The effect of liposome treatment on the quality of hypothermically stored red blood cells.

Recent studies have demonstrated that liposome treatment of red blood cells (RBCs) leads to improved recovery and membrane integrity following cryopreservation protocols. However, the effect of liposome treatment on hypothermically stored RBCs has not been previously investigated. The current study has investigated whether liposome treatment could modify the membrane quality and deformability of hypothermically stored RBCs. Unilamellar liposomes were synthesized using an extrusion protocol. Three lipid bilayer compositions were investigated: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC):PE:PS (8:1:1); 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC):PE:PS (8:1:1); and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC):PE:PS (8:1:1). RBCs were treated with liposomes and subsequently stored for 42 days in HEPES-NaCl buffer and saline-adenine-glucose-mannitol. RBC quality was assessed by percent hemolysis, mean corpuscular volume (MCV), and RBC deformability (ektacytometry). DOPC and DMPC liposome treatment resulted in destabilization of the RBC membrane. Percent hemolysis values for DMPC-treated RBCs were higher than untreated controls throughout storage (P<0.05). DOPC-treated RBCs showed elevated levels of hemolysis compared to controls from day 21 of storage onward (P<0.05). In addition, DOPC and DMPC-treated RBCs were less deformable than untreated controls from days 21(P=0.02) and 14 (P<0.001) of storage onward respectively. [We suggest that these changes in RBC hemolysis and deformability are due to cholesterol extraction from the RBC membrane into the liposome fraction.] In contrast, DPPC-treated RBCs maintained hemolysis, MCV, and deformability values comparable to untreated controls. Future research addressing the optimal liposome composition for stabilizing the RBC membrane at cold temperatures could lead to effective strategies to combat the RBC membrane hypothermic storage lesion and ultimately improve the quality of hypothermically preserved blood.

Freeze-thaw induced biomechanical changes in arteries: role of collagen matrix and smooth muscle cells.

Applications involving freeze-thaw, such as cryoplasty or cryopreservation can significantly alter artery biomechanics including an increase in physiological elastic modulus. Since artery biomechanics plays a significant role in hemodynamics, it is important to understand the mechanisms underlying these changes to be able to help control the biomechanical outcome post-treatments. Understanding of these mechanisms requires investigation of the freeze-thaw effect on arterial components (collagen, smooth muscle cells or SMCs), as well as the components' contribution to the overall artery biomechanics. To do this, isolated fresh swine arteries were subjected to thermal (freeze-thaw to -20 degrees C for 2 min or hyperthermia to 43 degrees C for 2 h) and osmotic (0.1-0.2 M mannitol) treatments; these treatments preferentially altered either the collagen matrix (hydration/stability) or smooth muscle cells (SMCs), respectively. Tissue dehydration, thermal stability and SMC functional changes were assessed from bulk weight measurements, analyses of the thermal denaturation profiles using Fourier transform infrared (FTIR) spectroscopy and in vitro arterial contraction/relaxation responses to norepinephrine (NE) and acetylcholine (AC), respectively. Additionally, Second Harmonic Generation (SHG) microscopy was performed on fresh and frozen-thawed arteries to directly visualize the changes in collagen matrix following freeze-thaw. Finally, the overall artery biomechanics was studied by assessing responses to uniaxial tensile testing. Freeze-thaw of arteries caused: (a) tissue dehydration (15% weight reduction), (b) increase in thermal stability (approximately 6.4 degrees C increase in denaturation onset temperature), (c) altered matrix arrangement observed using SHG and d) complete SMC destruction. While hyperthermia treatment also caused complete SMC destruction, no tissue dehydration was observed. On the other hand, while 0.2 M mannitol treatment significantly increased the thermal stability (approximately 4.8 degrees C increase in denaturation onset), 0.1 M mannitol treatment did not result in any significant change. Both 0.1 and 0.2 M treatments caused no change in SMC function. Finally, freeze-thaw (506+/-159 kPa), hyperthermia (268+/-132 kPa) and 0.2 M mannitol (304+/-125 kPa) treatments all caused significant increase in the physiological elastic modulus (Eartery) compared to control (185+/-92 kPa) with the freeze-thaw resulting in the highest modulus. These studies suggest that changes in collagen matrix arrangement due to dehydration as well as SMC destruction occurring during freeze-thaw are important mechanisms of freeze-thaw induced biomechanical changes.

Diffusion of dimethyl sulfoxide in tissue engineered collagen scaffolds visualized by computer tomography.

Cryopreservation is a convenient method for long-term preservation of natural and engineered tissues in regenerative medicine. Homogeneous loading of tissues with CPAs, however, forms one of the major hurdles in tissue cryopreservation. In this study, computer tomography (CT) as a non-invasive imaging method was used to determine the effective diffusion of Me2SO in tissue-engineered collagen scaffolds. The dimensions of the scaffolds were 30 x 30 x 10 mm3 with a homogeneous pore size of 100 microm and a porosity of 98%. CT images were acquired after equilibrating the scaffolds in phosphate buffered saline (PBS) and transferring them directly in 10% (v/v)Me2SO. The Me2SO loading process of the scaffold could thus be measured and visualized in real time. The experimental data were fitted using a diffusion equation. The calculated effective diffusion constant for Me2SO in the PBS loaded scaffold was determined from experimental diffusion studies to be 2.4 x 10(-6) cm2/s at 20 degrees C.