GRADIENT HYDROGELS FOR HIGH THROUGHPUT DRUG SCREENING

In this study we demonstrate that hydrogels loaded with a concentration gradient of an active molecule can be used as analytical tools for high throughput screening (HTS) and drug discovery applications. We used a microfluidic mixer to fabricate poly(ethylene glycol) (PEG) hydrogels that contained chemical concentration gradients. Each hydrogel was placed in culture medium in the vicinity of preosteoblast MC3T3-E1 cell layers. The controlled release of the toxin from the hydrogel in a gradient manner was used to study the effect of various chemical concentrations on cell viability. Given its simplicity, ease of storage and portability, this platform is a potentially useful approach to screen a range of drug concentra- tions simultaneously. Microdevice fabrication: The microfluidic device was fabricated by using soft-lithography and replica molding techniques. The SU-8 mold master patterned with a microchannel network (12mm x 10mm x 0.18mm) and a chamber (15mm x 10mm x 0.18mm) was generously provided by Dr Hongkai Wu from Hong Kong University of Science and Technology, Hong Kong. PDMS prepolymer mixed with curing reagent (10:1 mass ratio) was poured in the master mold and cured at 70 ° C for 1.5 h after degassing in a vacuum chamber. The PDMS microstructured mold was then peeled off from the master and holes were drilled at the inlets and outlets. To allow the gel fixation on its surface, a glass slide was partially treated with 2% (v/v) TMSPMA solution in methanol and baked during 30 min at 100°C before bonding to the PDMS mold. The microfluidic mixer part of the microstructured PDMS layer was permanently bonded to the glass slide after treatment with an oxygen plasma for 10 sec using a plasma cleaner (Harrick Plasma/model PDC-001). Silicone tubes were then fixed at the inlets and outlet with drops of PDMS prepolymer plus curing agent solution cured at 70 ° C for 1.5 h. PEG hydrogel and OA gradient formation: FITC-dex was used to visualize the concentration gradients. Two PEGDA 4000 prepolymer solutions with different FITC-dex and okadaic acid (OA) concentrations were injected at the microdevice inlets. Solution injected through the top inlet contained 40%(w/v) PEGDA, 1%(w/v) photoinitiator Irgacure, 1%(w/v) FITC- dex and 2M OA in 1 ml MilliQ water. Solution injected through the bottom inlet contained only 40%(w/v) PEGDA and 1%(w/v) photoinitiator. Flow rates of 0.5 µl/min for both solutions were controlled by using two syringe pumps (World Precision Instruments/Aladdin syringe pump/ Saratosa, FL/USA). Hydrogel precursor containing FITC and OA concentration gradients was then photopolymerized (UVP/code UVGL-48/Upland, CA/USA) for 5 min at 356 nm. Images were acquired by using an inverted fluorescent microscope (Axio Observer.Z1/ Carl Zeiss Corporation/Tokyo/ Japan) and processed with an Axio Vision Imaging management system. OA gradient and cell viability: After removing the PDMS layer with scalpel in a clean bench, a coverslip loaded with MC3T3-E1 cell layer was placed on the OA-gradient hydrogel, fixed with four clips and the whole construct was put in 25 ml culture medium for 24 h. The coverslip was then removed and cells were analyzed for viability with a live/dead kit assay (Invitrogen/ code L3224/Tokyo/Japan).