This project was funded by the National Science Foundation ECCS grant 10-02165, and the Interdisciplinary Innovation Initiative Program, University of Illinois grant 12035. M.Y. A. was funded at UIUC from NIH National Cancer Institute Alliance for Nanotechnology in Cancer &#8216;Midwest Cancer Nanotechnology Training Center&#8217; Grant R25 CA154015A. Immnunostaining and confocal microscopy imaging were carried out at the Institute for Genomic Biology (IGB), UIUC. M.Y.A. acknowledges the discussions with B. J. Williams of UIUC regarding the isolation experiments. M.Y.A. acknowledges C. Nemeh and Abdul Bhuiya of UIUC for assistance in schematic and materials list preparation.

Le protocole décrit ci-dessous suit les lignes directrices de l&#39;UIUC comité d&#39;éthique de la recherche humaine. 1. Collecte et digestion de l&#39;échantillon de tissu chirurgical <ol><li> Recueillir l&#39;échantillon de tissu tumoral juste après la résection du côlon (figure 1A et 1B). Prélever des tissus sur un site sain adjacent ainsi. </li><li> Transférer le tissu immédiatement dans un flacon de 15 ml contenant 12 ml de solution HBSS. Conserver le flacon sur la glace à l&#39;intérieur d&#39;une boîte de mousse isolante. </li><li> Transporter le tissu contenant flacon à une culture capot de tissu pour un traitement ultérieur dans les 45 min. Conserver le flacon sur un bloc de glace à l&#39;intérieur du capot. </li><li> Verser le tissu fourni dans une plaque à 6 puits contenant 6-7 ml de solution HBSS aide d&#39;une pipette. Remarque: Montant de tissu par puits est pas critique dans cette étape de rinçage. </li><li> Conservez la plaque à 6 puits sur un bloc de glace. Rincer deux fois le tissu dans une solution HBSS. </li><li> Coupez le tissu proprement en sections séparées avec scis stérilessor. Mince tissu en sections plus petites ne dépassant pas 1 à 2 mm de taille 3 avec une lame de scalpel stérile. </li><li> Peser 0,1% de trypsine un flacon étiqueté (contenant 1 ml de solution de trypsine 0,1%) avant de transférer le tissu. Transférer ~ 20 mg de tissu dans chacune des fioles de trypsine 0,1% de avec une pipette. </li><li> Essayer de minimiser le transfert de HBSS dans le flacon de la trypsine afin d&#39;éviter une dilution supplémentaire de la trypsine. </li><li> Peser les flacons de la trypsine après transfert de tissu; documenter la différence que la masse tissulaire. </li><li> Verser ~ 20 mg de tissu et de 1 ml de trypsine dans chaque puits d&#39;une plaque à 24 puits. </li><li> Mettez la plaque à 24 puits sur un agitateur dans le réfrigérateur à 4 ° C pendant 16-20 h. Pendant cette période d&#39;attente ou à une date antérieure, préparer polyacrylamide substrats de gel et de les fonctionnaliser avec des protéines d&#39;ECM comme décrit dans la section 3. </li></ol> 2. Dissociation enzymatique de l&#39;échantillon de tissu dans une cellule unique Suspension et culture de cellules isolées sur ECM fonctionnalisés élastique Substrates et des boîtes en polystyrène rigide <ol><li> Après 16-20 h d&#39;incubation dans de la trypsine et à 4 ° C sur agitateur, transférer à partir de tissu 4 ° C trypsine à 4 ° C complet flacon de milieu de croissance (contenant 3.2 ml de milieu de croissance complet) à l&#39;aide d&#39;une pipette. Assurer le transfert minimale de trypsine dans des milieux de croissance. Remarque: formulation complète de milieu de croissance est la suivante: milieu RPMI 1640 additionné de base avec du sérum de cheval à une concentration finale de 10% et de la pénicilline-streptomycine à 1% de la solution totale. </li><li> Placez le tissu contenant flacon de médias dans un bain d&#39;eau chaude à 37 ° C pendant 12-15 min. </li><li> Transférer le tissu à un flacon de 15 ml contenant une solution HBSS aide d&#39;une pipette, secouez doucement. </li><li> Répétez 2.3. </li><li> Retirer le tissu de la solution HBSS et transfert à 0,1% solution de collagénase (dans HBSS) flacon contenant 1 ml de solution. Incuber pendant 45 min à 37 ° C et 5% de CO2 dans un environnement humidifié incubateur de culture cellulaire. Pendant la période d&#39;attente, growt chaudeh médias dans un flacon de 15 ml à 37 ° C dans un bain d&#39;eau. </li><li> Tirez sur tous les fragments de tissus en pipette minimisation collagénase report. Ejecter seulement les fragments de tissus dans des flacons de milieux de culture préchauffées. </li><li> Placer un filtre de 40 um bien insert dans chaque puits d&#39;une plaque à 6 puits. Humidifier le filtre avec 1 ml de milieu de culture cellulaire. </li><li> Tissus triturer dans les médias avec une pipette de 10 ml en verre plusieurs fois jusqu&#39;à ce que le tissu est complètement dissocié. </li><li> Maintenir l&#39;embout de pipette le plus près possible de la base du flacon. </li><li> Déposer la solution sur le filtre pour éliminer les débris et les parties non dissociées. </li><li> Centrifugeuse filtré suspension de cellules dans un milieu à 150 g pendant 5 min à température ambiante. </li><li> Eliminer le surnageant et remettre en suspension après culot de cellules avec 2 ml de milieu de culture frais. </li><li> Compter les cellules en utilisant un hémocytomètre (si nécessaire). Graine isolé des cellules primaires sur la matrice extracellulaire (ECM) fonctionnalisés et des gels de polystyrène plats à la concentration souhaitée.</li></ol> 3. Préparation et fonctionnalisation des différentes raideurs de polyacrylamide (PA) Gels et polystyrène substrats Remarque: Pour la démonstration visuelle de l&#39;article 3, les téléspectateurs / lecteurs sont appelés à une récente Journal of Visualized Experiments (JoVE) l&#39;article 17. <ol><li> L&#39;adoption de protocoles publiés 18, 19, 12 mm 2 activent couvercle en verre glisse chimiquement pour assurer la liaison covalente de l&#39;hydrogel. <ol><li> Tout d&#39;abord, couvercle en verre glisse avec plaisir 3- Aminopropyltrymethoxysilane (ATS) pour 7 min à température ambiante. </li><li> Retirer l&#39;ATS complètement avec DI rinçage à l&#39;eau et traiter couvercle glisse avec 0,5% de glutaraldéhyde (dilué dans de la solution PBS Glutaraldehyde boursier de 70%) pour 30 min. </li></ol></li><li> Obtenir deux solutions de gel kPa en mélangeant 5% en poids / solution d&#39;acrylamide v et 0,05% de N, -methylenebisacrylamide (bis) solution N &#39;10 mM dans une solution saline tamponnée au HEPES 20. Utilisation 8% p / v d&#39;acrylamide et 0,13% N, -methylenebisacrylamide (bis) solution N &#39;dans 10 mM de solution saline tamponnée HEPES 10 kPa 20 gel. <ol><li> Dans les deux cas, utiliser 1: 200 persulfate d&#39;ammonium (10% p / v) et 1: 2000 N, N, N &#39;, N&#39; -tetramethylethylenediamine (TEMED) comme initiateur et de catalyseur pour le procédé de polymérisation, respectivement. </li><li> En outre, ajouter 100 ul perles fluorescentes à 2 solution de gel kPa comme marqueurs fiduciaires 21, 22. </li></ol></li><li> Déposez une goutte de 20 pi de solution de gel PA pré-polymère sur le 12 mm 2 activé couvercle en verre de glissement. Placez un autre 12 mm 2 régulière lamelle de verre sur la chute. Assurez-vous que la baisse se propage entre les lamelles par capillarité. Inverser le sandwich à deux gels kPa afin d&#39;assurer que la plupart des billes viennent à proximité de la surface de culture cellulaire. </li><li> Guérir le gel PA pendant 45 min à température ambiante. </li><li> Décoller la lamelle supérieure en utilisant un seul rasoir de bord. Remarque: Lors de l&#39;épluchage, le détachement procède d&#39;un borddu sandwich. Le gel reste adhérente à la lame de verre activée. </li><li> Stocker les gels dans une solution de PBS avant utilisation. </li><li> Fonctionnaliser gels PA et le verre avec des molécules d&#39;ECM, la fibronectine humaine à une concentration de 50 pg / ml 23 méthodes suivantes publiées. <ol><li> En bref, des substrats incuber avec 2 ml d&#39;hydrazine hydrate pur O / N. </li><li> Retirer l&#39;hydrate d&#39;hydrazine et bien rincer les substrats avec de l&#39;eau DI. </li><li> Laver les substrats avec de l&#39;acide acétique à 5% pendant 30 min. </li><li> Éliminer l&#39;acide acétique et bien rincer les substrats avec de l&#39;eau DI. </li><li> Gardez les substrats immergés dans l&#39;eau DI pendant 30 min. </li><li> Incuber les substrats avec la fibronectine oxydé pendant 35 min à une concentration de 50 ug / ml. </li><li> Rincer les substrats avec PBS sur agitateur à bas régime pendant 10 min. </li><li> Incuber tous les substrats à 37 ° C dans 2-3 ml de milieu de culture pendant 30 min avant l&#39;étalement des cellules. Remarque: cellules de la plaque dans un peu peuplerd manière (1.000-3.000 cellules / cm 2). Chaque bordereau de verre gel-couvert doit être contenue dans une boîte de Pétri de 35 mm. Permettre aux cellules d&#39;adhérer complètement avant toute microscopie (au moins O / N). </li></ol></li></ol> 4. Traction microscopie à force et immunofluorescence Microscopie dosages <ol><li> Traction Force Microscopy Assay <ol><li> Trypsine-EDTA / solution chaude de 0,25% à 10% de SDS à 37 ° C dans un bain d&#39;eau pendant 10 minutes avant le début des essais de traction. </li><li> Retirer un gel de l&#39;incubateur de culture cellulaire à un moment et placer sur la platine du microscope fluorescent inversé (grossissement 32X). </li><li> Trouver une seule cellule dans le champ de vision. Retirez le couvercle plat Petri. </li><li> Prenez une image de contraste de phase de cellule (par exemple, la figure 3). </li><li> Changez le mode d&#39;imagerie à fluorescence et de sélectionner un filtre approprié. Ne déplacez pas la platine du microscope ou de l&#39;échantillon pendant ce temps. </li><li> Prenez une image de l&#39;perles fluorescentes displaced par traction cellulaire (figure 4C). </li><li> Ajouter la solution / SDS 1 ml de trypsine à boîte de Pétri pour détacher la cellule de gel. Ne déplacez pas la platine du microscope ou de l&#39;échantillon pendant ce temps. Aussi, prenez une image de contrôle de la cellule pour assurer l&#39;élimination complète du gel. </li><li> Prenez une image de référence (force nulle) des billes après la cellule est supprimée. </li><li> Répétez les étapes 4.1.2-4.1.8 pour tous les autres gels. </li><li> Faire une pile d&#39;images utilisant ImageJ des deux images acquises à l&#39;étape 4.1.6 et 4.1.8 pour chaque cas. Pour générer la pile d&#39;images, utilisez la séquence suivante de commandes dans ImageJ après l&#39;ouverture des images: Image → → Stacks Images à empiler. </li><li> Pour obtenir le champ de déplacement et de traction, utiliser des plugins ImageJ suivante méthodes publiées 21, 22 Obtenir des codes et des tutoriels détaillés pour ces plugins en utilisant le lien suivant:. <a href="https://sites.google.com/site/qingzongtseng/imagejplugins">https://sites.google.com/site/qingzongtseng/ima</a>gejplugins. <ol><li> Alignez les images de la pile en utilisant le plugin &#39;Matching Template&#39;. Utilisez la séquence suivante de commandes dans ImageJ après l&#39;ouverture de la pile de l&#39;image générée à l&#39;étape 4.1.10: Plugins → Template Matching → Aligner les tranches dans Stack → OK. Enregistrer la pile de l&#39;image en conséquence. Utilisez cette nouvelle pile d&#39;images dans les étapes suivantes. </li><li> Obtenir le champ de déplacement en utilisant la PIV (vélocimétrie par images de particules) plugin (Figure 4D). Utilisez la séquence suivante de commandes après l&#39;ouverture de la pile de l&#39;image sauvegardée à l&#39;étape 4.1.11.1: Plugins → → itératif PIV PIV (de base) → OK → Accepter cette PIV et la sortie → OK. Enregistrer la sortie de PIV dans le même répertoire que dans la pile de l&#39;image originale. Utilisez-le comme entrée à l&#39;étape suivante. </li><li> Enfin utiliser (transformée de Fourier traction cytométrie) le FTTC plug-in pour obtenir la carte de traction (figure 4E). Utilisez la séquence suivante de commandes: Plugins → → FTTC Insert les propriétés des matériaux, à savoir, le module de Young et le coefficient de Poisson de PA gel → OK → Sélectionnez le fichier de sortie de PIV enregistré à l&#39;étape 4.1.11.2 → OK. Sauvegarder FTTC résulte dans le même répertoire que dans la pile d&#39;image et sortie PIV. </li></ol></li></ol></li><li> Immunofluorescence Microscopie dosages <ol><li> Prenez les substrats à immunocolorées à la hotte laminaire et retirez le support de culture. </li><li> Rincer les substrats avec du PBS et on fixe les cellules avec du paraformaldehyde à 4% dans du PBS pendant 20 min à température ambiante. </li><li> Laver les substrats avec du PBS pendant 3 fois (5 min à chaque fois). Par conséquent, incuber les substrats avec 500 ul Amplificateur de signal pendant 30 minutes et rincer avec du PBS. </li><li> Incuber les cellules avec un anticorps monoclonal anti-vinculine à une dilution de 1: 250 dans du PBS pendant 45 min à température ambiante. Laver les substrats avec du PBS pendant 3 fois (5 min à chaque fois). </li><li> Incuber les échantillons avec un anticorps secondaire Alexa Fluor 488 IgG de chèvre anti-souris à une dilution de 1: 200dans du PBS à température ambiante pendant 30 min. Laver les substrats avec du PBS pendant 3 fois (5 min à chaque fois). </li><li> Pour visualiser la structure F-actine, incuber les cellules avec la phalloïdine TRITC conjugués à une concentration de 50 ug / ml pendant 45 min à température ambiante. Laver les substrats avec du PBS pendant 3 fois (5 min à chaque fois). </li><li> Image les échantillons en utilisant microscope confocal à balayage laser (figure 5A - 5D). </li></ol></li></ol>

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The authors have nothing to disclose.

Effort de traction cellulaire a récemment émergé comme un indicateur potentiel biophysique de l&#39;état métastatique 13. Cependant, aucune donnée expérimentale de traction avec des cellules tumorales primaires existe dans la littérature à ce jour. En outre, la culture directement isolées les cellules du côlon primaire sur différents gels rigidité de polyacrylamide est pas encore signalé. Par conséquent, nous établissons un système optimisé côlon primaire conditions de culture cellulaire su...

Au cours des dernières années, il est devenu de plus en plus évident que les micro-environnement mécanique, en plus des facteurs biochimiques, joue un rôle important dans la régulation des fonctions cellulaires. Les cellules peuvent détecter et de répondre à la rigidité du substrat sur ​​lequel elles sont respectées (comme dans la culture 2D) ou entourées par (comme dans la culture 3D) 7.3. En faisant cela, les cellules peuvent moduler leur différenciation 3, 4 morphologie, la migration / mobilité 5, biophysiques propriétés 6, 7 croissance, et d&#39;autres processus. Les cellules cancéreuses répondent également à la rigidité de la matrice 2D et 3D en utilisant une combinaison coordonnée hiérarchique mécano-chimique de récepteurs d&#39;adhésion et les protéines de membrane de transduction de signal associées, l&#39;architecture du cytosquelette et moteurs moléculaires 1, 2. Par exemple, les cellules epitheliales mammaires (CEM) former parenchyme acineuses normale lorsqu&#39;elles sont cultivées sur des substrats 150 Pa qui est similaire à la rigiditédes tissus mammaires sains. Fait intéressant, ils présentent les caractéristiques d&#39;une tumeur en développement, à la fois structurelles et de transcription, lorsqu&#39;elles sont cultivées sur des substrats rigides (&gt; 5000 Pa) qui imitent la rigidité d&#39;un stroma tumoral 8. En outre, une autre expérience montre que la tumorigenèse mammaire est accompagné par une réticulation du collagène et de raidissement 9 ECM. Des expériences récentes montrent que carcinome du côlon humain HCT-8 () cellules présentent des métastases phénotype (MLP) lorsqu&#39;elles sont cultivées sur des substrats 2D ayant la rigidité physiologiquement pertinente (20-47 kPa), mais pas sur très raide (3,6 GPa) substrats 10- 12 .Ces premières cellules forment des amas de cellules tumorales comme dissocient et puis de l&#39;autre, à partir de la périphérie. Comme cette épithéliale à arrondie morphologique (E à R transition) changement se produit, elles prolifèrent, réduire cellule-cellule et cellule-ECM adhérence, et devenir migratoire. HCT-8 cellules cultivées sur des substrats de polystyrène très durs ne pas exposer ceux-citraits malignes. Ainsi, il a été émis l&#39;hypothèse que HCT-8 cellules deviennent métastatique en raison de leur exposition aux micro-environnement approprié. Il est à noter que ces expériences sont réalisées avec des lignées cellulaires de cancer immortalisées ou des cellules primaires de souris, dérivé avec des cellules non cancéreuses humaines primaires. Une étude récente propose que le stress augmentée de traction cellulaire peut être utilisé comme une signature biophysique potentiel pour les cellules métastatiques 13 .Le étude consiste à mesurer la force de traction de différentes lignées de cellules cancéreuses humaines sur des gels de Polyacrylamide. On constate que les cellules cancéreuses métastatiques peuvent exercer une contrainte sensiblement plus élevé de traction par rapport aux cellules non métastatiques 13 dans tous les cas. Cependant, ces résultats contredisent directement les résultats publiés précédemment sur ​​murins lignées cellulaires dérivées du cancer du sein 14. En outre, une étude récente met en évidence des différences notables entre les cellules humaines immortalisées et primaires dans leur remodelage du cytosquelette proprofilage protéine et la survie cellulaire expression de la protéine 15. Par conséquent, il est important de revenir sur la plupart des tests biophysiques traction, y compris pour les cellules cancéreuses humaines primaires. Cela permettra de répondre à la question de savoir si les cellules primaires récapitulent immortalisée lignées cellulaires de cancer tendance de traction. Le protocole décrit ici est optimisée pour l&#39;isolement de cellules primaires humaines du côlon (à la fois saines et cancéreuses), et pour les cultiver sur des substrats mous (hydrogels de Polyacrylamide), ainsi que sur des boîtes de Pétri. Le protocole est basé sur la digestion enzymatique et dissociation consécutive de l&#39;échantillon de tissu chirurgical en suspension de cellules individuelles 16. A notre connaissance, ceci est la première démonstration de la culture isolée tumeur primaire du colon et de cellules normales directement sur des substrats souples d&#39;hydrogel avec microbilles fluorescentes embarqués pour la traction cytométrie. Substrats de gel transparent permettent également immunocoloration. Cette analyse a révélé des différences dans l&#39;organisation F-actine etadhésions focales dans les cellules du côlon humaines primaires que les changements de rigidité du substrat. Cette plate-forme de culture cellulaire ouvre la possibilité d&#39;explorer diverses propriétés biophysiques des cellules humaines primaires telles que la rigidité des cellules et de traction en tant que paramètres de pronostic du cancer.

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isolation of primary human colon tumor cells from surgical tissues and culturing them directly on soft elastic substrates for traction cytometry

Les cellules cancéreuses réagissent à la matrice de rigidité mécanique d&#39;une manière complexe en utilisant un hiérarchique système coordonné mécano-chimique composée de récepteurs d&#39;adhésion et les protéines de membrane de transduction de signal associées, l&#39;architecture du cytosquelette et moteurs moléculaires 1, 2. mécanosensibilité de cellules cancéreuses différentes in vitro sont étudiée principalement avec des lignées cellulaires immortalisées ou des cellules primaires de souris, dérivé avec des cellules non cancéreuses humaines primaires. Par conséquent, on en sait peu sur la mécanosensibilité de cellules de cancer du côlon humains primaires in vitro. Ici, un protocole optimisé est développé qui décrit l&#39;isolement de cellules du colon à partir d&#39;échantillons humains primaires saines et cancéreuses tissus humains chirurgicales. Les cellules du côlon isolés sont ensuite cultivés avec succès sur le doux (2 kPa raideur) et rigide (10 kPa raideur) hydrogels de polyacrylamide et polystyrène rigide (~ 3,6 GPa de rigidité) substrats fonctionnalisés par une matrice extracellulaire (fibronectinedans ce cas). Les microbilles fluorescentes sont incorporées dans des gels mous à proximité de la surface de culture cellulaire, et essai de traction est effectué pour évaluer les contraintes de contraction cellulaire en utilisant le logiciel d&#39;accès ouvert libre. En outre, la microscopie d&#39;immunofluorescence sur des substrats différents de rigidité fournit des informations utiles sur la morphologie de la cellule primaire, l&#39;organisation du cytosquelette et la vinculine contenant adhésions focales en fonction de la rigidité substrat.

Le protocole décrit ci-dessus est utilisé avec succès pour plusieurs échantillons de tissus (n = 12) de quatre patients différents selon les lignes directrices de la commission d&#39;examen institutionnel. Figure 1A illustre une tumeur colorectale représentant tout de suite après la chirurgie à partir de laquelle on obtient des coupes de tissus pour les cultures de cellules. Une section typique de tissu dans une solution HBSS après le transfert de la hotte laminaire pour un traitement suppléme...

Watch this Scientific Journal Video about Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry at JoVE.com

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

Isolation des droits de cellules primaires de tumeurs du côlon à partir de tissus chirurgicaux et les cultivant directement sur élastique souple substrats pour Traction cytométrie

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

isolation-primary-human-colon-tumor-cells-from-surgical-tissues

Research

JoVE Journal

Bioengineering

15.4K vues.  University of Illinois at Urbana-Champaign. A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses. 

Vidéo: Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary tumors at distant sites. CTC count can be used to follow disease progression based on the correlation between CTC concentration in blood and disease severity1. As a treatment tool, CTC could be studied in the laboratory to develop personalized therapies. To this end, CTC isolation must cause no cellular damage, and contamination by other cell types, particularly leukocytes, must be avoided as much as possible2. Many of the current techniques, including the sole FDA-approved device for CTC enumeration, destroy CTC as part of the isolation process (for more information see Ref. 2). A microfluidic device to capture viable CTC is described, consisting of a surface functionalized with E-selectin glycoprotein in addition to antibodies against epithelial markers3. To enhance device performance a nanoparticle coating was applied consisting of halloysite nanotubes, an aluminosilicate nanoparticle harvested from clay4. The E-selectin molecules provide a means to capture fast moving CTC that are pumped through the device, lending an advantage over alternative microfluidic devices wherein longer processing times are necessary to provide target cells with sufficient time to interact with a surface. The antibodies to epithelial targets provide CTC-specificity to the device, as well as provide a readily adjustable parameter to tune isolation. Finally, the halloysite nanotube coating allows significantly enhanced isolation compared to other techniques by helping to capture fast moving cells, providing increased surface area for protein adsorption, and repelling contaminating leukocytes3,4. This device is produced by a straightforward technique using off-the-shelf materials, and has been successfully used to capture cancer cells from the blood of metastatic cancer patients. Captured cells are maintained for up to 15 days in culture following isolation, and these samples typically consist of &gt;50% viable primary cancer cells from each patient. This device has been used to capture viable CTC from both diluted whole blood and buffy coat samples. Ultimately, we present a technique with functionality in a clinical setting to develop personalized cancer therapies.

Circulating tumor cells are isolated from the blood of cancer patients without inflicting cellular damage. Isolation of tumor cells is accomplished using a bimolecular surface of E-selectin in addition to antibodies against epithelial markers. A nanotube coating specifically promotes cancer cell adhesion resulting in high capture purities.

Isolement rapide des cellules tumorales circulantes viables partir d&#39;échantillons sanguins des patients

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary ...

Recherche

Bio-ingénierie

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

Les tissus d&#39;ingénierie muscle squelettique de myoblastes murins cellules progénitrices et application d&#39;une stimulation électrique

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

Cytométrie de masse: Protocole pour Tuning quotidien et échantillons cellulaires course sur un cytomètre de masse CyTOF

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

Cellule structuration par photolithographie sur Défini Parylene-C: SiO<sub&gt; 2</sub&gt; Substrats

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical deficiencies that impair function and stability. Current options for the treatment of partial ACL tears range from nonoperative, conservative management to multiple surgical options, such as: thermal modification, single-bundle repair, complete reconstruction, and reconstruction of the damaged portion of the native ligament. Few studies, if any, have demonstrated any single method for management to be consistently superior, and in many cases patients continue to demonstrate persistent instability and other comorbidities.
The goal of this study is to identify a potential cell source for utilization in the development of a tissue engineered patch that could be implemented in the repair of a partially torn ACL. A novel protocol was developed for the expansion of cells derived from patients undergoing ACL reconstruction. To isolate the cells, minced hACL tissue obtained during ACL reconstruction was digested in a Collagenase solution. Expansion was performed using DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). The cells were then stored at -80 &#186;C or in liquid nitrogen in a freezing medium consisting of DMSO, FBS and the expansion medium. After thawing, the hACL derived cells were then seeded onto a tissue engineered scaffold, PLAGA (Poly lactic-co-glycolic acid) and control Tissue culture polystyrene (TCPS). After 7 days, SEM was performed to compare cellular adhesion to the PLAGA versus the control TCPS. Cellular morphology was evaluated using immunofluorescence staining. SEM (Scanning Electron Microscope) micrographs demonstrated that cells grew and adhered on both PLAGA and TCPS surfaces and were confluent over the entire surfaces by day 7. Immunofluorescence staining showed normal, non-stressed morphological patterns on both surfaces. This technique is promising for applications in ACL regeneration and reconstruction.

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

For future applications as a patch to repair partial tears of the Anterior Cruciate Ligament (ACL), human ACL derived cells were isolated from tissue obtained during reconstructive procedures, expanded in vitro and grown on tissue engineered scaffolds. Cellular adhesion and morphology was then performed to confirm biocompatibility on scaffold surface.

Extraction chirurgicale, isolement et<em&gt; In vitro</em&gt; Extension de antérieure humaine Cruciate cellules du ligament dérivés pour les applications d&#39;ingénierie tissulaire

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical ...

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should mimic the human tissues they are aiming to replace; to provide the required anatomical shape, the materials must be able to sustain the mechanical forces they will experience when implanted at the defect site. Although the mechanical properties of tissue-engineered scaffolds are of great importance, many human tissues that undergo restoration with engineered materials have not been fully biomechanically characterized. Several compressive and tensile protocols are reported for evaluating materials, but with large variability it is difficult to compare results between studies. Further complicating the studies is the often destructive nature of mechanical testing. Whilst an understanding of tissue failure is important, it is also important to have knowledge of the elastic and viscoelastic properties under more physiological loading conditions.
This report aims to provide a minimally destructive protocol to evaluate the compressive and tensile properties of human soft tissues. As examples of this technique, the tensile testing of skin and the compressive testing of cartilage are described. These protocols can also be directly applied to synthetic materials to ensure that the mechanical properties are similar to the native tissue. Protocols to assess the mechanical properties of human native tissue will allow a benchmark by which to create suitable tissue-engineered substitutes.

Tissue biomechanics is important for maintaining cell shape and function and for determining phenotype. This report demonstrates non-destructive mechanical protocols for characterizing elastic and viscoelastic properties of human soft tissues, which can be directly applied to tissue-engineered substrates to allow a close matching of engineered materials to native tissue.

Caractérisation biomécanique des tissus mous humains à l&#39;aide d&#39;essais indentation et traction

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should ...

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

Isolement des cellules de ganglion rétiniennes murines primaires (RGC) par cytométrie de flux

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

Isolement des cellules souches mésenchymateuses du périoste alvéolaire humaine et les effets de la vitamine D sur activité ostéogène du périoste dérivées des cellules

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been proven to be useful substrates for culturing cells in a physical microenvironment that partially mimics in vivo conditions. Here, we present a simple protocol for characterizing the Young's moduli of isotropic linear elastic substrates typically used for mechanobiology studies. The protocol consists of preparing a soft silicone substrate on a Petri dish or stiff silicone, coating the top surface of the silicone substrate with fluorescent beads, using a millimeter-scale sphere to indent the top surface (by gravity), imaging the fluorescent beads on the indented silicone surface using a fluorescence microscope, and analyzing the resultant images to calculate the Young's modulus of the silicone substrate. Coupling the substrate's top surface with a moduli extracellular matrix protein (in addition to the fluorescent beads) allows the silicone substrate to be readily used for cell plating and subsequent studies using traction force microscopy experiments. The use of stiff silicone, instead of a Petri dish, as the base of the soft silicone, enables the use of mechanobiology studies involving external stretch. A specific advantage of this protocol is that a widefield fluorescence microscope, which is commonly available in many labs, is the major equipment necessary for this procedure. We demonstrate this protocol by measuring the Young's modulus of soft silicone substrates of different elastic moduli.

Substrates with stiffness in the kilopascal-range are useful to study the response of cells to physiologically relevant micro-environment stiffness. Using just a widefield fluorescence microscope, the Young's modulus of soft silicone gels can be determined using an indentation with a suitable sphere.

Mesure de la rigidité des substrats souples en Silicone pour mécanobiologie études utilisant un Microscope à Fluorescence Widefield

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.