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Klaus Schulze-Osthoff and Marek Los share senior authorship. ABSTRACTCryopreserved cells and tissues are increasingly used for stem cell transplantation and tissue engineering. However, their freezing, storage, and thawing is associated with severe damage, suggesting the need for better cryopreservation methods. Here, we show that activation of caspase-3 is induced during the freeze-thaw process. Moreover, we demonstrate that prevention of caspase activation by the caspase inhibitor zVAD-fmk strongly improves the recovery and survival of several cryopreserved cell types and hematopoietic progenitor cells. A short preincubation with the caspase inhibitor after thawing also enhances the colony-forming activity of hematopoietic progenitor cells up to threefold. Furthermore, overexpression of Bcl-2, but not the blockade of the death receptor signaling, confers protection, indicating that cryoinjuryassociated cell death is mediated by a Bcl-2-controlled mitochondrial pathway. Thus, our data suggest the use of zVAD-fmk as an efficient cryoprotective agent. The addition of caspase inhibitors may be an important tool for the cryopreservation of living cells and advantageous in cell transplantation, tissue engineering, and other genetic technologies.Keywords: apoptosis • cryopreservation • stem cells • cathepsins • calpains W ith the recent advances in cell and tissue transplantation, tissue engineering, in vitro fertilization, and other genetic technologies, preservation of biological materials has become increasingly important in medical care and biotechnological industries. Cryopreservation is the only reliable form of long-term storage of viable cells and tissues. However, both the freeze and the thaw process result in considerable cell and tissue injury (1, 2). Various mechanisms, including oxidative stress, mechanical injury due to ice crystal formation, altered physical properties of cellular structures, osmotic injury, and disturbed ion homeostasis due to Na + /K + -ATPase inhibition are responsible for cell damage during hypothermia and freezethaw processes (3)(4)(5)(6)(7)(8). Traditional protocols rely on supplementation of the freeze medium with penetrating cryoprotectants such as dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, or hydroxyethyl starch (2). Their major role is the prevention of lethal ice formation and osmotic injury. Stabilization of cellular structures with small carbohydrate sugars such as trehalose has recently been shown to also markedly improve cell recovery (9). Despite significant improvements of cryopreservation protocols, clinical procedures are often not satisfactory, and up to 50% of the cryopreserved cells may die within the first 24 h after the thaw process (10). There are some indications that hypothermia is associated with apoptosis (9, 11), although the role of apoptosis in cryoinjury has not been studied in detail. Because inhibition of proteases of the caspase family protects against apoptosis in various experimental settings (12-15), we examined whether addition of the ...

These findings indicate an influence of blood group differences in PBPCT on survival and must be studied in further detail.

Data of 507 granulocyte donations from 183 donors were evaluated. No severe granulocyte colony-stimulating factor (G-CSF)-related side-effects were observed. Three donors complained of severe itching following infusion of hydroxyethyl starch (HES). A high proportion (85%) of the donors stated that they would donate granulocytes again. The mean granulocyte yield was 4.3 x 10(10). High-molecular-weight HES resulted in a significantly higher yield compared with low-molecular-weight HES. Mild, but no severe, adverse transfusion reactions were observed in 16% of the recipients. A leucocyte alloimmunization rate of 24% was found. G-CSF stimulation and transfusion of G-CSF-mobilized granulocytes were well tolerated by donors and recipients, respectively.