A Low-cost Easy-to-Fabricate Sandwich-Structured Microdevice for Controllable Removal of Extracellular Cryoprotective Agents with High Efficiency.

　　BACKGROUND: Osmotic shock upon the addition and removal of cryoprotectant agent (CPA) is a major source of cell damage during cryopreservation. OBJECTIVE: Microfluidic device offers a new platform for CPA loading and unloading. The micro scale dimension makes possible to perform a detailed analysis and controllable removal of CPA with many advantages. MATERIALS AND METHODS: A microfluidic device was developed for extracting dimethyl sulfoxide (DMSO) from the sample streamline. The device has two parallel channels separated by a polytetrafluoroethylene (PTFE) membrane and serves as the stable environment for CPA removal. A diffusion-based simulation model was used to characterize the CPA extraction. To support the experimental design and device optimization we developed analogous scheme to simulate by COMSOL Multiphysics. RESULTS AND CONCUSION: The device can extract cryoprotectant in a mesoscale volume from cells and simplify the post-thaw sample handling. It has sufficient control on loading/unloading of CPAs by controlling the flow rate of cell stream/wash stream solutions via syringe pumps. Compared to other customary devices, this device is easy to fabricate and assemble, with features of high precision, reusability and low cost.

Water-transport and Intracellular Ice Formation of Porcine Adipose-derived Stem Cells during Freezing.

BACKGROUND: Water transport and intracellular ice formation are important processes that relate to cryoinjury of cells upon freezing. To date, no study is reported on the characteristics of water transport and intracellular ice formation in porcine adipose-derived stem cells (pADSC). OBJECTIVE: To study water transport and intracellular ice formation upon freezing of pADSCs at different cooling rates. MATERIALS AND METHODS: The pADSCs were isolated using collagenase digestion from a subcutaneous adipose tissue of a 28-day-old Landrace pig. Freeze experiments were performed in a gas tight chamber of cryomicroscopy stage at different cooling rates between 40°C and -150°C. RESULTS: Water permeability coefficient Lpg and the activation energy ELP decrease with increasing cooling rates for pADSCs. The probability of intracellular ice formation increases with increasing cooling rates, being 0.35, 0.4 and 0.5 for cooling rates at 20, 30 and 60 °C/min respectively. CONCLUSION: Based on the characteristics of water transport and intracellular ice formation in pADSCs, slow freezing is perhaps more suitable for pADSC cryopreservation.