69. All that’s needed is inside the cell: New method to measure permeability of red blood cell membrane using intrinsic fluorescence

Abstract

To design effective cryopreservation procedures for cells and tissues, it is critical to know permeability of cell membrane to water and solutes. To determine the latter, one needs to measure the rate of cell volume changes when placed in anisotonic environment. Red blood cells (RBCs) respond very quickly to changes in extracellular solutes concentration, which makes it difficult to measure the rate of RBC volume change using traditional methods. Stopped-flow method captures rapid changes of RBC volume and is a common method to measure RBC osmotic permeability. There are two types of stopped-flow methods: the first measures RBC volume based on changes in the intensity of light scattered by RBCs (Sidel and Solomon, 1957), the second measures it based on the self-quenching of fluorescent dye entrapped inside RBCs (Chen et al., 1988). However, both techniques have flaws. A number of factors other than cell volume influence the intensity of scattered light (Chen et al., 1988). Absorbance of a significant portion of emitted fluorescent light by the broad spectrum of hemoglobin complicates the fluorescent quenching technique. Literature shows that RBC hemoglobin has autofluorescent properties (Hirstch, 2003). This work explored the possibility of using intrinsic fluorescence of intracellular hemoglobin to measure RBC permeability to water and solutes. Network Center for Applied Development in Vancouver provided adult RBCs for this study. We used SX20 stopped-flow reaction analyser (Applied Photophysics) to measure changes in RBC autofluorescence when mixed with sodium chloride or glycerol solutions of different concentration (excitation 280 nm, emission 314 nm). Equilibrium volumes of RBCs in sodium chloride solutions were measured on Coulter Counter Model ZBI (Coulter Electronics, Inc.). Lysed RBCs served as a negative control to verify that changes in autofluorescence intensity are caused by the changes in volume of intact RBCs. We also measured absolute values of autofluorescence as a function of hemoglobin concentration in the solution using SpectraMax GEMINI EM dual-scanning microplate spectrofluorometer (Molecular devices). Our results showed that the intensity of RBC autofluorescence and RBC volume measured on Coulter Counter were directly related. Specifically, mixing RBCs with hypotonic solution caused increase in cell volume and gradual increase in autofluorescence; cell shrinking in hypertonic solution resulted in autofluorescence decrease. On the contrary, tonicity of solution had no effect on autofluorescence of lysed RBCs. When RBCs were injected in glycerol solution, autofluorescence first dropped, then gradually increased, and finally reached a plateau. This response is similar to the well-known pattern of cell volume changes upon mixing with permeable cryoprotectant. We also found that at high (intracellular) hemoglobin concentrations fluorescence intensity and hemoglobin concentration are inversely related. Combined, our data shows that RBC autofluorescence is directly translated to RBC volume, and self-quenching of intracellular hemoglobin is responsible for the cell volume driven changes in RBC autofluorescence. Based on these findings, we are working on developing a new method to determine osmotic permeability of RBCs, which will significantly help with future design and optimization of cryopreservation protocols.