Humant Dupuytrens<em> Ex vivo</em> Kultur for Studiet af myofibroblaster og ekstracellulære matrix-interaktioner
Summary
Dupuytrens sygdom (DD) er en fibroproliferativ sygdom i håndfladen. Her præsenterer vi en protokol til kultur resektion prøver fra DD i en tredimensional (3D) dyrkningssystem. Sådanne kortsigtede kultur system tillader bevarelse af 3D-struktur og molekylære egenskaber af fibrøst væv.
Abstract
Organ fibrosis or “scarring” is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic.
To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells.
As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first “organ” culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.
Introduction
Dupuytrens sygdom (DD), en godartet fiberproliferativ sygdom forårsager permanent bøjning af fingrene som følge af dannelsen af knuder og ledninger i håndfladen. Selvom sygdomsspredning er særlig høj blandt kaukasiere i Nordeuropa, det underliggende genetiske ætiologi af sygdommen er stadig ukendt 1. Den vigtigste egenskab ved DD er overskydende produktion af ekstracellulær matrix (ECM) proteiner (fx collagen), som danner en hård fibrøst væv optager rummet mellem sener og hud håndfladen og fingrene, permanent forstyrre de fine bevægelser af hånden 2, 3. Den gentagelse af sygdommen tyder underliggende genetiske ændringer som årsag til fibrose 1, 4. En effektiv behandling kunne være at målrette direkte de ukontrollable fibrotiske mekanismer på det cellulære og molekylære niveau.
Vores seneste arbejde på fibrose har ført os til at udvikle etroman 3D kultur system, der tillader kortsigtet kultur af human fibrøst væv med potentiale narkotika testning. Dette system har været med til at overvinde den begrænsende tilgang af 2D fibroblastkulturer og definere en rolle for den delvise nedreguleringen, opnået ved exon skipping, af TGF-vejen aktivering i mediering fibrose 5.
Vi har udviklet en metode til dyrkning ex vivo human resektion prøver fra DD patienter at studere interaktionen mellem myofibroblaster og den omgivende ECM 5, 6. Studiet af DD bindevæv fibrose samt andre fibrotiske sygdomme beror på histopatologisk analyse af det udskårne kirurgiske prøver, isolering af fibroblaster fra vævet og etablering af primære kulturer eller cellesortering procedurer. Disse metoder er ret statisk, da de ikke tillader eksogen manipulation af ejendomme sygdommen eller terapeutisk intervention ved forsøgslederen. Derudover kan primære cell kulturer en tendens til at tilpasse sig de dyrkningsbetingelser og deres genekspression egenskaber hovedsageligt fra in vivo situationen adskiller sig på hver passage, selv under tidlige passager (blandt passage 3 og 6) 7, 8. Det er lykkedes at fastholde affaldet kirurgisk materiale i ex vivo dyrkningsbetingelser i et tidsrum, der tillader undersøgelse af patient-specifikke egenskaber og screening af anti-fibrotiske eller antiinflammatoriske lægemiddelstoffer.
Systemet er baseret på en nitrocellulosemembran som tillader kontakt af vævet med mediet, men ikke med plast, således at forhindre ændring af vævet ved fastgørelse, som tidligere observeret, når dyrkning DD fibroblaster samt andre celletyper 9. Nr kollagen gel eller andre ECM proteinsubstrat er påkrævet, da DD væv selv producerer store mængder af disse proteiner. Dette er fordelagtigt for vedligeholdelse af native ECM mikromiljø og omsætningen since matrixsubstrater er vigtig regulator af væv arkitektur og funktion 10, 11. For eksempel ECM-proteiner, såsom fibronectin, laminin og kollagen, kan påvirke forreste-bageste polaritet af fibroblaster som tilsvarende vist for apikal-basal polaritet i epitelceller 12, 13 . polariserede celler har asymmetrisk fordeling af ekstracellulære molekyler, som bestemmer cellevandring og genekspression, f.eks α1β1 integrin tilgængelighed på membranen påvirker celleadhæsion til type I collagen 14. Da et primært mål for denne 3D-model var at bevare den indfødte mikromiljø, blev der ikke kunstig ECM matrix anvendte substrat.
Kort: resektion prøver lige skåret i et sterilt miljø og anbringes på nitrocellular membraner. Hvis behandling administreres via injektion er påkrævet væv injiceres, efter at de er blevet placeret på membranen. Hvis behandlingen ikke kræver at administreres via injfdeling derefter forbindelsen tilsættes til dyrkningsmediet (Dulbeccos Modified Eagles Medium (DMEM), med 1% føtalt kalveserum (FCS), 1% penicillin-streptomycin (P / S)). Kulturerne opretholdes højst ti dage, hvorefter vævet fikseret i 4% paraformaldehyd (PFA), behandles gennem 30% saccharoseopløsning, indlejret i OCT-forbindelse og opbevaret ved -80 ° C, som tidligere beskrevet 5.
Protocol
Representative Results
Discussion
De mest kritiske trin i dyrkning ex vivo humane bindevæv er den umiddelbare anvendelse af vævet efter kirurgisk fjernelse for at sikre levedygtighed vævet bør forblive i medium eller saltvandsopløsning på alle tidspunkter; opretholde en steril kultur; tværsnit af væv skal have maksimalt 200 um tykkelse; opsætning af ex vivo kultur system er optimal, når vævet er i kontakt med mediet, men ikke fuldt neddykket. Medium bør tilføjes kun på ydersiden kammer transwell i et lille volumen (f….
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors are thankful to the nurses at the AMC that have facilitated the tissue collection. We would also like to acknowledge A.M.A. van der Laan for excellent technical support with the SHG and the two-photon imaging.
Materials
| Dulbecco’s Modified Eagle’s Medium | Invitrogen | 11965-084 | |
| fetal calf serum (FCS) | Gibco | high glucose, heat inactivated | |
| penicillin-streptomycin | Invitrogen | 15070-063 | |
| Cell culture inserts | Millicell | PIHA01250 | 0.45um pore size, 12mm diameter |
| anti-collagen type I | Southern Biotech | 1310-08 | |
| anti- α smooth muscle actin | Sigma | A2547 | |
| anti-collagen type III | Southern Biotech | 1330-01 | |
| Alexa Fluor555 Donkey Anti-Mouse IgG (H+L) | Invitrogen | A-31570 | |
| Alexa Fluor 488 Donkey Anti-Goat IgG (H+L) Antibody | Invitrogen | A-11055 | |
| TOPRO-3 | Invitrogen | T3605 | |
| methylsalicylate | Sigma | M6752 | |
| paraformaldehyde | Sigma | P6148 | |
| Tissue Tek OCT | Sakura | 25608-930 | |
| Microsccope glass coverslips | Menzel-Glaser | BB024060A1 | 24x60mm |
| Microscope SuperFrost slides | Menzel-Glaser | AA00000102E | 76x26mm |
| VECTASHIELD HardSet Mounting Medium | Vector laboratories | H-1400 | |
| Leica TCS SP5 II confocal microscope | Leica Microsystems | Argon-488, 514 nm and HeNe-633 nm laser lines | |
| Zeiss 710 NLO upright confocal microscope | Jena, Germany | equipped with femtosecond Spectra – Physics Deep See MP laser (Santa Clara, United States) using a Plan -Apochromat 20x/1.0 NA water-immersion objective. |
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