Membrane stabilization versus perturbation by aromatic monoamine-modified γ-PGA for cryopreservation of human RBCs with high intracellular trehalose

Abstract

As a nonreducing disaccharide, trehalose can be used as a biocompatible cryoprotectant for solvent-free cell cryopreservation, but the membrane-impermeability limits its cryoprotective efficiency. Herein, a series of aromatic monoamines with...

Integration of Trehalose Lipids with Dissociative Trehalose Enables Cryopreservation of Human RBCs

“…After incubation and cryopreservation, the samples were centrifuged at 1000 g for 7 min, and the absorbance ( A ) of the supernatant was measured via a spectrophotometer (Tecan, Switzerland) at 540 nm. The hemolysis was calculated according to eq − hemolysis 0.25em ( % ) = A sample A positive × 100 % …”

“…For the frozen samples, cells were collected before and after cryopreservation, lysed with Triton X-100, the absorbance of the lysate was measured, and the cryosurvival was calculated according to eq − cryosurvival false( % false) = cell 0.25em count 0.25em after 0.25em freeze − thaw cell 0.25em count 0.25em after 0.25em incubation × 100 % …”

“…After incubation and cryopreservation, the osmotic stability of hRBCs was measured by the osmotic fragility test. ,, The hemolysis of samples was determined by suspending 0.1 mL of concentrated hRBCs in 1 mL of NaCl solution at ∼9% HCT in different osmolarities at room temperature for 30 min and centrifuged at 1000 g for 7 min. The fragility index (C 50 ) was referred to the osmolarity at 50% hemolysis.…”

“…Along with the aforementioned hypotheses, it has been demonstrated that trehalose offers optimal cryoprotection when dispersed on both sides of the cell membrane . Consequently, there have been considerable works exploring strategies to promote intracellular delivery of nonpermeable trehalose. − For erythrocytes without the ability of endocytosis, different methods such as osmotic stress, electroporation, polymers, and peptides have been employed to facilitate intracellular trehalose uptake. For instance, an electroporation technology was attempted to introduce trehalose into hRBCs by allowing electrical pulses to pass through the cell membrane .…”

“…Synthetic polymers have been investigated as facilitators of trehalose entry into the cytoplasm for their capacity to interact with cell membranes, giving rise to transient permeability. Especially, a pH-responsive polymer, poly­( l -lysine iso -phthalamide) grafted with phenylalanine, called PP-50, was studied that could improve the membrane permeability to trehalose. − Our previous studies suggested that hydrophobically modified glycopeptides and ε-poly­( l -lysine) could effectively enhance intracellular trehalose uptake. − It should be emphasized that the suitable value of intracellular trehalose is still unknown, and adverse effects could happen during intracellular trehalose loading at 37 °C in ovine or human erythrocytes. , Because the temperature-dependent cell metabolism can be reduced by about 40 times, a series of our previous works on intracellular trehalose uptake were carried out at 4 °C via incubation of hRBCs with amphiphilic polymers or biosurfactants. − The incubation time was generally prolonged to around 24 h to achieve an effective interaction of facilitators with cell membranes to promote trehalose entry into cells. − …”