Successful cryopreservation of purified autologous CD34+ cells: influence of freezing parameters on cell recovery and engraftment.

Conventional hematopoietic stem cell cryopreservation methods use a DMSO concentration of 10%. However, cells manipulated ex vivo may require more refined freezing protocols adapted to the specific cell suspension. In this retrospective study, we evaluated the results obtained with CD34+ cells purified from peripheral blood of 39 patients on the CEPRATE SC System and frozen in 7.5% DMSO with a view to transplantation. The post-freezing recovery of progenitor cells was 89.4 +/- 27.87% for CD34+ cells, 59.13 +/- 36.93% for CFU-GM, and 53.49 +/- 40.71 for BFU-E. Neither the purity of the suspension nor the nucleated cell density during freezing was predictive of cell recovery. No difference was observed between cells stored in vials and bags. Thirty-seven patients transplanted with the concentrated CD34+ fraction received 4.46 x 10(6) CD34+ cells/kg and 33.04 x 10(4) CFU-GM/kg. The median time to granulocyte (>0.5 x 10(9)/l) and platelet (>50 x 10(9)/l) engraftment was 11 and 13 days, respectively. Only cell density and the infused number of CD34+ cells and CFU-GM were significantly related to hematological recovery. Our data suggest that purified CD34+ cells can be successfully cryopreserved in 7.5% DMSO and may represent a first step in establishing freezing parameters for selected CD34+ cells.

Characteristics of peripheral blood progenitor cells frozen after 24 hours of liquid storage.

Peripheral blood progenitor cells (PBPC) harvested for autologous transplantation are usually cryopreserved within 2-4 h of collection. However, there are conditions in which it would be useful to freeze PBPC after a liquid storage period. This study was performed to evaluate whether prior storage for just 24 h damages frozen PBPC. First, leukapheresis products were obtained from 9 patients and divided into three fractions. The first fraction was frozen within 2 h and used as a control. The second and the third fractions were stored either at 4 degrees C or at 22 degrees C for 24 h before freezing. Cell counts, CFU-GM values, and pH were studied after collection, after storage, and after cryopreservation. Similar results were obtained at 4 degrees C and 22 degrees C. However, pH decreased most markedly at 22 degrees C. Mean postcryopreservation CFU-GM recoveries were not significantly different and were, respectively, 74.03% (control), 96.39% (4 degrees C), and 80.33% (22 degrees C). These observations were confirmed in an additional study of frozen PBPC collected from 12 patients and previously stored for 24 h at 4 degrees C. These data indicate that blood progenitors may be stored for 24 h and subsequently frozen without quantitative and qualitative impairment.

Successful engraftment after autologous transplantation of 10-day cultured bone marrow activated by interleukin 2 in patients with acute lymphoblastic leukemia.

Disease recurrence remains the major problem in autologous bone marrow transplantation (BMT) for hematologic malignancies. To improve the therapeutic efficiency of autologous BMT, we investigated the use of autologous marrow activated in vitro with interleukin 2 (IL-2) to generate killer cells for in vivo purging. A feasibility trial was initiated in 5 patients with poor prognosis acute lymphoblastic leukemia, who were transplanted, after marrow ablative therapy, with autologous marrow cultured for 10 days with 10(3) units of IL-2/ml. A highly significant increase in NK activity and an induction of LAK activity were observed after incubation. Patients received 0.64 to 1.56 X 10(8) cultured BM cells/kg and 1.87 to 44.8 x 10(4) CFU-GM/kg. Four patients engrafted and achieved granulocyte counts > 0.5 x 10(9)/l on days 35, 24, 36 and 22 after transplant. Three of these patients showed platelet recovery to > 50 x10(9)/l on days 25, 42 and 40 after transplant. One patient remained thrombocytopenic until relapse. One patient died on day 12 after transplant. This study demonstrates that cultured BM activated with IL-2 can be used successfully for hematological rescue in the clinical setting.

Effect of human granulocyte colony stimulating factor on neuroblastoma cell growth.

We have studied the effect of recombinant human granulocyte colony-stimulating factor (G-CSF) on the growth of three different human neuroblastoma (NB) cell lines, using a clonal cell culture and a short proliferation test. The results have shown a lack of effect of G-CSF on the three cell lines. These data promote the use, in clinical trials, of G-CSF as adjunctive treatment in children with NB receiving intensive chemotherapy.

A simple, efficient washing procedure for cryopreserved human hematopoietic stem cells prior to reinfusion.

We describe our experience with a washing procedure used on cryopreserved, thawed bone marrow (BM) and peripheral blood stem cell (PBSC) grafts prior to autologous transplantation in 50 and 12 patients respectively. The procedure consists of a stepwise dilution with 2% human serum albumin and centrifugation performed either manually or using a blood cell processor (Cobe 2991). In vitro studies showed mean recoveries of 80.8% for BM nucleated cells and 73.9% for BM hematopoietic progenitors (CFU-GM). The corresponding recoveries for PBSC were 89.1 and 93.9%. After 4 h storage at +20 degrees C of the manipulated grafts, no significant loss of CFU-GM was observed. We conclude that the technique is simple and efficient for washing large numbers of hematopoietic stem cells. This method may avoid the clinical complications often arising with unwashed grafts.

Effects of irradiation on human platelets stored at 20 degrees C.

The morphologic and functional properties of platelets after irradiation with 2500 rads and storage, in first-generation containers, for 48 h in a liquid phase at 20 degrees C with continuous horizontal agitation have been analyzed and compared with a control group of the same platelets which were not irradiated. The preservation technique induced changes in the morphology and aggregation stimulated by ADP and collagen. However, no significant differences were found between the irradiated and non-irradiated groups. Irradiation is not a conditioning factor to add to the hazards of preserving platelets in a liquid phase.

Separation of large quantities of mononuclear cells from human blood using a blood processor.

A blood processor (IBM 2991) was used to separate lymphocytes from large volumes of blood. The procedure included the centrifugation of 200 ml whole blood on a density gradient. The results of this procedure were compared with those obtained with a manual procedure. Mononuclear cell (MNC) viability was preserved well in the two methods. But with the processor, recovery of MNC was better (63.5 +/- 2.5%) than with manual separation (26.5 +/- 4.1%). Monoclonal antibodies were used to identify the various cell subsets in the MNC fractions. No particular cell selection was observed when MNC fractions were obtained by the separator. In conclusion, the use of a cell separator provided an efficient technique for rapid isolation of large quantities of lymphocytes.

Successful infusion of 40 cryopreserved autologous bone-marrows. In vitro studies of the freezing procedure.

Cryopreservation of human bone marrow may be helpful to use supralethal chemoradiotherapy in order to cure malignant diseases. We report here a cryopreservation procedure from large volumes of human bone marrow which can be applied in routinal use. Whole bone marrow in 10% Dimethylsulfoxide (ME2SO) was frozen at an 1 degree C/min. controlled rate and was stored into liquid nitrogen. After thawing and before infusion, both ME2SO and free hemoglobin were removed. The in vitro recovery of nucleated cells and the myeloid stem cell assay (CFC) were performed as quality control. About 60% of the marrow cells and 40% of the total CFC number were recovered at the end of the procedure. Using this technique, 40 patients (25 children with malignant lymphoma and 15 adults with lymphoma and solid tumors) were transplanted with autologous cryopreserved bone marrow after receiving intensive chemotherapy. Three of them received both chemotherapy and a total body irradiation. Engraftment was achieved in all patients. Rise in leucocyte count (greater than 1.10(9)/l) occurred within an average of 17 days. In conclusion, in autologous bone marrow transplantation, this method of cryopreservation is effective to obtain rapid hematological reconstitution in patients treated for malignant diseases by intensive cytoreductive regimens.

[Red cell preservation by freezing at -25 degrees C]. / Conservation des hématies par congélation à -25 degr'es C.

A method of red cell preservation by freezing at -25 degrees C is described. Glycerol is added to the red blood cells in the primary polyvinyl chloride plastic collection bag to achieve a concentration of 28 pr cent (W/V). The blood cells are concentrated by centrifugation and the supernatant glycerol is discarded. Glycerolized red cells are frozen and stored at -25 degrees C for 1 to 6 months. After thawing, Sodium chloride solutions are used to wash the red cells in the IBM Blood Processor 2991. The following parameters have been investigated before freezing, after thawing and washing and after storage of red blood cells at 4 degrees C for 24 hours: --Hemoglobin level --leukocytes and platelets --amount of 2-3 DPG and ATP. Preliminary data show that the in vitro quality of erythrocytes stored at -25 degrees C is well preserved for 4 months and that this simple method can be applied to blood preservation in any Blood Center.