Bone marrow processing using the fenwal CS-3000 plus blood cell separator: results of 99 procedures.

BMT is used as an established therapy for patients with malignant and nonmalignant diseases. Many techniques for ex vivo treatment have been developed, but these techniques must be preceded by BM processing. We report our experience in processing 99 BM using the Fenwal CS-3000 Plus cell separator using the 1-special program. Ninety-nine procedures were performed in BM harvested from 73 patients and 26 healthy donors. The number of nucleated cells (NC), mononuclear cells (MNC), RBC, platelets, colony-forming units-granulocyte-macrophage (CFU-GM), CD34+ cells, relative purity of MNC and PMN, and volume were determined in the unprocessed BM and in the final product. BM processing resulted in NC, MNC, CFU-GM, and CD34+ cell recoveries of 31%, 82.2%, 117.6%, and 97.8%, respectively. RBC, PMN, platelets, and volume removal, respectively, were 96%, 92%, 37.2%, and 85.1%. In pediatric patients, the volume reduction was significantly lower than in adult patients (79.6% versus 88.8%). No other significant differences were found between pediatric and adult results. We conclude that BM processing with the Fenwal CS-3000 Plus cell separator provides a product that can undergo further ex vivo treatments or cryopreservation.

Ex-vivo expansion and maturation of CD34-positive hematopoietic progenitors optimization of culture conditions.

The aim of this study was to look for an ex vivo culture system for clinical application. We evaluated the ex vivo expansion of peripheral blood CD34+ cells in gas-permeable bags and whether or not an exogenous protein source would be required in these kind of cultures. We also evaluated maturation of the cells during culture. Cells were cultured for 15 days in medium supplemented with SCF, G-CSF, IL3 and IL6. The bags supported the expansion of hematopoietic cells in a similar manner to small scale flasks system: (a) the expansion means of total nucleated cells on day +5 were 12.5-fold for bag versus 5-fold for flask, on day +10 were 44.12-fold for bag versus 41-fold for flask and on day +15 were 67.7-fold for bag versus 84.2-fold for flask, (b) the peak values of CFU-GM were reached on day +10 (9.2-fold for bag vs. 12-fold for flask), and (c) maximal expansion of CD15+/CD11b- population occurred on day +10 (517.5-fold for bag vs. 2959.2-fold for flask). So, we did not find any advantages by culturing further than day +10. We subsequently investigated the use of serum-free medium. The study showed better results when we used medium supplemented with autologous plasma versus serum-free system. In summary, these data described a strategy of culture clinically feasible and safe, using gas-permeable bags, and the kinetics and differentiation of neutrophils and neutrophil precursors from selected CD34+ cells in liquid cultures. Ex vivo expansion of this population might result in earlier engraftment as compared with that for selected stem cells alone.

[Evaluation of bone-marrow cell cryopreservation using long-term cultures]. / Valoración de la criopreservación de células de medula ósea mediante los cultivos celulares a largo término.

PURPOSE: Autologous bone marrow transplantation (ABMT) is frequently used in the treatment of neoplastic diseases. It involves several manipulations of bone marrow cells in vitro that can damage the stem cells responsible for grafting. Long-term marrow cultures (LTBMC) support hematopoiesis in vitro for several weeks. We analyzed the effect of bone marrow cryopreservation on haematopoiesis when using this technique. PATIENTS AND METHODS: 9 bone marrow from healthy donors (Group A) and 15 from patients who were about to undergo ABMT (Group B) were assayed in LTBMC. In all cases, cultures were initiated after cell concentration and also after cryopreservation in group B patients. Adherent cell layer formation, supernatant nucleated cell counts and CFU-GM growth from the non-adherent fraction were assessed. Statistical analysis were evaluated using Wilcoxon test for paired results and Mann-Whitney test for unpaired results. RESULTS: No significant statistical differences were observed when the LTBMC from group A controls were compared to those from group B patients prior to cell cryopreservation. There was a significant statistical difference between cumulative cell recoveries among the cultures developed prior to and after cryopreservation of BM cells from group B patients. CONCLUSION: The results supports the use of LTBMC to obtain information on the extent of the injury of BM cells during manipulations.