C-2016: A study of fundamental cryobiological characteristics of human vaginal immune cells for optimizing their functional cryopreservation

Abstract

Cryopreservation of collected human vaginal mucosal immune specimens is vital to the successful analysis of mucosal immunity and HIV trials. However, the mucosal immune specimens cannot be well cryopreserved with current empirical procedures. The optimum cryopreservation protocol is determined by the cryobiological features of the cells. Thus, a cryobiological study of human female genital mucosal immune cells (FG-MIC) is crucial towards the successful specimen cryopreservation. In this work, CD3 + T cells and CD19 + macrophages were investigated. The intracellular osmotically inactive volume (V b ) was determined by exposing the cells to hypertonic saline solutions, evaluating the volume changes after equilibrium and applying the Boyle–van’t Hoff plot. At room temperature, the cell membrane permeability to water (L p ) and to four different CPAs (dimethyl sulfoxide (Me 2 SO), glycerol, propylene glycol (PG) and ethylene glycol (EG)) were measured with a microfluidic perfusion channel. Osmotic tolerance limits of the cells to CPAs and the CPA toxicities were also examined. At sub-zero temperatures (0 to −40 °C), the L p (T) and the associated activation energy E a of cell membranes were determined using a Differential Scanning Calorimetry (DSC). The “Slow–Fast–Fast–Slow” DSC scanning program, similar to the method of Devireddy and Bischof, was applied. Some preliminary cryopreservation trials were also conducted. Results showed that mucosal immune cells have relatively small V b (14.1% for T cells and 23.8% for macrophages). T cells are more vulnerable to stresses of osmotic change and freezing compared to macrophages. Among the four CPAs, Me 2 SO and PG have higher P s values and lower toxicity to the cells, which indicates that Me 2 SO and PG potentially can be better CPA options to avoid lethal IIF and severe cell dehydration with less toxicity during freezing. Based on the measured cell properties, a freezing protocol with slow cooling rate in a critical temperature range was predicted and experimentally demonstrated by preliminary cryopreservation results.