A Simplified Method for Cryopreservation of Hematopoietic Stem Cells with -80dGC Mechanical Freezer with Dimethyl Sulfoxide as the Sole Cryoprotectant

Abstract

A simplified method for cryopreservation was developed with 10% dimethylsulfoxide (DMSO) as the sole cryoprotectant without rate-controlled freezing. This method produced high recovery rate for mononucleated cells (87%) and elevated trypan blue viability (90%). Autologous peripheral blood stem cells (PBSCs) and bone marrow cells with plasma and 10% DMSO were frozen and stored in a -80 degrees C mechanical freezer. Eleven patients with solid and hematological malignancies were transplanted with autologous bone marrow or PBSCs. The median number of infused mononuclear cells (MNC) and CD34+ cells were 3.63 x 10(8)/Kg and 4.80 x 10(6)/Kg, respectively. The median number of infused post-thawing CFU-GM was 20 x 10(4)/Kg. All patients showed a rapid and sustained engraftment. The mean times to reach a neutrophil count of 0.5 x 10(9)/L and a platelet count of 50 x 10(9)/L were 11 and 13 days, respectively. All patients are alive and 10 in unmaintained complete remission for 3-9 months after transplantation. These results show the efficacy of this simplified cryopreservation technique that will be useful for institutions without rate-controlled freezing facilities.

Impact of uncontrolled freezing and long-term storage of peripheral blood stem cells at −80 °C on haematopoietic recovery after autologous transplantation. Report from two centres

“…The median time required to reach 0.5 × 10 9 /L neutrophils was 12 (1-43), 11 (7-28) and 11 (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) days in groups I, II and III, respectively. We did not observe any negative impact of storage duration on neutrophil recovery.…”

“…[2][3][4][5] Successful engraftment with bone marrow stem cells stored at − 80°C after uncontrolled-rate freezing was first described in 1987 by Stiff et al 6 This simplified procedure was later used successfully at other centres. [7][8][9][10][11][12][13][14][15][16][17] We recently reported the results of a prospective study that include a large number of autologous PBSCTs performed after freezing and cryopreservation at − 80°C for o6 months. 18 In this study both short-term and long-term haematologic and immunologic recoveries seemed not to differ from recoveries observed after conventional freezing approach.…”

Cell culture bags allow a large extent of ex vivo expansion of LTC-IC and functional mature cells which can subsequently be frozen: interest for large-scale clinical applications

“…Overnight freezing of the tubes in a mechanical freezer at Ϫ80°C in a polystyrene box was followed by their transfer the next day to a plastic rack, still at Ϫ80°C for 1 to 3 weeks, until thawing. 16 Thawing was carried out by immersing the cryotubes in a waterbath at 39°C. The thawed cells were immediately diluted 1/20 in IMDM containing 5% bovine serum albumin and then washed once at 4°C.…”

Long-term hematologic reconstitution and clinical evaluation of autologous peripheral blood stem cell transplantation after cryopreservation of cells with 5% and 10% dimethylsulfoxide at 80 C in a mechanical freezer

“…This protocol involved storage in the same mechanical freezer in solutions containing 5% or 10% DMSO as the sole cryoprotectant without HES. [7][8][9] Most studies seem to indicate that this simplified procedure is associated with a reduction in infusion toxicity and lower costs, with a similar hematopoietic reconstitution and clinical outcome to more standard protocols. However, there is little data regarding the long-term hematologic recovery and clinical course when such protocols are used.…”