L&#39;auteur aimerait remercier le Dr Dongxuan Jia pour l&#39;assistance technique. Certaines des images figurant dans le présent article ont été en collaboration avec le Dr Malcolm S. Steinberg, Département de Biologie Moléculaire, Université de Princeton. L&#39;auteur tient également à remercier le ministère de la Défense de la prostate Cancer Research Programme (subventions PC-030482-991552 et PC) et le pour le NCI / NIH (subvention R01CA118755) leur généreux soutien.

1. Préparation d&#39;une suspension cellulaire unique <ol><li> Cultures de cellules adhérentes doivent être cultivés à 90% de confluence, les monocouches quoi doivent être rincés deux fois avec du PBS. Après la vidange bien, ajoutez 2 ml (pour les plaques de 100 mm) de 0,05% de trypsine-1 mM EDTA, et incuber à 37 ° C jusqu&#39;à ce que les cellules se détacher. Arrêtez trypsinisation en ajoutant 2 ml de milieu complet et doucement l&#39;utilisation d&#39;une pipette 5 ml de triturer le mélange jusqu&#39;à cellules sont en suspension. Transfert des cellules dans un tube de 15 ml conique. </li><li> Ajouter 40 ul d&#39;un stock de 10 mg / ml de DNAse et incuber pendant 5 minutes à température ambiante. Brièvement au vortex et centrifuger à 200 xg pendant 5 minutes. </li><li> Rejeter le surnageant et laver culot avec 1 ml de culture de tissus milieu complet. Répéter, puis remettre les cellules en 2 ml de milieu de culture de tissu complet. </li><li> Compter les cellules en utilisant un hématimètre, ou un compteur de cellules automatisé et d&#39;ajuster la concentration de 2,5 x 10 6 cellules / ml. Pour cette démonstration, une BioRad TC10 compteur de cellules automatisé a été utilisé. </li></ol> 2. Formation de gouttes suspendues. <ol><li> Retirez le couvercle d&#39;une boîte de culture de tissu de 60 mm et placer 5 ml de PBS dans le fond du plat. Ceci agira comme une chambre de l&#39;hydratation. </li><li> Inverser le couvercle et l&#39;utilisation d&#39;une pipette 20 ul de déposer 10 gouttes ul sur le fond du couvercle. Assurez-vous que des gouttes sont placées suffisamment éloignés afin de ne pas toucher. Il est possible de placer au moins 20 gouttes par plat. </li><li> Inverser le couvercle sur le PBS-remplie chambre inférieure et incuber à 37 ° C CO / 5% 2 / 95% d&#39;humidité, de surveiller l&#39;gouttes par jour et incuber jusqu&#39;au feuilles cellule soit ou agrégats sont formés. Un microscope stéréo peut être utilisé pour évaluer la formation d&#39;agrégats. </li><li> Une fois la forme des feuilles, ils peuvent être transférés à fond rond en verre Agitateur flacons contenant 3 ml de milieu complet et incubés dans un bain d&#39;eau agité à 37 ° C et 5% de CO 2 jusqu&#39;à la formation de sphéroïdes. </li></ol> 3. Remarques <ol><li> Selon la taille des cellules, la concentration peut être ajusté en amont ou vers le bas. </li><li> Les cellules peuvent être colorées avec membrane intercalant des colorants fluorescents avant la formation de goutte suspendue. </li><li> Deux différents types de cellules peuvent être différemment colorés et mélangés dans un rapport de 1:1 (ou autre) Le ratio avant la formation de gouttes suspendues. Elles peuvent être des cellules cancéreuses et stromales, les tissus embryonnaires, ou cellules génétiquement modifiées pour avoir des signatures adhésive spécifique. </li><li> Les cellules peuvent se détacher de boîtes de culture tissulaire en utilisant la trypsine 0,05% / 2 mM de calcium afin de préserver la fonction cadhérine. </li><li> Selon l&#39;expérience, la taille des feuilles peut être mesurée, ou des agrégats peuvent être utilisés pour les essais mécaniques ou pour l&#39;analyse biochimique ou moléculaire. </li></ol> 4. Les résultats représentatifs: Fiche ou formation sphéroïde se produit généralement dans les 24 heures, mais peut prendre plus de temps selon le type de cellule. La figure 1 représente des images capturées après 18 heures d&#39;incubation du cancer de la prostate non traité MAT-LyLu (MLL), les cellules de culture en suspension de chute (figure 1A) ou des cellules traitées avec l&#39;inhibiteur de MEK 25 um PD98059. Notez que les résultats du traitement Meki dans un compactage marqués de la feuille de cellule. Le compactage peut être quantifiée en mesurant la taille de 10-20 (ou plus) des gouttes pendantes et en comparant la taille moyenne entre les groupes de traitement. Nous généralement d&#39;analyser les images en utilisant le logiciel ImageJ par seuillage chaque image puis en convertissant les images en mode binaire, après quoi nous appliquons l&#39;analyse des particules de révéler la zone de l&#39;image totale, en pixels. Cela peut ensuite être facilement converti en microns carrés. La figure 2 représente une comparaison de taille pour les cellules non traitées et MLL Meki-traitée. Comme indiqué, le traitement réduit significativement la taille Meki feuille par rapport à t de Student-test (p &lt;0,0001). Pour cette comparaison, 10 pour chacune des agrégats de cellules traitées et non traitées ont été mesurés. La figure 3 représente un sphéroïde de foie de poulet embryonnaire formé après 24 heures dans la culture suspendus chute et 2 jours dans un bain de shaker. Le dosage de goutte suspendue peut également être modifié pour inclure plus d&#39;un type cellulaire pour révéler le positionnement spatial adopté. Par exemple, les cellules de poulet foie embryonnaire peut être isolé, colorées avec une lampe fluorescente à membrane intercalant colorant et mélangé avec des cellules chiches coeur embryonnaire qui ont été colorés avec un marqueur fluorescent différent. Montre la figure 4A que plutôt que de rester entremêlés, les cellules du foie et le cœur ont tendance à «trier-out&quot; d&#39;un autre-et à adopter une sphère-dans-un-sphère configuration dans laquelle les cellules cardiaques sont enveloppés par des cellules du foie. Cette configuration peut être expliquée par les différences dans l&#39;adhésion intercellulaire entre les cellules du foie et du cœur. Ce concept est codifié dans l&#39;hypothèse adhésion différentielle qui a été utilisé pour expliquer le comportement de tri cellulaire entre les tissus embryonnaires pour la couche germe d&#39;amphibiens et de poissons téléostéens 3 4 embryons, pour l&#39;organisation des cellules des îlots pancréatiques 5 et pour la cellule de tri entre deux populations de cellules identiques in ce qui concerne tous les autres que pour les niveaux d&#39;expression de même type de cadhérine (6 et la figure 4B). Ce résultat est particulièrement important pour démontrer comment une simple différence d&#39;ingénierie de l&#39;adhérence entre les populations cellulaires peut entraîner la génération de la structure des organes, par exemple, un îlot 5 pancréatiques. <img src="/files/ftp_upload/2720/2720fig1.jpg" alt="Figure 1" /> Figure 1. Images de cellules incubées en culture en suspension déposer pendant 18 heures soit en l&#39;absence (A) ou en présence (B) de 25 microns de l&#39;inhibiteur de MEK PD98059. Les images ont été capturées à l&#39;aide d&#39;un Nikon Eclipse TE 300 microscope à épifluorescence et un Photometrics Coolsnap ES appareil photo numérique. <img src="/files/ftp_upload/2720/2720fig2.jpg" alt="Figure 2" /> Figure 2. Quantification de Meki-induites chez le rat compactage des cellules de la prostate MLL cancer. Cultures de cellules goutte suspendue MLL non traitées et traitées à Meki ont été incubées pendant 18 heures, après quoi la taille des agrégats a été déterminée comme décrit ci-dessus. L&#39;analyse statistique de la taille des agrégats a été par la Student t-test. L&#39;astérisque représente une différence statistiquement significative de la taille globale (p &lt;0,0001) entre les cellules non traitées et traitées à Meki et suggère que les résultats de Meki-traitement dans les agrégats nettement plus petit et plus compact. <img src="/files/ftp_upload/2720/2720fig3.jpg" alt="Figure 3" /> Figure 3. Un sphéroïde formé en incubant les cellules du foie de poulet embryonnaire en culture en suspension déposer pendant 18 heures puis dans un incubateur / secousses de bain pour 48 heures. Cette image a été capturée à l&#39;aide d&#39;un microscope Nikon stéréo SMZ800 et une caméra CCD Sony Noire. <img src="/files/ftp_upload/2720/2720fig4.jpg" alt="Figure 4" /> Figure 4. Hanging culture déposer révèle le tri-out comportement entre différents types cellulaires. Dans la partie A, les cellules de poulet foie embryonnaire ont été colorées avec un colorant intercalant PKH-2 membrane et mélangés dans des proportions égales avec PKH-26 cellules chiches tachés coeur embryonnaire. Dans le panel B, deux N-cadhérine-clones cellulaires transfectées L (LN4 et LN2a), exprimant la N-CAD à leurs surfaces dans le ratio de 2,4:1, ont également été colorés avec des colorants fluorescents membrane intercalant PKH-2 et PKH-26 (Sigma-Aldrich), mélangés dans des proportions égales et cultivées comme des gouttes pendantes 7. Après 18 heures de culture, des feuilles de cellules ont été transférées à des flacons secouant et incubé pendant 48 heures. Agrégats ont été fixés dans du paraformaldéhyde à 2% dans un tampon phosphate salin et visualisées avec un système laser BioRad MRC600 microscope confocal à balayage attaché à un Nikon Optiphot-2 microscope. Sections optiques des deux canaux vert et rouge ont été recueillis et une Silicon Graphix Crimson VGX poste de travail équipé de Vital Images Voxel Ultra View logiciel d&#39;imagerie a été utilisée pour attribuer des fausses couleurs pour les deux canaux et de fusionner les images, révélant des configurations anatomiques générées (A). confocale section optique grâce à un enveloppement global démontrant des cellules cardiaques (ici en jaune pseudo-couleurs) par les cellules du foie dans pseudocolor bleu (de 8). (B) l&#39;article optique confocal par un enveloppement global démontrant de haute N-cadhérine des cellules exprimant (vert) par ceux qui expriment des niveaux inférieurs de la N-cadhérine (en rouge) (à partir de 6).

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Des études ont montré que les cellules en culture dans un contexte en trois dimensions (3D) produit distinctes morphologie cellulaire et de signalisation par rapport à une démarche rigide en deux dimensions du système de culture (2D) 9. Par exemple, des gels de collagène des fibroblastes peuplées de démontrer que la morphologie des fibroblastes en 3D est tout à fait distincte de celle observée en 2D 10,11. De même, la culture 3D peut induire tissu-spécifique de différenciation des cell...

<ul><li> compteur de cellules automatique (BioRad TC10) </li><li> secouant bain d&#39;eau avec du CO 2 Modèle couvercle de pignon 3540 (Lab-line) </li></ul>

a simple hanging drop cell culture protocol for generation of 3d spheroids

Les études sur la cohésion de cellule à cellule et cellule-substrat d&#39;adhérence ont historiquement été réalisées sur des cultures en monocouche adhérente sur des substrats rigides. Cellules dans un tissu, cependant, sont généralement enfermés dans une masse de tissu très dense dans lequel les cellules d&#39;établir des connexions intimes avec de nombreux quasi-voisins et avec des composants de la matrice extracellulaire. En conséquence, le milieu chimique et les forces physiques vécus par les cellules dans un tissu 3D sont fondamentalement différentes de celles vécues par les cellules en culture monocouche. Cela a été démontré que l&#39;impact nettement la morphologie cellulaire et de signalisation. Plusieurs méthodes ont été conçues pour générer des cultures cellulaires en 3D, y compris l&#39;encapsulation des cellules dans une des gels de collagène ou biomatériau échafaudages 2. Ces méthodes, bien qu&#39;utile, ne pas rappeler les intimes architecture Direct adhérence cellule-cellule dans les tissus normaux. Plutôt, ils plus étroitement les systèmes de culture approximative dans laquelle des cellules individuelles sont vaguement dispersés au sein d&#39;un maillage 3D de produits d&#39;ECM. Ici, nous décrivons une méthode simple dans lequel les cellules sont placées dans la culture suspendus baisse et incubés dans des conditions physiologiques jusqu&#39;à ce qu&#39;ils forment vraie sphéroïdes 3D dans lequel les cellules sont en contact direct avec eux et avec les composants de la matrice extracellulaire. La méthode ne nécessite pas d&#39;équipement spécialisé et peut être adapté pour inclure plus d&#39;un agent biologique en très petites quantités qui peuvent être d&#39;intérêt à élucider les effets sur la cellule-cellule ou cellule-ECM interaction. La méthode peut également être utilisé pour la co-culture de deux (ou plusieurs) des populations de cellules différentes, afin d&#39;élucider le rôle des interactions cellule-cellule ou cellule-ECM en précisant les relations spatiales entre les cellules. Cellule-cellule et cellule-cohésion ECM d&#39;adhésion sont les pierres angulaires d&#39;études du développement, de l&#39;interaction de cellules embryonnaires de tumeurs stromales malignes dans l&#39;invasion, la cicatrisation, et pour les applications d&#39;ingénierie tissulaire. Cette méthode simple vous fournir un moyen de générer des tissus tels que les agrégats cellulaires pour la mesure des propriétés biomécaniques ou pour l&#39;analyse moléculaire et biochimique dans un modèle physiologiquement pertinents.

Watch this Scientific Journal Video about A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids at JoVE.com

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Nous décrivons une méthode simple et rapide de générer des tissus 3D-like sphéroïdes et leur application potentielle de quantifier les différences dans les interactions cellule-cellule.

Un protocole simple Hanging culture cellulaire pour la production chute de sphéroïdes 3D

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

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56.6K vues.  UMDNJ-Robert Wood Johnson Medical School. Nous décrivons une méthode simple et rapide de générer des tissus 3D-like sphéroïdes et leur application potentielle de quantifier les différences dans les interactions cellule-cellule.

Vidéo: A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Préparation de la 3D pour les applications de fibrine Échafaudages CULTURE DE CELLULES SOUCHES

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

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Bio-ingénierie

Generation and Recovery of &beta;-cell Spheroids From Step-growth PEG-peptide Hydrogels

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional microenvironment with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, in part due to their tunable and cytocompatible properties. In this protocol, we utilized thiol-ene step-growth photopolymerizations to fabricate PEG-peptide hydrogels for encapsulating pancreatic MIN6 b-cells. The gels were formed by 4-arm PEG-norbornene (PEG4NB) macromer and a chymotrypsin-sensitive peptide crosslinker (CGGYC). The hydrophilic and non-fouling nature of PEG offers a cytocompatible microenvironment for cell survival and proliferation in 3D, while the use of chymotrypsin-sensitive peptide sequence (CGGY&darr;C, arrow indicates enzyme cleavage site, while terminal cysteine residues were added for thiol-ene crosslinking) permits rapid recovery of cell constructs forming within the hydrogel. The following protocol elaborates techniques for: (1) Encapsulation of MIN6 &beta;-cells in thiol-ene hydrogels; (2) Qualitative and quantitative cell viability assays to determine cell survival and proliferation; (3) Recovery of cell spheroids using chymotrypsin-mediated gel erosion; and (4) Structural and functional analysis of the recovered spheroids.

Generation and Recovery of &amp;#946;-cell Spheroids From Step-growth PEG-peptide Hydrogels

The following protocol provides techniques for encapsulating pancreatic &#x03B2;-cells in step-growth PEG-peptide hydrogels formed by thiol-ene photo-click reactions. This material platform not only offers a cytocompatible microenvironment for cell encapsulation, but also permits user-controlled rapid recovery of cell structures formed within the hydrogels.

Génération et récupération des cellules β-Sphéroïdes partir de l&#39;étape de croissance peptidiques hydrogels de PEG-

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional  microenvironment with tissue-like elasticity and high permeability for  ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

3D hydrogel échafaudages pour articulaire chondrocytes Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

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 ...

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

Un protocole pour un suivi en temps réel 3D seule particule

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

Construction d’une feuille les cellules souches mésenchymateuses multicouche avec un système de Culture dynamique 3D

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

Un modèle de culture matrix-adipocyte extracellulaire humaine 3D pour étudier matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

Production automatisée à grande échelle de sphéroïdes de cellules souches dérivées de cellules adipeuses pour la bioimpression 3D

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

Generation of Multicue Cellular Microenvironments by UV-Photopatterning of Three-Dimensional Cell Culture Substrates

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand distribution and tissue geometry, have been increasingly shown to play critical roles in governing cell phenotype and behavior. However, these environmental cues and their effects on cells are often studied separately using in vitro platforms that isolate individual cues, a strategy that heavily oversimplifies the complex in vivo situation of multiple cues. Engineering approaches can be particularly useful to bridge this gap, by developing experimental setups that capture the complexity of the in vivo microenvironment, yet retain the degree of precision and manipulability of in vitro systems.
This study highlights an approach combining ultraviolet (UV)-based protein patterning and lithography-based substrate microfabrication, which together enable high-throughput investigation into cell behaviors in multicue environments. By means of maskless UV-photopatterning, it is possible to create complex, adhesive protein distributions on three-dimensional (3D) cell culture substrates on chips that contain a variety of well-defined geometrical cues. The proposed technique can be employed for culture substrates made from different polymeric materials and combined with adhesive patterned areas of a broad range of proteins. With this approach, single cells, as well as monolayers, can be subjected to combinations of geometrical cues and contact guidance cues presented by the patterned substrates. Systematic research using combinations of chip materials, protein patterns, and cell types can thus provide fundamental insights into cellular responses to multicue environments.

Traditionally, cell culture is performed on planar substrates that poorly mimic the natural environment of cells in vivo. Here we describe a method to produce cell culture substrates with physiologically relevant curved geometries and micropatterned extracellular proteins, allowing systematic investigations into cellular sensing of these extracellular cues.

Génération de microenvironnements cellulaires multicue par photomodèlement UV de substrats de culture cellulaire tridimensionnels

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand ...

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

Presented here is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human-induced pluripotent stem cell-derived cardiomyocytes cultured in a high-throughput, multidimensional format. This three-dimensional model functions as a robust platform for disease modeling, high-throughput screenings, and maintains its functionality after cryopreservation.

Génération, criblage à haut débit et biobanque de sphéroïdes cardiaques dérivés de cellules souches pluripotentes d’origine humaine

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We ...