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On-chip characterization of cryoprotective agent mixtures using an EWOD-based digital microfluidic device.

ABSTRACT: For tissue engineering and regenerative medicine, cryopreservation, a technique for preserving biomaterials in the frozen state with cryoprotective agents (CPAs), is critically important for preserving engineered tissues (ETs) as well as cells necessary to create ETs. As more diverse ETs are produced using various cell types, CPAs and corresponding freeze/thaw (F/T) protocols need to be developed cell/tissue-type specifically. This is because CPAs and F/T protocols that have been successful for one cell/tissue type have proven to be difficult to adapt to other cell/tissue types. The most critical barrier to address this challenge is the inability to screen and identify CPA or CPA mixtures efficiently. In this paper, we developed an "electro-wetting-on-dielectic" (EWOD) based digital microfluidic platform to characterize and screen CPA mixtures cell-type specifically. The feasibility of the EWOD platform was demonstrated by characterizing and optimizing a mixture of dimethlysulfoxide (DMSO) and PBS for human breast cancer cell line as model CPA mixture and cell line. The developed platform multiplexed droplets of DMSO and PBS to create an array of DMSO-PBS mixtures, and mapped the phase change diagram of the mixture. After loading cell suspensions on the platform, the mixture was further screened on-chip for toxicity and cryoprotection. The results were discussed to illustrate the capabilities and limitations of the EWOD platform for cell and tissue-type specific optimization of CPA mixtures and F/T protocols.

SUBMITTER: Park S

PROVIDER: S-EPMC3138493 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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On-chip characterization of cryoprotective agent mixtures using an EWOD-based digital microfluidic device.

Park Sinwook S Wijethunga Pavithra A L PA Moon Hyejin H Han Bumsoo B

Lab on a chip 20110520 13

For tissue engineering and regenerative medicine, cryopreservation, a technique for preserving biomaterials in the frozen state with cryoprotective agents (CPAs), is critically important for preserving engineered tissues (ETs) as well as cells necessary to create ETs. As more diverse ETs are produced using various cell types, CPAs and corresponding freeze/thaw (F/T) protocols need to be developed cell/tissue-type specifically. This is because CPAs and F/T protocols that have been successful for ...[more]

PMID: 21603697

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Project description:Access to diverse PET tracers for preclinical and clinical research remains a major obstacle to research in cancer and other disease research. The prohibitive cost and limited availability of tracers could be alleviated by microfluidic radiosynthesis technologies combined with a high-yield microscale radiosynthetic method. In this report, we demonstrate the multistep synthesis of 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) with high yield on an electrowetting-on-dielectric (EWOD) microfluidic radiosynthesizer, previously developed in our group. We have identified and established several parameters that are most critical in the microscale radiosynthesis, such as the reaction time, reagent concentration, and molar ratios, to successfully synthesize (18)F-FLT in this compact platform.(18)F-FLT was synthesized from the 3-N-Boc-1-[5-O-(4,4'-dimethoxytrityl)-3-O-nosyl-2-deoxy-?-D-lyxofuranosyl] thymine precursor on the EWOD chip starting from the first solvent exchange and (18)F-fluoride ion activation step to the final deprotection step. The fluorination reaction was performed in a mixture of thexyl alcohol and dimethyl sulfoxide. The crude product after deprotection was collected from the chip and purified on a custom-made solid-phase extraction cartridge and subjected to quality control testing. The purified (18)F-FLT was suitable for small-animal PET studies in multiple nude mice xenografted with the A431 carcinoma cell line.(18)F-FLT was successfully synthesized on the EWOD microdevice coupled with an off-chip solid-phase extraction purification with a decayed-corrected radiochemical yield of 63% ± 5% (n = 5) and passed all of the quality control tests required by the U.S. Pharmacopeia for radiotracers to be injected into humans. We have successfully demonstrated the synthesis of several batches of (18)F-FLT on EWOD, starting with approximately 333 MBq of radioactivity and obtained up to 52 MBq (non-decay-corrected) of (18)F-FLT on cartridge purification. The specific activity of 2 representative preparations of (18)F-FLT synthesized on the EWOD chip were measured to be 1,800 and 2,400 GBq/?mol.The EWOD microchip and optimized synthesis method in combination represent an effective platform for synthesizing (18)F-FLT with high yield and of good quality for imaging. This compact platform, with configurable synthesis steps, could potentially form the basis of a stand-alone system that decouples PET probe production from the cyclotron and specialized radiochemistry facilities and increases diversity and flexibility in probe production.

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On-chip characterization of cryoprotective agent mixtures using an EWOD-based digital microfluidic device.

Publications

On-chip characterization of cryoprotective agent mixtures using an EWOD-based digital microfluidic device.

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