Study On Membrane Permeability Of Cell By Using Calorimetry And Microfluidic Perfusion Method

Cell cryopreservation mainly consists of the following steps: cryoprotective agents(CPAs)addition,cryogenically freezing,cryogenically preservation,rewarming and CPAs dilution.The permeability parameters of cell membrane can be used to predict the change of cell volume during freezing,rewarming,CPAs addition and dilution,thus optimize the effectiveness of cryopreservation.Measuring permeability parameters is one of the fundamental studies in the field of cryobiology.Currently the available methods to measure these parameters includes thermal analysis and microfluidic perfusion method.Thermal analysis method determines the membrane permeability of cell indirectly by analyzing thermal signal measured via differential scanning calorimeter(DSC).It is always used to measure permeability of cell on large scale and primarily falls into SlowFast-Slow(SFS)freezing and Single-Slow(SS)freezing method.Permeability parameters measured by these two methods were very similar and fell in reasonable range.The measured permeability parameters were used to predict the optimal cooling rate and simulate the change of cell volume at several cooling rates.Then the verified experiment indicated that the simulated water transport behavior was in good agreement with experimental data,and the measured parameter can predict the change of cell volume effectively.It was found that though the temperature procedures of SS method are simplified,the response of cell volume at different cooling rates showed no distinct difference.Also,SFS method involves no requirement of samples with different cytocrits,and the experimental process and data processing are much easier to perform.For comparative purpose,the microfluidic perfusion method was also employed to measure membrane permeability.It directly observes the change of cell volume by analyzing images and is always applied to few samples.The microperfusion system was used to determine membrane permeability during one-and three-step addition of CPA.The results of one-step addition was used to predict the change of cell volume during three-step addition.It was indicated that CPA molecules can dramatically reduce the water conductivity.Also,three-step addition of CPA can facilitate the transmembrane transport of water and CPA molecules,wherein the facilitation of CPA molecules is more significant.Moreover,the prediction based on parameters measured by one-step addition shows smaller volume of shrinkage and wider excursion of cell volume.