Quantitative Measurements of Cryobiological Characteristics of Mouse Dendritic Cells and Its Evaluation Using Commercialized Coulter Counter

Abstract

Quantitative measurements of cell osmotic behavior and membrane transport properties is critical for the development of cell-type-specific, optimal cryopreservation conditions. A microfluidic perfusion system has been developed here to measure the kinetic changes of cell volume under various extracellular conditions, in order to determine cell osmotic behavior and membrane transport properties. The system is fabricated using soft lithographic techniques and is composed of inlets, outlets, micchannels and a perfusion chamber for trapping cells. In this study Dendritic cells (DCs) are using as a model to validate our microfluidic system with a commercialized Beckham Coulter Counter (Multisizer 3). DCs are antigen presenting cells that have been increasingly used in immunotherapy for the treatment of various diseases. Cryopreservation and banking of DCs is critical to facilitate flexible and effective immunotherapy treatment. Using mouse DCs (MDC), membrane transport properties were first investigated using our microfluidic perfusion system. Cells in the microfluidic system were perfused with 3x phosphate buffer solution. The kinetics of cell volume changes under the specific extracellular conditions were monitored by a digital camera and analyzed using a biophysical model to determine water and cryoprotectant transport properties of the cell membrane. DCs were later tested using Beckman Coulter Counter, where the kinetic osmotic behaviors of cells were quantified through the correlation between electric pulses and their corresponding cell sizes. It was shown from this study that the cryobiological characteristics of DCs determined using microfluidic perfusion system and Coulter Counter agreed well.