Name: generation and recovery of &beta;-cell spheroids from step-growth peg-peptide hydrogels
Uploaded: 2012-12-06T13:00:00
Description: Watch this Scientific Journal Video about Generation and Recovery of ÃŽÂ²-cell Spheroids From Step-growth PEG-peptide Hydrogels at JoVE.com

Dette projekt blev finansieret af NIH (R21EB013717) og IUPUI OVCR (RSFG). Forfatteren takker Ms Han Shih for hendes teknisk bistand.

A. makromeren og Peptide Synthesis <ol><li> Syntetisere 4-armet PEG-norbornen (PEG4NB) og fotoinitiator Lithium arylphosphanate (LAP) ved anvendelse af etablerede protokoller. 18,19 </li><li> Syntetisere bis-cysteinholdige chymotrypsin-sensitive peptid CGGY ↓ C (pil angiver chymotrypsin spaltningssted) ved anvendelse af standard fast-fase peptidsyntese i en mikrobølgeovn peptidsynteseapparat (CEM Discover SPS). <ol class="lalpha"><li> Beregne mængden af ​​harpiks (Rink-amid-MBHA-harpiks) nødvendig ...

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Den beskrevne protokol præsenterer oplysninger om nem indkapsling af celler i thiol-hydrogeler dannet ved trin-vækst fotopolymerisation. Mens et støkiometrisk forhold på 1:1 af norbornen til thiol-funktionelle grupper blev anvendt i denne protokol, kan forholdet justeres afhængigt af eksperimenterne. Foruden en korrekt formulering, er det vigtigt at opretholde homogenitet i præ-polymeropløsningen. Især ved anvendelse forsigtig pipettering at sikre, at cellerne er godt fordelt i den præ-polymeropløsningen for a...

Hydrogeler er hydrofile tværbundne polymerer med enestående potentiale som stilladser materialer til reparation og regenerering af væv. 1-3 Den høje vandindhold af hydrogeler muliggør let diffusion af oxygen og udveksling af næringsstoffer og metaboliske produkter, som alle er afgørende for opretholdelse af cellelevedygtighed. Desuden er hydrogeler fremragende bærere til reguleret frigivelse og celle produktionstid på grund af deres høje justerbarhed. 2 Syntetiske hydrogeler, såsom dem fremstillet ud fra poly (ethylenglycol) (PEG) anvendes i stigende grad i vævsdyrkningsapplikationer, hovedsagelig på grund af deres cytocompatibility, vævs- såsom elasticitet og høj justerbarhed i materiale fysiske og mekaniske egenskaber. 4-6 Selv om en almindeligt anvendt hydrogel platform, har undersøgelser vist, at PEG-diacrylat (PEGDA) hydrogeler dannet ved kæde-vækst photopolymerizations har en tendens til skade indkapslede celler During netværk tværbinding og in situ celleindkapsling. 7 Den cellulære skader blev i vid udstrækning tilskrives radikalspecies genereret af fotoinitiator molekyler, som udbreder sig gennem vinylgrupper på PEGDA at tværbinde polymerkæder til hydrogeler. Desværre er disse radikalspecies også forårsage spændinger og cellulære beskadigelse under celleindkapsling, især for radikal-følsomme celler, såsom pancreas-β-celler. 8-10 For at opnå en højere maskestørrelse for bedre diffusion og celleoverlevelse, højere molekylvægte PEGDA anvendes ofte til celleindkapsling. Dette vil imidlertid gøre polymerisationskinetik og forårsager suboptimale gel biofysiske egenskaber. 7,11,12 Ud over de ovennævnte ulemper, er det meget vanskeligt at genvinde cellestrukturer fra PEGDA hydrogeler på grund af heterogenitet og ikke-nedbrydelige natur de tværbundne netværk. Medens protease-sensitive peptider kan inkorporeresi PEG makromer backbone at gøre de ellers inerte PEGDA hydrogeler følsomme for enzymatisk spaltning, ofte konjugeringen anvender dyre reagenser, og de ​​resulterende net stadig indeholder høj grad af heterogenitet på grund af karakteren af kæde-vækst polymerisation. 13-15 For nylig er PEG-peptid-hydrogeler dannet via trin-vækst thiol-en fotopolymerisation vist sig at udvise fordelagtige egenskaber til celleindkapsling i hydrogeler dannet ved kæde-vækst fotopolymerisation. 7 De overlegne geleringsegenskaber kinetik thiol-hydrogeler tilskrives &#39;klik &quot;karakter reaktion mellem thiol og ØNØ funktionaliteter. Sammenlignet med kæde-vækst polymerisation af PEGDA, er thiol-reaktion mindre oxygen inhiberet, hvilket resulterer i hurtigere gelering rate. 16,17 thiol-hydrogeler også højere polymerisation effektivitet og bedre gel biofysiske egenskaber sammenlignet med kæde-vækst PEGDA hydrogeler, 7 , 18 &lt;/ Sup&gt;, som resulterer i begrænset cellulær skader forårsaget af radikale arter i fotopolymerisation. Tidligere thiol-hydrogeler dannet af 4-armet PEG-norbornen (PEG4NB) makromer og bis-cysteinholdige peptid-tværbindere, såsom protease-sensitive peptider er blevet anvendt til celleindkapsling. 7,18 High justerbarhed af PEG-hydrogel-net tilbyder en fleksibel og styrbar 3D mikromiljø til undersøgelse af celleoverlevelse og-aktivitet, mens anvendelsen af ​​protease-sensitive peptidsekvens tilvejebringer en mild måde til genvinding af cellekonstruktioner dannes naturligt i hydrogeler. I denne protokol anvender vi trinvis vækst fotopolymeriserede thiol-hydrogeler fremstillet under anvendelse af 4-armet PEG-norbornen (PEG4NB) og en chymotrypsin-følsom peptid tværbinder (CGGY ↓ C) til indkapsling af MIN6 β-celler. Denne protokol systematisk uddyber teknikker til at studere overlevelse, spredning og klumpformet dannelse af MIN6β-celler i thiol-hydrogeler. Vi tilvejebringer endvidere metode til β-celle sphæroide genopretning og biologisk karakterisering af genvundne sfæroider.

<table border="1"><tbody><tr><td> Navn </td><td> Firma </td><td> Catalog Number </td><td> Kommentarer </td></tr><tr><td> 4-armet PEG (20 kDa) </td><td> Jenkem Teknologi USA </td><td> 4ARM-PEG-20K </td><td></td></tr><tr><td> Fmoc-aminosyrer </td><td> Anaspec </td><td></td><td></td></tr><tr><td> Live / Dead cellelevedygtighed kit </td><td> Invitrogen </td><td> L3224 </td><td> Omfatter Calcein AM og ethidium-homodimer-1 </td></tr><tr><td> AlamarBlue reagens </td><td> Abd Serotec </td><td> BUF012 </td><td></td></tr><tr><td> CellTiter Glo reagens </td><td> Promega </td><td> G7570 </td><td></td></tr><tr><td> DPBS </td><td> Lonza </td><td> 17-512F </td><td> Uden Ca +2 og Mg +2 </td></tr><tr><td> HBSS </td><td> Lonza </td><td> 10547F </td><td> UdenCa +2 og Mg +2 </td></tr><tr><td> High Glucose DMEM </td><td> Hyclone </td><td> SH30243.01 </td><td></td></tr><tr><td> FBS </td><td> Gibco </td><td> 16000-044 </td><td></td></tr><tr><td> Antibiotika-antimykotisk </td><td> Invitrogen </td><td> 15240-062 </td><td></td></tr><tr><td> β-mercaptoethanol </td><td> Sigma-Aldrich </td><td> M7522-100ML </td><td></td></tr><tr><td> Trypsin-EDTA </td><td> Invitrogen </td><td> 15400-054 </td><td></td></tr><tr><td> Trypsin-free α-chymotrypsin </td><td> Worthington Biochemical Corp </td><td> LS001432 </td><td></td></tr><tr><td> Muse Inusin ELISA kit </td><td> Mercodia </td><td> 10-1247-01 </td><td></td></tr><tr><td> 1 ml engangssprøjte </td><td> BD Biosciences </td><td></td><td></td></tr></tbody></table>

generation and recovery of &beta;-cell spheroids from step-growth peg-peptide hydrogels

Hydrogeler er hydrofile tværbundne polymerer, som giver et tredimensionelt mikromiljø med vævslignende elasticitet og høj permeabilitet til dyrkning af terapeutisk relevante celler eller væv. Hydrogeler fremstillet ud fra poly (ethylenglycol) (PEG)-derivater anvendes i stigende grad til mange forskellige vævsdyrkningsapplikationer, delvis på grund af deres justerbare og cytocompatible egenskaber. I denne protokol, udnyttede vi thiol-step-vækst photopolymerizations at fabrikere PEG-peptid hydrogeler til indkapsling pancreas MIN6 B-celler. Gelerne blev dannet ved 4-armet PEG-norbornen (PEG4NB) makromeren og en chymotrypsin-følsom peptid tværbinder (CGGYC). Den hydrofile og ikke-fouling karakter af PEG har en cytocompatible mikromiljø for celleoverlevelse og proliferation i 3D, mens anvendelsen af chymotrypsin-sensitive peptidsekvens (C GGY ↓ C, pilen angiver enzym spaltningssted, mens terminal cysteeine rester blev tilføjet til thiol-tværbinding) muliggør hurtig genopretning af cellekonstruktioner danner i hydrogelen. Følgende protokol uddyber teknikker til: (1) Indkapsling af MIN6 β-celler i thiol-hydrogeler, (2) Kvalitativ og kvantitativ cellelevedygtighedsassays at bestemme celleoverlevelse og proliferation, (3) Udvinding af celle sfæroider ved hjælp af chymotrypsin-medieret gel erosion, og (4) Strukturel og funktionel analyse af de inddrevne sfæroider.

<html> 1-4 viser repræsentative resultater for indkapsling, overlevelse, proliferation, kugleformet dannelse og sfæroide opsving i thiol-hydrogeler. Figur 1 viser reaktionen skematisk (1) trin-vækst thiol-en fotopolymerisation ved hjælp PEG4NB og CGGYC, og ( 2) chymotrypsin medieret gel erosion, der følger en overfladeerosion mekanisme. figur 2 og 3 foreliggende levedygtighed opnåede resultater med Live / Dead-farvning og alamarBlue assay. Vi obser...

Watch this Scientific Journal Video about Generation and Recovery of ÃŽÂ²-cell Spheroids From Step-growth PEG-peptide Hydrogels at JoVE.com

Generation and Recovery of ÃƒÅ½Ã‚Â²-cell Spheroids From Step-growth PEG-peptide Hydrogels

Den følgende protokol giver teknikker til indkapsling af bugspytkirtlen β-celler i trin-vækst PEG-peptid hydrogeler dannet af thiol-foto-klik reaktioner. Dette materiale platform tilbyder ikke kun en cytocompatible mikromiljø for celleindkapsling, men også tillader brugeren kontrolleret, hurtig genopretning af cellestrukturer dannet inden for hydrogeler.

Generation og indvinding af β-celle Sfæroider Fra Step-vækst PEG-peptid hydrogeler

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional  microenvironment with tissue-like elasticity and high permeability for  ...

Research

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Bioengineering

Generation and Recovery of &beta;-cell Spheroids From Step-growth PEG-peptide Hydrogels

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

Syntesen af ​​RGD-funktionaliserede Hydrogeler som værktøj for terapeutiske applikationer

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

Robotic Mirror Therapy System for Functional Recovery of Hemiplegic Arms

Robotic Mirror Therapy System for Funktionel Inddrivelse af hemiplegisk Arms

Mirror therapy has been performed as effective occupational therapy in a clinical setting for functional recovery of a hemiplegic arm after stroke. It is ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Tunable Hydrogeler fra Pulmonal ekstracellulære matrix for 3D cellekultur

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Produktionen af Elastin-lignende Protein Hydrogels til indkapsling og immunfarvning af celler i 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

Kontrolleret stamme af 3D Hydrogels under Live Mikroskop imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

Injectable Supramolecular Polymer-Nanoparticle Hydrogels for Cell and Drug Delivery Applications

Injicerbare supramolelekylære polymer-nanopartikelhydrogeler til anvendelse til levering af celler og lægemidler

These methods describe how to formulate injectable, supramolecular polymer-nanoparticle (PNP) hydrogels for&#160;use as biomaterials. PNP hydrogels are ...

Affordable Oxygen Microscopy-Assisted Biofabrication of Multicellular Spheroids

Overkommelig iltmikroskopiassisteret biofabrikation af multicellulære sfæroider

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue ...

Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for Biomedical Applications

Fragmentering af bulkhydrogeler og forarbejdning til granulære hydrogeler til biomedicinske applikationer

Granular hydrogels are jammed assemblies of hydrogel microparticles (i.e., &#34;microgels&#34;). In the field of biomaterials, granular hydrogels have ...

Human Liver Spheroids from Peripheral Blood for Liver Disease Studies

Humane leversfæroider fra perifert blod til undersøgelser af leversygdomme

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to ...