Vurdering af funktionelle målinger af skeletmuskulatursundhed i humane skeletmuskulaturmikrotisser
Summary
Dette manuskript beskriver en detaljeret protokol til fremstilling af arrays af 3D menneskelige skeletmuskulaturmikrotiss og minimalt invasive downstream in situ-analyser af funktion, herunder kontraktilkraft og calciumhåndteringsanalyser.
Abstract
Tre-dimensionelle (3D) in vitro modeller af skeletmuskulatur er et værdifuldt fremskridt i biomedicinsk forskning, da de giver mulighed for at studere skeletmuskulatur reformation og funktion i et skalerbart format, der er modtagelige for eksperimentelle manipulationer. 3D-muskelkultursystemer er ønskelige, da de gør det muligt for forskere at studere skeletmuskulatur ex vivo i forbindelse med menneskelige celler. 3D in vitro modeller nøje efterligne aspekter af den indfødte vævsstruktur af voksne skeletmuskulatur. Men deres universelle anvendelse er begrænset af tilgængeligheden af platforme, der er enkle at fremstille, omkostninger og brugervenlige, og giver relativt store mængder af human skelet muskelvæv. Da skeletmuskulaturen spiller en vigtig funktionel rolle, der er svækket over tid i mange sygdomstilstande, er en eksperimentel platform for mikrotissuestudier mest praktisk, når minimalt invasive calciumtransient- og kontraktile kraftmålinger kan udføres direkte inden for selve platformen. I denne protokol beskrives fremstillingen af en 96-velkendt platform kendt som ‘MyoTACTIC’ og en masseproduktion af 3D-humane skeletmuskulaturmikrotiss (hMMT’er). Derudover rapporteres metoderne til en minimalt invasiv anvendelse af elektrisk stimulation, der muliggør gentagne målinger af skeletmuskulaturkraft og calciumhåndtering af hver mikrotissue over tid.
Introduction
Skeletmuskulatur er en af de mest rigelige væv i den menneskelige krop og understøtter centrale kropsfunktioner såsom bevægelse, varme homøostase og stofskifte1. Historisk set er dyremodeller og todimensionelle (2D) cellekultursystemer blevet brugt til at studere biologiske processer og sygdomspatogenese samt til test af farmakologiske forbindelser til behandling af skeletmuskulatursygdomme2,3. Mens dyremodeller i høj grad har forbedret vores viden om skeletmuskulatur i sundhed og sygdom, er deres translationelle virkning blevet hæmmet af høje omkostninger, etiske overvejelser og interspecies forskelle2,4. Ved at henvende sig til menneskelige cellebaserede systemer til at studere skeletmuskulatur er 2D-cellekultursystemer gunstige på grund af deres enkelhed. Der er dog en begrænsning. Dette format undlader ofte at generobre de cellecelle- og celle-ekstracellulære matrixinteraktioner, der forekommer naturligt i kroppen5,6. I løbet af de sidste mange år har tredimensionelle (3D) skeletmuskulaturmodeller vist sig som et stærkt alternativ til hele dyremodeller og konventionelle 2D-kultursystemer ved at tillade modellering af fysiologisk og patologisk relevante processer ex vivo7,8. Faktisk har en overflod af undersøgelser rapporteret strategier til at modellere menneskelige skeletmuskulatur i et bioartificialt 3D-kulturformat1. En begrænsning for mange af disse undersøgelser er, at aktiv kraft kvantificeres efter fjernelse af muskelvæv fra kulturplatformene og tilknytningen til en krafttransducer, som er destruktiv og dermed begrænset til at fungere som en endpoint assay9,10,11,12,13,14,15,16,17,18 ,19,20,21. Andre har designet kultursystemer, der giver mulighed for ikke-invasive metoder til måling af aktiv kraft, men ikke alle er modtagelige for molekyletestapplikationer med højt indhold7,8,9,10,14,18,22,23,24,25,26,27,28 ,29.
Denne protokol beskriver en detaljeret metode til fremstilling af humane muskelmikrotiss (hMMTs) i skeletmuskulaturen (Myo) microTissue Array deviCe To Investigate forCe (MyoTACTIC) platform; en 96 brønd plade enhed, der understøtter bulk produktion af 3D skeletmuskulatur microtissues30. MyoTACTIC pladefremstillingsmetoden muliggør generering af en 96 brøndpolylsiloxan (PDMS) kulturplade og alle tilsvarende brøndfunktioner i et enkelt støbetrin, hvorved hver brønd kræver et relativt lille antal celler til mikrotissuedannelse. Microtissues dannet i MyoTACTIC indeholder justeret, striated, og multinukleated myotubes, der er reproducerbare fra godt til brønd af enheden, og ved modning, kan reagere på kemiske og elektriske stimuli in situ30. Heri, teknikken til at fremstille en PDMS MyoTACTIC kultur plade enhed fra en polyurethan (PU) replika, en optimeret metode til at gennemføre udødeliggjorte menneskelige myogene stamfader celler til at fremstille hMMTs, og den funktionelle vurdering af manipuleret hMMT kraft generation og calcium håndtering egenskaber er skitseret og diskuteret.
Protocol
Representative Results
Discussion
Dette manuskript beskriver metoder til at fremstille og analysere en 3D hMMT kulturmodel, der kan anvendes til undersøgelser af grundlæggende muskelbiologi, sygdom modellering, eller for kandidat molekyle test. MyoTACTIC-platformen er omkostningsvenlig, nem at fremstille og kræver et relativt lille antal celler til at producere skeletmuskulaturmikrotiss. hMMTs dannet inden for MyoTACTIC kulturplatformen består af justerede, flerkernede og striated myotubes og reagerer på elektriske stimuli ved at indlede calciumtran…
Declarações
The authors have nothing to disclose.
Acknowledgements
Vi vil gerne takke Mohammad Afshar, Haben Abraha, Mohsen Afshar-Bakooshli og Sadegh Davoudi for at have bidraget til opfindelsen af myoTACTIC-kulturplatformen og for at etablere de fremstillings- og analysemetoder, der er beskrevet heri. HL modtaget støtte fra en Naturvidenskab og Engineering Research Council (NSERC) Training Program i Organ-on-a-Chip Engineering and Entrepreneurship Scholarship og en University of Toronto Wildcat graduate stipendier. PMG er Canada Research Chair i Endogenous Repair og modtog støtte til denne undersøgelse fra Ontario Institute for Regenerativ Medicin, Stem Cell Network, og fra Medicine by Design, en Canada First Research Excellence Program. Skemadiagrammer blev oprettet med BioRender.com.
Materials
| 0.9% Saline Solution, Sterile | House Brand | 1010 | 10 mL aliquots of the solution are made and stored at 4°C |
| 25G Needle | BD, Medstore, University of Toronto | 2548-CABD305127 | |
| 6-Aminocaproic Acid, ≥99% (titration), Powder | Sigma – Aldrich | A2504-100G | A 50 mg / mL stock solution is generated by dissolving 5 mg of 6-aminocaproic acid powder in 100 mL of autoclaved, distilled water. The solution is vaccum filtered and 10 mL aliquots are stored at 4°C |
| 6.35 mm ID Tubing | VWR | 60985-528 | |
| AB1167 Myoblast Cell Line | Institut de Myologie (Paris, France) | ||
| Arbitrary Waveform Generator | Rigol | DG1022Z | |
| Basement Membrane Extract (Geltrex) | Thermo Fisher Scientific | A14132-02 | Stored as aliquots of 50 µL or 100 µL at -80°C |
| Benchtop Vacuum Chamber | Sigma – Aldrich | D2672 | |
| BNC to Aligator Clip Cable | Ordered from Amazon | ||
| Culture Plastics | Sarstedt | Includes culture plates, serological pipettes, etc | |
| Dimethyl Sulfoxide | Sigma – Aldrich | D8418-250ML | |
| DPBS, Powder, No Calcium, No Magnesium | Thermo Fisher Scientific | 21600069 | |
| Dulbecco's Modified Eagle Medium (DMEM) (1X) | Gibco | 11995-065 | This is a high glucose DMEM with L-glutamine and sodium pyruvate |
| Fetal Bovine Serum | Fisher Scientific | 10437028 | |
| Fibrinogen from Bovine Plasma | Sigma – Aldrich | F8630-5G | Aliquots ranging from 7 – 10 mg of fibrinogen powder are made and stored at -20°C |
| Filtropur Syringe Filter, 0.22um Pore Size | Sarstedt | 83.1826.001 | |
| Horse Serum | Gibco | 16050-122 | |
| Human Recombinant Insulin | Sigma – Aldrich | 91077C | Stock solution is 100X and made by dissolving 1 mg of human recombinant insulin in 1 mL of DMEM and 1 µL of NaOH 10N. Solution is filtered and stored as 1 mL aliquots at 4°C |
| Image Acquisition Software | Olympus | cellSens Dimension | |
| Image Processing Software | National Institutes of Health | ImageJ | |
| Isotemp Oven | Thermo Fisher Scientific | 201 | |
| Microscope | Olympus | IX83 | |
| Microscope – Camera Mount | Labcam | Labcam for iPhone | Ordered from Amazon |
| Penicillin-Streptomycin (10,000 U/mL) | Gibco | 15140-122 | |
| Plastic Disposable Syringes, 1cc | BD | 2606-309659 | |
| Plastic Disposable Syringes, 50cc | BD | 2612-309653 | |
| Pluronic F-127, Powder, BioReagent | Sigma – Aldrich | P2443-250G | A 5% stock solution of pluronic acid is made by dissolving 5 g of pluronic acid powder in 100 mL of chilled, autoclaved, distilled water. The solution is vaccum filtered and 10 mL aliquots are stored at 4°C |
| Polydimethylsiloxane (Sylgard 184 Silicone Elastomer Kit) | Dow | 4019862 | Kits are also available at Thermo Fisher Scientific, Sigma – Aldrich, etc. |
| Polyurethane Negative Mold | In House | ||
| Release Agent | Mann Release Technologies | 200 | |
| Rotary Vane Vacuum Pump | Edwards | A65401906 | |
| Scalpel | Almedic, Medstore, University of Toronto | 2586-M36-0100 | |
| Single Edge Razor Blade | VWR | 55411-050 | |
| Skeletal Muscle Cell Basal Medium | Promocell | C-23260 | 30 mL aliquotes are generated and at stored at 4°C. |
| Skeletal Muscle Cell Growth Medium (Ready-to-use) | Promocell | C-23060 | 42 mL aliquots are generated and stored at 4°C. |
| Smartphone (iPhone) | Apple | SE | |
| Standard Duty Dry Vacuum Pump | Welch | 2546B-01 | |
| Sterilization Bag | Alliance | 211-SCM2 | |
| Thimble | Igege | Ordered from Amazon | |
| Thrombin from human plasma | Sigma – Aldrich | T6884-250UN | 100 units of thrombin is dissolved in 1 mL of a 0.1% BSA solution. 10 µL aliquots are prepared and stored at – 20°C. |
| Tin coated copper wire | Arco | B8871K48 | Ordered from Amazon |
| Trypan Blue Solution, 0.4% | Thermo Scientific | 15250061 | |
| Trypsin-EDTA, 0.25% | Thermo FIsher Scientific | 25200072 | |
| Vacuum Chamber 2 | SP Bel-Art | F42027-0000 |
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