Comparative Study on Two Different Methods for Determination of Hydraulic Conductivity of HeLa Cells During Freezing

The measurement of hydraulic conductivity of the cell membrane is very important for optimizing the protocol of cryopreservation and cryosurgery. There are two different methods using differential scanning calorimetry (DSC) to measure the freezing response of cells and tissues. Devireddy et al. presented the slow-fast-slow (SFS) cooling method, in which the difference of the heat release during the freezing process between the osmotically active and inactive cells is used to obtain the cell membrane hydraulic conductivity and activation energy. Luo et al. simplified the procedure and introduced the single-slow (SS) cooling protocol, which requires only one cooling process although different cytocrits are required for the determination of the membrane transport properties. To the best of our knowledge, there is still a lack of comparison of experimental processes and requirements for experimental conditions between these two methods. This study made a systematic comparison between these two methods from the aforementioned aspects in detail.

The SFS and SS cooling methods mentioned earlier were utilized to obtain the reference hydraulic conductivity (L_pg) and activation energy (E_Lp) of HeLa cells by fitting the model to DSC data.

With the SFS method, it was determined that L_pg = 0.10 μm/(min·atm) and E_Lp = 22.9 kcal/mol; whereas the results obtained by the SS cooling method showed that L_pg = 0.10 μm/(min·atm) and E_Lp = 23.6 kcal/mol.

The results indicated that the values of the water transport parameters measured by two methods were comparable. In other words, the two parameters can be obtained by comparing the heat releases between two slow cooling processes of the same sample according to the SFS method. However, the SS method required analyzing heat releases of samples with different cytocrits. Thus, more experimental time was required.