The effects of a simplified method for cryopreservation and thawing procedures on peripheral blood stem cells

Abstract

Summary:initially reported by Stiff et al 1 in 1983. Makino et al 2 modified this method and evaluated its clinical use for peripheral blood stem cell transplantation (PBSCT) in 1991. A simplified method for cryopreservation at −80؇C of peripheral blood stem cells (PBSC) has been increasThey reported that rates of CFU-GM remained at more than 70% during 18 months of cryopreservation and that rapid ingly used for autologous PBSC transplantation in Japan. Although this method, using 6% hydroxyethyl and sustained trilineage engraftment was obtained in 10 patients who received marrow-ablative chemotherapy and starch (HES) and 5% dimethyl sulfoxide (DMSO) as a cryoprotectant without rate-controlled freezing, has sevautotransplantation of PBSC cryopreserved by this method. While this simplified method has several advantages eral advantages over the conventional method using 10% DMSO with rate-controlled freezing, little is such as being a quick and inexpensive procedure and less clumping of the thawed cells compared with the convenknown about effects of long-term cryopreservation for years and thawing process on hematopoietic progenitional method using rate-controlled freezing with 10% DMSO and storage in liquid nitrogen, 2 little is known about tors. We examined the recovery rates of BFU-E and CFU-GM in sample tubes cryopreserved by the simplithe influence of long-term cryopreservation for years and of the thawing process on frozen-thawed hematopoietic fied method under various conditions as follows: (1) long-term storage for 1-5 years; (2) DMSO exposure stem cells. In order to assess the clinical efficacy of this simplified for 1 h after rapid thawing; and (3) thawing at a lower temperature other than 37؇C. In our study, we found method, we investigated effects of long-term cryopreservation (1-5 years), exposure of frozen-thawed cells to that the recovery rates of BFU-E and CFU-GM were not affected by the length of cryopreservation period; DMSO, and slow thawing at room temperature on hematopoietic activity of PBSC when they were cryopreserved by they remained at more than 70% on average for 16-61 months. In our hands, a 1-h exposure to DMSO after this method. rapid thawing was not toxic for hematopoietic progenitors. Furthermore, there was no significant difference in the recovery rates of BFU-E and CFU-GM between Materials and methods thawing at 37؇C and 20؇C. These observations indicate that PBSC cryopreserved for at least 5 years by the simPreparation of peripheral blood mononuclear cells plified method can be used clinically without losing hematopoietic activity, and suggest that hematopoietic Peripheral blood mononuclear cells (PBMNC) were collected by apheresis from patients with hematologic maligactivity of the thawed PBSC may be unaffected when PBSC are infused slowly within 60 min or even when nancies in remission during hematologic recovery from consolidation chemotherapy with or without G-CSF. Aph-PBSC are thawed gradually at room temperature.

Storage of PBSC at -80°C

“…The fall in progenitor viability during cryopreservation for 1 year contrasts with the experience of storage in vapour phase nitrogen. 8 Furthermore, in contrast to the present study and that of Galmes et al, 7 Katayama et al 9 have recently reported minimal loss of colony-forming cell viability for up to 5 years storage at −80°C. The reasons for the difference are not clear, but emphasise the necessity for each laboratory to carry out its own quality control tests for long-term storage.…”

Hematopoietic stem cells from peripheral blood the perspective of non-mobilized peripheral blood

“…The viability and clonogenic potential were comparable, with a trend towards less infectious contamination in the dry method [25]. Different studies examined the preservation of function when thawed units were incubated at 0-37˚C [19,26].…”

“…Hence, the use of uncontrolled rate freezing in which the specimen is first cooled down to -4˚C and then directly deposited into a freezer at -80˚C or put into liquid phase nitrogen has been evaluated. Several reports [17][18][19] established that the uncontrolled method is safe and reveals comparable results to the controlled rate process for PBSCs. A controlled study performed by Perez-Oteyza et al [20] showed that the controlled and uncontrolled rate freezing approach are comparable in terms of viability testing and that only a statistically significant decrease in the CFU-GM clonality assay could be detected in the uncontrolled freezing situation.…”

“…The standard method is warming in a water bath at 37˚C until all ice crystals disappear [19]. A German study compared the thawing of cryopreserved units in a warm water bath with dry heat applied by gel pads at 37˚C.…”

Double High-dose Chemotherapy Supported by Autologous Transplantation of Peripheral Blood Stem Cells for Treatment of an Elderly Patient with Small-Cell Lung Cancer.

“…The total numberof mononuclear cells collected was 8.22xlO10 cells; CD34 positive cells and myeloid progenitor cells (CFU-GM) harvested were 1 1.2xlO6 and 16.8xl05 per kilogram of the patient's body weight, respectively. These harvested cells were cryopreserved at -80°C with a simplified method as previously reported ( 12).…”