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Effects of incubation temperature and time after thawing on viability assessment of peripheral hematopoietic progenitor cells cryopreserved for transplantation.

Bone Marrow Transplantation (2003) 32, 1021–1026 & 2003 Nature Publishing Group All rights reserved 0268-3369/03 $25.00

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Technical Reports Effects of incubation temperature and time after thawing on viability assessment of peripheral hematopoietic progenitor cells cryopreserved for transplantation H Yang1, JP Acker1,2, M Cabuhat1 and LE McGann1,2 1

Canadian Blood Services, Edmonton, Alberta, Canada; and 2Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada

Summary: Three widely used viability assessments were compared: (1) membrane integrity of nucleated cells using trypan blue (TB) exclusion and a fluorometric membrane integrity assay (SYTO 13 and propidium iodide), (2) enumeration of viable CD34 þ cells, and (3) clonogenic assay (granulocyte-macrophage colony-forming units, CFU-GM). Post thaw peripheral hematopoietic progenitor cells (HPC) were incubated at 0, 22, and 371C for 20min intervals before assessment. The recovery of viable nucleated cells assessed by TB and SYTO/PI decreased significantly with time at incubation temperatures of 22 and 371C (Po0.05), and correlated with the concentration of mononuclear cells (MNC) (r ¼ 0.936, Po0.05). The decrease in recovery of viable nucleated cells was slower when thawed cells were incubated at 01C compared with 221C or 371C. The recovery, measured by absolute viable CD34 þ or CFU-GM, was not affected by 2 h post thaw incubation (P40.05) at 0, 22, and 371C (P40.05). There were no significant differences in the measured recovery of viable CD34 þ cells and CFU-GM at all incubation times (P40.05) and temperatures (P40.05). Both CFU-GM and absolute CD34 þ cells can be used as post thaw viability assays for HPC cryopreserved for transplantation. Bone Marrow Transplantation (2003) 32, 1021–1026. doi:10.1038/sj.bmt.1704247 viability; hematopoietic progenitor cells; Keywords: cryopreservation

The minimum CD34 þ cell dose for hematopoietic progenitor cell (HPC) transplant is based on the number of HPC (42  106 CD34 þ cells/kg body weight) before cryopreservation.1 As the number of viable CD34 þ cells at the time of transplant is not equal to the number of cells

Correspondence: Dr H Yang, Canadian Blood Services, Edmonton Centre 8249-114, Street, Edmonton, Alberta, Canada T6G 2R8; E-mail: [email protected] Received 24 February 2003; accepted 8 June 2003

before cryopreservation,2–4 there will be a minimum number of viable HPC post thaw that is crucial to a successful autologous HPC transplant. The reason that the post thaw viability of cells is not accepted as a determinant for HPC transplant is that there is no validated post thaw assay available for accurate assessment of viable cells for transplant of HPC. Three types of assays are commonly used for pre- and post cryopreservation assessment – membrane integrity of nucleated cells,2,5,6 enumeration of viable CD34 þ cells,2,7,8 and clonogenic assays.3,5,9 Trypan blue (TB) is the most common membrane integrity stain used to distinguish between viable and nonviable nucleated cells. Cells with damaged plasma membranes absorb TB and appear blue. Results of this assay are sometimes difficult to interpret because the outcome changes as cells absorb TB over time after thawing. Red blood cells in an HPC product can also affect the accurate enumeration of cells using TB. It has been reported that viability above 50% assayed by TB does not correlate with a high ratio of granulocyte–macrophage colony-forming units (CFUGM) before and after cryopreservation – only TB viability below 50% predicted a high loss of in vitro proliferative capacity.2 A dual-fluorescent method using acridine orange (AO)5 or SYTO10 combined with propidium iodide (PI) has also been used to distinguish viable and nonviable nucleated cells based on membrane integrity. SYTO, a live-cell nucleic acid stain, readily penetrates cells and binds to nucleic acids. Cells retain high green fluorescence intensity for up to 2 h after staining.10 PI only penetrates cells with damaged membranes and binds to DNA and RNA. Unlike TB, this nucleic acid assay is not affected by the presence of red blood cells. Clonogenic assays, mostly CFU-GM, have been used as a viability assay for decades, but their use is in decline as the assay is time consuming and the results are highly variable (0–304%).2 The variability occurs even when standardized media and growth factor combinations are used.11 In addition, the 2-week incubation period for a clonogenic assay renders this method impractical for post cryopreservation assessment as there is typically a short time period between the request for release of cryopreserved HPC and the reinfusion into a patient.

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Several studies on enumeration of viable mobilized peripheral blood CD34 þ cells have been reported.2–4 In two studies, the viability of CD34 þ cells increased from 36 to 94% after cryopreservation using dual-platform flow cytometric measurements.2,3 One study using singleplatform flow cytometric measurements reported that the number of viable CD34 þ cells reinfused predicts engraftment in autologous hematopoietic stem cell transplantation.3 It is obvious that the methodology for the enumeration of viable CD34 þ cells is critical for ensuring the accuracy of CD34 þ cell viability testing. A singleplatform flow cytometric absolute CD34 þ cell count technique has been increasingly used to quantify viable CD34 þ cells from post thaw samples of HPC.7 This methodology has been validated in our previous study of thawed cord blood samples, and demonstrated that the accuracy of CD34 þ cell enumeration and viability testing is extremely high.6 This method can count absolute CD34 þ cells directly from a flow cytometer by measuring the ratio of a known amount of fluorescent microbeads and CD34 þ cells in a sample, eliminating the need to use the total nucleated cell count as denominator in two-platform analysis.8 It is well known that the ratio of nucleated cells to CD34 þ cells changes following cryopreservation;6 therefore, the enumeration of CD34 þ cells will vary when based on a nucleated cell count in a post thaw HPC product. This method, however, has not replaced membrane integrity and clonogenic methods for post thaw assessment of HPC used in clinical transplantation because of the lack of a systemic study to compare the available assays. It is difficult for a stem cell laboratory or transplant physician to decide which assay or assays should be used to evaluate the quality of the post thaw HPC because the available assays have never been extensively investigated, validated, or standardized. This study focuses the investigation of viability assessment on two major factors affecting the post thaw viability of HPC samples – incubation time and temperature – with the aim of standardizing a post thaw assay for HPC samples cryopreserved for transplantation.

Materials and methods Cells HPC were collected by leukapheresis from five patients, who were eligible for autologous donation. Of the five patients, two donations were from patients with multiple myeloma (MM), and three with Hodgkin’s disease. After informed consent was obtained, 2 ml was withdrawn from the apheresis component, which was cryopreserved for later transplantation. Cell counts and differentials were obtained using an electronic counter (Coulter AcT Diff, Beckman Coulter, Hialeah, FL, USA). The components were diluted with Iscove’s modified Dulbecco’s medium (IMDM) with 2% fetal bovine serum (Stem Cell Technologies, Vancouver, Canada) to obtain 1.50  108 nucleated cells/ml. Without any further manipulation 100 ml of cells were transferred into a tube, and 50 ml IMDM was added to get a final concentration of 1.00  108 nucleated cells/ml. These Bone Marrow Transplantation

samples were used as controls for the post thaw viability assays.

Cryopreservation Immediately after preparation of the control sample, 1 ml of a plasmaLyte A (Baxter, Toronto, Canada) solution with 30% (v/v) dimethyl sulfoxide (DMSO) was slowly added to 2 ml of the experimental sample (1.50  108 cells/ ml) at 221C, for a final cell concentration of 1.00  108 nucleated cells/ml in 10% (v/v) DMSO. After approximately 5 min, aliquots of 0.6 ml were placed into each of the five freezing vials, which were then cooled at 11C/min to –601C, using a controlled-rate freezer (CryoMed1010, Forma Scientific, Marjetta, OH, USA), stored in liquid nitrogen vapor phase for over 48 h, and then rapidly warmed in water at 371C. The thawed samples were slowly diluted with IMDM and 2% fetal bovine serum to the corresponding pre-cryopreservation concentration required for the assays (5.0  105/ml for CFU-GM and 2.0  107/ml for remaining assays). The concentration of DMSO in the diluted post thaw samples was 2% or lower. The diluted aliquots were incubated at 0, 22, or 371C in a temperaturecontrolled bath (70.51C) and viability was assessed every 20 min for 2 h.

Viability assays All viability assays were performed at 221C. Nucleated cell concentrations for each assay was based on those from precryopreservation samples. Post thaw samples were subjected to the same manipulation as the control samples regardless of the concentration of nucleated cells in the post thaw samples, thereby avoiding errors introduced by changes to the cell population during cryopreservation. As different assessment techniques evaluate the structure or function of different biological elements, each method will give different measures of cell ‘viability’. Viability is a generic term that is a function of the assessment technique performed and is not a precise indicator of the overall status of a cell.12 In this study, a viable cell is defined based on the assessment technique that is performed. The TB assay was performed using a standard method13 with some modifications. Briefly, 20 ml of cell suspension was mixed with 80 ml 1  lysing solution (Beckman Coutler, Marseille Cedex, France) for 5 min to lyse the red blood cells that interfere with the leukocyte count, and then mixed with 100 ml TB solution (0.4% w/v, GIBCO). The samples were loaded into a Neubauer hemocytometer, and the cells lying within a 1 mm2 area (about 200 cells) were counted using a light microscope. Blue-stained cells were counted as nonviable and nonstained cells as viable. The dual-fluorescent assay was described in our previous paper.10 The assay used an SYTO 13 (Molecular Probes, Eugene, OR, USA) stock solution (1.25 mm in DMSO) and a stock PI solution (15 mm in H2O). The SYTO/PI working solution was prepared daily by diluting the stock solutions with PBS to final concentrations of 12.5 mm SYTO and 150 mm PI. Samples of the cell suspension (180 ml) were mixed with 20 ml of the SYTO/PI working solution and incubated for 5 min. The stained cells were then loaded into

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Two indices of cell viability were calculated from the experimental data to compare the different assessment techniques. The percent recovery is defined as the absolute number of viable cells (or colonies) that are retained following cryopreservation and is calculated as follows: % recovery ¼ Number of viable cells in thawed sample 100 Number of viable cells in unfrozen control The percent survival is the proportion of viable cells to the total number of cells that remain following cryopreservation: % survival ¼ Number of viable cells in thawed sample 100 Total number of cells remaining in thawed sample

Statistical analysis The data were analyzed using commercial software programs (Excel version 7, Microsoft, Redmond, WA, USA; SigmaStat version 2.03,. SPSS Science, Chicago, IL, USA). The results were expressed as mean7standard error (s.e.m.) unless specified. The results were considered to be significantly different when the P-value was below 0.05. The regression coefficients of determination of concentration of mononuclear cells (MNC) in pre-cryopreservation samples and viability from post thaw-treated samples were computed. Stepwise linear regression was performed to

Results The post thaw percent recovery of nucleated cells assayed with TB decreased with incubation time from 20 to 120 min (Po0.05) as shown in Figure 1a. The rate of decrease of viable cells was significantly slower when post thaw cells were incubated at 01C than at 22 and 371C (Po0.05). When assessed with SYTO/PI (Figure 2a), the post thaw recovery significantly decreased with incubation time between 40 and 120 min at incubation temperatures of 22

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determine the suitability of the membrane integrity and enumeration of viable CD34 þ cells to predict the viability of CFU-GM. The recovery and survival measures performed with the four viability assays were analyzed by oneway ANOVA, and the Student–Newman–Keuls (SNK) test was applied if the P-value was less than 0.05.

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a Neubauer hemocytometer and counted (cells within a 1 mm2 area), using a fluorescent microscope. Green cells were recorded as viable and red cells as nonviable. A standardized clonogenic progenitor assay9 was performed using a progenitor assay medium (MethoCult GF HC4434, Stem Cell Technologies, Vancouver, Canada). Duplicate aliquots corresponding to 5  104 cells from control samples were plated in 35-mm culture dishes. Equal volumes from the post thaw samples were also plated in duplicate, in 35-mm culture dishes, which were incubated at 371C in a humidified 5% CO2 incubator for 14 days. CFUGM colonies were counted using an inverted microscope. Single-platform flow cytometric absolute CD34 þ cell counts were performed as described by Keeney et al.7 Briefly, 100 ml of the nucleated cell suspension (corresponding to 2  107 cells/ml in the control samples) from control or post thaw samples were treated with 20 ml of either CD34-PE (580 clone)/CD45-FITC (J33 clone) or CD45FITC/Isoclonic PE followed by 20 ml of 7-amino-actinomycin D (7-AAD, 1 mg/ml, Molecular Probes, Eugene, OR, USA), and then incubated at room temperature for 20 min in the dark. A volume of 2 ml 10% ammonium chloride was then added to the tubes and the tubes were incubated for an additional 10 min in the dark. After incubation, 100 ml of Coulter Stem-Count beads was added, and flow cytometric analysis was performed immediately on a Coulter EPICS XL/MCL cytometer.

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Incubation time (min) Figure 1 Nucleated cells measured by TB after post thaw cells incubated at 0, 22, and 371C, at intervals of 20 min. (a) Percent recovery of viable nucleated cells. The recovery significantly decreased with incubation time from 20 to 120 min (Po0.05). The rate of decreasing was slower at 01C than at 22 and 371C (Po0.05). Mean7s.e.m. (n ¼ 5). (b) Percent survival of nucleated cells. Incubation time (P40.05) and temperature (P40.05) did not affect the survival. The survival was significantly higher than that of recovery (Po0.05). Mean7s.e.m. (n ¼ 5). Bone Marrow Transplantation

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Nucleated cells measured by SYTO/PI after post thaw cells incubated at 0, 22, and 371C, at intervals of 20 min. (a) Percent recovery of viable nucleated cells. The recovery significantly decreased with incubation at 22 and 371C at time from 40 to 120 min (Po0.05). The rate of decreasing was slower at 01C than at 22 and 371C (Po0.05). Mean7s.e.m. (n ¼ 5). (b) Percent survival of nucleated cells. Incubation time (P40.05) and temperature (P40.05) did not affect the survival. Survival of cells was significantly higher than that of recovery (Po0.05). Mean7s.e.m. (n ¼ 5).

and 371C compared with 01C (Po0.05). In contrast, Figures 1b and 2b show that percent survival following cryopreservation was significantly higher than that of recovery (Po0.05), and did not change significantly with increasing incubation time and temperature (P40.05). Neither percent recovery (Figure 3a) nor percent survival (Figure 3b) of CD34 þ cells was affected by incubation time (P40.05) or temperature (P40.05). The survival of CD34 þ cells following cryopreservation was significantly higher than percent recovery as shown by Figure 3b (Po0.05). Figure 4 shows that growth of CFU-GM from post thaw samples was not affected by incubation time (P40.05) or temperature (P40.05). Data summarized in Table 1 show that when cells were incubated at 371C for 2 h, the percent recovery of nucleated cells assayed by SYTO/PI significantly correlated with the concentration of MNC in pre-cryopreservation samples (r ¼ 0.936, Po0.05). However, neither recovery of

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Figure 3 CD34 þ cells measured by single-platform flow cytometric technique incorporated with 7-AAD after post thaw cells incubated at 0, 22, and 371C, at intervals of 20 min. (a) Percent recovery of viable CD34 þ cells. Incubation time (P40.05) and temperature (P40.05) did not affect the recovery. (b) Percent survival of CD34 þ cells. Incubation time (P40.05) and temperature (P40.05) did not affect the percent survival. Survival was significantly higher than that of recovery (Po0.05). Mean7s.e.m. (n ¼ 5).

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Incubation time (min) Figure 4 Post thaw recovery of HPC CFU-GM incubated at 0, 22, and 371C at intervals of 20 min. Incubation time (P40.05) and temperature (P40.05) did not affect the recovery. Mean7s.e.m. (n ¼ 5).

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1025 Table 1 Concentration of MNCs pre-cryopreservation, and recovery of NC, CFU-GM, and CD34+ cells in post thaw samples incubated at 371C for 2 h Recovery in post thaw samples (%) Sample no. 1 2 3 4 5 Mean7s.e.m. (range) a

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26.0 45.0 57.3 67.7 84.8

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Concentration of MNCs in pre-cryopreservation samples. Assessed by SYTO/PI.

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nucleated cells from post thaw samples nor concentration of MNC in pre-cryopreservation samples correlated with CFU-GM (rp0.05, P40.05) or viable CD34 þ cells (rp0.12, P40.05). The recovery of viable CD34 þ cells was not significantly different to that of CFU-GM in the post thaw samples (P40.05).

Discussion TB and fluorescent assays are the most commonly used viability assays for pre- and post-cryopreservation assessments of membrane integrity of nucleated cells. It has been reported that fluorescent assays have a better correlation with CFU-GM frequency than that of TB and therefore are superior viability assays.5 Our study, however, shows that incubation time (Figures 1a, 2a), incubation temperature (Figures 1a, 2a), and the cell differential of the HPC component (Table 1) significantly affect the percent recovery assessed by both the membrane integrity assays. As Table 1 shows, of the five HPC samples, sample no. 1 with 26.0% MNC gave 24.1% recovery of nucleated cells and a CFU-GM recovery of 66.0%. In contrast, another sample with 84.8% MNC resulted in 75.4% recovery of nucleated cells but had a CFU-GM recovery of 62.9%. As only 1.7870.73% (mean7s.e.m.) of the nucleated cell populations were CD34 þ cells, it is not surprising that the recovery of nucleated cells did not correlate with CFUGM. The nucleated cell membrane-integrity assays are therefore unreliable for assessment of the quality of HPC cryopreserved for transplantation. We therefore recommend that nucleated cell membrane-integrity assessments should not be used as post thaw viability assays of HPC. CFU-GM assay has been reported as being a good indicator of cell viability with a strong correlation to engraftment.14,15 It must be emphasized that the specific technique used for CFU-GM is critical for the assessment of post thaw HPC samples. In this study, 5  104 nucleated cells from an apheresis product and the same volume equal to 5  104 nucleated cells from post thaw samples without further manipulation were plated in each culture dish. This culture technique significantly reduced variability in post thaw samples that result from either separation or washing

procedures. The post thaw recovery of CFU-GM was 67.371.0% (range, 54.4–90.7%), showing significantly lower variability compared with results of 0–304% reported elsewhere.2 Importantly, unlike the membrane integrity assays of TB and SYTO/PI, the recovery of CFU-GM was consistent and unaffected by incubation time (P40.05), temperature (P40.05), and concentration of MNC (P40.05). We conclude that the CFU-GM assay could be used to determine the quality of cryopreserved HPC component for transplant. It, however, is not always possible to have sufficient time to complete the assay prior to an autologous transplantation. Compared with growth of CFU-GM, enumeration of viable CD34 þ cells by flow cytometry is easier and faster to perform. Percent recovery and survival of CD34 þ cells, like growth of CFU-GM, was consistent and unaffected by incubation time (P40.05), temperature (P40.05), and concentration of MNC (P40.05). Membrane damage of CD34 þ cells assayed by 7-AAD, unlike total nucleated cells, did not progress with incubation for 2 h at 0, 22, and 371C. This suggests that the membrane of CD34 þ cells may be the primary site of freezing injury during cryopreservation. While there was no significant difference between temperatures, incubation of post thaw samples at 221C (Po0.05) was selected as the best predication of viability of CFU-GM based on forward stepwise linear regression. It should be stressed that the methodology for enumeration of viable CD34 þ cells is critical to accurately measure the recovery and survival of CD34 þ cells. Single-platform flow cytometric measurements of absolute CD34 þ counts based on the ISHAGE guidelines4 have been validated to accurately enumerate CD34 þ cells in thawed cord blood samples.3 The recovery of CD34 þ cells from this study was 67.472.0%. In contrast, studies using a dual-platform flow cytometric measurements have shown a 36–94% increase in viable CD34 þ cells after cryopreservation.1,16 Recovery of CD34 þ cells (67.472.0%) compared to CFU-GM (67.371.0%, P40.05) demonstrates that the recovery of CD34 þ cells assayed by 7-AAD accurately reflects the HPC proliferative capacity. Therefore, enumeration of CD34 þ cells and CFU-GM could be used as reliable and reproducible post thaw assays for HPC. Since there is no significant difference between recovery of CD34 þ cells and recovery of CFU-GM over all incubation times (P40.05) and temperatures (P40.05), assessment of CD34 þ cells can be used as the sole viability assay. We suggest that thawed samples be incubated at 221C for the best correlation with CFU-GM. Our results demonstrate the need for detailed documentation of the viability calculation used in published reports. This is clearly exemplified in our study by the fact that our TB, SYTO/PI, and CD34 þ enumeration results were significantly different depending on the calculation used. The difference between the recovery and survival of nucleated cells increased with increasing incubation time and incubation at higher temperature (Figures 1 and 2). The percent recovery, which depends on the total number of viable cells in the unfrozen control, decreased as the number of viable cells in the cryopreserved samples decreased with time and temperature. Conversely, as the percent survival is based on the proportion of viable cells to Bone Marrow Transplantation

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total cells remaining in the thawed sample, cells lost by lysis or aggregation16,17 during the post thaw incubation would not be counted. Therefore, while the percent survival remains relatively constant, the absolute number of viable and recovered cells decreases. Similarly, percent survival of CD34 þ cells (93.070.4%) was significantly higher than the percent recovery of CD34 þ cells (67.472.0%, Po0.05) since the former does not consider the loss of CD34 þ cells during cryopreservation. Owing to this, the survival of CD34 þ cells, when gated on recovered CD34 þ cells, should not be used as a post thaw viability assay for HPC. When the membrane integrity of nucleated cells is assessed, the incubation time and temperature must be defined, as well as the concentration of MNC, in order to compare post thaw recovery of HPC apheresis products. In conclusion, both CFU-GM and single-platform flow cytometric enumeration of viable CD34 þ cells from quality control specimens can be used as post thaw viability assays for HPC cryopreserved for transplantation. Single-platform flow cytometry could be used as the sole post thaw viability assay if the post thaw samples are incubated at 221C and assessed within 2 h. It is important to note that viable CD34 þ cells from a pre-cryopreservation sample is required as a control to obtain a meaningful post thaw viability measure.

Acknowledgements We gratefully acknowledge the expert technical work of Hong Zhao. This research was supported by a Grant XE00007 from the Canadian Blood Services Research & Development program.

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