Simple Polyacrylamid-baserede Multiwell Stivhed Assay for Studiet af Stivhed afhængige celleresponser
Summary
Here, a method that enables quick, efficient, and inexpensive preparation of polyacrylamide gels in a multiwell plate format is described. The method does not require any specialized equipment and could be easily adopted by any research laboratory. It would be particularly useful in research focused on understanding stiffness-dependent cell responses.
Abstract
Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a matrix that is elastic and much softer (0.1 – 100 kPa). Since such stiffness mismatch greatly affects cell responses, there is a strong interest in developing hydrogel materials that span a wide range of stiffness to serve as cell substrates. Polyacrylamide gels, which are inexpensive and cover the stiffness range of all soft tissues in the body, are the hydrogel of choice for many research groups. However, polyacrylamide gel preparation is lengthy, tedious, and only suitable for small batches. Here, we describe an assay which by utilizing a permanent flexible plastic film as a structural support for the gels, enables the preparation of polyacrylamide gels in a multiwell plate format. The technique is faster, more efficient, and less costly than current methods and permits the preparation of gels of custom sizes not otherwise available. As it doesn’t require any specialized equipment, the method could be easily adopted by any research laboratory and would be particularly useful in research focused on understanding stiffness-dependent cell responses.
Introduction
De fleste væv i kroppen er bløde viskoelastiske materialer med en Youngs modul i området fra 0,1 kPa for hjernen til 100 kPa for blød brusk, endnu, de fleste in vitro celle forskning foregår på vævskultur polystyren (TCP), som har et modul på ~ 1 GPa . 1 Dette stivhed mismatch i høj grad påvirker den måde celler reagerer på deres omgivelser. En voksende mængde forskning er således dedikeret til at belyse effekten af substrat stivhed om skæbne forskellige celletyper, 2,3 herunder stamceller. 4 Som følge heraf har flere hydrogeler blevet udviklet til at hjælpe med forståelsen af stivhed-afhængig celle biologi herunder polyacrylamid (PA), 5-7 polyethylenglycol (PEG), 8,9 polydimethylsiloxan (PDMS), 10 og alginat. 11 Mens bevis for, at underlaget stivhed har en væsentlig indvirkning på celle skæbne er stigende, er de fleste undersøgelser på en lille skala med et lille antal ssempler. Systematiske, multidimensionale undersøgelser af virkningen af substrat stivhed for en række celletyper eller miljømæssige forhold er sjældne. 12
Der er udviklet flere lovende high-throughput hydrogel teknologier, herunder PEG-baserede microarrays, 13 mikrofluidenheder til produktion af agarose hydrogel mikroperler, 14 eller mikro- og nano-stænger hvor stivhed moduleres af diameter og højde mikrostavene. 15. Dog , de teknologier til fremstilling af sådanne substrater er sofistikeret og tilgængelig for begrænset antal laboratorier. Megen forskning involverer stivhed moduleret cellereaktioner udnytter polyacrylamid (PA) geler, som ikke kun er billig og enkel at gennemføre, men også udviser en fysiologisk relevant række Youngs modul, nemlig 0,3 -. 300 kPa 16-22 Men de eksisterende metoder til at fremstille PA geler til cellekultur er arbejdskrævende og dermed prepARED i små partier. Nogle af de vanskeligheder, der er forbundet med udarbejdelsen af PA geler som cellesubstrater skyldes kravet om, at gelerne skal være forberedt: 1) i fravær af ilt for at tillade fuldstændig polymerisering, 2) med en flad og jævn overflade for at muliggøre en ensartet celle fastgørelse og spredning, og 3) permanent fastgjort til bunden af cellekultur skålen for at forhindre flydende.
Adskillige grupper har forsøgt at producere PA geler til cellekultur i store partier. Semler et al. Forberedte tykke plader af PA geler, der var så "cut" med en hulning og anbringes i 96-brønds plader. 23. Men denne metode er begrænset til stivere geler, dvs.> 1 kPa i Youngs modul, fordi blødere geler "sticky", svært at skære og beskadiges let. MIH et al. Udviklet en mere sofistikeret teknik, som gør det muligt geler direkte polymeriseret i en glasbund multibrøndsplade. <sop> 6 Dette blev opnået ved at hælde gelen løsninger i funktionaliserede glasbund plader og danner geler med "sandwich" dem med en brugerdefineret dækglas array. 6 Selvom meget lovende, lille kant effekter stadig observeret med denne teknik. Derudover teknikken kræver en specialdesignet opstilling ikke umiddelbart tilgængelige for mange laboratorier samt dyre glasbund flerbrøndsplader.
Dette papir beskriver en enkel og billig måde at samle PA geler i en flerbrøndsplade, der let kunne vedtages af alle laboratorier. Her er en fleksibel plastik støtte anvendes, der har to sider – en hydrofob en, som er afvisende PA geler og et hydrofilt, som kovalent binder PA gel ved deposition. Når PA gel ark aflejres og permanent fastgjort til den fleksible plastik støtte, gør det muligt at håndtere geler af enhver tykkelse eller stivhed og skære dem i enhver ønsket form. Denne ca.oach ikke kun producerer tilpassede plast «dækglas« i størrelser ellers ikke kommercielt tilgængelige, men også overflødiggør behovet for at forbehandle glasoverflader, enten dækglas eller brøndene i dyre glasbund flerbrøndsplader med en PA bindende løsning, som er en kedelig og tidskrævende trin. Endelig kan ensartede PA gels plader fremstilles på store partier og opbevares de-hydreret i flere måneder.
Sammenfattende assay præsenteres her, er en forbedring i forhold til eksisterende metoder i flere aspekter. Først processen flerbrøndspladen samling er effektiv, og de samlede omkostninger ved de nødvendige materialer er lav. For det andet er de hydrogeler som produceres i store partier i en enkelt homogen gel film. Endelig er kun materialer, der er kommercielt tilgængelige påkrævet. Anvendeligheden af assayet er vist ved at undersøge virkningen af substrat stivhed på cellemorfologi og spredning område.
Protocol
Representative Results
Discussion
Polyacrylamidgeler oprindelig er udviklet til elektroforese 28 nu rutinemæssigt anvendes som celledyrkningssubstrater at undersøge virkningerne af substrat stivhed på cellemorfologi, motilitet og kommunikation 3,24,29 blandt andre celletyper egenskaber. Polyacrylamid tillader manipulation af substrat stivhed til at omfatte stivheden af alle bløde væv i kroppen (0,3-300 kPa) 1 med en simpel ændring i koncentration polymerpræcursor (figur 2, tabel 1, se også refere…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was funded by start-up funds provided to Dr. Silviya Zustiak by Saint Louis University as well as by a President’s Research Fund (PRF) grant awarded to Dr. Silviya Zustiak by Saint Louis University. We thank Naveed Ahmed and Keval Shah for technical assistance.
Materials
| Reagents | |||
| 40% Acrylamide | Bio-Rad | 161-0140 | |
| 2% Bis-acrylamide | Bio-Rad | 161-0142 | |
| Ammonium Persulfate | Bio-Rad | 161-07000 | |
| TEMED | Sigma Aldrich | T9281 | |
| Sulfo-SANPAH | Thermo Scientific | 22589 | |
| Collagen Type 1, from Rat tail, 3.68 mg/mL | BD Biosciences | 354236 | |
| Dimethyl sulfoxide (DMSO) | Fisher Scientific | BP231-100 | |
| Hydrophobic solution – Repel Silane | GE Healthcare Bio-Sciences | 17-1332-01 | |
| PBS (1x), pH 7.4 | HyClone | SH30256.01 | |
| Polydimehylsiloxane (PDMS) [Slygard 182 Elastomer Kit] | Elsworth Adhesives | 3097358-1004 | |
| Tyrpsin/EDTA (10x) | Sigma Aldrich | 44174 | |
| RPMI-1640 Medium (1x) | HyClone | SH30027-02 | |
| Fetal Bovine Serum | HyClone | SH30073-03 | |
| Penicillin Streptomycin | MP Biomedicals | 1670046 | |
| Detergent – Triton-X | Sigma Aldrich | T8787 | |
| Formaldehyde 37% Solution | Sigma Aldrich | F1635 | |
| Bovine Serum Albumin (BSA) | Sigma Aldrich | A2153 | |
| BSA-based cell adhesion blocking kit – ECM Cell Adhesion Array Kit | Chemicon International | ECM540 | |
| Disposable lab equipment | |||
| flexible plastic support – GelBond PAG Film for Polyacrylamide Gels | GE Healthcare Bio-Sciences | 309819 | |
| Glass Plates | Slumpys | GBS4100SFSL | |
| 50 mL Conicals | Fisher Scinetific | 3181345107 | |
| 15 mL Conicals | FALCON | 352097 | |
| Micro centrifuge tubes | Fisher Scinetific | 2 mL: 02681258 | |
| 96-well plate (flat bottom) | Fisher Scinetific | 12565501 | |
| Disposable Pipettes (1 mL, 2mL, 5mL, 10mL, 25 mL, 50mL) | Fisher Scinetific | 1 mL: 13-678-11B, 2mL: 05214038, 5mL(FALCON): 357529, 10mL: 13-678-11E, 25mL: 13-678-11, 50mL: 13-678-11F | |
| Glass Transfer Pipettes | Fisher Scinetific | 5 3/4": 1367820A, 9":136786B | |
| Pipette Tips (1-200uL, 101-1000uL) | Fisher Scinetific | 2707509 | |
| Plastic Standard Disposable Transfer Pipettes | Fisher Scientific | 13-711-9D | |
| Parafilm | PARAFILM | PM992 | |
| Powder Free Examination Gloves | Quest | 92897 | |
| Silicone spacers – Silicone sheet, 0.5 mm thick/13 cm x 18 cm | Grace Bio-Labs | JTR-S-0.5 | |
| Large/non-disposable lab equipment | |||
| Light and Flourescent Microscope (Axiovert 200M) | Zeiss | 3820005619 | |
| Microscope Software | Zeiss | AxioVision Rel. 4.8.2 | |
| UV oven | UVITRON | UV1080 | |
| Vacuum chamber/degasser | BelArt | 999320237 | |
| Vacuum pump for degasser | KNF Lab | 5097482 | |
| Tissue Culture Hood | NUAIRE | NU-425-600 | |
| Chemical Fume Hood | KEWAUNEE | 99151 | |
| Inverted Microscope (Axiovert 25) | Zeiss | 663526 | |
| Incubator | NUAIRE | NU-8500 | |
| Pipette Aid | Drummond Scientific Co. | P-76864 | |
| Hemacytometer | Bright-Line | 383684 |
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