Automated dry thawing of cryopreserved haematopoietic cells is not adversely influenced by cryostorage time, patient age or gender.

Cell therapies are becoming increasingly widely used, and their production and cryopreservation should take place under tightly controlled GMP conditions, with minimal batch-to-batch variation. One potential source of variation is in the thawing of cryopreserved samples, typically carried out in water baths. This study looks at an alternative, dry thawing, to minimise variability in the thawing of a cryopreserved cell therapy, and compares the cellular outcome on thaw. Factors such as storage time, patient age, and gender are considered in terms of cryopreservation and thawing outcomes. Cryopreserved leukapheresis samples from 41 donors, frozen by the same protocol and stored for up to 17 years, have been thawed using automated, water-free equipment and by conventional wet thawing using a water bath. Post-thaw viability, assessed by both trypan blue and flow cytometry, showed no significant differences between the techniques. Similarly, there was no negative effect of the duration of frozen storage, donor age at sample collection or donor gender on post-thaw viability using either thawing method. The implication of these results is that the cryopreservation protocol chosen initially remains robust and appropriate for use with a wide range of donors. The positive response of the samples to water-free thawing offers potential benefits for clinical situations by removing the subjective element inherent in water bath thawing and eliminating possible contamination issues.

Efficient human cytomegalovirus reactivation is maturation dependent in the Langerhans dendritic cell lineage and can be studied using a CD14+ experimental latency model.

Studies from a number of laboratories have shown that the myeloid lineage is prominent in human cytomegalovirus (HCMV) latency, reactivation, dissemination, and pathogenesis. Existing as a latent infection in CD34(+) progenitors and circulating CD14(+) monocytes, reactivation is observed upon differentiation to mature macrophage or dendritic cell (DC) phenotypes. Langerhans' cells (LCs) are a subset of periphery resident DCs that represent a DC population likely to encounter HCMV early during primary infection. Furthermore, we have previously shown that CD34(+) derived LCs are a site of HCMV reactivation ex vivo. Accordingly, we have utilized healthy-donor CD34(+) cells to study latency and reactivation of HCMV in LCs. However, the increasing difficulty acquiring healthy-donor CD34(+) cells--particularly from seropositive donors due to the screening regimens used--led us to investigate the use of CD14(+) monocytes to generate LCs. We show here that CD14(+) monocytes cultured with transforming growth factor ß generate Langerin-positive DCs (MoLCs). Consistent with observations using CD34(+) derived LCs, only mature MoLCs were permissive for HCMV infection. The lytic infection of mature MoLCs is productive and results in a marked inhibition in the capacity of these cells to promote T cell proliferation. Pertinently, differentiation of experimentally latent monocytes to the MoLC phenotype promotes reactivation in a maturation and interleukin-6 (IL-6)-dependent manner. Intriguingly, however, IL-6-mediated effects were restricted to mature LCs, in contrast to observations with classical CD14(+) derived DCs. Consequently, elucidation of the molecular basis behind the differential response of the two DC subsets should further our understanding of the fundamental mechanisms important for reactivation.

Autologous infusion of expanded mobilized adult bone marrow-derived CD34+ cells into patients with alcoholic liver cirrhosis.

OBJECTIVES: Recent advances in regenerative medicine, including hematopoietic stem cell (HSC) transplantation, have brought hope for patients with severe alcoholic liver cirrhosis (ALC). The aim of this study was to assess the safety and efficacy of administering autologous expanded mobilized adult progenitor CD34+ cells into the hepatic artery of ALC patients and the potential improvement in the liver function. METHODS: Nine patients with biopsy-proven ALC, who had abstained from alcohol for at least 6 months, were recruited into the study. Following granulocyte colony-stimulating factor (G-CSF) mobilization and leukapheresis, the autologous CD34+ cells were expanded in vitro and injected into the hepatic artery. All patients were monitored for side effects, toxicities, and changes in the clinical, hematological, and biochemical parameters. RESULTS: On average, a five-fold expansion in cell number was achieved in vitro, with a mean total nucleated cell count (TNCC) of 2.3 x 10(8) pre infusion. All patients tolerated the procedure well, and there were no treatment-related side effects or toxicities observed. There were significant decreases in serum bilirubin (P < 0.05) 4, 8, and 12 wk post infusion. The levels of alanine transaminase (ALT) and aspartate transaminase (AST) showed improvement through the study period and were significant (P < 0.05) 1 wk post infusion. The Child-Pugh score improved in 7 out of 9 patients, while 5 patients had improvement in ascites on imaging. CONCLUSION: It is safe to mobilize, expand, and reinfuse autologous CD34+ cells in patients with ALC. The clinical and biochemical improvement in the study group is encouraging and warrants further clinical trials.