The viability of cryopreserved PBPC depends on the DMSO concentration and the concentration of nucleated cells in the graft.

BACKGROUND DMSO is widely used as a cryoprotectant for PBPC. It is desirable to reduce the amount of DMSO without jeopardizing the quality of the stem cell product. The present study was undertaken to investigate whether recovery and survival of CD34+ cells would be significantly altered when PBPC used for autologous transplantations were cryopreserved with four different DMSO concentrations. METHODS Apheresis samples of PBPC from 20 consecutive patients were mixed in parallel with 2%, 4%, 5% and 10% DMSO, frozen with identical cell concentrations at a controlled rate, and stored in liquid nitrogen for 6-8 weeks. PBPC samples from 11 consecutive patients were also cryopreserved with two different cell concentrations (150 and 300 x 10(6) nucleated cells/mL) to investigate the effect of increasing the cell concentrations while decreasing the DMSO concentration. The flow cytometric absolute count method, based on ISHAGE guidelines, was used to measure the absolute count of total and viable CD34+ cells in the post-thaw samples. RESULTS PBPC cryopreserved at 150 x 10(6) cells/mL with 2% DMSO yielded significantly inferior CD34+ cell recovery (P < 0.001) and survival (P < 0.001) compared with cryopreservation with 4% and 5% DMSO. This was also observed when comparing higher cell concentrations. However, a reduced cell survival (P = 0.02) was observed when the nucleated cell concentration was increased from 150 to 300 x 10(6) cells/mL in samples cryopreserved with 5% DMSO. DISCUSSION We conclude that 5% DMSO may be the optimal dose for cryopreserving PBPC as long as the cells have not been concentrated at much more than 200 x 10(6) nucleated cells/mL.

[1]  Øystein Bruserud,et al.  Cryopreserving human peripheral blood progenitor cells with 5‐percent rather than 10‐percent DMSO results in less apoptosis and necrosis in CD34+ cells , 2002, Transfusion.

[2]  H. Szmant.  PHYSICAL PROPERTIES OF DIMETHYL SULFOXIDE AND ITS FUNCTION IN BIOLOGICAL SYSTEMS , 1975, Annals of the New York Academy of Sciences.

[3]  S. Inaba,et al.  The effects of a simplified method for cryopreservation and thawing procedures on peripheral blood stem cells , 1997, Bone Marrow Transplantation.

[4]  T. Ruutu,et al.  Collection, Cryopreservation and Subsequent Viability of Haemopoietic Stem Cells Intended for Treatment of Chronic Granulocytic Leukaemia in Blast‐Cell Transformation , 1978, British journal of haematology.

[5]  Ø. Bruserud,et al.  Better preservation of early hematopoietic progenitor cells when human peripheral blood progenitor cells are cryopreserved with 5 percent dimethylsulfoxide instead of 10 percent dimethylsulfoxide , 2004, Transfusion.

[6]  D. Stroncek,et al.  Adverse reactions in patients transfused with cryopreserved marrow , 1991, Transfusion.

[7]  S. Piantadosi,et al.  Clinical toxicity of cryopreserved bone marrow graft infusion. , 1990, Blood.

[8]  S. Heimfeld,et al.  A randomized phase III clinical trial of autologous blood stem cell transplantation comparing cryopreservation using dimethylsulfoxide vs dimethylsulfoxide with hydroxyethylstarch , 2003, Bone Marrow Transplantation.

[9]  J. Hows,et al.  Optimal cryopreservation of human umbilical cord blood. , 1996, Bone marrow transplantation.

[10]  T. Gooley,et al.  Effect of Cell Concentration on Bone Marrow and Peripheral Blood Stem Cell Cryopreservation , 1994 .

[11]  J. M. Guerra,et al.  Long‐term storage at −80°C of hematopoietic progenitor cells with 5‐percent dimethyl sulfoxide as the sole cryoprotectant , 1999 .

[12]  G. Poggi,et al.  Clinical toxicity of cryopreserved circulating progenitor cells infusion. , 1998, Anticancer research.

[13]  O. Tournilhac,et al.  Uncontrolled‐rate freezing and storage at –80°C, with only3.5‐percent DMSO in cryoprotective solution for 109 autologous peripheral blood progenitor cell transplantations , 2001, Transfusion.

[14]  S. Goldmann,et al.  5% Me2SO is sufficient to preserve stem cells derived from cord blood. , 1998, Bone marrow transplantation.

[15]  H. Johnsen,et al.  Acute and Late Toxicity Following Adjuvant High-Dose Chemotherapy for High-Risk Primary Operable Breast Cancer A Quality Assessment Study , 2002, Acta oncologica.

[16]  J. Akabutu,et al.  Damage and protection of UC blood cells during cryopreservation. , 2001, Cytotherapy.

[17]  S. Rives,et al.  Cryopreservation of HPCs with high cell concentration in 5‐percent DMSO for transplantation to children , 2002, Transfusion.

[18]  D. Branch,et al.  Hematopoietic progenitor cells are resistant to dimethyl sulfoxide toxicity , 1994, Transfusion.

[19]  T. Wentzel‐Larsen,et al.  Autologous transplantation: the viable transplanted CD34+ cell dose measured post-thaw does not predict engraftment kinetics better than the total CD34+ cell dose measured pre-freeze in patients that receive more than 2x10(6) CD34+ cells/kg. , 2004, Cytotherapy.

[20]  O. Bruserud,et al.  No differences in colony formation of peripheral blood stem cells frozen with 5% or 10% dimethyl sulfoxide. , 2003, Journal of hematotherapy & stem cell research.

[21]  J. Lakota,et al.  Autologous stem cell transplantation with stem cells preserved in the presence of 4.5 and 2.2% DMSO. , 1996, Bone marrow transplantation.

[22]  H. Jouault,et al.  Successful cryopreservation of purified autologous CD34+ cells: influence of freezing parameters on cell recovery and engraftment , 1998, Bone Marrow Transplantation.

[23]  D. Sutherland,et al.  Single platform flow cytometric absolute CD34+ cell counts based on the ISHAGE guidelines. International Society of Hematotherapy and Graft Engineering. , 1998, Cytometry.