Opdigte Komplekse kultur Underlag Brug Robotic mikrokontakttrykning (R-μCP) og sekventielle nukleofil substitution
Summary
Cell culture substrates functionalized with microscale patterns of biological ligands have immense utility in the field of tissue engineering. Here, we demonstrate the versatile and automated manufacture of tissue culture substrates with multiple, micropatterned poly(ethylene glycol) brushes presenting orthogonal chemistries that enable spatially precise and site-specific immobilization of biological ligands.
Abstract
In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates presenting grafted poly(ethylene glycol) (PEG) brushes can be used to achieve this task by creating microscale, non-fouling and cell adhesion resistant regions as well as regions where cells participate in biospecific interactions with covalently tethered ligands. To engineer complex tissues using such substrates, it will be necessary to sequentially pattern multiple PEG brushes functionalized to confer differential bioactivities and aligned in microscale orientations that mimic in vivo niches. Microcontact printing (μCP) is a versatile technique to pattern such grafted PEG brushes, but manual μCP cannot be performed with microscale precision. Thus, we combined advanced robotics with soft-lithography techniques and emerging surface chemistry reactions to develop a robotic microcontact printing (R-μCP)-assisted method for fabricating culture substrates with complex, microscale, and highly ordered patterns of PEG brushes presenting orthogonal ‘click’ chemistries. Here, we describe in detail the workflow to manufacture such substrates.
Introduction
Evnen af PEG-podede overflader for at vise kovalent bundne biokemiske ligander samtidig bevare iboende ikke-fouling egenskaber gør dem til et ideelt valg for engineering brugerdefinerede mikroskala miljøer på kultur substrater 1,2,3. De biospecifikke interaktioner medieret af ligand konjugeret PEG pensler muliggør reduktionistisk analyse af virkningerne af biokemiske signaler inden kompleks in vivo væv mikromiljøerne på individuelle cellefænotyper. Endvidere kan bio-ortogonale "klik" kemiske anvendes til at lette retningsbestemt immobilisering af ligander, således at de præsenteres i native konformationer 4-6. Således mikroskala rumlig mønsterdannelse af PEG børster er et alsidigt værktøj til at skabe designer in vitro nicher at undersøge cellesignalering induceret af immobiliserede biokemiske 6,7 signaler.
En almindelig fremgangsmåde til generering af rumlige mønstre af biokemisk cues indebærer mikrokontakttrykning (μCP) guld-substrater med mønstre af PEG konjugerede alkanthioler. Derefter de mikromønstrede selvsamlede monolag (SAM'er) af PEG-ylated alkanthioler begrænser fysisk adsorption af biokemiske molekyler, fx proteiner, kun til ikke-mønstrede regioner af substratet 8,9. Men SAMS genereret ved denne teknik er følsomme over for oxidation i langsigtet celledyrkningsmedier. Således μCP'd alkanthiol SAMs ofte yderligere podet med PEG polymer børster hjælp overflade-initierede atom transfer radikalpolymerisation (SI-ATRP) for at øge regionens ikke-tilsmudsning stabilitet 10. Specifikt μCP af alkanthiol polymerisation initiator, ω-meraptoundecyl bromisobutyrat på guld overflader efterfulgt af SI-ATRP af poly (ethylenglycol) methylether-methacrylat (PEGMEMA) monomerer genererer overflader med mikromønstrede langsigtet, stabil og ikke- begroning PEG børster. Desuden er disse er i stand til at blive yderligere modificeret til at præsentere forskellige kemiske dele 11.
Drage fordel af denne egenskab, Sha et. al. udviklet en metode til at konstruere kultur substrater med flerkomponent PEGMEMA børster præsenterer ortogonale "klik" kemi. Ved denne metode de bruger en række μCP / SI-ATRP trin spækket med sekventiel natriumazid, ethanolamin, og propargylamin nukleofile substitutioner skabe kultur substrater frembyder mikroskala mønstre af flere immobiliserede ligander 6. Mens potentialet for anvendelse af sådan kemi i forbindelse med manuel μCP at konstruere nye kultur substrater er enorm, er det begrænset af præcision og nøjagtighed med, som kan tilpasses flere μCP skridt på et enkelt substrat. En høj grad af præcision og nøjagtighed vil være forpligtet til at reproducerbart fremstille komplekse in vitro nicher ved hjælp af disse alsidige teknikker.
e_content "> For at løse denne begrænsning, har flere automatiske og halvautomatiske μCP systemer blevet genereret. Chakra et. al. udviklet et μCP system, hvor brugerdefinerede stempler er placeret på et skinnesystem og bringes i overensstemmende kontakt med guld-belagte objektglas med en computer-kontrolleret pneumatisk aktuator. Men denne metode kræver en præcis fremstilling af brugerdefineret stempel designs og rapporterer en 10 um præcision med nogen rapport af den opnåede, når du udfører flere μCP trin 12 nøjagtighed. For nylig en fremgangsmåde, der anvender et integreret kinematisk koblingssystem rapporteret præcision under 1 um ved anvendelse af et enkelt mønster, men var ikke i stand til præcist at tilpasse flere mønstre på grund af mangel på præcis styring af stempel funktioner fra formen for at forme 13. Derudover begge foregående metoder kræver substratet at forblive fastgjort mellem mønsterdannende trin og dermed væsentligt begrænse mangfoldigheden af overflademodificering kemi, der kan væreudnyttes. Her beskriver vi en automatiseret R-μCP system, der kan nøjagtig og præcis justering af flere μCP skridt, samtidig med at maksimal fleksibilitet i stempel design og fabrikation. Endvidere kan de mønstrede substrater gentagne gange fjernes fra systemet mellem udstansning, hvilket tillader anvendelse af forskellige substrat modifikation kemier, herunder sekventielle nukleofile substitutioner. Substrater manipuleret ved hjælp af sådan kemi er blevet anvendt til celledyrkning tidligere af både os 6,14 og andre 7. Således har vi fusioneret R-μCP og sekventielle nukleofile substitutionsreaktioner at udvikle en metode til skalerbar fremstilling af kultur substrater med komplekse og mikromønstrede biokemiske signaler.Protocol
Representative Results
Discussion
Ideelle substrater for tissue engineering ville blive bioinspirerede og derved rekapitulere den rumlige fordeling af kritiske bioaktive ligander inden de indfødte væv. De ville også have dynamiske egenskaber, der muliggør tidsmæssige justeringer af ligander og de rumlige mønstre, som de præsenteres for at tillade rettet vævsmorfogenese og rumligt begrænsede induktion af celle skæbne. Fabrikation af sådanne substrater kræver immobilisering af flere biokemiske signaler i komplekse og stærkt bestilte retningsl…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Funding for this work, GTK, TK, and JDM were provided by the Wisconsin Institute for Discovery and the Wisconsin Alumni Research Foundation.
Materials
| Name | Company | Catalog Number | Comments |
| SCARA | Epson | LS3-401ST | Higher end models with increased precision are available if desired. |
| (TRIDECAFLUORO-1,1,2,2-TETRAHYDROOCTYL)TRICHLOROSILANE | Gelest | SIT8174.0 | CAUTION, Should only be handled in a chemical fume hood. When silanizing wafers no one should enter the hood until all silane has been evaporated. |
| Sylgard 184 Silicone Elastomer Kit | Ellsworth Adhesive Co | NC9020938 | Thouroughly degass solutions via vacuum exposure before use. Alternative kits such as Kit 182 are acceptable. |
| 24mm X 50 mm #1 Cover Glass Slides | Fisher Scientific | 48393106 | These can be purchased from a number of suppliers with varying dimensions to suit need. |
| CHA-600 Telemark Electron Beam Evaporator | Telemark | SEC-600-RAP | Requries specialized training. |
| EPSON LS3 SCARA | EPSON | LS3-401ST | |
| ω-mertcaptoundecyl bromoisobutyrate | Prochimia | FT 015-m11-0.2 | Store at -20°C. Other ATRP initiators may be used as this R-μCP platform is applicable to all micropatterning modalities. |
| Schlenk Tube Flask 50 mL | Synthware | 60003-078 | Requires rubber stoppers with diaphram. |
| Poly(ethylene glycol) methyl ether methacrylate | Sigma Aldrich | 447943 | Shipped containing MEHQ and BHT free readical inhibitors. |
| Methanol (Certified ACS) | Fisher Scientific | A412-4 | CAUTION, only handle in chemical fume hood. |
| Copper(II) Bromide | Sigma Aldrich | 437867 | CAUTION, limit exposure with surgical mask. |
| 2',2-Bipyridine | Sigma Aldrich | D216305 | CAUTION, limit exposure with surgical mask. |
| Sodium L-Ascorbate | Sigma Aldrich | A4034 | |
| 20mL Borosilicate Glass Scintillation Vials | Fisher Scientific | 03-340-4E | |
| Sodium Azide | Sigma Aldrich | S2002 | CAUTION, limit exposure with surgical mask. |
| N,N-dimethyformamide | Sigma Aldrich | 227056 | CAUTION, only handle in chemical fume hood. |
| Ethanolamine | Sigma Aldrich | 398136 | CAUTION, only handle in chemical fume hood. |
| Triethylamine | Sigma Aldrich | T0886 | CAUTION, only handle in chemical fume hood. |
| Dimethylsulfoxide | Sigma Aldrich | 276855 | CAUTION, only handle in chemical fume hood. |
| Propargylamine | Sigma Aldrich | P50900 | CAUTION, only handle in chemical fume hood. |
| 200 Proof Ethanol | University of Wisconsin Material Distribution Services | 2292 | CAUTION, only handle in chemical fume hood. |
| Azide-PEG3-Biotin | ClickChemistryTools | AZ104-100 | Solubilized in DMF |
| Copper(II) Sulfate | Sigma Aldrich | C1297 | CAUTION, limit exposure with surgical mask. |
| Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) | Sigma Aldrich | 678937 | |
| L-Ascorbic Acid | Sigma Aldrich | A7506 | |
| Phosphate Buffer Saline | Invitrogen | 14190144 | |
| Donkey Serum | Sigma Aldrich | D9663 | Donkey serum contaminated items are considered bio-hazardous material and should be disposed of accordingly. Various other compounds (e.g. BSA) are available and serve this purpose. |
| 12-Well Polystyrene Plate | Thermo Scientifit – NUNC | 07-200-81 | Plates can be purchased form a number of suppliers with varying dimensions. |
| DBCO-PEG4-Biotin | Clickchemistytools | A105P4-10 | Solubilized in DMF |
| Streptavidin, Alexa Fluor 488 Conjugate | Life Technologies | S-11223 | Solubilized in PBS |
| Streptavidin, Alexa Fluor 546 conjugate | Life Technologies | S-11225 | Solubilized in PBS |
| Nikon A1-R Confocal Microscope | Nikon | Nikon Eclipse Ti, A1R | An epifluorescent microscope is sufficient to image functionalized micropatterned substrates. |
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