Name: engineering skeletal muscle tissues from murine myoblast progenitor cells and application of electrical stimulation
Uploaded: 2013-03-19T12:00:00
Description: Watch this Scientific Journal Video about Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation at JoVE.com

Forfatterne vil gerne takke Yabin Wu til dyrkning af væv præsenteret i figur 2, blev billedet taget af Bart van Overbeeke. Arbejdet blev støttet af SenterNovem, tilskud ISO 42.022.

1. Kultur af murine myoblast progenitorceller eller C2C12-celler <ol><li> Isolere celler ifølge protokollen oprindeligt offentliggjort af Shefer og kolleger 10 og senere tilpasset ved Collins et al. 11 og Boonen et al. 12 og lagre dem i flydende nitrogen. Dette kræver mus, f.eks C57BL / 6. Alternative metoder anvendes i andre laboratorier, fx en metode offentliggjort i Journal of visualiseret Eksperimenter med Li Y et al. 13. Fo...

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Den teknik af muskelvæv har et stort potentiale for anvendelse som en sygdom model, til drug screening, regenerativ medicin og til kødproduktion. Men kravene til disse applikationer varierer. Vi valgte at arbejde med en blanding af kollagen og Matrigel, fordi collagen tillader cellejustering og fordi myoblast progenitorceller kræver tilstedeværelse af basalmembran afledte proteiner som bestemt i tidligere 2D studier 12. Desuden har fibringeler blevet testet i vores laboratorium og synes ikke at støtte de...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer (valgfrit) </td></tr><tr><td> Matrigel-vækstfaktor reduceres </td><td> Beckton og Dickinson </td><td></td><td></td></tr><tr><td> DMEM (høj glucose) * </td><td> Gibco </td><td> 42.430 </td><td></td></tr><tr><td> Advanced DMEM </td><td> Gibco </td><td> 12.491 </td><td></td></tr><tr><td> Hesteserum </td><td> Gibco </td><td> 65050-122 </td><td></td></tr><tr><td> Føtalt bovint serum </td><td> Greiner </td><td> 758.075 </td><td></td></tr><tr><td> 0,45 og 0,22 um sprøjtefilter * </td><td> Whatmann (Schleicher og Scheull) </td><td> 10462100 </td><td></td></tr><tr><td> L-glutamin </td><td> Gibco </td><td> 25030024 </td><td></td></tr><tr&gt; <td> Penicillin / streptomycin </td><td> Gibco </td><td> 10378016 </td><td></td></tr><tr><td> Amphotericin </td><td> Gibco </td><td> 15290-018 </td><td></td></tr><tr><td> Kultur plast </td><td> Greiner </td><td></td><td> Inkluderer dyrkningskolber og pipetter </td></tr><tr><td> Hønseembryo ekstrakt </td><td> USA Biologisk </td><td> C3999 </td><td></td></tr><tr><td> Pasteurpipette * </td><td> Hilgenberg </td><td></td><td> Pasteur pipetter, med konstriktion, med bomuld, åben tip L: 230 mm med spids diameter på 0,9 - 1,1 mm </td></tr><tr><td> Pasteurpipette * </td><td> Hilgenberg </td><td></td><td> Pasteur pipetter, med konstriktion, med bomuld, åbne spids L: 230 mm med spids diameter på 1,4 - 1,6 mm </td></tr><tr><td> Pasteurpipette </td><td> VWR </td><td> 612-1702 </td><td></td></tr><tr><td> Collagenase type I* </td><td> Sigma </td><td> C0130-16 </td><td></td></tr><tr><td> 40 um cell si * </td><td> BD Falcon </td><td> 352.340 </td><td></td></tr><tr><td> 19G nål </td><td></td><td></td><td></td></tr><tr><td> Elastomer </td><td> Dow Corning Corporation </td><td> 3097358-1004 </td><td> Silastic MDX 4-4210 # </td></tr><tr><td> Hærder </td><td> Dow Corning Corporation </td><td></td><td> Silastic MDX 4-4210 # </td></tr><tr><td> Velcro </td><td> Regelmæssig butik </td><td></td><td> Du kan købe dette på en almindelig butik, kun bruge den bløde side </td></tr><tr><td> Collagen type I, rotte hale </td><td> BD Biosciences </td><td> 3544236 </td><td></td></tr><tr><td> C-Pace EP Culture Pacer </td><td> Ionoptix </td><td></td><td></td></tr><tr><td> 6-brønds dyrkningsskåle for elektrisk stimulering </td><td> Beckton Dickinson-Falcon </td><td> BD Falcon # 353.846 </td><td></td></tr><tr><td> C-Dish dyrkningsskål elektroder </td><td> Ionoptix </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> * Nødvendig til isolering af celler (punkt 1,1) # Sammen i ét sæt </td></tr></tbody></table>

engineering skeletal muscle tissues from murine myoblast progenitor cells and application of electrical stimulation

Manipuleret muskelvæv kan anvendes til flere formål, der omfatter fremstilling af væv til anvendelse som en sygdomsmodel in vitro, fx til at studere tryksår, for regenerativ medicin og som kød alternativ 1. De første rapporterede 3D ​​muskel konstruktioner er blevet gjort for mange år siden og pionerer på området er Vandenburgh og kolleger 2,3. Fremskridtene i muskelvæv engineering er ikke kun resultatet fra det store gevinst i viden om biokemiske faktorer, stamceller og stamceller, men er især baseret på indsigt opnået af forskere, at fysiske faktorer spiller afgørende roller i kontrollen af ​​cellernes adfærd og vævsudvikling. State-of-the-art manipuleret muskel konstruktioner består i øjeblikket af celle-befolkede hydrogel konstruktioner. I vores laboratorium Disse består af murine myoblast progenitorceller, isoleret fra murine bagben muskler eller en murin myoblast cellelinje C2C12, miXED med en blanding af collagen / Matrigel og udpladet mellem to forankringspunkter, efterligne den muskel ledbånd. Andre celler kan betragtes som godt, f.eks alternative cellelinjer, såsom L6-myoblaster fra rotter 4, neonatal muskel progenitorceller 5, celler afledt fra voksne muskelvæv fra andre arter, såsom human 6 eller endda induceret pluripotente stamceller (iPS celler) 7 . Cellesammentrækningsevne bevirker tilpasning af cellerne langs den lange akse af konstruktionen 8,9 og differentiering af muskel progenitorceller efter cirka en uge i kultur. Endvidere kan anvendelsen af elektrisk stimulering forbedre processen til differentiering til en vis grad 8. På grund af sin begrænsede størrelse (8 x 2 x 0,5 mm) den komplette væv kan analyseres ved hjælp af konfokal mikroskopi til at overvåge fx levedygtighed, differentiering og cellejustering. Afhængigt af den specifikke anvendelse kravene til engineerøde muskelvæv vil variere, fx anvendelse til regenerativ medicin kræver opskalering af væv størrelse og vaskularisering, medens at tjene som en kød alternativ oversættelse til andre arter er nødvendig.

Slutproduktet vil være muskel konstruktioner som vist i figur 3.. Størrelsen af ​​vævet vil være ca 8 mm lang, 2 mm bred og 0,5 mm tykke. Elektrisk stimulering under differentiering vil ændre ekspressionen af ​​myosin tung kæde isoformer, men ikke i høj grad forbedre differentieringsprocessen som induceres af differentiering medium 8, men elektrisk stimulering kan også anvendes ved afslutningen af ​​processen for at kontrollere funktionaliteten af ​​musklen , idet en muskel med f...

Watch this Scientific Journal Video about Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation at JoVE.com

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

Engineered muskelvæv har et stort potentiale i regenerativ medicin, som sygdommen model og også som en alternativ kilde til kød. Her beskriver vi konstruktionen af ​​en muskel-konstruktion, i dette tilfælde fra muse myoblast progenitorceller, og en stimulering af elektriske impulser.

Engineering skeletmuskel Væv fra murine myoblast progenitorceller og anvendelse af elektrisk stimulering

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