrition diffusion pathway to the center of the IVD. The presence of growth plates on both sides of the vertebral bone has to be avoided and to be removed prior to culture. In this video, we outline the crucial steps during preparation and demonstrate the key to a successful organ culture maintaining high cell viability for 14 days under free swelling culture. The culture time could be extended when appropriate mechanical environment can be maintained by using mechanical loading bioreactor. The technique demonstrated here can be extended to other animal species such as porcine, ovine and leporine caudal and lumbar IVD isolation.

This protocol illustrates a harvesting technique for coccygeal bovine intervertebral discs for organ culture for in vitro organ culture.

Préparation d&#39;Intact disques intervertébraux Queue bovine pour la culture d&#39;organe

The intervertebral disc (IVD) is the joint of the spine connecting  vertebra to vertebra. It functions to transmit loading of the spine and give  ...

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

Tridimensionnelle modèle de culture cellulaire pour mesurer les effets de l&#39;écoulement des fluides interstitiels sur l&#39;invasion tumorale

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

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.

Analyse de la migration cellulaire dans un tridimensionnelle Matrice Collagène

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

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

Génération d&#39;une épaisseur en trois dimensions équivalent de peau et automatisé Blessant

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper composition, targeted modulus, and well-defined architectural features. Biomaterials that recapitulate the intrinsic architecture of in vivo tissue are vital for studying diseases as well as to facilitate the regeneration of lost and malformed soft tissue. A novel biofabrication technique was developed which combines state of the art imaging, three-dimensional (3D) printing, and selective enzymatic activity to create a new generation of biomaterials for research and clinical application. The developed material, Bovine Serum Albumin rubber, is reaction injected into a mold that upholds specific geometrical features. This sacrificial material allows the adequate transfer of architectural features to a natural scaffold material. The prototype consists of a 3D collagen scaffold with 4 and 3 mm channels that represent a branched architecture. This paper emphasizes the use of this biofabrication technique for the generation of natural constructs. This protocol utilizes a computer-aided software (CAD) to manufacture a solid mold which will be reaction injected with BSA rubber followed by the enzymatic digestion of the rubber, leaving its architectural features within the scaffold material.

An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

Trois dimensions technologie biomimétique: Roman Biorubber Crée Défini micro et macro-échelle Architectures en collagène hydrogels

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

Fabrication de Inverted colloïdal cristal Poly (éthylène glycol) Échafaudages: Une cellule plate-forme en trois dimensions de la culture pour l&#39;ingénierie tissulaire du foie

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

Layered alginate Constructs: Une plate-forme pour la coopération en culture des populations de cellules hétérogènes

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Formation et Patterning Light-médiée hydrogels pour la culture cellulaire Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Accordable hydrogels de matrice extracellulaire pulmonaire pour la 3D Culture Cellulaire

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...