3D Bioprinting Phototunable Hydrogels til undersøgelse af fibroblastaktivering
Summary
Denne artikel beskriver, hvordan man 3D bioprinter fototjusterbare hydrogeler for at studere ekstracellulær matrixafstivning og fibroblastaktivering.
Abstract
Fototjusterbare hydrogeler kan transformere rumligt og tidsmæssigt som reaktion på lyseksponering. Inkorporering af disse typer biomaterialer i cellekulturplatforme og dynamisk udløsende ændringer, såsom øget mikromiljømæssig stivhed, gør det muligt for forskere at modellere ændringer i den ekstracellulære matrix (ECM), der opstår under fibrotisk sygdomsprogression. Heri præsenteres en metode til 3D-bioprint af et fototunable hydrogelbiomateriale, der er i stand til to sekventielle polymerisationsreaktioner i et gelatinestøttebad. Teknikken med Freeform Reversible Embedding of Suspended Hydrogels (FRESH) bioprint blev tilpasset ved at justere pH i støttebadet for at lette en Michael-additionsreaktion. Først blev bioblækket indeholdende poly(ethylenglycol)-alfamethacrylat (PEGαMA) omsat off-støkiometri med en cellenedbrydelig tværbinding til dannelse af bløde hydrogeler. Disse bløde hydrogeler blev senere udsat for fotoinitator og lys for at inducere homopolymerisation af ureagerede grupper og stive hydrogelen. Denne protokol dækker hydrogelsyntese, 3D-bioprint, fotostiffening og slutpunktskarakteriseringer til vurdering af fibroblastaktivering inden for 3D-strukturer. Metoden, der præsenteres her, gør det muligt for forskere at 3D-bioprinte en række materialer, der gennemgår pH-katalyserede polymerisationsreaktioner og kunne implementeres for at konstruere forskellige modeller af vævshomeostase, sygdom og reparation.
Introduction
3D-bioprint er en transformativ teknologi, der gør det muligt for forskere præcist at deponere celler og biomaterialer inden for 3D-volumener og genskabe den komplekse hierarkiske struktur af biologisk væv. I løbet af det sidste årti har fremskridt inden for 3D-bioprint skabt bankende humant hjertevæv1, funktionelle modeller af nyrevæv2, modeller for gasudveksling i lungen3 og tumormodeller til kræftforskning4. Opfindelsen af indlejrede 3D-bioprintteknikker, såsom Freeform Reversible Embedding of Suspended Hydrogel (FRESH) bioprinting, har gjort det muligt at reproducere komplekse bløddelsstrukturer såsom lungeblodkar5 og endda menneskehjerte6 i 3D. FRESH 3D-bioprinting letter lag-for-lag-udskrivning af blødt bioblæk med lav viskositet gennem ekstrudering i et forskydningsfortyndende støttebad. Støttebadet består af et materiale såsom tæt pakkede gelatinemikropartikler, der fungerer som en Bingham-plast og opretholder den tilsigtede form og struktur af bioblækket efter udskrivning. Når den trykte konstruktion er størknet, kan støttebadet opløses væk ved at øge temperaturen til 37 °C7.
En nylig oversigtsartikel opsummerede de materialer, der er blevet 3D-bioprintet i forskellige publikationer ved hjælp af FRESH-teknik. Disse naturligt afledte materialer spænder fra kollagen type I til methacryleret hyaluronsyre og repræsenterer flere forskellige geleringsmekanismer7. De fleste forskningsundersøgelser udført ved hjælp af denne 3D-bioprintteknik anvender statiske biomaterialer, der ikke ændres som reaktion på eksterne stimuli. Dynamiske fototunable hydrogel biomaterialer er blevet brugt af vores laboratorium og andre 8,9,10,11,12 til at modellere en række fibrotiske sygdomme. I modsætning til statiske biomaterialer giver fototjusterbare bioblæk mulighed for at skabe en blødgjort model med lavere elastisk modulværdi og senere stivne for at udforske cellulære reaktioner på stigninger i mikromiljømæssig afstivning.
Fibrotiske sygdomme er kendetegnet ved en stigning i den ekstracellulære matrixproduktion, der kan forårsage ardannelse og stivhed13. Vævsafstivning kan indlede yderligere skade og ødelæggelse af det påvirkede væv, hvilket forårsager permanent organskade og endda død; Fibrotiske lidelser er ansvarlige for en tredjedel af dødeligheden på verdensplan. Fibroblaster producerer overskydende og afvigende ekstracellulær matrix i denne sygdomstilstand14,15. Øget fibroblastproliferation og ekstracellulær matrixaflejring stiver vævet yderligere og aktiverer en profibrotisk positiv feedback-sløjfe16,17,18,19. At studere fibroblastaktivering er afgørende for at forstå fibrotiske sygdomme. Her præsenterer vi human pulmonal arteriel hypertension (PAH) som et eksempel på en fibrotisk lidelse, hvor det er vigtigt at efterligne blodkarets 3D-geometri ved hjælp af 3D-bioprint og introducere de dynamiske afstivningsevner hos fototjusterbare hydrogeler. PAH er en tilstand, hvor trykket i de vigtigste lungearterier overstiger normale niveauer og lægger pres på hjertet, øger human pulmonal arterie adventitial fibroblast (HPAAF) aktivering og stiver blodkarrenevæv 16,17,18,19. En fototunable poly(ethylenglycol)-alpha methacrylat (PEGαMA) bioink-formulering giver mulighed for tidsmæssig afstivning i konstruktioner og hjælper med at modellere både sundt væv og sygdomsprogression 5,8,9,10. Udnyttelse af denne unikke funktion muliggør kvantificering af HPAAF-aktivering og spredning som reaktion på mikromiljømæssig afstivning i 3D og kan give værdifuld indsigt i de cellulære mekanismer, der er involveret i denne sygdom. Protokollen beskrevet her vil give forskere mulighed for at oprette 3D-modeller, der rekapitulerer ændringer i det ekstracellulære mikromiljø under sygdomsprogression eller vævsreparation og studerer fibroblastaktivering.
Protocol
Representative Results
Discussion
Totrinspolymerisationsreaktioner som reaktion på kontrolleret lyseksponering kan stivne biomaterialer med rumlig og tidsmæssig kontrol. Flere undersøgelser har udnyttet denne teknik til at evaluere cellematrixinteraktioner i forskellige platforme 5,8,9,10,11,21,22,23.<sup class…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Forfatterne vil gerne anerkende Dr. Adam Feinberg (Carnegie Mellon University) og dem, der var vært for 3D Bioprinting Open-Source Workshop. Disse personer gjorde det muligt at lære teknikkerne til FRESH bioprint og bygge den 3D-bioprinter, der blev brugt til disse undersøgelser. Derudover vil forfatterne gerne anerkende Biorender.com, som blev brugt til at producere figurer i dette manuskript. Dette arbejde blev støttet af flere grupper eller finansieringskilder, herunder Rose Community Foundation (DDH og CMM), en Colorado Pulmonary Vascular Disease Research Award (DDH og CMM), National Science Foundation under Award 1941401 (CMM), Department of the Army under Award W81XWH-20-1-0037 (CMM), National Cancer Institute of NIH under Award R21 CA252172 (CMM), Ludeman Family Center for Women’s Health Research ved University of Colorado Anschutz Medical Campus (DDH og CMM), National Heart, Lung and Blood Institute of National Institutes of Health under Awards R01 HL080396 (CMM), R01 HL153096 (CMM), F31 HL151122 (DDH) og T32 HL072738 (DDH og AT).
Materials
| AccuMax Radiometer/Photometer Kit | Spectronics Corporation | XPR-3000 | To measure light intensity, used for photostiffening |
| Acetic Acid | Fisher Scientific | BP2401-500 | Used during PEGaMA synthesis |
| Acetone | Fisher Scientific | A184 | Used with the cryosections |
| ActinGreen 488 ReadyProbes | Fisher Scientific | R37110 | Used for staining |
| Aluminum Foil | Reynolds | F28028 | |
| Anhydrous Tetrahydrofuran (THF) | Sigma-Aldrich | 401757-1L | Used during PEGaMA synthesis |
| Argon Compressed Gas | Airgas | AR R300 | Used during PEGaMA synthesis |
| 8 Arm Poly(ethylene glycol)-hydroxyl (PEG-OH) | JenKem Technology | 8ARM-PEG-10K | Used during PEGaMA synthesis |
| 365 nm Bandpass Filter | Edmund Optics | 65-191 | Used for photostiffening |
| Bovine Serum Albumin (BSA) | Fisher Scientific | BP9700-100 | Used during staining process |
| Buchner Funnel | Quark Glass | QFN-8-14 | Used during PEGaMA synthesis |
| Calcein AM | Invitrogen | 65-0853-39 | Used during staining process |
| Celite 545 (Filtration Aid) | EMD Millipore | CX0574-1 | Used during PEGaMA synthesis |
| Charged Microscope Slides | Globe Scientific | 1358W | |
| Chloroform-d | Sigma-Aldrich | 151823-10X0.75ML | Used to characterize PEGaMA |
| Click-iT Plus EdU Cell Proliferation Kit | Invitrogen | C10637 | Used for staining |
| 50 mL Conical Tubes | CELLTREAT | 667050B | |
| Cryogenic Safety Kit | Cole-Parmer | EW-25000-85 | |
| Cryostat | Leica | CM 1850-3-1 | |
| Dialysis Tubing | Repligen | 132105 | |
| 4’,6-Diamidino-2-Phylindole (DAPI) | Sigma-Aldrich | D9542-1MG | Used for staining |
| Diethyl Ether | Fisher Scientific | E1384 | Used during PEGaMA synthesis |
| 1,4-Dithiothreitol (DTT) | Sigma-Aldrich | 10197777001 | Bioink component |
| Dulbecco's Modified Eagle's Medium (DMEM) | Cytiva | SH30271.FS | |
| Ethyl 2-(Bromomethyl)Acrylate (EBrMA) | Ambeed Inc. | A918087-25g | Used during PEGaMA synthesis |
| Filter Paper | Whatman | 1001-090 | Used during PEGaMA synthesis |
| Freezone 2.5L Freeze Dry System | Labconco | LA-2.5LR | Lyophilizer |
| Fusion 360 | Autodesk | N/A | Software download |
| 2.5 mL Gastight Syringe | Hamilton | 81420 | Used for bioprinting |
| 15 Gauge 1.5" IT Series Tip | Jensen Global | JG15-1.5X | Used for bioprinting |
| 30 Gauge 0.5" HP Series Tip | Jensen Global | JG30-0.5HPX | Used for bioprinting |
| Goat Anti-Mouse Alexa Fluor 555 Antibody | Fisher Scientific | A21422 | Used for staining |
| Glycine | Fisher Scientific | C2H5NO2 | Used during staining process |
| Hemocytometer | Fisher Scientific | 1461 | |
| Hoechst | Thermo Scientific | 62249 | Used during staining process |
| Human Pulmonary Artery Adventitial Fibroblasts (HPAAFs) | AcceGen | ABC-TC3773 | From a 2-year-old male patient |
| Hydrochloric Acid (HCl) | Fisher Scientific | A144-500 | Used to pH adjust solutions |
| ImageJ | National Institutes of Health (NIH) | N/A | Free software download |
| ImmEdge® Pen | Vector Laboratories | H-4000 | Used during staining process |
| Incubator | VWR | VWR51014991 | |
| LifeSupport Gelatin Microparticle Slurry (Gelatin Slurry) | Advanced Biomatrix | 5244-10GM | Used for bioprinting |
| Light Microscope | Olympus | CKX53 | Inverted light microscope |
| Lithium Phenyl-2,4,6-Trimethylbenzoylphosphinate (LAP) | Sigma-Aldrich | 900889-5G | Photoinitiator used for photostiffening |
| Liquid Nitrogen | N/A | N/A | |
| LulzBot Mini 2 | LulzBot | N/A | Bioprinter adapted |
| Methacryloxyethyl Thiocarbamoyl Rhodamine B | Polysciences Inc. | 669775-30-8 | |
| 2-Methylbutane | Sigma-Aldrich | M32631-4L | |
| Microman Capillary Pistons CP1000 | VWR | 76178-166 | Positive displacement pipette tips |
| MMP2 Degradable Crosslinker (KCGGPQGIWGQGCK) | GL Biochem | N/A | Bioink component |
| Mouse Anti-Human αSMA Monoclonal Antibody | Fisher Scientific | MA5-11547 | Used for staining |
| OmniCure Series 2000 | Lumen Dynamics | S2000-XLA | UV light source used for photostiffening |
| Paraformaldehyde (PFA) | Electron Microscopy Sciences | 15710 | Used to fix samples |
| pH Meter | Mettler Toledo | FP20 | |
| pH Strips | Cytiva | 10362010 | |
| Phosphate Buffered Saline (PBS) | Hyclone Laboratories, Inc. | Cytiva SH30256.FS | |
| Pipette Set | Fisher Scientific | 14-388-100 | |
| 10 µL Pipette Tips | USA Scientific | 1120-3710 | |
| 20 µL Pipette Tips | USA Scientific | 1183-1510 | |
| 200 µL Pipette Tips | USA Scientific | 1111-0700 | |
| 1000 µL Pipette Tips | USA Scientific | 1111-2721 | |
| Poly(Ethylene Glycol)-Alpha Methacrylate (PEGαMA) | N/A | N/A | Refer to manuscript for synthesis steps |
| Poly(Ethylene Oxide) (PEO) | Sigma-Aldrich | 372773-250G | Bioink component |
| Positive Displacement Pipette | Fisher Scientific | FD10004G | 100-1000 µL |
| Potassium Hydroxide (KOH) | Sigma-Aldrich | 221473-500G | Used to pH adjust solutions |
| ProLong Gold Antifade Reagent | Invitrogen | P36930 | Used during staining process |
| Pronterface | All3DP | N/A | Software download |
| Propidium Iodide | Sigma-Aldrich | P4864-10ML | Used for staining |
| RGD Peptide (CGRGDS) | GL Biochem | N/A | Bioink component |
| Rocker | VWR | 10127-876 | |
| Rotary Evaporator | Thomas Scientific | 11100V2022 | Used during PEGaMA synthesis |
| Rubber Band | Staples | 808659 | |
| Schlenk Flask | Kemtech America | F902450 | Used during PEGaMA synthesis |
| Slic3r | Slic3r | N/A | Software download |
| Smooth Muscle Cell Growth Medium-2 (SmGM-2) BulletKit | Lonza | CC-3182 | Kit contains CC-3181 and CC-4149 components |
| Sodium Hydride | Sigma-Aldrich | 223441-50G | Used during PEGaMA synthesis |
| Sorvall ST 40R Centrifuge | Fisher Scientific | 75-004-525 | |
| Stir Bar | VWR | 58948-091 | |
| Syringe Filter | VWR | 28145-483 | Used to sterile filter solutions |
| T-75 Tissue-Cultured Treated Flask | VWR | 82050-856 | Used for cell culture work |
| Tissue-Tek Cyromold | Sakura | 4557 | |
| Tissue-Tek O.C.T Compound (OCT) | Sakura | 4583 | |
| Tris(2-Carboxyethyl) Phosphine (TCEP) | Sigma-Aldrich | C4706-2G | |
| Triton X-100 | Fisher Bioreagents | C34H622O11 | Used during staining process |
| Trypan Blue | Sigma-Aldrich | T8154-20ML | Used for cell culture work |
| 0.05% Trypsin-EDTA | Gibco | 25-300-062 | Used for cell culture work |
| Tween 20 | Fisher Bioreagents | C58H114O26 | Used during staining process |
| Upright Microscope | Olympus | BX63F | Fluorescent microscope capabilities |
| Water Bath | PolyScience | WBE20A11B | |
| 24-Well Tissue Culture Plates | Corning | 3527 |
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