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Bioengineering

Изготовление Перевернутый коллоидный кристалл поли (этиленгликоль) строительные леса: трехмерная Cell Culture платформа для печени тканевой инженерии

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.

Способность поддерживать функцию гепатоцитов в пробирке, для целей тестирования цитотоксичности ксенобиотиков, изучение вирусной инфекции и разработки лекарств , нацеленных на печень, требует платформу , в которой клетки получают соответствующие биохимические и механические сигналы. Последние ткани печени инженерные системы использовали трехмерные (3D) каркасы, состоящие из синтетических или натуральных гидрогели, учитывая их высокую задержку воды и их способность обеспечивать механические стимулы, необходимые клетками. Там было растущий интерес к перевернутой коллоидный кристалл (ICC) эшафот, новейшая разработка, которая обеспечивает высокую пространственную организацию, однотипны и взаимодействие гетеротипичной клеток, а также матрицу взаимодействия (ECM) клеточно-внеклеточный. В данном случае мы опишем протокол для изготовления строительных лесов ICC с использованием поли (этиленгликоль) диакрилат (PEGDA) и метод выщелачивания частиц. Если коротко, то решетка изготовлена ​​из частиц микросфер, после чего предварительно polymeR раствор добавляют, правильно полимеризуется, а частицы затем удаляются, или выщелоченные, с использованием органического растворителя (например, тетрагидрофуран). Растворение результатов решетки в сильно пористом помост с контролируемым размером пор и interconnectivities, которые позволяют медиа достичь клетки более легко. Эта уникальная структура позволяет большую площадь поверхности для клеток придерживаться, а также легкой связи между порами, а также возможность покрыть ICC эшафот PEGDA с белками также показывает заметное влияние на производительность клеток. Мы анализируем морфологии эшафот, а также гепатокарциномой клетки поведения (Хух-7,5) с точки зрения жизнеспособности и функции для изучения влияния структуры ICC и ECM покрытий. В целом, этот документ содержит подробный протокол формирующейся строительных лесов, который имеет широкое применение в тканевой инженерии, особенно печени тканевой инженерии.

Печень является насыщена сосудами орган с множеством функций, в том числе детоксикацию крови, метаболизма ксенобиотиков, а также при производстве сывороточных белков. Ткань печени имеет сложную трехмерную (3D) микроструктуру, состоящий из нескольких типов клеток, желчные канальцы, синусоиды и зон различного состава BIOMATRIX и различных концентраций кислорода. Учитывая эту сложную структуру, было трудно создать правильную модель печени в пробирке 1. Тем не менее, существует растущий спрос на функционал в моделях пробирке хостинг гепатоцитов человека в качестве платформ для тестирования лекарственной токсичности 2 и изу....

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1. МТП Эшафот Fabrication (Рисунок 1)

  1. Подготовьте полистирол (PS) решетки (диаметр = 6 мм; 8-13 слои шариков).
    1. Чтобы подготовить почву, отрезать кончики от от 0,2 мл кипячения доказательство микропробирок на уровне 40 мкл. Приклейте верхнюю часть срезанных труб до 24 х 60 мм 2 микроскопа п?.......

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Представительные результаты для структурной характеристики эшафота ICC и сравнения эффективности каждого ICC подмости условие в культивировании печеночных клеток показаны и объяснены ниже. Условия подмости МТП, используемые в этих результатов являются коллагеновые ?.......

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Тканевая инженерия каркасы быстро развиваются , чтобы обеспечить все физические и биохимические сигналы , необходимые для восстановления, поддержания или ремонта тканей для применения замены органов, изучение болезней, разработки лекарств, и многие другие 57. В печени тканевой и?.......

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Авторы хотели бы выразить признательность поддержку от Национального исследовательского фонда стипендий (NRF -NRFF2011-01) и конкурентоспособных исследований программы (СРН-CRP10-2012-07).

....

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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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