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Bioengineering

Ters Kolloidal Kristal Poli (etilen glikol) İskele imalatı: Karaciğer Doku Mühendisliği için Üç boyutlu Hücre Kültürü Platformu

Published: August 27th, 2016

DOI:

10.3791/54331

1School of Materials Science and Engineering, Nanyang Technological University, 2School of Chemical and Biomedical Engineering, Nanyang Technological University

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.

, Ksenobiyotiklerin 'sitotoksisite testi virüs enfeksiyonu okuyan ve karaciğer hedefleyen ilaçların geliştirilmesi amacıyla, in vitro hepatosit fonksiyonunu korumak için yeteneği, hücreler uygun biyokimyasal ve mekanik ipuçları aldığınız bir platform gerektirir. Son karaciğer doku mühendisliği sistemleri, yüksek su tutma ve hücreler tarafından ihtiyaç duyulan mekanik uyarıcılara sağlamak için kendi özellikleri göz önüne alındığında, sentetik ya da doğal hidrojeller oluşan üç boyutlu (3D) iskeleleri kullanmışlardır. ters koloidal kristal (ICC) iskele artan bir ilgi olmuştur, yüksek uzaysal organizasyon, homotipik ve heterotypic hücre etkileşimi yanı sıra hücre dışı matriks (ECM) etkileşimi sağlayan bir son gelişme. Burada, poli (etilen glikol) diakrilat (PEGDA) ve partikül ayrıştırma yöntemi kullanılarak ICC iskele imal etmek için bir protokol açıklar. Kısaca, bir örgü mikrosfer parçacıklarından yapıldığı bir ön polimer daha sonraR solüsyonu doğru polimerize ilave edilir ve tanecikler daha sonra organik bir çözücü (ör tetrahidrofuran) kullanılarak kaldırılır veya yıkanarak ekstre edilir. Kontrollü gözenek boyutları ve ortam daha kolay hücrelere ulaşmasına izin interconnectivities ile çok gözenekli iskele kafes sonuçlarının çözünme. Bu benzersiz yapı hücreleri hem de kolay gözenekler arasındaki iletişim ve proteinlerle PEGDA ICC skafold, hücre performansı üzerinde belirgin bir etki gösterir kat yeteneği yapışması için yüksek bir yüzey alanı sağlar. Biz canlılığı açısından iskele morfolojisi yanı sıra hepatokarsinom hücre (Huh-7.5) davranış analiz ve ICC yapısı ve ECM kaplamaların etkisini araştırmak için çalışır. Genel olarak, bu kağıt doku mühendisliği geniş bir uygulama, özellikle karaciğer doku mühendisliği sahip gelişmekte olan bir iskele ayrıntılı bir protokol sağlar.

Karaciğer kan detoksifikasyon, ksenobiyotiklerin metabolizması ve serum proteinlerinin üretimi dahil fonksiyonları çok sayıda, bir çok kanlanan bir organdır. Karaciğer dokusu birden fazla hücre tipleri, safra kanalcıklarının, sinüzoidler ve farklı biyomatris kompozisyon ve farklı oksijen konsantrasyonlarına bölgelerinin içeren karmaşık bir üç boyutlu (3D) mikro-yapıya sahiptir. Bu ayrıntılı yapısı göz önüne alındığında, in vitro 1 uygun bir karaciğer model oluşturmak için zor olmuştur. Ancak, in vitro modellerde fonksiyonel test ilaç toksisitesi 2 platformları gibi insan hepatositleri barındırma ve karaciğer 3

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1. ICC İskele İmalatı (Şekil 1)

  1. polistiren (PS) menfezler (; boncuk 8-13 katmanları çapı = 6 mm) hazırlayın.
    1. 40 ul seviyesinde 0.2 ml kaynatın geçirmez mikrosantrifüj tüpler ipuçları kesti, kalıp hazırlamak. Su geçirmez yapıştırıcı ile 24 x 60 mm 2 mikroskop cam kapak fişleri kesme tüplerin üst uyun.
    2. dikkatle 20 ml'lik bir şişe bir su süspansiyonu içinde bulunan PS küreleri (çap = 140 mm) konulan su süspansiyonu üzerinden pipet ve şişe içi.......

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ICC iskele yapısal karakterizasyon ve hepatositler kültüre her ICC iskele condition en etkinliğinin karşılaştırılması için temsili sonuçlar gösterilmiştir ve aşağıda açıklanmıştır. Bu sonuçlar kullanılan ICC iskele şartları 0 ug / ml (çıplak), 20 ug / ml (Kollajen 20), 200 ug / ml (Kollajen 200) ve 400 ug / ml (400 kolajen) ve ilk kollajen kaplamalar huh-7.5 hücre tohum sayısı 1x10 6.

.......

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Doku mühendisliği iskeleleri hızla tüm yeniden korumak veya ilaçlar, hastalığın okuyan gelişen organ değiştirme uygulaması için dokuları onarmak için gerekli olan fiziksel ve biyokimyasal ipuçları, ve diğerleri 57 sağlamak için gelişmektedir. karaciğer doku mühendisliği, birincil insan hepatositler hızla kez vücuttan izole onların metabolik fonksiyonları, mühendislik iskeleleri için büyük bir ihtiyaç yaratmak ve karaciğer fonksiyon korumak için platformlar geliştirme kaybed.......

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Yazarlar Ulusal Araştırma Vakfı Bursu (NRF -NRFF2011-01) ve Rekabet Araştırmaları Programı (NRF-CRP10-2012-07) dan destek için teşekkürlerimizi sunarız.

....

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NameCompanyCatalog NumberComments
0.2 mL PCR tubeAxygen ScientificPCR-02D-CBoil-proof
Gorilla GlueGorilla Glue, Inc.Depends on vendor. This was purchased from a local store.
Glass slidesVWR 631-1575Dimensions: 24×60 mm
Polystyrene spheres Fisher ScientificTSS#4314ADiameter = 140 um; 3x10^4 particles per milliliter and 1.4% size distribution
EthanolMerck1.00983.1011absolute for analysis EMSURE; Dilute to 70% with Milli-Q water
Ultrasonic BathElmaS10HEquiment
FurnaceNaberthermN7/HEquipment
200 µL pipette tipAxygen ScientificT-210-Y-R-S
Rocking shakerVWR444-0142
Polyethylene Glycol (PEG)Merck1.09727.0100Mw= 4kDa; acrylation of PEG monomers and purification of the resulting precipitate produces a PEGDA macromer with Mw = 4.6kDa
CentrifugeBeckman Coulter392932Equipment
Acrylate-Poly (Ethylene Glycol) - Succinimidyl Valerate Laysan BioACRL-PEG-SVA-3400-1gMw = 3.4 kDa
2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenoneSigma Aldrich410896
VortexVWR58816-123Equipment
MicrocentrifugeEppendorf5404 000.413
Paraffin Film Parafilm M #PM996Kept at 9" with allows intensity of 10.84 mW/cm^2
Bluewave 200 UV spotlightBlaze Technology 120008, 122300
Tetrahydrofuran (THF)Merck107025
Orbital shakerHeidolph543-123120-00-5From rat
Collagen Type ISigma AldrichC3867-1VL1X, w/o CaCl & MgCl; Ph = 7.2
Phosphate Buffered Saline (PBS) Gibco20012-02716% W/V AQ. 10x10ml
ParaformaldehydeVWR43368.9MEquipment
Freezone 4.5 freeze drierLabconco7750020Equipment
Sputter coaterJeol Ltd.JFC-1600Equipment
Scanning Electron MicroscopeJeol Ltd.JSM 5310
Anti-mouse primary antibodies against Collagen type IAbcamab6308
Anti-mouse secondary antibody conjugated with Alexa Fluor 488Life TechnologiesA21121
Plate, Tissue Culture 24 Well, Flat Bottom (Nunclon) Bio-Rev PTE LTD3820-024
Dulbecco's Modified Eagle's Medium(DMEM)
2.5 g/L Glucose w/ L-Gln		Lonza12-604F
Fetal Bovine Serum (FBS)GibcoA15-151
Penicillin-Streptomycin (P/S)Life Tchnologies15140-122 E
APC49‐Huh ‐7.5 Cell LineApath
100 mm Corning non-treated culture dishesSigma AldrichCLS430591
0.25% Trypsin-EDTAGibco25200-056Equipment; 37°C, 5% Humidity
Forma Steri-Cycle CO2 IncubatorsThermofisher Scientific371
Hausser Bright-Line Phase HemacytometerThermofisher Scientific02-671-6
Live/Dead Viability/Cytotoxicity Kit 'for mammalian cellsLife TechnologiesL3224 
CCK-8 AssayDojindo LaboratoriesCK04-11Monosodium-salt reagent (MSR)
Infinite 200 PRO microplate reader Tecan
Albumin Human ELISA kitAbcamab108788
Triton X-100Bio-Rad#1610407
Bovine Serum Albumin (BSA)Sigma-AldrichA2153-50G
Anti-mouse primary antibodies (against CYP3A4, albumin)Santa Cruz Biotechnologysc-53850; sc-271605
DAPILife TechnologiesD3571
Alexa Fluor 555 labelled PhalloidinLife TechnologiesA34055
TrizolLife Technologies15596-026
ChloroformVWR22706.326
IsopropanolFisher Scientific67-63-0
DPEC waterThermofisher ScientificAM9916
Nanodrop 2000c SpectrophotometerThermofisher ScientificND-2000
iScript Reverse Transcription Supermix Bio-Rad Laboratories1708840
SYBR select Master Mix for CFXLife Technology4472937
Primers (to be chosen)
CFX96 Real-Time System, C-1000 Touch Thermal CyclerBio Rad LaboratoriesSOFT-CFX-31-PATCH 

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