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.

Il protocollo descritto di seguito segue le linee guida del UIUC comitato etico della ricerca umana. 1. Raccolta e digestione di Campione di tessuto chirurgico <ol><li> Raccogliere il campione di tessuto tumorale dopo la resezione del colon (Figura 1A e 1B). Raccogliere il tessuto da un sito sano adiacente pure. </li><li> Trasferire immediatamente il tessuto ad un flaconcino da 15 ml contenente 12 ml di soluzione HBSS. Conservare il flaconcino sul ghiaccio all&#39;interno di una scatola di schiuma isolante. </li><li> Trasportare la fiala contenente il tessuto di una cappa coltura di tessuti per l&#39;ulteriore elaborazione in 45 min. Conservare il flaconcino su un blocco di ghiaccio all&#39;interno della cappa. </li><li> Versare il tessuto fornito in un 6 pozzetti contenenti 6-7 ml di soluzione HBSS con una pipetta. Nota: quantità di tessuto per pozzetto non è critico in questa fase di risciacquo. </li><li> Mantenere la piastra 6 bene su un blocco di ghiaccio. Lavare il tessuto in soluzione HBSS due volte. </li><li> Tagliare il tessuto in modo pulito in sezioni separate con SIC sterilisor. Tessuto Mince in sezioni più piccole non superiore a 1-2 mm 3 dimensioni, con una lama di bisturi sterile. </li><li> Pesare una etichetta 0,1% tripsina flacone (contenente 1 ml di 0,1% soluzione di tripsina) prima di trasferire il tessuto. Trasferire ~ 20 mg di tessuto in ciascuna delle fiale tripsina 0,1% con una pipetta. </li><li> Cercare di ridurre al minimo il trasferimento di HBSS nel flaconcino tripsina per evitare ulteriore diluizione della tripsina. </li><li> Pesare le fiale tripsina dopo il trasferimento dei tessuti; documentare la differenza come la massa di tessuto. </li><li> Versare ~ 20 mg di tessuto e 1 ml di tripsina in ciascun pozzetto di una piastra ben 24. </li><li> Mettere la piastra ben 24 su shaker in frigorifero a 4 ° C per 16-20 ore. Durante questo periodo di attesa o in un momento precedente, preparare i substrati gel di poliacrilamide e funzionalizzare con proteine ​​ECM come descritto nel paragrafo 3. </li></ol> 2. enzimatica dissociazione di Campione di tessuto in sospensione singola cella e coltura isolati Celle ECM funzionalizzato elastico Substrates e piatti polistirolo rigido <ol><li> Dopo 16-20 ore di incubazione in tripsina ben a 4 ° C su agitatore, trasferire il tessuto da 4 ° C tripsina a 4 ° C completa flacone mezzi di crescita (contenente 2-3 ml di terreni di crescita completo) usando una pipetta. Garantire minimo trasferimento di tripsina in terreni di crescita. Nota: Complete formulazione mezzi di crescita è la seguente: RPMI 1640 medium di base integrato con siero di cavallo ad una concentrazione finale del 10% e la penicillina-streptomicina al 1% di soluzione totale. </li><li> Posizionare il tessuto contenente mezzi provetta in un bagno di acqua calda a 37 ° C per 12-15 min. </li><li> Trasferire il tessuto di una fiala da 15 ml contenente soluzione HBSS con una pipetta, agitare delicatamente. </li><li> Ripetere 2.3. </li><li> Rimuovere il tessuto dalla soluzione HBSS e trasferimento al 0,1% soluzione di collagenasi (in HBSS) flaconcino contenente 1 ml di soluzione. Incubare per 45 minuti a 37 ° C e 5% CO 2 ambiente in una coltura cellulare incubatore umidificato. Durante il periodo di attesa, growt caldoh media in un flaconcino da 15 ml a 37 ° C a bagnomaria. </li><li> Estrarre tutti i frammenti di tessuto in pipetta minimizzazione collagenasi riporto. Espellere solo i frammenti di tessuto in flaconi di coltura pre-riscaldato. </li><li> Mettere un filtro ben inserto 40 micron in ciascun pozzetto di una piastra ben 6. Inumidire il filtro con 1 ml di coltura cellulare multimediale. </li><li> Tessuto triturare in media con una pipetta di vetro da 10 ml più volte fino a quando il tessuto è completamente dissociato. </li><li> Mantenere la punta della pipetta il più vicino possibile alla base della fiala. </li><li> Depositare la soluzione sul filtro per rimuovere i detriti e parti indissociate. </li><li> Centrifugare filtrato sospensione cellulare in mezzi a 150 xg per 5 minuti a temperatura ambiente. </li><li> Rimuovere il surnatante dopo e risospendere pellet cellulare con 2 ml di terreni di coltura fresco. </li><li> Contare le cellule utilizzando un emocitometro (se richiesto). Seed isolato cellule primarie di matrice extracellulare (ECM) gel funzionalizzati e piatti di polistirene al concentrazione desiderata.</li></ol> 3. Preparazione e funzionalizzazione di diversi Rigidità Poliacrilammide (PA) Gel e polistirolo substrati Nota: Per la dimostrazione visiva del punto 3, gli spettatori / lettori sono riferite ad una recente ufficiale di esperimenti visualizzati (JoVE) articolo 17. <ol><li> L&#39;adozione di protocolli pubblicati 18, 19, attivano 12 millimetri 2 copertura di vetro scivola chimicamente per garantire vincolante dell&#39;idrogel covalente. <ol><li> In primo luogo, la copertura di vetro scivola trattare con 3- Aminopropyltrymethoxysilane (ATS) per 7 minuti a temperatura ambiente. </li><li> Rimuovere la ATS completamente con acqua deionizzata risciacquo e trattare copertura scivola con lo 0,5% glutaraldeide (diluito in PBS dal 70% soluzione glutaraldeide magazzino) soluzione per 30 minuti. </li></ol></li><li> Ottenere 2 soluzioni gel kPa miscelando 5% w / v di acrilammide e 0,05% N, soluzione N &#39;-methylenebisacrylamide (bis) in soluzione salina 10 mM HEPES-buffered 20. Utilizzare 8% w / v di acrilamide e 0,13% N, soluzione N &#39;-methylenebisacrylamide (bis) in 10 mM HEPES soluzione salina tamponata per 10 kPa gel 20. <ol><li> In entrambi i casi, usare 1: 200 persolfato di ammonio (10% w / v) e 1: 2.000 N, N, N &#39;, N&#39; -tetramethylethylenediamine (TEMED) come iniziatore e catalizzatore per il processo di polimerizzazione, rispettivamente. </li><li> Inoltre, aggiungere 100 microlitri perline fluorescenti a 2 kPa soluzione di gel come marcatori fiduciari 21, 22. </li></ol></li><li> Depositare una goccia di 20 ml di pre-polimero soluzione di gel PA il 12 mm 2 attivato antiscivolo copertura in vetro. Posizionare un altro 12 millimetri 2 regolare polizza di copertura in vetro sulla goccia. Assicurarsi che il calo si diffonde tra i coprioggetto per capillarità. Capovolgere il panino per 2 gel kPa per garantire che la maggior parte delle perle provengono vicino alla superficie di coltura cellulare. </li><li> Curare il gel PA per 45 minuti a RT. </li><li> Staccare la polizza di copertura superiore utilizzando un unico rasoio bordo. Nota: Durante peeling, il distacco procede da un bordodel sandwich. Il gel rimane aderente al vetrino attivato. </li><li> Conservare i gel in soluzione PBS prima dell&#39;uso. </li><li> Funzionalizzare gel PA e vetro con le molecole ECM, fibronectina umana ad una concentrazione di 50 mg / ml metodi seguito pubblicati 23. <ol><li> In breve, incubare substrati con 2 ml puro idrato di idrazina O / N. </li><li> Rimuovere idrato di idrazina e lavare accuratamente i substrati con acqua deionizzata. </li><li> Lavare le substrati con 5% di acido acetico per 30 min. </li><li> Rimuovere acido acetico e lavare accuratamente i substrati con acqua deionizzata. </li><li> Mantenere i substrati immersi in acqua deionizzata per 30 min. </li><li> Incubare i substrati con fibronectina ossidato per 35 minuti ad una concentrazione di 50 ug / ml. </li><li> Sciacquare i substrati con PBS in agitatore a basso numero di giri per 10 minuti. </li><li> Incubare tutti i substrati a 37 ° C in 2-3 ml di coltura per 30 minuti prima della placcatura le cellule. Nota: le cellule piastra in un popolano scarsamented maniera (1.000-3.000 cellule / cm 2). Ogni bicchiere slittamento gel-rivestito deve essere contenuta in una capsula Petri da 35 mm. Consentono alle cellule di aderire completamente prima di qualsiasi microscopia (almeno O / N). </li></ol></li></ol> 4. trazione Forza microscopia e immunofluorescenza Microscopia saggi <ol><li> Trazione Force Microscopy Assay <ol><li> Tripsina-EDTA soluzione calda 0,25% / 10% SDS a 37 ° C a bagnomaria per 10 minuti prima di iniziare esperimenti di trazione. </li><li> Rimuovere un gel dalla incubatore coltura cellulare alla volta e immettere sul palco microscopio a fluorescenza invertito (32X di ingrandimento). </li><li> Trovare una singola cella nel campo visivo. Togliere il coperchio piastra di Petri. </li><li> Dai contrasto di fase immagine di cellule (ad esempio, figura 3). </li><li> Attivare la modalità di imaging per fluorescenza e selezionare filtro appropriato. Non spostare il tavolino del microscopio o del campione durante questo periodo. </li><li> Prendete l&#39;immagine della perline fluorescenti CILINDRATAcato da trazione cellulare (Figura 4C). </li><li> Aggiungere 1 ml di soluzione tripsina / SDS per capsula di Petri staccare la cella dal gel. Non spostare il tavolino del microscopio o del campione durante questo periodo. Inoltre, prendere un&#39;immagine di controllo della cella per assicurare la rimozione completa dal gel. </li><li> Prendere una immagine di riferimento (forza null) delle perline dopo che la cella è rimossa. </li><li> Ripetere i passaggi 4.1.2-4.1.8 per tutti gli altri gel. </li><li> Fare una serie di immagini utilizzando ImageJ dalle due immagini acquisite nella fase 4.1.6 e 4.1.8 per ciascun caso. Per generare la pila di immagini, utilizzare seguente sequenza di comandi in ImageJ dopo l&#39;apertura delle immagini: Immagine → Stack → Immagini per impilare. </li><li> Per ottenere il campo di spostamenti e la trazione, utilizzare i plugin ImageJ seguente metodi pubblicati 21, 22 Ottenere i codici e tutorial dettagliati per questi plugin utilizzando il seguente link:. <a href="https://sites.google.com/site/qingzongtseng/imagejplugins">https://sites.google.com/site/qingzongtseng/ima</a>gejplugins. <ol><li> Allineare le immagini in stack utilizzando il plugin &#39;template matching&#39;. L&#39;uso seguente sequenza di comandi in ImageJ dopo l&#39;apertura della serie di immagini generate al punto 4.1.10: Plugin → Modello di corrispondenza → Allinea Fette di Pila → OK. Salvare l&#39;serie di immagini di conseguenza. Usa questa nuova serie di immagini nei seguenti passi. </li><li> Ottenere il campo di spostamento con il PIV (particle image velocimetry) plug (Figura 4D). L&#39;uso seguente sequenza di comandi dopo l&#39;apertura della serie di immagini salvata in fase 4.1.11.1: Plugin → PIV → iterativo PIV (Basic) → OK → Accetta questo PIV e uscita → Ok. Salvare l&#39;uscita PIV nella stessa directory nella serie di immagini originale. Utilizzare questo come input nel passaggio successivo. </li><li> Infine utilizzare il FTTC (trasformata di Fourier trazione citometria) plugin per avere la mappa di trazione (figura 4E). L&#39;uso seguente sequenza di comandi: Plugin → FTTC → Insproprietà dei materiali ERT, vale a dire, il modulo di Young e il rapporto di Poisson di PA gel → OK → Selezionare il file di output PIV salvato nel passaggio 4.1.11.2 → OK. Salva FTTC risulta nella stessa directory nella serie di immagini e di uscita PIV. </li></ol></li></ol></li><li> Immunofluorescenza Microscopia Assays <ol><li> Prendere substrati da immunostained alla cappa laminare e rimuovere il mezzo di coltura. </li><li> Risciacquare i substrati con PBS e fissare le cellule con 4% paraformaldeide in PBS per 20 minuti a RT. </li><li> Lavare le substrati con PBS per 3 volte (5 min ogni volta). Di conseguenza, incubare i substrati con segnale 500 microlitri enhancer per 30 minuti e risciacquare con PBS. </li><li> Incubare le cellule con anticorpi anti-vinculin monoclonale ad una diluizione 1: 250 in PBS per 45 minuti a RT. Lavare le substrati con PBS per 3 volte (5 min ogni volta). </li><li> Incubare i campioni con anticorpo secondario Alexa Fluor IgG anti-topo 488 capra una diluizione 1: 200in PBS a temperatura ambiente per 30 min. Lavare le substrati con PBS per 3 volte (5 min ogni volta). </li><li> Per visualizzare la struttura F-actina, incubare le cellule con TRITC falloidina coniugati ad una concentrazione di 50 ug / ml per 45 minuti a RT. Lavare le substrati con PBS per 3 volte (5 min ogni volta). </li><li> Immagine i campioni utilizzando microscopio confocale a scansione laser (Figura 5A - 5D). </li></ol></li></ol>

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The authors have nothing to disclose.

Lo stress di trazione Cellular è recentemente emerso come un potenziale indicatore biofisico di stato metastatico 13. Tuttavia, i dati non trazione sperimentale con cellule del tumore primario esiste in letteratura fino ad oggi. Inoltre, la coltura direttamente isolate cellule del colon primario su gel differenti rigidità poliacrilammide non è ancora segnalato. Quindi, noi stabiliamo un ottimizzato colon primario condizioni di coltura cellulare su gel e polistirene (Figura 2). Incapsulamen...

Negli ultimi anni è diventato sempre più evidente che meccanico micro-ambiente, oltre a fattori bio-chimici, svolge un ruolo importante nella regolazione funzionalità cellulari. Le cellule possono rilevare e rispondere alla rigidità del substrato su cui sono rispettate (come in coltura 2D) o circondati da (come nella cultura 3D) 3-7. In questo modo, le cellule possono modulare la loro differenziazione 3, morfologia 4, migrazione / motilità 5, bio-fisiche 6, di crescita del 7, e altri processi. Le cellule tumorali rispondono anche alla rigidezza della matrice 2D e 3D con una coordinata, gerarchico combinazione meccano-chimica di recettori di adesione e proteine ​​di membrana trasduzione del segnale associate, l&#39;architettura del citoscheletro, e motori molecolari 1, 2. Ad esempio, le cellule epiteliali mammarie (MECs) formare normale parenchima acinose quando coltivate su substrati 150 Pa che è simile alla rigidezzadi tessuto mammario sano. È interessante notare che essi mostrano i tratti distintivi di un tumore in via di sviluppo, sia strutturali che di trascrizione, quando coltivate su supporti rigidi (&gt; 5.000 Pa) che imitano la rigidità di uno stroma tumorale 8. Inoltre, un altro esperimento dimostra che tumorigenesi seno è accompagnata da collagene reticolazione e ECM irrigidimento 9. Recenti esperimenti mostrano che di carcinoma del colon umano (HCT-8), le cellule mostrano metastasi come fenotipo (MLP) quando sono coltivate su substrati 2D che hanno rigidità fisiologicamente rilevanti (20-47 kPa), ma non molto rigida (3,6 GPa) substrati 10- 12 .Queste cellule prima forma gruppi di cellule tumorali-simili e poi dissociano l&#39;una dall&#39;altra, partendo dalla periferia. Dato che questo epiteliale a arrotondata morfologica (E transizione R) cambiamento si verifica, proliferano, ridurre cellula-cellula e cellula-ECM adesione, e diventare migratori. HCT-8 celle coltivate su substrati di polistirolo molto duri non presentano questetratti maligni. Così è stato ipotizzato che HCT-8 cellule diventano metastatiche a causa della loro esposizione ai appropriato microambiente. Vale la pena notare che questi esperimenti sono condotti con linee cellulari tumorali murine immortalizzate o derivati ​​pile, non con cellule primarie tumorali umane. Uno studio recente propone aumentata sforzo di trazione cellulare può essere utilizzato come potenziale firma biofisico per cellule metastatiche 13 studio .Il comporta la misurazione della forza di trazione per diverse linee cellulari tumorali umane su gel di poliacrilammide. Si è trovato che le cellule tumorali metastatiche possono esercitare significativamente maggiore sforzo di trazione rispetto alle cellule non-metastatiche in tutti i casi 13. Tuttavia, questi risultati contraddicono direttamente i risultati precedentemente pubblicate su murine derivate linee cellulari di cancro al seno 14. Inoltre, un recente studio mette in luce notevoli differenze tra le cellule umane immortalizzate e primarie nel loro rimodellamento pro citoscheletroprofiling TEIN e la sopravvivenza delle cellule proteina espressione 15. Quindi, è importante rivisitare molti dei saggi biofisici compreso trazione per cellule primarie tumorali umane. Questo affronterà la questione se le cellule primarie ricapitolano immortalato linee cellulari tumorali tendenza trazione. Il protocollo qui descritto è ottimizzato per l&#39;isolamento di cellule primarie umane colon (sani e cancerosi), e per loro coltura su substrati molli (idrogeli poliacrilammide) nonché su piastre di Petri. Il protocollo si basa sulla digestione e la conseguente dissociazione enzimatica di campione di tessuto chirurgico in sospensione singola cella 16. A nostra conoscenza, questa è la prima dimostrazione di coltura isolato tumore primitivo del colon e cellule normali direttamente su substrati idrogel morbide con microsfere fluorescenti incorporati per trazione citometria. Substrati gel trasparenti consentono anche immunocolorazione. Questo test ha rivelato differenze nell&#39;organizzazione F-actina eadesioni focali in cellule del colon umani primari come cambiamenti substrato rigidità. Questa piattaforma coltura cellulare apre la possibilità di esplorare le varie proprietà biofisiche di cellule umane primarie come la rigidità delle cellule e la trazione come parametri per pronostici cancro.

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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

Le cellule tumorali rispondono alla matrice rigidità meccanica in modo complesso con un sistema coordinato, gerarchico meccano-chimico composto di recettori di adesione e proteine ​​di membrana trasduzione del segnale associati, l&#39;architettura del citoscheletro, e motori molecolari 1, 2. Mechanosensitivity delle cellule tumorali in vitro sono differenti indagato principalmente linee cellulari immortali o murine derivate cellule primarie, non con cellule primarie tumorali umane. Quindi, poco si sa circa la mechanosensitivity di pile tumorali di colon umano in vitro. Qui, un protocollo ottimizzato è sviluppato che descrive l&#39;isolamento di cellule del colon umane primarie da campioni di tessuti umani sani e tumorali chirurgici. Isolate cellule del colon sono poi successo coltivate su substrati funzionalizzati da una matrice extracellulare morbido (2 kPa rigidità) e rigida (10 kPa rigidità) idrogel di poliacrilamide e polistirolo rigido (~ 3.6 GPa rigidità) (fibronectinain questo caso). Microsfere fluorescenti sono incorporati in morbido gel vicino alla superficie di coltura cellulare, e il dosaggio di trazione viene eseguita per valutare le sollecitazioni contrattili cellulari che utilizzano il software gratuito di accesso aperto. Inoltre, immunofluorescenza su diversi substrati rigidezza fornisce informazioni utili sul morfologia cellulare primaria, organizzazione citoscheletro e vinculina contenente adesioni focali in funzione del substrato rigidità.

Il protocollo di cui sopra è impiegata con successo per i campioni di tessuto multipli (n = 12) provenienti da quattro diversi pazienti secondo le linee guida del comitato istituzionale di revisione. La figura 1A illustra un tumore del colon-retto rappresentante destra dopo l&#39;intervento chirurgico, da cui si ottengono le sezioni di tessuto per colture cellulari. Una tipica sezione di tessuto in soluzione HBSS dopo il trasferimento alla cappa laminare per l&#39;ulteriore elaborazione è mostrato in ...

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.

Isolamento di primarie cellule del colon tumorali umane dai tessuti chirurgici e Coltura direttamente su più elastica e flessibile substrati per trazione citometria

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 Views.  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. 

Video: 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.

Isolamento rapido di cellule tumorali circolanti da campioni di sangue del paziente

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

Ricerca

Bioingegneria

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.

Ingegneria tessuti muscolo scheletrico di cellule murine progenitrici mioblasti e di applicazione di stimolazione elettrica

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

Massa Citometria: Protocollo per l&#39;ottimizzazione giornaliera e campioni di cellule in esecuzione su un citometro Messa 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.

Patterning cellulare su fotolitograficamente Definito parylene-C: SiO<sub&gt; 2</sub&gt; Substrati

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.

Recupero chirurgico, Isolamento e<em&gt; In vitro</em&gt; Ampliamento anteriore umana crociati Cells Legamento di derivazione per applicazioni di ingegneria tissutale

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.

Caratterizzazione biomeccanica di morbidi tessuti umani usando una indentazione e prove di trazione

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.

Isolamento delle cellule della ganglioma della retina primaria (RGC) mediante citometria a flusso

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.

Isolamento di cellule staminali mesenchimali da periostio alveolare umano e gli effetti della vitamina D su attività osteogenica delle cellule derivate dal periostio

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.

Misura di rigidità dei substrati in morbido Silicone per gli studi di Mechanobiology usando un microscopio a fluorescenza 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.