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Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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Bioengineering

Fabrikation af Inverted Kolloid Crystal Poly (ethylenglycol) Stillads: En Tre-dimensionel Cell Culture Platform for levervæv Engineering

Published: August 27, 2016
doi:
10.3791/54331
, , , , , , ,
1School of Materials Science and Engineering,Nanyang Technological University, 2School of Chemical and Biomedical Engineering,Nanyang Technological University

Summary

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

Abstract

Evnen til at opretholde hepatocyt funktion in vitro, med det formål at teste xenobiotika 'cytotoksicitet, studerer virusinfektion og udvikling af lægemidler rettet mod leveren, kræver en platform, hvor celler modtager passende biokemiske og mekaniske signaler. Nylige levervæv tekniske systemer har ansat tre-dimensionelle (3D) stilladser sammensat af syntetiske eller naturlige hydrogeler, da deres høje væskeophobning og deres evne til at levere de mekaniske stimuli nødvendige for cellerne. Der har været en stigende interesse for den omvendte kolloide krystal (ICC) stillads, en ny udvikling, der giver høj rumlig organisation, homotypisk og heterotypisk celle interaktion, samt celle-ekstracellulær matrix (ECM) interaktion. Heri beskriver vi en protokol til fremstilling ICC stillads anvendelse af poly (ethylenglycol) diacrylat (PEGDA) og partiklen udvaskning metode. Kort fortalt et gitter fremstillet af mikrokugle-partikler, hvorefter en præ-polymetilsættes r opløsning, korrekt polymeriseret, og partiklerne fjernes derefter, eller udvaskes, anvendelse af et organisk opløsningsmiddel (f.eks tetrahydrofuran). Opløsningen af ​​gitter resulterer i en meget porøs scaffold med kontrollerede porestørrelser og interconnectivities der tillader medier til at nå celler lettere. Denne unikke struktur muliggør højt overfladeareal for at cellerne kan klæbe til samt nem kommunikation mellem porerne, og evnen til at belægge PEGDA ICC stillads med proteiner viser også en markant virkning på celle ydeevne. Vi analyserer morfologien af ​​stilladset samt leverkarcinom celle (Huh-7,5) adfærd med hensyn til levedygtighed og funktion at udforske effekten af ​​ICC struktur og ECM overtræk. Samlet set dette papir giver en detaljeret protokol af en spirende stillads, der har brede anvendelser i tissue engineering, især levervæv engineering.

Introduction

Leveren er et stærkt vaskulariseret organ med et væld af funktioner, herunder afgiftning af blod, metabolisme af xenobiotika, og produktionen af ​​serumproteiner. Levervæv har en kompleks tredimensional (3D) mikrostruktur, bestående af flere celletyper, galde-canaliculi, sinuskurver, og zoner med forskellig biomatrix sammensætning og forskellige oxygenkoncentrationer. I betragtning af denne omfattende struktur, har det været vanskeligt at skabe en ordentlig liver model in vitro 1. Der er imidlertid en stigende efterspørgsel efter funktionelle in vitro-modeller hosting humane hepatocytter som platforme for testning lægemiddeltoksicitet 2 og studere sygdomme forbundet med leveren 3.

Nuværende liver tissue engineering platforme har forenklet kompleksiteten af leveren ved at isolere én eller fokusere på et par, af leverens parametre, nemlig co-dyrkning af celler 4, biokemiske sammensætning af zonal mikromiljøer 5, strømningsdynamik 6,7 og konfigurationen af biomatrix 8. Konfiguration af biomatrix kan opdeles i parametre som stillads materialer, sammensætning af ekstracellulær matrix (ECM) proteiner, matrix stivhed samt konstruktion og struktur af stilladset. Der har været en stigning i vævsmanipulation studier under anvendelse af syntetiske hydrogeler, især poly (ethylenglycol) (PEG) hydrogeler 9, får mulighed for at tune hydrogelens mekaniske egenskaber, bioaktivitet, og nedbrydningshastighed. Med hensyn til lever-relateret forskning, blev biokompatible hydrogel ansøgt om virusinfektion undersøgelse af leversygdom 3. Som en hepatocyt platform design, har talrige undersøgelser udnyttet hepatocyt sandwich kulturer 10,11 og celleindkapsling inden en hydrogel 12,13 til tilvejebringelse af 3D-miljø og celle-ECM og celle-celle-interaktion, som er væsentlige for at efterligne in vivo mikromiljø. However, behøver disse platforme ikke besidder en høj grad af kontrol og rumlig organisation, hvilket fører til ikke-ensartede egenskaber gennem stilladset 14.

Den omvendte krystal kolloid (ICC) 14 stillads er et velorganiseret 3D stillads for cellekultur, som først blev introduceret i begyndelsen af 2000'erne. Stilladset unikke struktur kan tilskrives den enkle fremstillingsproces ved anvendelse af en kolloid krystal, en ordnet gitter af kolloide partikler med variabel diameter. Kort fortalt, for at opsummere processen, partikler pænt arrangeret og udglødet ved anvendelse af varme til dannelse af et gitter. Udvaskningen af dette gitter, som et organisk opløsningsmiddel, i en polymeriseret hydrogel resulterer i Hexagonal pakket sfæriske hulrum 15 med stort overfladeareal. Denne yderst ordnet stillads er tidligere blevet fremstillet med både syntetiske og naturlige materialer, herunder, men ikke begrænset til poly (acrylamid) 16-21, poly (mælke-co-glycolsyre) 15,22-30, Poly (ethylenglycol) 31,32, poly (2-hydroxyethylmethacrylat) 21,33-35, og chitosan 36-39. ICC stilladser fremstillet af ikke-fouling materialer har en tendens til at fremme cellulære sfæroider inde i hulrummene 14,23,40. Flere celletyper har vist sig at kunne formere sig, differentiere og funktion inden denne konfiguration, herunder chondrocytter 41, stromale knoglemarvsceller 42, og stamceller 43,44. Med hensyn hepatocyt, har undersøgelserne været udført med ICC stilladser lavet af Na 2 SiO 3 og poly (acrylamid), men ikke PEG. Med enkle bioconjugation strategier (dvs. aminkobling gennem EDC / NHS), kan ECM-proteiner-konjugerede PEG-baserede scaffolds skal fremstilles, der kan vise sig mere cellebindende sider for at være en mere in vivo lignende miljø og forbedre leverfunktion.

I dette manuskript og den tilhørende video, we detaljer fremstillingen af ​​ICC stilladsanvendelse af poly (ethylenglycol) diacrylat (PEGDA) hydrogel og en polystyren mikrosfære gitter, optimeret til leverkarcinom (Huh-7,5) kultur. Vi demonstrerer forskellene mellem de generelt ikke-klæbende nøgne PEGDA ICC stilladser og collagen-coatede PEGDA ICC stillads i form af stillads topologi og celleydelsen. Cellernes levedygtighed og funktion måles kvalitativt og kvantitativt at vurdere Huh-7.5 celle adfærd.

Protocol

1. ICC Stillads Fabrication (figur 1) Forbered polystyren (PS) gitre (diameter = 6 mm, 8-13 lag af perler). For at forberede formen, skæres de tips af fra 0,2 ml koge-bevis mikrocentrifugerør på 40 pi niveau. Overhold toppen af cut-rør til 24 x 60 mm 2 mikroskop dække glas glider med vandtæt lim. Sæt PS kugler (diameter = 140 um) indeholdt i en vandsuspension i et 20 ml hætteglas, omhyggeligt pipette vandet ud suspensionen, og der tilsættes 18 ml 70% ethanol-opløsning i…

Representative Results

De repræsentative resultater for den strukturelle karakterisering af ICC stillads og sammenligningen af ​​hver ICC stillads tilstand s effekt i dyrkning hepatocytter er vist og forklaret nedenfor. ICC stillads betingelser anvendt i disse resultater er kollagen belægninger af 0 ug / ml (Bare), 20 ug / ml (Collagen 20), 200 ug / ml (Collagen 200) og 400 ug / ml (Kollagen 400) og den første huh-7.5 celle seeding nummer er 1×10 6. <p class="jove_content" fo:keep-togethe…

Discussion

Tissue engineering stilladser er i rivende udvikling til at give alle de fysiske og biokemiske signaler, der er nødvendige for at regenerere, vedligeholde eller reparere væv for anvendelsen af orgel udskiftning, studere sygdom, at udvikle lægemidler, og mange andre 57. I levervæv engineering, primære, humane hepatocytter hurtigt mister deres metaboliske funktioner gang isoleret fra kroppen, hvilket skaber et stort behov for engineering scaffolds og udvikle platforme til at opretholde den leverfunktion. D…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Forfatterne ønsker at anerkende støtte fra en Grundforskningsfonden Fellowship (NRF -NRFF2011-01) og konkurrencedygtig forskning program (NRF-CRP10-2012-07).

Materials

0.2 mL PCR tube Axygen Scientific PCR-02D-C Boil-proof
Gorilla Glue Gorilla Glue, Inc. Depends on vendor. This was purchased from a local store.
Glass slides VWR  631-1575 Dimensions: 24×60 mm
Polystyrene spheres  Fisher Scientific TSS#4314A Diameter = 140 um; 3×10^4 particles per milliliter and 1.4% size distribution
Ethanol Merck 1.00983.1011 absolute for analysis EMSURE; Dilute to 70% with Milli-Q water
Ultrasonic Bath Elma S10H Equiment
Furnace Nabertherm N7/H Equipment
200 µL pipette tip Axygen Scientific T-210-Y-R-S
Rocking shaker VWR 444-0142
Polyethylene Glycol (PEG) Merck 1.09727.0100 Mw= 4kDa; acrylation of PEG monomers and purification of the resulting precipitate produces a PEGDA macromer with Mw = 4.6kDa
Centrifuge Beckman Coulter 392932 Equipment
Acrylate-Poly (Ethylene Glycol) – Succinimidyl Valerate  Laysan Bio ACRL-PEG-SVA-3400-1g Mw = 3.4 kDa
2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone Sigma Aldrich 410896
Vortex VWR 58816-123 Equipment
Microcentrifuge Eppendorf 5404 000.413
Paraffin Film  Parafilm M  #PM996 Kept at 9" with allows intensity of 10.84 mW/cm^2
Bluewave 200 UV spotlight Blaze Technology  120008, 122300
Tetrahydrofuran (THF) Merck 107025
Orbital shaker Heidolph 543-123120-00-5 From rat
Collagen Type I Sigma Aldrich C3867-1VL 1X, w/o CaCl & MgCl; Ph = 7.2
Phosphate Buffered Saline (PBS)  Gibco 20012-027 16% W/V AQ. 10x10ml
Paraformaldehyde VWR 43368.9M Equipment
Freezone 4.5 freeze drier Labconco 7750020 Equipment
Sputter coater Jeol Ltd. JFC-1600 Equipment
Scanning Electron Microscope Jeol Ltd. JSM 5310
Anti-mouse primary antibodies against Collagen type I Abcam ab6308
Anti-mouse secondary antibody conjugated with Alexa Fluor 488 Life Technologies A21121
Plate, Tissue Culture 24 Well, Flat Bottom (Nunclon)  Bio-Rev PTE LTD 3820-024
Dulbecco's Modified Eagle's Medium(DMEM)
2.5 g/L Glucose w/ L-Gln		 Lonza 12-604F
Fetal Bovine Serum (FBS) Gibco A15-151
Penicillin-Streptomycin (P/S) Life Tchnologies 15140-122 E
APC49‐Huh ‐7.5 Cell Line Apath
100 mm Corning non-treated culture dishes Sigma Aldrich CLS430591
0.25% Trypsin-EDTA Gibco 25200-056 Equipment; 37°C, 5% Humidity
Forma Steri-Cycle CO2 Incubators Thermofisher Scientific 371
Hausser Bright-Line Phase Hemacytometer Thermofisher Scientific 02-671-6
Live/Dead Viability/Cytotoxicity Kit 'for mammalian cells Life Technologies L3224 
CCK-8 Assay Dojindo Laboratories CK04-11 Monosodium-salt reagent (MSR)
Infinite 200 PRO microplate reader  Tecan
Albumin Human ELISA kit Abcam ab108788
Triton X-100 Bio-Rad #1610407
Bovine Serum Albumin (BSA) Sigma-Aldrich A2153-50G
Anti-mouse primary antibodies (against CYP3A4, albumin) Santa Cruz Biotechnology sc-53850; sc-271605
DAPI Life Technologies D3571
Alexa Fluor 555 labelled Phalloidin Life Technologies A34055
Trizol Life Technologies 15596-026
Chloroform VWR 22706.326
Isopropanol Fisher Scientific 67-63-0
DPEC water Thermofisher Scientific AM9916
Nanodrop 2000c Spectrophotometer Thermofisher Scientific ND-2000
iScript Reverse Transcription Supermix  Bio-Rad Laboratories 1708840
SYBR select Master Mix for CFX Life Technology 4472937
Primers (to be chosen)
CFX96 Real-Time System, C-1000 Touch Thermal Cycler Bio Rad Laboratories SOFT-CFX-31-PATCH 
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Tags

Inverted Colloidal CrystalPoly(ethylene Glycol)3D Cell CultureExtracellular MatrixDrug TestingStem CellsPolystyrene MicrospheresPEGDAPhoto-initiator

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Shirahama, H., Kumar, S. K., Jeon, W., Kim, M. H., Lee, J. H., Ng, S. S., Tabaei, S. R., Cho, N. Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering. J. Vis. Exp. (114), e54331, doi:10.3791/54331 (2016).
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