Stephen A. Livesey

Biochemical stabilization enhances red blood cell recovery and stability following cryopreservation

Glycerolized red blood cells (RBC) are approved for long-term cryopreservation. However, the need to remove the glycerol cryoprotectant prior to transfusion has limited the usefulness of this cryopreservation method. This report describes using non-cryoprotectant biochemical stabilization techniques to substitute for the standard glycerol cryoprotectant. The glycerolized RBC method was compared to a newly developed LC-V method that combines transfusable cryoprotectants (hydroxyethyl starch and dextran) and specific non-cryoprotectant biochemical stabilizers (nicotinamide, nifedipine, and flurbiprofen). Results demonstrate that the biochemical stabilizers significantly reduce cryopreservation-induced hemolysis compared to cryopreservation in their absence and that thaw hemolysis levels approach those of standard 40% (w/v) glycerolized RBC (3.1+/-0.2% for 40% glycerol compared to 8.7+/-0.9% for the LC-V protocol). Furthermore, LC-V cryopreserved RBC exhibit a significantly enhanced post-thaw stability compared to glycerolized RBC as determined by osmotic fragility index (0.557+/-0.034 for 40% glycerol compared to 0.478+/-0.016 for the LC-V protocol). Analysis of biochemically stabilized RBC proteins revealed a transient translocation of carbonic anhydrase to the membrane fraction. However, the enhanced RBC recovery and stability could not be attributed to this event. Finally, DSC analysis demonstrated that the biochemical stabilizers of the LC-V process were not functioning as surrogate cryoprotectants in that they did not affect the quantity or quality of ice formed. Overall, this work demonstrates that cryopreservation-induced RBC damage may be corrected or prevented through specific biochemical stabilization and represents a significant step toward a directly transfusable cryopreserved RBC product.

The dynamics of adult haematopoiesis in the bone and bone marrow environment

This review explores the dynamic relationship between bone and bone marrow in the genesis and regulation of adult haematopoiesis and will provide an overview of the haematopoietic hierarchical system. This will include the haematopoietic stem cell (HSC) and its niches, as well as discuss emerging evidence of the reciprocal interplay between bone and bone marrow, and support of the pleiotropic role played by bone cells in the regulation of HSC proliferation, differentiation and function. In addition, this review will present demineralized bone matrix as a unique acellular matrix platform that permits the generation of ectopic de novo bone and bone marrow and provides a means of investigating the temporal sequence of bone and bone marrow regeneration. It is anticipated that the utilization of this matrix‐based approach will help researchers in gaining deeper insights into the major events leading to adult haematopoiesis in the bone marrow. Furthermore, this model may potentially offer new avenues to manipulate the HSC niche and hence influence the functional output of the haematopoietic system.

Freeze-Dried, Decellularized Goat Arterial Allograft

We investigated the fate of decellularized, 3-4-mm-diameter, arterial allografts transplanted in the goat carotid. Eighteen goats received a decellularized, carotid allograft in one carotid and a fresh autologous cephalic vein graft as a control in the other carotid. Half the grafts were explanted at 3 months, and the remainder at 6 months. Patency was 100% for both graft types at both time points. At explant the processed arterial grafts were uniform in diameter without evidence of dilation. In contrast, all autologous vein grafts exhibited dilation with aneurysm formation in a majority of the grafts. Cells lining the graft lumen were positive for Factor VIII, an endothelial cell marker; α-smooth muscle cell actin-positive cells were present in the media, and cells positively staining for vimentin (marker for fibroblasts) were located in the adventitia. The midportion of the processed grafts had significantly less intimal thickness at both time points as compared with vein grafts (48 ± 92 μm vs. 274 ± 21...

Instability of Frozen Human Erythrocytes at Elevated Temperatures

Frozen cells are known to be unstable at elevated subzero temperatures; however, the kinetics of cell damage as a function of storage temperature and time are not well understood. The present study investigated the instability of frozen human erythrocytes during isothermal storage at elevated subzero temperatures. The relationship between the instability of frozen cells and the temperature-dependent state/phase transitions in frozen domains was examined. Human erythrocytes were cryopreserved with 12% (wt/vol) hydroxyethyl starch in phosphate-buffered saline solution by plunging into liquid nitrogen, and were then isothermally stored at elevated subzero temperatures. Hemolysis following thawing and dilution was used as an indicator of cell damage during isothermal storage. The instability of frozen cells was found to conform to a special form of the Johnson–Mehl–Avrami model, H(T, t) = H(T)[1 - exp( -kt)], where H(T, t) represented the percent hemolysis at temperature T after time t, H(T) was the maximal h...