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

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1 Bone Marrow Transplantation, (1998) 22, Stockton Press All rights reserved /98 $ Successful cryopreservation of purified autologous CD34 cells: influence of freezing parameters on cell recovery and engraftment F Beaujean 1, J-H Bourhis 2, Ch Bayle 2, H Jouault 3, M Divine 3, C Rieux 3, M Janvier 4, Ch Le Forestier 1 and JL Pico 2 1 Laboratoire de Thérapie Cellulaire, Centre de Transfusion Sud Est Francilien, Hôpital Henri Mondor, Créteil; 2 Département d Hématologie Clinique et Biologique, Institut Gustave Roussy, Villejuif; 3 Département d Hématologie Clinique et Biologique, Hôpital Henri Mondor, Créteil; and 4 Département d Hématologie Clinique, Centre René Huguenin, Saint-Cloud, France Summary: 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 % for CD34 cells, % for CFU-GM, and 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 CD34 cells/kg and CFU-GM/kg. The median time to granulocyte ( /l) and platelet ( /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. Keywords: purified CD34 cells; cryopreservation; hematopoietic recovery Successful stem cell-based therapies, including autologous hematopoietic rescue, generally require an efficient method of long-term preservation. Current cryobiology techniques were defined many years ago and generally involve the use of dimethylsulfoxide (DMSO) as cryoprotectant, a slow cooling rate, and storage below 120 C. 1,2 Available methods can be used successfully to reconstitute normal hematopoiesis in heavily myelosuppressed patients. How- Correspondence: F Beaujean, Laboratoire de Thérapie Cellulaire, Etablissement de Transfusion Sanguine du Sud-Francilien, Hôpital Henri Mondor, 51, Avenue du Maréchal de Lattre de Tassigny, Créteil, France Received 12 May 1998; accepted 21 July 1998 ever, hematopoietic stem cell processing for transplantation has changed considerably in recent years. Hematopoietic cells can now be obtained from a variety of sources and ex vivo manipulation of hematopoietic grafts, by means of tumor purging, CD34 enrichment or T cell depletion has become relatively common. These techniques have introduced novel cellular products into clinical trials and routine practice. 3 There have been few studies aimed at determining if conventional freezing protocols are appropriate for such manipulated cells and ensure sufficient numbers of viable cells for reliable engraftment. In autologous transplantation, positive selection of CD34 cells reduces the amount of contaminating tumor cells in apheresis products without impairing hematopoietic recovery. 4 Isolation of CD34 cells results in a 2-log reduction in the total nucleated cell count, especially of mature blood elements, resulting in a highly purified population at a low cell density, thereby reducing the volume of the graft. 5 In this retrospective study, peripheral blood stem cells (PBSC) were positively selected by immunoadsorption using the CEPR- ATE SC Stem Cell Concentration System (CellPro, Bothell, WA, USA) and then frozen with a view to transplantation. In keeping with the company s guidelines, purified CD34 cells were cryopreserved with 7.5% DMSO and stored, depending on the clinical protocol, in either vials or bags. These conditions differed from our conventional protocol established for unmanipulated stem cell grafts (cryoprotectant concentration of 10% and a bag storage system). In order to evaluate cell cryosurvival in these new conditions and to determine the specific effect of a low cell density during freezing, we studied the post-freezing results of CD34 cells isolated from 39 patients, with regard to cell viability and engraftment kinetics after treatment with severely myelosuppressive regimens. Materials and methods Patient populations and mobilization procedures Between April 1993 and February 1997, 39 consecutive patients from three institutions were eligible for PBSC collection and selection. The diagnoses comprised multiple myeloma (16 patients), non-hodgkin s lymphoma (16 patients), breast cancer (6 patients), and chronic lymphoid leukemia (1 patient). The age range was 37 to 62 years

2 1092 (median 51). The study protocol was approved by the institutional review board and all the patients gave their written informed consent. PBSC were collected after high-dose chemotherapy followed by G-CSF (filgrastim; Amgen, Thousand Oaks, CA, USA). The timing of PBSC collection was based on an absolute white blood cell count exceeding /l. All the patients underwent daily leukapheresis sessions (12 l of blood each) on a Cobe Spectra cell separator (Cobe BCT, Lakewood, CO, USA). Anticoagulation was obtained with acid-citrate-dextrose (ACD.A; Baxter, Maurepas, France) at a ratio of 1 ml to 10 ml of blood. PBSC processing Two leukapheresis products were processed to positively select CD34 cells. The first apheresis product was stored overnight at 4 C and pooled with the second before further processing. Forty procedures were performed on 39 patients, one patient having two column separations because the threshold dose of CD34 cells ( /kg) was not achieved after the first selection. CD34 immunoselection was performed according to the instructions of the CEPRATE SC Stem Cell Concentration System manufacturer. Briefly, pooled PBSC were washed and resuspended in 150 ml of PBS (phosphate-buffered saline without Ca or Mg (CellPro)) containing 0.1% human serum albumin (HSA) (Laboratoire Français du Fractionnement et des Biotechnologies, Les Ulis, France). Cells were incubated for 25 min at room temperature with 20 g/ml of the biotinylated anti-cd34 monoclonal antibody The treated cells were washed with PBS to remove unbound antibody and processed through the avidin-coated column of the CEPRATE SC device. After washing, CD34 cells were recovered by mechanical agitation, resuspended in 80 ml of PBS containing 5 ml of 20% HSA and concentrated. Cryopreservation protocol Cryopreservation solution, consisting of 15% dimethyl sulfoxide (DMSO) in 8% HSA, was added to the cell products at an equal volume, to achieve a final concentration of 7.5% DMSO. The cryoprotectant solution was chilled to 4 C before use. Two freezing containers were used during the study. In 22 cases, cells from patients enrolled in specific protocols were transferred into cryovials (4 ml each). In the other 18 procedures, cells (40 ml) were placed in a minimum of two freezing bags (Hemofreeze bag DF 200; Gambro, Heckingen, Germany). Because of the different storage volumes, cell density in the bags was approximately half that in the vials. The cells, in vials or bags, were frozen using appropriate programs with a controlled cooling rate of 1 C/min to 50 C and then 5 C/min to 80 C, using a Planer Kryo 10 rate controller and chamber (Planer Biomed, Sunbury, UK). The vials were stored in the vapor phase of liquid nitrogen and the bags in the liquid phase. Before reinfusion the cells were rapidly thawed in a water bath at 40 C. Cells in vials were diluted with five volumes of PBS in the first nine procedures, and with 4% HSA in the remaining 13 procedures. An equal volume of 4% HSA was added to the cell suspension in bags. The cells were then promptly re-infused over 5 10 min. Quality control Samples were taken before and after cryopreservation. Quality control procedures for all the products included total nucleated cell counts, viability, CD34 cell counts and measurement of granulocyte macrophage colony-forming units (CFU-GM) and burst-forming unit-erythroid (BFU-E) content. Nucleated cell counts were done manually with a Malassez chamber (Ateliers Cloup, Champigny sur Marne, France). Trypan blue dye exclusion was used to estimate the percentage viability of nucleated cells. All results are expressed in terms of viable cells. Flow cytometry was used to estimate the number of CD34 cells with a phycoerythrin (PE) or fluorescein isothiocyanate (FITC) conjugated anti-cd34 monoclonal antibody HPCA 2 (Becton Dickinson, San Jose, CA, USA). For each sample, cells were incubated with 20 l of HPCA 2-PE or FITC or an irrelevant isotype control monoclonal antibody at 4 C for 30 min. After washing, samples were analyzed in a FacScan (Becton Dickinson) or Epics-Profile II (Coulter, Miami, FL, USA) flow cytometer and events were acquired. The resulting percentage of CD34 cells was multiplied by the number of nucleated cells to obtain the total number. Granulocyte macrophage colony-forming units (CFU- GM) and burst-forming units-erythroid (BFU-E) were simultaneously evaluated in ready-to-use culture medium (Methocult H4431; Stem Cell Technologies, Vancouver, Canada) consisting of IMDM (Iscove s modified Dulbecco s medium), 0.9% methyl cellulose, 30% FCS, 1% bovine serum albumin, 10 4 m 2-mercaptoethanol, 2 mm l-glutamine, 10% agar leukocyte conditioned medium and 3 U/ml human recombinant erythropoietin. Cells were plated in triplicate in 35-mm plastic culture dishes at densities of /ml and /ml. The dishes were maintained at 37 C in 100% humidified air with 5% CO 2. Colonies containing more than 50 cells were scored after 14 days of culture. CD34 cell infusion and hematopoietic recovery Thirty-seven of the 39 patients were transplanted with autologous selected CD34 cells alone. Conditioning regimens consisted of chemotherapy with (n = 31) or without (n = 6) total body irradiation (TBI). All the patients received G- CSF at a dose of 5 g/kg, starting 1 day after the infusion of selected cells and continuing until the neutrophil count was at least 10 9 /l on 3 consecutive days. The time required for engraftment was defined as the number of days required for patients to achieve neutrophils/l and platelets/l. Statistical analysis The results are expressed as means and standard deviations (s.d.) unless otherwise indicated. Post-cryopreservation recovery of progenitor cells (CD34 cells, CFU-GM and BFU-E) was obtained by dividing the absolute number of progenitor cells after thawing by the corresponding value before freezing ( 100). Correlations between cell density, recovery of myeloid (CFU-GM), erythroid (BFU-E) and

3 CD34 cells, and the time to engraftment were studied by using the non-parametric Spearman test. Two-way factorial analysis of variance (ANOVA) was used to compare the results obtained with the two types of freezing container. Significance was set at P Results In vitro studies Forty positive selection procedures were performed on 39 patients. All selected products were frozen, thawed and evaluable for the study. After CD34 cell selection, the mean ( s.d.) number of viable cells was in a final mean volume of ml ( 30.34). The purity of CD34 cells ranged from 33.9% to 97%, with a mean value of 84.65% Purity was above 80% in 30 of the 40 samples. After the addition of cryoprotectant, the nucleated cell density ranged from to /ml, with an average of /ml. After thawing and dilution, the recovery of nucleated cells and CD34 cells was respectively 88.62% (range: ) and 89.44% (range: ). Thirty-seven of the 40 samples had better than 60% of CD34 cell recovery. The mean purity and viability of the thawed suspensions were respectively 84.65% and 81.73% The progenitor recovery rates were 59.13% for CFU-GM and 53.49% for BFU-E. CD34 cell recovery correlated well with the recovery of nucleated cells (r = 0.94, P ), CFU- GM (r = 0.53, P = ) (Figure 1) and BFU-E (r = 0.45, P = ). Initial CD34 cell purity was not predictive of any of these parameters (r = 0.09, P = 0.54). We also explored the predictive value of nucleated cell density on progenitor cell survival. No correlation was found between % CFU-GM recovery % CD34+ cell recovery Figure 1 Correlation between CD34 cell and CFU-GM recoveries after cryopreservation (r = 0.53, P = ). this parameter and the recovery of CD34 cells (r = 0.23), CFU-GM (r = 0.22) or BFU-E (r = 0.22). The effect of the type of freezing container on cell and progenitor recovery was then explored (Table 1). The mean cell density in vials was nucleated cells/ml (range ), whereas cell density in the bags was nucleated cells/ml (range: ). Although cell density was significantly different (P = 0.02), the results obtained with the two types of container were of the same order. Cells frozen in vials yielded post-thaw nucleated cell and CD34 cell recoveries of % and %, respectively. Cells frozen in bags yielded slightly but not significantly better recovery ( % for nucleated cells and % for CD34 cells). CFU-GM and BFU-E activities after cryopreservation were similar regardless of whether cells were stored in vials or bags. Although the number of samples was limited, the effects of PBS and HSA as post-thaw solutions were compared (Table 2). No significant difference was observed (P = 0.3), although there was a trend towards better CD34 cell yields after dilution with 4% HSA. Clinical data Thirty-seven patients were reinfused with a median of 4.46 ( ) 10 6 /kg CD34 cells and ( ) 10 4 /kg CFU-GM. No side-effects occurred during infusion of selected CD34 cells. However, one patient became febrile within 24 h of transfusion, and blood culture yielded Pseudomonas rickettii. The same organism was isolated from the post-thaw sample of the graft (frozen in a cryovial). The infection resolved on appropriate antibiotic therapy. All patients showed successful neutrophil engraftment, taking a median of 11 days (range 6 to 20) to recover a count of /l. Thirty-five patients achieved a platelet count above /l a median of 13 days after transplantation (range 9 to 125 days). Thirty-one of these patients showed platelet engraftment within 21 days. Two patients did not achieve platelet recovery, because of relapses on days 85 and 90 (they had been reinfused with /kg and /kg CD34 cells). The longest case of thrombocytopenia (125 days) was observed in a patient who developed hepatic veno-occlusive disease and who had received the lowest dose of CD34 cells ( /kg). The time to neutrophil and platelet engraftment correlated negatively with the CD34 cell number infused (r = 0.58, P = and r = 0.38, P = , respectively). We also found a negative correlation (r = 0.40, P = 0.013) between the CFU-GM dose and the speed of neutrophil recovery. The number of BFU-E was not predictive of engraftment. The predictive value of cell density during cryopreservation on engraftment kinetics was also studied. Lower cell densities were associated with longer times to achieve granulocytes/l (r = 0.45, P = ) and to achieve platelet recovery (r = 0.36, P = 0.03). No differences in hematological recovery were observed between patients receiving grafts frozen and stored in vials or in bags (Table 3). 1093

4 1094 Table 1 Effects of the freezing container on cell recovery Vials (n = 22) Bags (n = 18) Cell density ( 10 6 /ml) P 0.05 Volume frozen (ml) Recovery post-freezing (%) Nucleated cells NS CD34 cells NS CFU-GM NS BFU-E NS Values are expressed as mean s.d. NS = not significant. Table 2 Cell recovery through the post-thaw dilution step Dilution in PBS Dilution in HSA (n = 9) (n = 13) Recovery of CD34 cells (%) NS Recovery of CFU-GM (%) NS Recovery of BFU-E (%) NS Values are expressed as mean s.d. NS = not significant. Discussion There is little published information on the best storage conditions for CD34 cell products. This study provides evidence that autologous CD34 cell concentrates isolated with the CellPro device can be efficiently cryopreserved with a final DMSO concentration of 7.5%. This concentration was defined as optimal in preliminary experiments in which the CD34 cell yield post-freezing exceeded 75%. 6 Our data confirm these results and indicate that over 89% of CD34 cells can be recovered. Moreover, cell viability and CD34 cell and clonogenic progenitor (CFU-GM, BFU-E) recoveries were similar to those obtained with unmanipulated hematopoietic products cryopreserved with 10% DMSO. 7,8 These cryopreserved CD34 cells rapidly reconstituted hematopoiesis in our study, even after myeloablative regimens such as total body irradiation. All patients engrafted, and the rates of neutrophil and platelet recovery were similar to those in other published reports with purified CD34 cells. 9 In these studies the methods used to preserve CD34 cells were heterogeneous. The final DMSO concentration varied from 7.5% 10,11 to 10% Hematopoietic reconstitution occurred as rapidly in our patients as in those whose CD34 cells were cryopreserved in 10% DMSO. A DMSO concentration of 10% has been established as optimal for the preservation of hematopoietic stem cells Lower concentrations have mainly been used when DMSO is combined with the extracellular cryoprotectant hydroxyethyl starch. 18 The use of hematopoietic grafts cryopreserved with 10% DMSO is often associated with specific side-effects apparently related to cell lysis debris and the volume of cryoprotectant. 19 Therefore, the use of cryoprotectant solutions containing lower proportions of DMSO is desirable. It was recently reported that blood hematopoietic cells could be cryopreserved with 5% DMSO as the sole cryoprotectant. 20,21 Other teams recommended a concentration of 7.5% for optimal recovery of hematopoietic stem cells in umbilical cord blood depleted of red cells. 22 In our study 7.5% DMSO also preserved efficiently purified CD34 cells. Regarding the clonogenic potential of CD34 cells, a good correlation between CD34 cell content and clonogenic activities was observed. However, CFU-GM and BFU-E cryosurvival (respectively 59.1% and 53.5%) was lower than that of CD34 cells. With the same cryopreservation parameters, Gorin et al 23 reported post-freezing CFU-GM and BFU-E recovery of more than 70% with purified CD34 bone mar- Table 3 Impact of the freezing container on time to engraftment Storage in vials Storage in bags (n = 19) n = 18 CD34 cells 10 6 /kg infused NS ( ) ( ) CFU-GM 10 5 /kg infused NS ( ) ( ) Days post-transplant to: Neutrophils /l NS (7 20) (6 16) Platelets /l NS (9 125) (9 41) The values are given as the mean s.d. with the range in parentheses. NS = not significant.

5 row cells. This disparity may in part be explained by differing sources of selected cells and/or stimulating factors. Our culture conditions were more specific for mature myeloid and erythroid progenitors and were suboptimal. Other investigations are needed to determine the reasons for the defective proliferative potential of CD34 cells, such as an intrinsic defect with increased sensitivity to cryoinjury, or inappropriate culture conditions. Freezing and storage conditions, ie cell density, the type of container, and the post-thaw diluent do not appear to influence the quality of the process. There have been some investigations concerning the effect of high cell density during cryopreservation. 9,24 Few data are available on the very low cell densities used in this study (range /ml to /ml). In previous murine studies, Dicke et al 25 found a significant deterioration of progenitor cell survival when marrow cells were frozen at densities of /ml. In this study, the lower concentrations were obtained with freezing bags, as the products were routinely split between two bags for freezing. We found no detrimental effect of relatively low cell densities on the recovery of CD34 cells or hematopoietic cells. Our data show the feasibility of using vials or bags for freezing cells. Similar data have previously been described. 26,27 However, in routine laboratory practice, bags are more convenient for handling and most manipulations can be performed using closed systems. The only graft contamination we documented in this study concerned cells frozen and stored in vials. Univariate analysis of our results showed that the cell density used for freezing, the CD34 cell number, and the CFU-GM content infused influenced the time required to recover peripheral blood cells. Cell density did not correlate with the total CD34 cell number recovered before freezing and was not therefore related to particularly poor grafts. Thus the mechanism of its adverse effect on engraftment are difficult to explain. This must be confirmed and analyzed in a larger number of procedures. Concerning the relationship between the number of cells infused and the speed of engraftment, our results are consistent with those of other clinical reports. In summary, hematopoietic cell storage conditions must be adapted as new cell processing modalities are developed. This routine study of purified CD34 cell cryopreservation efficiency may represent a first step towards the establishment of freezing parameters for selected CD34 cells. Acknowledgements The authors wish to express their gratitude to Claude Santiano for careful typing of the manuscript and David Young for editorial assistance. This work was supported in part by a grant from PHRC AP-HP (1994). References 1 Sputtek A, Körber Ch. Cryopreservation of red blood cells, platelets, lymphocytes, and stem cells. In: Fuller BJ, Grout BWW (eds). 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