ORIGINAL ARTICLE Optimization of the freezing process for hematopoietic progenitor cells: effect of precooling, initial dimethyl sulfoxide concentration, freezing program, and storage in vapor-phase or liquid nitrogen on in vitro white blood cell quality Margriet J. Dijkstra-Tiekstra, Airies C. Setroikromo, Marcha Kraan, Effimia Gkoumassi, and Janny de Wildt-Eggen
BACKGROUND: Adding dimethyl sulfoxide (DMSO) to hematopoietic progenitor cells (HPCs) causes an exothermic reaction, potentially affecting their viability. The freezing method might also influence this. The aim was to investigate the effect of 1) precooling of DMSO and plasma (D/P) and white blood cell (WBC)-enriched product, 2) DMSO concentration of D/P, 3) freezing program, and 4) storage method on WBC quality. STUDY DESIGN AND METHODS: WBC-enriched product without CD34+ cells was used instead of HPCs. This was divided into six or eight portions. D/P (20 or 50%; precooled or room temperature [RT]) was added to the WBC-enriched product (precooled or RT), resulting in 10% DMSO, while monitoring temperature. The product was frozen using controlled-rate freezing (“fastrate” or “slow-rate”) and placed in vapor-phase or liquid nitrogen. After thawing, WBC recovery and viability were determined. RESULTS: Temperature increased most for precooled D/P to precooled WBC-enriched product, without influence of 20 or 50% D/P, but remained for all variations below 30°C. WBC recovery for both freezing programs was more than 95%. Recovery of WBC viability was higher for slow-rate freezing compared to fast-rate freezing (74% vs. 61%; p < 0.05) and also for 50% compared to 20% D/P (two test variations). Effect of precooling D/P or WBC-enriched product and of storage in vapor-phase or liquid nitrogen was marginal. CONCLUSION: Based on these results, precooling is not necessary. Fifty percent D/P is preferred over 20% D/P. Slow-rate freezing is preferred over fast-rate freezing. For safety reasons storage in vapor-phase nitrogen is preferred over storage in liquid nitrogen. Additional testing using real HPCs might be necessary.
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o protect hematopoietic progenitor cells (HPCs) during cryopreservation, a cryoprotectant like dimethyl sulfoxide (DMSO) is added. The DMSO penetrates cells and binds water molecules in solution thereby preventing cellular dehydration.1,2 However, a disadvantage of DMSO is that it may cause cell injury before freezing and after thawing.2-4 Other disadvantages are that by adding DMSO the final volume frozen is increased and sometimes DMSO needs to be washed away before transplantation of the HPCs, causing a delayed platelet engraftment.5 If DMSO is transplanted together with the HPCs it may cause mild reactions like nausea, vomiting, chills, and fever or the more severe but less frequent adverse events, like anaphylactic reactions, respiratory problems, renal and hepatic dysfunction, cardiac complications, and neurologic toxicity.6-9 DMSO is diluted to a certain concentration, mostly 20%, before addition to HPCs. To reduce storage volume and absolute amount of DMSO, a higher preconcentration DMSO can be used, for example, 50%.10 Because addition of DMSO to HPCs causes an exothermic reaction, which can cause cell damage, the DMSO solution and HPCs are
ABBREVIATIONS: D/P = DMSO and plasma; RT = room temperature. From the Division of Research, Department of Transfusion Monitoring, Sanquin Blood Supply, Groningen, the Netherlands. Address reprint requests to: Margriet J. Dijkstra-Tiekstra, Division of Research, Department of Transfusion Monitoring, Sanquin Blood Supply, Hanzeplein 1, PO Box 1191, NL-9701 BD Groningen, the Netherlands; e-mail: [email protected]. Received for publication March 14, 2014; revision received May 9, 2014, and accepted May 9, 2014. doi: 10.1111/trf.12756 © 2014 AABB TRANSFUSION **;**:**-**. Volume **, ** **
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often precooled. Furthermore, the used DMSO concentration may be of importance in cell survival after thawing, since it is expected that a higher concentration or faster addition of DMSO causes more local overheating resulting in protein denaturation.2,11 Another point of attention is the freezing rate. Cells can be frozen using controlled-rate freezing as well as uncontrolled-rate freezing in a −80 or −135°C mechanical freezer.12-15 The freezing program is important. During too-fast cooling, intracellular crystallization may occur, which could result in cell membrane rupture. During tooslow cooling, extracellular ice formation can occur, which could result in dehydration of the cell due to osmosis.1,16 Storage of HPCs can be done at temperatures between −80 and −196°C.12-15 Until about 10 years ago, HPCs were stored in liquid nitrogen. Because of the possibility of cross-contamination of microbial pathogens when HPCs are stored in liquid nitrogen, it is advised to store HPCs in the vapor phase of nitrogen,17,18 although the vapor phase of nitrogen might also be a potential source of pathogen contamination.19-21 However, literature for studying differences between storage in vapor-phase or liquid nitrogen is scarce. One study described the effect on temperature,22 and another described the effect on quality of HPCs in provials,15 which might deviate from results of HPCs in bags.15,23 Because it is difficult to obtain HPC products for research purposes, in this study a white blood cell (WBC)enriched product with comparable cell counts and hematocrit (Hct), but absence of CD34+ and immature cells, was used. The aim of this study was to investigate the effect of 1) precooling of DMSO/plasma (D/P) and WBC-enriched product compared to room temperature (RT), 2) the DMSO concentration of D/P (20 or 50%), and 3) the freezing program (“fast-rate” or “slow-rate”), on temperature course, total cell recovery (WBCs, mononuclear cells [MNCs], and lymphocytes) and WBC viability. Besides this, the effect of storage of WBC-enriched product in either liquid nitrogen or vapor-phase nitrogen on total WBC recovery and viability was studied.
MATERIALS AND METHODS The WBC-enriched product Because HPCs are not readily available for research purposes, a WBC-enriched product, with comparable cell counts and Hct, but absence of CD34+ and immature cells, was used in this study. The WBC-enriched product was prepared from buffy coats and plasma. In short, plasma was added to a residual buffy coat pool via a used leukoreduction filter (Compostop, Fresenius Hemocare, Emmer Compascuum, the Netherlands) in the opposite direction to increase the WBC count. The used plasma and residual buffy coat pool derived from whole blood collection of 1 day previously. Six of these plasma-remaining 2
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buffy coat pool units were pooled and diluted using plasma to a WBC concentration of approximately 10 × 109/L and a Hct of about 10%. This was divided over several 400-mL bags and allowed to sediment by gravity for 105 minutes. The supernatant including the buffy coat layer was pressed to a satellite bag, sampled, and centrifuged at 3900 × g for 14 minutes. From the sample the WBC count was determined. The supernatant was pressed from the pellets. The pellets were resuspended in supernatant plasma and pooled resulting in approximately 150 mL of WBC-enriched product with a WBC concentration of approximately 200 × 109/L.
Preparing D/P For preparation of 20% D/P, four volume parts of plasma were placed into in two bags. Subsequently the probe of a data logger (Escort Junior, −40 to 70°C, Escort Data Logging Systems LTD, New Lynn, Auckland, New Zealand) was placed in each bag and was activated (one measurement/5 sec). One bag was stored at RT and the other was placed between two precooled gel packs (Cool Gel, SCA Cool Logistics, Leighton Buzzard, UK) at 2 to 6°C for 15 minutes. When temperatures of the bags were 20 to 24°C (RT) and 6 to 10°C, respectively, one volume part of DMSO (WAK-Chemie Medical GmbH, Steinbach, Germany) was added slowly under continuous agitation to each bag. After DMSO was added, the bags were stored for at least 15 minutes again at RT or at 2 to 6°C between precooled gel packs. For 50% D/P the procedure for 20% D/P as described was followed but with equal volumes of plasma and DMSO.
Study effect of precooling, initial DMSO concentration, and freezing program The processing of the WBC-enriched product with the various study variations is shown in a flow chart (Fig. 1). After overnight storage at 2 to 6°C, the WBC-enriched product was divided into six portions of 20 mL in 150-mL transfer bags (Fresenius Hemocare), in which the probe of a data logger (Escort Junior, −40 to 70°C) was placed and activated (one measurement/5 sec). To three bags 12 mL plasma was added. Two bags with only WBC-enriched product and two bags with additional plasma were kept at RT (20-24°C) and the remaining two bags were precooled between precooled gel packs at 2 to 6°C for at least 15 minutes. The 20 and 50% D/P at RT were sterile connected to the WBC-enriched product stored at RT and the precooled 20 and 50% D/P were sterile connected to the remaining two bags with WBC-enriched product stored at RT and also to two bags with precooled WBC-enriched product. The 50% D/P was docked on the bags containing the additional plasma, to keep final volumes equal. Twenty milliliters of 20% D/P or 8 mL of 50% D/P was
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± 150 mL WBC-enriched product (cells)
Reference 20 mL cells in freezing bag
Var I 20 mL cells + 0 mL plasma (20-24°C)
Var II 20 mL cells + 0 mL plasma (20-24°C)
Var III 20 mL cells + 0 mL plasma (6-10°C)
Var IV 20 mL cells + 12 mL plasma (20-24°C)
Var V 20 mL cells + 12 mL plasma (20-24°C)
Var VI 20 mL cells + 12 mL plasma (6-10°C)
20 mL 20% D/P (20-24°C)
20 mL 20% D/P (20-24°C)
20 mL 20% D/P (6-10°C)
20 mL 20% D/P (6-10°C)
8 mL 50% D/P (20-24°C)
8 mL 50% D/P (6-10°C)
8 mL 50% D/P (6-10°C)
Add D/P to cells
Add D/P to cells and transfer to freezing bag
Add D/P to cells and transfer to freezing bag
data logger (Escort Precision with PT 300 adaptor, -200 to +300°C, Escort Data Logging Systems LTD) was placed in a 250-mL freezing bag. Subsequently for half of the experiments 20 mL of WBCenriched product and 20 mL 20% D/P were added slowly under continuous agitation. For the other half of the experiments 20 mL of WBC-enriched product, 12 mL of plasma, and 8 mL 50% D/P were added slowly under continuous agitation. The data logger was activated (one measurement/sec).
Freezing and thawing of the WBC-enriched product
All freezing bags (six study variations and a reference) were frozen using controlled-rate freezing with either a Reference cells + cells + cells + cells + cells + cells + cells + fast-rate or slow-rate freezing program D/P in D/P in D/P in D/P in D/P in D/P in D/P in in a controlled-rate freezer (Kryo 560freezing freezing freezing freezing freezing freezing freezing bag bag bag bag bag bag 16, Planer, Middlesex, UK). The fast-rate bag program was as follows: start temperature, 10°C; 5 minutes at 10°C; −4°C/min Freezing using either the fast-rate or the slow-rate program until 0°C; 2 minutes at 0°C; −1°C/min until −15°C, −4°C/min until −25°C; −5°C/min until −35°C; −8°C/min until Thawing −100°C; −15°C/min until −160°C; and 10 minutes at −160°C. The slow-rate Fig. 1. Flow chart for freezing and thawing of WBC-enriched product in six variations program was as follows: start tempera(Var). Experiments were performed separately, thus nonpaired, for fast-rate and ture, 10°C; 1 minute at 10°C; −1°C/min slow-rate freezing programs. The reference bag was prepared in half of the experiuntil −8°C; −20°C/min until −60°C; 2 ments using 20 mL of WBC-enriched product and 20 mL of 20% D/P (as is shown in minutes at −60°C; 15°C/min until −35°C; the flow chart) and in the other half of the experiments using 20 mL of WBC−0.5°C/min until −45°C; and −5.0°C/min enriched product, 12 mL of plasma, and 8 mL of 50% D/P. Temperature was until −140°C (Fig. 2). monitored for Variations I to VI for preparation of D/P and for adding D/P to Subsequently the products were WBC-enriched product. The temperature of the reference bag was monitored while placed in an aluminum racking system freezing the WBC-enriched product with D/P. into the vapor phase of nitrogen. After storage for at least 1 week, the products were thawed in a 37°C water bath and immediately fivefold diluted using phosphate-buffered added slowly under continuous agitation to the WBCsaline. enriched product. The WBC-enriched product had a final DMSO concentration of 10% and a final WBC concentration of 100 × 109/L. At the moment the temperature Study effect of storage in vapor-phase or did not increase further, the products were sampled liquid nitrogen (1 mL) and transferred to 250-mL freezing containers (CryoMACS, Miltenyi Biotec, Bergisch Gladbach, For this, eight variations (VII-XIV) were compared with Germany) and weighed. variations for 20 or 50% D/P, freezing using either the fastrate or slow-rate freezing program, and storage in vaporphase nitrogen or liquid nitrogen (see Table 1). For all Reference bag variations precooled D/P was added to precooled WBCenriched product. The followed procedure was as The reference bag was used to monitor the temperature described above. during the freezing of the cells. Therefore, the probe of a Volume **, ** **
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the figures or tables. A p value of less than 0.05 was used to indicate a significant difference.
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Temperature increase was lower for adding 20% DMSO to plasma compared to 50% DMSO (p < 0.05; Table 2). As expected, the maximum temperature -180 0 10 20 30 40 50 60 70 reached was lower when DMSO was added to precooled plasma (p < 0.05). Time, min For adding D/P to the WBCFig. 2. Temperature course of the fast-rate (gray line) and slow-rate (black line) enriched product, it was found that freezing programs. Temperature was measured in the reference bag that was frozen highest temperature was reached when simultaneously with the bags of the experiments (continuous lines). The curve as D/P and/or WBC-enriched products programmed is shown by the dotted lines. were at RT (p < 0.05; Table 2). However, the increase in temperature was highest when both D/P and WBC-enriched product were preTests cooled (p < 0.05). Tests were performed before adding D/P, after adding D/P, and after thawing. WBC count (including differentiation) WBCs was determined using a hematology analyzer (Model For all variations except for Variation I (20% D/P and XT1800i, Sysmex, Kobe, Japan). The MNCs consisted of WBC-enriched product at RT) total WBC recovery was monocytes and lymphocytes. WBC viability was measured higher for WBC-enriched product frozen using the fastusing a flow cytometric 7-aminoactinomycin D method rate program than when using the slow-rate program according to the prescriptions of the manufacturer (BD (p < 0.05; Fig. 3A). Total WBC recovery was approximately stem cell enumeration kit, BD Biosciences, San Jose, CA) 98% for freezing the fast-rate program and approximately on a flow cytometer (FACSCalibur, BD Biosciences). The 95% for the slow-rate freezing program. No influence on percentages of recovery after thawing compared to after total WBC recovery of precooling and 20 or 50% D/P was adding D/P for total WBCs, total MNCs, total lymphocytes, found. and WBC viability were calculated. -150
MNCs Statistical analysis Experiments for studying the effect of precooling, initial DMSO concentration, and freezing program using the fast-rate freezing program were independently performed from experiments using the slow-rate freezing program. The other variations were performed in a paired design. All experiments for studying the effect of storage in vapor-phase or liquid nitrogen were performed in a paired design. Experiments were performed with n = 6. For statistical analysis computer software (Microsoft Excel 2002, Microsoft Corp., Redmond, WA; and Instat, GraphPad, 2005, San Diego, CA) was used. Results are shown as mean (±SD). For comparison of data either a t test (paired or unpaired) or an analysis of variance (ANOVA), regular or for repeated measurements, was used followed by Tukey posttest as is indicated in the legends of 4
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Total MNC recovery was between 93 and 95% for the fastrate freezing program and between 90 and 92% for the slow-rate freezing program (Fig. 3B, p > 0.05). The absolute amounts of total MNCs per unit after adding D/P were higher for 20% D/P than for 50% D/P, Variation II versus Variation V (precooled D/P and WBC-enriched product at RT) for both freezing programs and for the fast-rate freezing program also for Variation I versus Variation IV (D/P and WBC-enriched product at RT; Table 3, p < 0.05). However, no influence on total MNC recovery of precooling and 20% or 50% D/P was found.
Lymphocytes Total lymphocyte recovery was between 94 and 98% for the fast-rate freezing program and between 90 and 92% for the slow-rate freezing program, with only significant differences for Variations II and III (Fig. 3C, p < 0.05). Amounts of total lymphocytes after adding D/P were
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TABLE 1. Results for testing effect of storage WBC-enriched product in vapor-phase or liquid nitrogen Variation VII VIII IX X XI XII XIII XIV
D/P (%) 20 20 20 20 50 50 50 50
Freezing program Fast-rate Fast-rate Slow-rate Slow-rate Fast-rate Fast-rate Slow-rate Slow-rate
Storage Vapor phase Liquid nitrogen Vapor phase Liquid nitrogen Vapor phase Liquid nitrogen Vapor phase Liquid nitrogen
Total WBC recovery* 95.8 ± 2.0 95.8 ± 1.3 96.0 ± 1.9 95.0 ± 2.9 96.5 ± 1.7 96.3 ± 1.8 96.2 ± 1.9 94.9 ± 2.9
WBC viability* 63.8 ± 4.3a 61.7 ± 2.8b,e 73.9 ± 3.6a 71.4 ± 7.0b 65.2 ± 5.4c 65.0 ± 4.0d,e 73.5 ± 4.3c 71.7 ± 5.8d
* Results show recovery after thawing compared to after adding D/P; results (%) are shown as mean ± SD. a, b, c, d p < 0.05, paired t test between freezing the fast-rate and slow-rate programs; ep < 0.05, paired t test between 20 and 50% D/P.
TABLE 2. Temperature course during adding DMSO to plasma and during adding D/P to WBC-enriched product* Adding DMSO to plasma Variation D/P (%) I 20 II/III 20 IV 50 V/VI 50
Temp DMSO (°C) 20-24 20-24 20-24 20-24
Adding D/P to WBC-enriched product Variation D/P (%) Temp D/P (°C) I 20 20-24 II 20 6-10 III 20 6-10 IV 50 20-24 V 50 6-10 VI 50 6-10
Start temp plasma (°C) 22.7 ± 0.8a 8.5 ± 1.1a 22.7 ± 0.6b 8.3 ± 0.6b
Max temp (°C) 32.8 ± 0.9a,c 21.4 ± 1.1a,d 43.3 ± 0.8b,c 34.7 ± 1.7b,d
Max increase (°C) 10.1 ± 0.5a,c 12.9 ± 1.2a,d 20.6 ± 0.4b,c 26.3 ± 1.6b,d
Start temp WBC-enriched product (°C) 23.0 ± 0.8e,f 20.7 ± 1.7e,g,j 9.2 ± 1.0f,g 23.0 ± 1.2h 23.1 ± 0.8i,j 9.1 ± 1.0h,i
Max temp (°C) 25.5 ± 0.9e,f 23.8 ± 1.3e,g,j 15.1 ± 1.2f,g 25.6 ± 0.7h 25.9 ± 1.3i,j 15.6 ± 1.8h,i
Max increase (°C) 2.5 ± 0.8e 3.1 ± 1.4f 5.9 ± 1.3e,f 2.7 ± 1.2h 2.8 ± 1.6i 6.6 ± 1.4h,i
* Results are shown as mean ± SD. Max temp = maximum temperature after adding DMSO or D/P; Max increase = the maximal increase in temperature. n = 12, except for adding DMSO to Plasma Variation IV with n = 9. Adding DMSO to plasma: ap < 0.05, paired t test between Variations I and II/III; bp < 0.05, unpaired t test, between Variations IV and V/VI; cp < 0.05, unpaired t test between 20% D/P (Variation I) and 50% D/P (Variation IV); dp < 0.05, paired t test between 20% D/P (Variation II/III) and 50% D/P (Variation V/VI). Adding D/P to WBC-enriched product: e, f, gp < 0.05, ANOVA for repeated measurements between Variations I to III; h, ip < 0.05, ANOVA between Variations IV to VI; jp < 0.05, paired t test between 20 and 50% D/P.
different between 20 and 50% only for experiments with the slow-rate freezing program, with higher amounts of lymphocytes per unit for 20% D/P, Variation II versus Variation V (Table 3, p < 0.05). However, no influence on total lymphocyte recovery of precooling and 20 or 50% D/P was found.
WBC viability The WBC viability was approximately 93% before adding D/P and decreased to 88% to 91% and 86% to 88% after adding D/P for experiments with the fast-rate and slowrate freezing programs, respectively. After thawing, WBC viability was further decreased to 53% to 57% for the fastrate freezing program and 60% to 68% for the slow-rate freezing program (Table 3). The recovery of the WBC viability showed even more pronounced differences between both freezing programs, which was significant for all variations except for Variation I (Fig. 3D, p < 0.05). Comparing 20 and 50% D/P, a lower recovery of the WBC viability was found for 20% D/P (p < 0.05), for Variation II
(59%) versus Variation V (63%) when using the fast-rate freezing program and for Variation III (73%) versus Variation VI (78%) when using the slow-rate freezing program. The absolute number of viable WBCs only shows a significant difference between both freezing programs for Variations III and VI (D/P and WBC-enriched product are precooled), with a lower number of viable WBCs for the fast-rate freezing program compared to the slow-rate freezing program, 1.80 × 109 versus 2.16 × 109 for Variation III and 1.93 × 109 versus 2.30 × 109 for Variation VI (Table 3, p < 0.05).
Effect of storage in vapor-phase or liquid nitrogen WBC recovery For these experiments no differences in total WBC recovery were found between storage in vapor phase or liquid nitrogen (Table 1). Neither differences were found between both freezing programs or between 20% D/P and Volume **, ** **
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Fig. 3. Recovery after thawing compared to after adding D/P for total WBCs (A), total MNCs (B), total lymphocytes (C), and WBC viability (D). ( ) Fast-rate freezing program; ( ) slow-rate freezing program. *p < 0.05, unpaired t test between both freezing programs; †p < 0.05, paired t-test between 20 and 50% D/P.
50% D/P. For all tested variations the mean total WBC recovery was between 95 and 96%.
WBC viability Recovery of WBC viability also did not differ between storage in vapor-phase or liquid nitrogen. Differences were found between both freezing programs, with higher values for the slow-rate freezing program (p < 0.05). A difference was also found between 20 and 50% D/P for Variation VIII versus Variation XII (fast-rate freezing program, liquid nitrogen; 62% vs. 65%; p < 0.05). For other variations no differences between 20% D/P and 50% D/P were found.
DISCUSSION In this article it was discussed that pre-cooling of either D/P or WBC-enriched product minimally affected recovery of total WBCs, total MNCs, total lymphocytes, and WBC viability after thawing. However, 20% D/P showed a 6
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lower WBC viability compared to 50% D/P (2%-5% difference, p < 0.05 for two of six variations tested), and slowrate freezing showed better viability results after thawing than fast-rate freezing (8%-15% difference; p < 0.05 for five of six variations tested). For this study a WBC-enriched product was used since real HPC products are not readily available for research purposes, due to ethical and practical considerations. Although this WBC-enriched product does not contain CD34+ cells or other immature cells we think this product can be used to optimize the freezing process of HPCs. Since WBCs are less stable than CD34+ cells,24,25 we think that when a method results in good WBC recovery and viability, it will be also the case for CD34+ cells. However, confirmation using real HPC product might still be desired. It can be expected that when products are precooled this will positively influence the results after thawing, because an exothermic reaction takes place when DMSO is added to plasma or HPCs. Our maximum temperature
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TABLE 3. Results for six variations of precooling D/P and WBC-enriched product using two freezing programs* Variation Fast-rate, fast-rate I II freezing program; D/P (%): 20 20 Slow-rate, slow-rate Temp D/P (°C): 20-24 2-6 freezing program Temp WBC-enriched product (°C): 20-24 20-24 Total WBCs, ×109/unit WBC-enriched product After adding D/P Fast-rate 3.63 ± 0.13 3.64 ± 0.15 Slow-rate 3.57 ± 0.33 3.67 ± 0.25a After thawing Fast-rate 3.56 ± 0.14 3.57 ± 0.17 Slow-rate 3.39 ± 0.24 3.46 ± 0.22 Total MNCs, ×109/unit WBC-enriched product After adding D/P 1.71 ± 0.20b Fast-rate 1.69 ± 0.19a Slow-rate 1.71 ± 0.20 1.77 ± 0.19c After thawing Fast-rate 1.60 ± 0.22 1.61 ± 0.24 Slow-rate 1.62 ± 0.24 1.66 ± 0.24d Total lymphocytes, ×109/unit WBC-enriched product After adding D/P Fast-rate 1.33 ± 0.22 1.33 ± 0.23 Slow-rate 1.37 ± 0.23 1.41 ± 0.22a After thawing Fast-rate 1.27 ± 0.24 1.30 ± 0.27 Slow-rate 1.26 ± 0.17 1.27 ± 0.17b WBC viability, % After adding D/P Fast-rate 89.0 ± 4.1 89.8 ± 3.5 Slow-rate 86.9 ± 7.6 86.25 ± 7.1a,l After thawing Fast-rate 54.3 ± 3.6 52.7 ± 3.5e Slow-rate 60.2 ± 7.0 62.9 ± 5.6e WBC viability, ×109/unit WBC-enriched product After adding D/P Fast-rate 3.34 ± 0.16 3.43 ± 0.24 Slow-rate 3.07 ± 0.43 3.21 ± 0.52 After thawing Fast-rate 1.89 ± 0.16 1.88 ± 0.12 Slow-rate 2.00 ± 0.33 2.22 ± 0.43
III 20 2-6 2-6
IV 50 20-24 20-24
V 50 2-6 20-24
VI 50 2-6 2-6
3.57 ± 0.18 3.59 ± 0.25
3.54 ± 0.18 3.64 ± 0.28
3.60 ± 0.13 3.61 ± 0.14 3.58 ± 0.21a 3.66 ± 0.12
3.53 ± 0.16 3.40 ± 0.23
3.47 ± 0.19 3.42 ± 0.13
3.56 ± 0.17 3.38 ± 0.19
1.69 ± 0.22 1.74 ± 0.21
1.57 ± 0.18a 1.66 ± 0.20b 1.69 ± 0.21 1.73 ± 0.18 1.70 ± 0.19c 1.77 ± 0.15
1.59 ± 0.23 1.64 ± 0.25
1.51 ± 0.18 1.65 ± 0.20
1.57 ± 0.21 1.58 ± 0.22 1.62 ± 0.22d 1.65 ± 0.22
1.32 ± 0.25 1.40 ± 0.24
1.25 ± 0.20 1.39 ± 0.20
1.31 ± 0.23 1.33 ± 0.24 1.36 ± 0.21a 1.40 ± 0.16
1.26 ± 0.24 1.27 ± 0.19
1.21 ± 0.19 1.26 ± 0.14
1.26 ± 0.23 1.25 ± 0.24 1.25 ± 0.16b 1.28 ± 0.16
90.1 ± 4.9 88.8 ± 6.8l
88.1 ± 5.4j 85.8 ± 7.1m
88.5 ± 5.0k 87.2 ± 6.6a
91.3 ± 2.5j,k 87.9 ± 7.3m
54.0 ± 3.5f 64.6 ± 5.6f
56.1 ± 3.2g 65.1 ± 8.2g
55.3 ± 1.3h 64.6 ± 9.1h
56.9 ± 4.7i 68.3 ± 5.7i
3.41 ± 0.10 3.21 ± 0.42
3.23 ± 0.34 3.18 ± 0.65
3.30 ± 0.15 3.17 ± 0.44
3.49 ± 0.16 3.21 ± 0.52
1.80 ± 0.06e 1.89 ± 0.21 2.16 ± 0.32e 2.19 ± 0.49
1.87 ± 0.09 2.17 ± 0.50
1.93 ± 0.16f 2.30 ± 0.31f
3.55 ± 0.15 3.45 ± 0.16
* Results are shown as mean ± SD. a, b, c, dp < 0.05 paired t test, 20% versus 50% D/P. e, f, g, h, ip < 0.05 unpaired t test, fast-rate versus slow-rate freezing program. j, k, l, mp < 0.05 ANOVA for repeated measurements, Variations I to III or Variations IV to VI.
increase was approximately 6°C for adding precooled D/P to precooled WBC-enriched product, which is comparable with that found by Nicoud and colleagues.11 The temperature increase was less when WBC-enriched product was at RT. An explanation for this might be the faster penetration of DMSO into the cell when the absolute temperature is higher,26 resulting in a lower increase in temperature of the product. Without precooling, the WBC-enriched product had a mean maximum temperature between 25 and 26°C, which appears to still be acceptable for a good cell recovery and viability after thawing. In the literature, it is suggested that addition of D/P to HPC causes a local overheating that causes protein denaturation and will be deleterious for the cells.2 However, in this study, this was not confirmed by results of total WBC recovery and WBC viability.
The temperature course of adding DMSO to plasma was also measured. For this a difference in maximum temperature of approximately 10°C was seen between 20 and 50% D/P. For adding 50% DMSO to plasma at RT, the maximum temperature was more than 40°C and the D/P became cloudy, indicating denaturation of proteins. Nicoud and colleagues11 described appearance of a “mushroom cloud” when adding DMSO solution quickly and this is probably the same phenomenon. However, in our study addition of a cloudy D/P did not affect total WBC recovery or viability after thawing, while Nicoud and colleagues11 found inferior results for fast compared to slow adding of DMSO. Although 20% D/P is most commonly used for freezing HPCs, 50% D/P did not show inferior results after thawing. These results are confirmed by Rubinstein and Volume **, ** **
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colleagues,10 although they used a 50% DMSO solution in 5% dextran 40. Therefore, it might be preferable to use higher concentrations D/P so that less volume is needed to be frozen. And even the volume of DMSO added to the patient will be a bit lower, which might reduce some adverse events caused by DMSO.7-9 In this study it was shown that the freezing curve influenced the postthaw results. A slow-rate freezing including a steep cooling rate to achieve vitrification and avoid nucleation had superior results above fast-rate freezing. Sputtek and coworkers13 showed that the optimum cooling rate was from 1 to 5°C/min from end of plateau phase until −45°C. Both tested programs in our study fall within these cooling rates. Tijssen and colleagues14 described a further optimization of the freezing curve, which is dependent of the final DMSO concentration. However, Tijssen and coworkers14 performed their experiments in cryotubes, while in our study cryobags were used. We did this, because in a previous study we showed that there might be a difference in thawing results between freezing in bags and tubes.23 Except for the steep cooling rate to achieve vitrification, the slow-rate freezing program is quite comparable to the optimized freezing program of Tijssen and colleagues.14 Further, freezing in a −80°C freezer instead of controlled-rate freezing has been described. It appeared that products in this case also have freezing rates in the 1 to 5°C/min range and result in good thawing results.15,24,27 From these studies, including our study, it appeared that a “slow-freezing” rate is preferred over a “fast-freezing” rate, but this depends on the final DMSO concentration. Furthermore, testing in tubes should be confirmed by testing in bags. One remarkable point is the contradiction in total WBC recovery and WBC viability. Total WBC recovery is optimal for fast-rate freezing (only shown for the experiments in which effect of precooling, initial DMSO concentration, and freezing program was studied) while WBC viability is optimal for the slow-rate freezing program. Total WBC recovery is calculated from WBC counts measured using the impedance technique of an automatic cell analyzer, while WBC viability is measured using a flow cytometric 7-aminoactinomycin D method. A possible explanation is that for the fast-rate freezing program more nonviable WBCs are present and for the slow-rate program these nonviable cells get lost. Since WBC viability is more important than total WBC recovery, slow-rate freezing is preferred over fast-rate freezing. Finally, the effect of storage in vapor-phase nitrogen versus liquid nitrogen was studied. Rowley and Byrne22 described this effect for temperature change during storage depending on the material of the storage racking system, distance from the liquid nitrogen, and opening of the lid of the tank. Their findings show that temperature of samples in an aluminum racking system barely increases at greater distance from liquid nitrogen in con8
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trast to samples in steel racking systems and that temperature of liquid nitrogen is more stable than that of vapor-phase nitrogen. Temperature of vapor-phase nitrogen remained for aluminum racking systems well below −150°C. Valeri and Pivacek15 described a comparison of storage of HPCs in provials in vapor-phase and liquid nitrogen and found no significant differences in recovery and viability and in culture assay. Our results of WBCenriched product in bags confirm these results as no difference in recovery and viability between both storage methods was found. Thus, for safety of the patient, because of the lower chance of cross-contamination, and for safety of the technician, because of the lower chance of spoiling liquid nitrogen, storage of HPCs in vaporphase nitrogen might be preferred over storage of HPCs in liquid nitrogen. In conclusion, precooling of HPCs or D/P seems not to be necessary, but 50% D/P showed for some variations a higher recovery of WBC viability after thawing than 20% D/P. The slow-rate freezing program is preferred above the fast-rate freezing program. Further, based on safety, storage in vapor-phase nitrogen is preferred over storage in liquid nitrogen. However, additional testing using real HPCs, and thus containing CD34+ and other immature cells, might be necessary to confirm these results. ACKNOWLEDGMENT We thank Paul Thijssen, head of the stem cell laboratory of Sanquin Blood Supply in Groningen, for critical reviewing of the manuscript.
CONFLICT OF INTEREST The authors have disclosed no conflicts of interest.
REFERENCES 1. Rowley SD. Hematopoietic stem cell processing and cryopreservation. J Clin Apher 1992;7:132-4. 2. Smagur A, Mitrus I, Giebel S, et al. Impact of different dimethyl sulphoxide concentrations on cell recovery, viability and clonogenic potential of cryopreserved peripheral blood hematopoietic stem and progenitor cells. Vox Sang 2013;104:240-7. 3. Baust JG, Gao D, Baust JM. Cryopreservation: an emerging paradigm change. Organogenesis 2009;5:90-6. 4. Rodriguez L, Velasco B, Garcia J, et al. Evaluation of an automated cell processing device to reduce the dimethyl sulfoxide from hematopoietic grafts after thawing. Transfusion 2005;45:1391-7. 5. Akkok CA, Holte MR, Tangen JM, et al. Hematopoietic engraftment of dimethyl sulfoxide-depleted autologous peripheral blood progenitor cells. Transfusion 2009;49:35461.
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6. Keung YK, Lau S, Elkayam U, et al. Cardiac arrhythmia after infusion of cryopreserved stem cells. Bone Marrow Transplant 1994;14:363-7. 7. Stroncek DF, Fautsch SK, Lasky LC, et al. Adverse reactions in patients transfused with cryopreserved marrow. Transfusion 1991;31:521-6. 8. Windrum P, Morris TC, Drake MB, et al. Variation in dimethyl sulfoxide use in stem cell transplantation: a survey of EBMT centres. Bone Marrow Transplant 2005;36: 601-3. 9. Zambelli A, Poggi G, Da PG, et al. Clinical toxicity of cryopreserved circulating progenitor cells infusion. Anticancer Res 1998;18(6B):4705-8. 10. Rubinstein P, Dobrila L, Rosenfield RE, et al. Processing and cryopreservation of placental/umbilical cord blood for unrelated bone marrow reconstitution. Proc Natl Acad Sci U S A 1995;92:10119-22. 11. Nicoud IB, Clarke DM, Taber G, et al. Cryopreservation of umbilical cord blood with a novel freezing solution that mimics intracellular ionic composition. Transfusion 2012; 52:2055-62. 12. McCullough J, Haley R, Clay M, et al. Long-term storage of peripheral blood stem cells frozen and stored with a conventional liquid nitrogen technique compared with cells frozen and stored in a mechanical freezer. Transfusion 2010;50:808-19. 13. Sputtek A, Jetter S, Hummel K, et al. Cryopreservation of peripheral blood progenitor cells: characteristics of suitable techniques. Beitr Infusionsther Transfusionsmed 1997; 34:79-83. 14. Tijssen MR, Woelders H, de Vries-van RA, et al. Improved postthaw viability and in vitro functionality of peripheral blood hematopoietic progenitor cells after cryopreservation with a theoretically optimized freezing curve. Transfusion 2008;48:893-901.
17. Foundation for the Accreditation of Cellular Therapy (FACT) and Joint Accreditation Committee ISCT and EBMT (JACIE). International standards for cellular therapy product collection, processing, and administration accreditation manual. 5.3 ed. Omaha (NE): FACT/JACIE; 2012. 18. Tedder RS, Zuckerman MA, Goldstone AH, et al. Hepatitis B transmission from contaminated cryopreservation tank. Lancet 1995;346:137-40. 19. Fountain D, Ralston M, Higgins N, et al. Liquid nitrogen freezers: a potential source of microbial contamination of hematopoietic stem cell components. Transfusion 1997;37: 585-91. 20. Grout BW, Morris GJ. Contaminated liquid nitrogen vapour as a risk factor in pathogen transfer. Theriogenology 2009; 71:1079-82. 21. Mirabet V, Alvarez M, Solves P, et al. Use of liquid nitrogen during storage in a cell and tissue bank: contamination risk and effect on the detectability of potential viral contaminants. Cryobiology 2012;64:121-3. 22. Rowley SD, Byrne DV. Low-temperature storage of bone marrow in nitrogen vapor-phase refrigerators: decreased temperature gradients with an aluminum racking system. Transfusion 1992;32:750-4. 23. Dijkstra-Tiekstra MJ, Setroikromo AC, de Wildt-Eggen J. Freezing “stem cells” in a bag and tube under various freezing conditions? Vox Sang 2012;102:273. 24. Almici C, Ferremi P, Lanfranchi A, et al. Uncontrolled-rate freezing of peripheral blood progenitor cells allows successful engraftment by sparing primitive and committed hematopoietic progenitors. Haematologica 2003;88:1390-5. 25. Humpe A, Beck C, Schoch R, et al. Establishment and optimization of a flow cytometric method for evaluation of viability of CD34+ cells after cryopreservation and comparison with trypan blue exclusion staining. Transfusion
15. Valeri CR, Pivacek LE. Effects of the temperature, the duration of frozen storage, and the freezing container on in
2005;45:1208-13. 26. Meryman HT. Cryopreservation of living cells: principles
vitro measurements in human peripheral blood mononuclear cells. Transfusion 1996;36:303-8. 16. Mazur P. The role of intracellular freezing in the death of cells cooled at supraoptimal rates. Cryobiology 1977;14: 251-72.
and practice. Transfusion 2007;47:935-45. 27. Solves P, Mirabet V, Planelles D, et al. Influence of volume reduction and cryopreservation methodologies on quality of thawed umbilical cord blood units for transplantation. Cryobiology 2008;56:152-8.
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BACKGROUND: Adding dimethyl sulfoxide (DMSO) to hematopoietic progenitor cells (HPCs) causes an exothermic reaction, potentially affecting their viability. The freezing method might also influence this. The aim was to investigate the effect of 1) precooling of DMSO and plasma (D/P) and white blood cell (WBC)-enriched product, 2) DMSO concentration of D/P, 3) freezing program, and 4) storage method on WBC quality. STUDY DESIGN AND METHODS: WBC-enriched product without CD34+ cells was used instead of HPCs. This was divided into six or eight portions. D/P (20 or 50%; precooled or room temperature [RT]) was added to the WBC-enriched product (precooled or RT), resulting in 10% DMSO, while monitoring temperature. The product was frozen using controlled-rate freezing (“fastrate” or “slow-rate”) and placed in vapor-phase or liquid nitrogen. After thawing, WBC recovery and viability were determined. RESULTS: Temperature increased most for precooled D/P to precooled WBC-enriched product, without influence of 20 or 50% D/P, but remained for all variations below 30°C. WBC recovery for both freezing programs was more than 95%. Recovery of WBC viability was higher for slow-rate freezing compared to fast-rate freezing (74% vs. 61%; p < 0.05) and also for 50% compared to 20% D/P (two test variations). Effect of precooling D/P or WBC-enriched product and of storage in vapor-phase or liquid nitrogen was marginal. CONCLUSION: Based on these results, precooling is not necessary. Fifty percent D/P is preferred over 20% D/P. Slow-rate freezing is preferred over fast-rate freezing. For safety reasons storage in vapor-phase nitrogen is preferred over storage in liquid nitrogen. Additional testing using real HPCs might be necessary.
T
o protect hematopoietic progenitor cells (HPCs) during cryopreservation, a cryoprotectant like dimethyl sulfoxide (DMSO) is added. The DMSO penetrates cells and binds water molecules in solution thereby preventing cellular dehydration.1,2 However, a disadvantage of DMSO is that it may cause cell injury before freezing and after thawing.2-4 Other disadvantages are that by adding DMSO the final volume frozen is increased and sometimes DMSO needs to be washed away before transplantation of the HPCs, causing a delayed platelet engraftment.5 If DMSO is transplanted together with the HPCs it may cause mild reactions like nausea, vomiting, chills, and fever or the more severe but less frequent adverse events, like anaphylactic reactions, respiratory problems, renal and hepatic dysfunction, cardiac complications, and neurologic toxicity.6-9 DMSO is diluted to a certain concentration, mostly 20%, before addition to HPCs. To reduce storage volume and absolute amount of DMSO, a higher preconcentration DMSO can be used, for example, 50%.10 Because addition of DMSO to HPCs causes an exothermic reaction, which can cause cell damage, the DMSO solution and HPCs are
ABBREVIATIONS: D/P = DMSO and plasma; RT = room temperature. From the Division of Research, Department of Transfusion Monitoring, Sanquin Blood Supply, Groningen, the Netherlands. Address reprint requests to: Margriet J. Dijkstra-Tiekstra, Division of Research, Department of Transfusion Monitoring, Sanquin Blood Supply, Hanzeplein 1, PO Box 1191, NL-9701 BD Groningen, the Netherlands; e-mail: [email protected]. Received for publication March 14, 2014; revision received May 9, 2014, and accepted May 9, 2014. doi: 10.1111/trf.12756 © 2014 AABB TRANSFUSION **;**:**-**. Volume **, ** **
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often precooled. Furthermore, the used DMSO concentration may be of importance in cell survival after thawing, since it is expected that a higher concentration or faster addition of DMSO causes more local overheating resulting in protein denaturation.2,11 Another point of attention is the freezing rate. Cells can be frozen using controlled-rate freezing as well as uncontrolled-rate freezing in a −80 or −135°C mechanical freezer.12-15 The freezing program is important. During too-fast cooling, intracellular crystallization may occur, which could result in cell membrane rupture. During tooslow cooling, extracellular ice formation can occur, which could result in dehydration of the cell due to osmosis.1,16 Storage of HPCs can be done at temperatures between −80 and −196°C.12-15 Until about 10 years ago, HPCs were stored in liquid nitrogen. Because of the possibility of cross-contamination of microbial pathogens when HPCs are stored in liquid nitrogen, it is advised to store HPCs in the vapor phase of nitrogen,17,18 although the vapor phase of nitrogen might also be a potential source of pathogen contamination.19-21 However, literature for studying differences between storage in vapor-phase or liquid nitrogen is scarce. One study described the effect on temperature,22 and another described the effect on quality of HPCs in provials,15 which might deviate from results of HPCs in bags.15,23 Because it is difficult to obtain HPC products for research purposes, in this study a white blood cell (WBC)enriched product with comparable cell counts and hematocrit (Hct), but absence of CD34+ and immature cells, was used. The aim of this study was to investigate the effect of 1) precooling of DMSO/plasma (D/P) and WBC-enriched product compared to room temperature (RT), 2) the DMSO concentration of D/P (20 or 50%), and 3) the freezing program (“fast-rate” or “slow-rate”), on temperature course, total cell recovery (WBCs, mononuclear cells [MNCs], and lymphocytes) and WBC viability. Besides this, the effect of storage of WBC-enriched product in either liquid nitrogen or vapor-phase nitrogen on total WBC recovery and viability was studied.
MATERIALS AND METHODS The WBC-enriched product Because HPCs are not readily available for research purposes, a WBC-enriched product, with comparable cell counts and Hct, but absence of CD34+ and immature cells, was used in this study. The WBC-enriched product was prepared from buffy coats and plasma. In short, plasma was added to a residual buffy coat pool via a used leukoreduction filter (Compostop, Fresenius Hemocare, Emmer Compascuum, the Netherlands) in the opposite direction to increase the WBC count. The used plasma and residual buffy coat pool derived from whole blood collection of 1 day previously. Six of these plasma-remaining 2
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buffy coat pool units were pooled and diluted using plasma to a WBC concentration of approximately 10 × 109/L and a Hct of about 10%. This was divided over several 400-mL bags and allowed to sediment by gravity for 105 minutes. The supernatant including the buffy coat layer was pressed to a satellite bag, sampled, and centrifuged at 3900 × g for 14 minutes. From the sample the WBC count was determined. The supernatant was pressed from the pellets. The pellets were resuspended in supernatant plasma and pooled resulting in approximately 150 mL of WBC-enriched product with a WBC concentration of approximately 200 × 109/L.
Preparing D/P For preparation of 20% D/P, four volume parts of plasma were placed into in two bags. Subsequently the probe of a data logger (Escort Junior, −40 to 70°C, Escort Data Logging Systems LTD, New Lynn, Auckland, New Zealand) was placed in each bag and was activated (one measurement/5 sec). One bag was stored at RT and the other was placed between two precooled gel packs (Cool Gel, SCA Cool Logistics, Leighton Buzzard, UK) at 2 to 6°C for 15 minutes. When temperatures of the bags were 20 to 24°C (RT) and 6 to 10°C, respectively, one volume part of DMSO (WAK-Chemie Medical GmbH, Steinbach, Germany) was added slowly under continuous agitation to each bag. After DMSO was added, the bags were stored for at least 15 minutes again at RT or at 2 to 6°C between precooled gel packs. For 50% D/P the procedure for 20% D/P as described was followed but with equal volumes of plasma and DMSO.
Study effect of precooling, initial DMSO concentration, and freezing program The processing of the WBC-enriched product with the various study variations is shown in a flow chart (Fig. 1). After overnight storage at 2 to 6°C, the WBC-enriched product was divided into six portions of 20 mL in 150-mL transfer bags (Fresenius Hemocare), in which the probe of a data logger (Escort Junior, −40 to 70°C) was placed and activated (one measurement/5 sec). To three bags 12 mL plasma was added. Two bags with only WBC-enriched product and two bags with additional plasma were kept at RT (20-24°C) and the remaining two bags were precooled between precooled gel packs at 2 to 6°C for at least 15 minutes. The 20 and 50% D/P at RT were sterile connected to the WBC-enriched product stored at RT and the precooled 20 and 50% D/P were sterile connected to the remaining two bags with WBC-enriched product stored at RT and also to two bags with precooled WBC-enriched product. The 50% D/P was docked on the bags containing the additional plasma, to keep final volumes equal. Twenty milliliters of 20% D/P or 8 mL of 50% D/P was
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± 150 mL WBC-enriched product (cells)
Reference 20 mL cells in freezing bag
Var I 20 mL cells + 0 mL plasma (20-24°C)
Var II 20 mL cells + 0 mL plasma (20-24°C)
Var III 20 mL cells + 0 mL plasma (6-10°C)
Var IV 20 mL cells + 12 mL plasma (20-24°C)
Var V 20 mL cells + 12 mL plasma (20-24°C)
Var VI 20 mL cells + 12 mL plasma (6-10°C)
20 mL 20% D/P (20-24°C)
20 mL 20% D/P (20-24°C)
20 mL 20% D/P (6-10°C)
20 mL 20% D/P (6-10°C)
8 mL 50% D/P (20-24°C)
8 mL 50% D/P (6-10°C)
8 mL 50% D/P (6-10°C)
Add D/P to cells
Add D/P to cells and transfer to freezing bag
Add D/P to cells and transfer to freezing bag
data logger (Escort Precision with PT 300 adaptor, -200 to +300°C, Escort Data Logging Systems LTD) was placed in a 250-mL freezing bag. Subsequently for half of the experiments 20 mL of WBCenriched product and 20 mL 20% D/P were added slowly under continuous agitation. For the other half of the experiments 20 mL of WBC-enriched product, 12 mL of plasma, and 8 mL 50% D/P were added slowly under continuous agitation. The data logger was activated (one measurement/sec).
Freezing and thawing of the WBC-enriched product
All freezing bags (six study variations and a reference) were frozen using controlled-rate freezing with either a Reference cells + cells + cells + cells + cells + cells + cells + fast-rate or slow-rate freezing program D/P in D/P in D/P in D/P in D/P in D/P in D/P in in a controlled-rate freezer (Kryo 560freezing freezing freezing freezing freezing freezing freezing bag bag bag bag bag bag 16, Planer, Middlesex, UK). The fast-rate bag program was as follows: start temperature, 10°C; 5 minutes at 10°C; −4°C/min Freezing using either the fast-rate or the slow-rate program until 0°C; 2 minutes at 0°C; −1°C/min until −15°C, −4°C/min until −25°C; −5°C/min until −35°C; −8°C/min until Thawing −100°C; −15°C/min until −160°C; and 10 minutes at −160°C. The slow-rate Fig. 1. Flow chart for freezing and thawing of WBC-enriched product in six variations program was as follows: start tempera(Var). Experiments were performed separately, thus nonpaired, for fast-rate and ture, 10°C; 1 minute at 10°C; −1°C/min slow-rate freezing programs. The reference bag was prepared in half of the experiuntil −8°C; −20°C/min until −60°C; 2 ments using 20 mL of WBC-enriched product and 20 mL of 20% D/P (as is shown in minutes at −60°C; 15°C/min until −35°C; the flow chart) and in the other half of the experiments using 20 mL of WBC−0.5°C/min until −45°C; and −5.0°C/min enriched product, 12 mL of plasma, and 8 mL of 50% D/P. Temperature was until −140°C (Fig. 2). monitored for Variations I to VI for preparation of D/P and for adding D/P to Subsequently the products were WBC-enriched product. The temperature of the reference bag was monitored while placed in an aluminum racking system freezing the WBC-enriched product with D/P. into the vapor phase of nitrogen. After storage for at least 1 week, the products were thawed in a 37°C water bath and immediately fivefold diluted using phosphate-buffered added slowly under continuous agitation to the WBCsaline. enriched product. The WBC-enriched product had a final DMSO concentration of 10% and a final WBC concentration of 100 × 109/L. At the moment the temperature Study effect of storage in vapor-phase or did not increase further, the products were sampled liquid nitrogen (1 mL) and transferred to 250-mL freezing containers (CryoMACS, Miltenyi Biotec, Bergisch Gladbach, For this, eight variations (VII-XIV) were compared with Germany) and weighed. variations for 20 or 50% D/P, freezing using either the fastrate or slow-rate freezing program, and storage in vaporphase nitrogen or liquid nitrogen (see Table 1). For all Reference bag variations precooled D/P was added to precooled WBCenriched product. The followed procedure was as The reference bag was used to monitor the temperature described above. during the freezing of the cells. Therefore, the probe of a Volume **, ** **
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the figures or tables. A p value of less than 0.05 was used to indicate a significant difference.
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-60 -90
Temperature course
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Temperature increase was lower for adding 20% DMSO to plasma compared to 50% DMSO (p < 0.05; Table 2). As expected, the maximum temperature -180 0 10 20 30 40 50 60 70 reached was lower when DMSO was added to precooled plasma (p < 0.05). Time, min For adding D/P to the WBCFig. 2. Temperature course of the fast-rate (gray line) and slow-rate (black line) enriched product, it was found that freezing programs. Temperature was measured in the reference bag that was frozen highest temperature was reached when simultaneously with the bags of the experiments (continuous lines). The curve as D/P and/or WBC-enriched products programmed is shown by the dotted lines. were at RT (p < 0.05; Table 2). However, the increase in temperature was highest when both D/P and WBC-enriched product were preTests cooled (p < 0.05). Tests were performed before adding D/P, after adding D/P, and after thawing. WBC count (including differentiation) WBCs was determined using a hematology analyzer (Model For all variations except for Variation I (20% D/P and XT1800i, Sysmex, Kobe, Japan). The MNCs consisted of WBC-enriched product at RT) total WBC recovery was monocytes and lymphocytes. WBC viability was measured higher for WBC-enriched product frozen using the fastusing a flow cytometric 7-aminoactinomycin D method rate program than when using the slow-rate program according to the prescriptions of the manufacturer (BD (p < 0.05; Fig. 3A). Total WBC recovery was approximately stem cell enumeration kit, BD Biosciences, San Jose, CA) 98% for freezing the fast-rate program and approximately on a flow cytometer (FACSCalibur, BD Biosciences). The 95% for the slow-rate freezing program. No influence on percentages of recovery after thawing compared to after total WBC recovery of precooling and 20 or 50% D/P was adding D/P for total WBCs, total MNCs, total lymphocytes, found. and WBC viability were calculated. -150
MNCs Statistical analysis Experiments for studying the effect of precooling, initial DMSO concentration, and freezing program using the fast-rate freezing program were independently performed from experiments using the slow-rate freezing program. The other variations were performed in a paired design. All experiments for studying the effect of storage in vapor-phase or liquid nitrogen were performed in a paired design. Experiments were performed with n = 6. For statistical analysis computer software (Microsoft Excel 2002, Microsoft Corp., Redmond, WA; and Instat, GraphPad, 2005, San Diego, CA) was used. Results are shown as mean (±SD). For comparison of data either a t test (paired or unpaired) or an analysis of variance (ANOVA), regular or for repeated measurements, was used followed by Tukey posttest as is indicated in the legends of 4
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Total MNC recovery was between 93 and 95% for the fastrate freezing program and between 90 and 92% for the slow-rate freezing program (Fig. 3B, p > 0.05). The absolute amounts of total MNCs per unit after adding D/P were higher for 20% D/P than for 50% D/P, Variation II versus Variation V (precooled D/P and WBC-enriched product at RT) for both freezing programs and for the fast-rate freezing program also for Variation I versus Variation IV (D/P and WBC-enriched product at RT; Table 3, p < 0.05). However, no influence on total MNC recovery of precooling and 20% or 50% D/P was found.
Lymphocytes Total lymphocyte recovery was between 94 and 98% for the fast-rate freezing program and between 90 and 92% for the slow-rate freezing program, with only significant differences for Variations II and III (Fig. 3C, p < 0.05). Amounts of total lymphocytes after adding D/P were
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TABLE 1. Results for testing effect of storage WBC-enriched product in vapor-phase or liquid nitrogen Variation VII VIII IX X XI XII XIII XIV
D/P (%) 20 20 20 20 50 50 50 50
Freezing program Fast-rate Fast-rate Slow-rate Slow-rate Fast-rate Fast-rate Slow-rate Slow-rate
Storage Vapor phase Liquid nitrogen Vapor phase Liquid nitrogen Vapor phase Liquid nitrogen Vapor phase Liquid nitrogen
Total WBC recovery* 95.8 ± 2.0 95.8 ± 1.3 96.0 ± 1.9 95.0 ± 2.9 96.5 ± 1.7 96.3 ± 1.8 96.2 ± 1.9 94.9 ± 2.9
WBC viability* 63.8 ± 4.3a 61.7 ± 2.8b,e 73.9 ± 3.6a 71.4 ± 7.0b 65.2 ± 5.4c 65.0 ± 4.0d,e 73.5 ± 4.3c 71.7 ± 5.8d
* Results show recovery after thawing compared to after adding D/P; results (%) are shown as mean ± SD. a, b, c, d p < 0.05, paired t test between freezing the fast-rate and slow-rate programs; ep < 0.05, paired t test between 20 and 50% D/P.
TABLE 2. Temperature course during adding DMSO to plasma and during adding D/P to WBC-enriched product* Adding DMSO to plasma Variation D/P (%) I 20 II/III 20 IV 50 V/VI 50
Temp DMSO (°C) 20-24 20-24 20-24 20-24
Adding D/P to WBC-enriched product Variation D/P (%) Temp D/P (°C) I 20 20-24 II 20 6-10 III 20 6-10 IV 50 20-24 V 50 6-10 VI 50 6-10
Start temp plasma (°C) 22.7 ± 0.8a 8.5 ± 1.1a 22.7 ± 0.6b 8.3 ± 0.6b
Max temp (°C) 32.8 ± 0.9a,c 21.4 ± 1.1a,d 43.3 ± 0.8b,c 34.7 ± 1.7b,d
Max increase (°C) 10.1 ± 0.5a,c 12.9 ± 1.2a,d 20.6 ± 0.4b,c 26.3 ± 1.6b,d
Start temp WBC-enriched product (°C) 23.0 ± 0.8e,f 20.7 ± 1.7e,g,j 9.2 ± 1.0f,g 23.0 ± 1.2h 23.1 ± 0.8i,j 9.1 ± 1.0h,i
Max temp (°C) 25.5 ± 0.9e,f 23.8 ± 1.3e,g,j 15.1 ± 1.2f,g 25.6 ± 0.7h 25.9 ± 1.3i,j 15.6 ± 1.8h,i
Max increase (°C) 2.5 ± 0.8e 3.1 ± 1.4f 5.9 ± 1.3e,f 2.7 ± 1.2h 2.8 ± 1.6i 6.6 ± 1.4h,i
* Results are shown as mean ± SD. Max temp = maximum temperature after adding DMSO or D/P; Max increase = the maximal increase in temperature. n = 12, except for adding DMSO to Plasma Variation IV with n = 9. Adding DMSO to plasma: ap < 0.05, paired t test between Variations I and II/III; bp < 0.05, unpaired t test, between Variations IV and V/VI; cp < 0.05, unpaired t test between 20% D/P (Variation I) and 50% D/P (Variation IV); dp < 0.05, paired t test between 20% D/P (Variation II/III) and 50% D/P (Variation V/VI). Adding D/P to WBC-enriched product: e, f, gp < 0.05, ANOVA for repeated measurements between Variations I to III; h, ip < 0.05, ANOVA between Variations IV to VI; jp < 0.05, paired t test between 20 and 50% D/P.
different between 20 and 50% only for experiments with the slow-rate freezing program, with higher amounts of lymphocytes per unit for 20% D/P, Variation II versus Variation V (Table 3, p < 0.05). However, no influence on total lymphocyte recovery of precooling and 20 or 50% D/P was found.
WBC viability The WBC viability was approximately 93% before adding D/P and decreased to 88% to 91% and 86% to 88% after adding D/P for experiments with the fast-rate and slowrate freezing programs, respectively. After thawing, WBC viability was further decreased to 53% to 57% for the fastrate freezing program and 60% to 68% for the slow-rate freezing program (Table 3). The recovery of the WBC viability showed even more pronounced differences between both freezing programs, which was significant for all variations except for Variation I (Fig. 3D, p < 0.05). Comparing 20 and 50% D/P, a lower recovery of the WBC viability was found for 20% D/P (p < 0.05), for Variation II
(59%) versus Variation V (63%) when using the fast-rate freezing program and for Variation III (73%) versus Variation VI (78%) when using the slow-rate freezing program. The absolute number of viable WBCs only shows a significant difference between both freezing programs for Variations III and VI (D/P and WBC-enriched product are precooled), with a lower number of viable WBCs for the fast-rate freezing program compared to the slow-rate freezing program, 1.80 × 109 versus 2.16 × 109 for Variation III and 1.93 × 109 versus 2.30 × 109 for Variation VI (Table 3, p < 0.05).
Effect of storage in vapor-phase or liquid nitrogen WBC recovery For these experiments no differences in total WBC recovery were found between storage in vapor phase or liquid nitrogen (Table 1). Neither differences were found between both freezing programs or between 20% D/P and Volume **, ** **
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110 *
*
*
*
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MNC recove recovery %
WBC recovery %
*
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70
I
A
100
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VI
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B
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*
*
*
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†
110 *
WBC viability recovery %
Lymphocyte recovery %
*
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*
*
II
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80
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C
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Fig. 3. Recovery after thawing compared to after adding D/P for total WBCs (A), total MNCs (B), total lymphocytes (C), and WBC viability (D). ( ) Fast-rate freezing program; ( ) slow-rate freezing program. *p < 0.05, unpaired t test between both freezing programs; †p < 0.05, paired t-test between 20 and 50% D/P.
50% D/P. For all tested variations the mean total WBC recovery was between 95 and 96%.
WBC viability Recovery of WBC viability also did not differ between storage in vapor-phase or liquid nitrogen. Differences were found between both freezing programs, with higher values for the slow-rate freezing program (p < 0.05). A difference was also found between 20 and 50% D/P for Variation VIII versus Variation XII (fast-rate freezing program, liquid nitrogen; 62% vs. 65%; p < 0.05). For other variations no differences between 20% D/P and 50% D/P were found.
DISCUSSION In this article it was discussed that pre-cooling of either D/P or WBC-enriched product minimally affected recovery of total WBCs, total MNCs, total lymphocytes, and WBC viability after thawing. However, 20% D/P showed a 6
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lower WBC viability compared to 50% D/P (2%-5% difference, p < 0.05 for two of six variations tested), and slowrate freezing showed better viability results after thawing than fast-rate freezing (8%-15% difference; p < 0.05 for five of six variations tested). For this study a WBC-enriched product was used since real HPC products are not readily available for research purposes, due to ethical and practical considerations. Although this WBC-enriched product does not contain CD34+ cells or other immature cells we think this product can be used to optimize the freezing process of HPCs. Since WBCs are less stable than CD34+ cells,24,25 we think that when a method results in good WBC recovery and viability, it will be also the case for CD34+ cells. However, confirmation using real HPC product might still be desired. It can be expected that when products are precooled this will positively influence the results after thawing, because an exothermic reaction takes place when DMSO is added to plasma or HPCs. Our maximum temperature
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TABLE 3. Results for six variations of precooling D/P and WBC-enriched product using two freezing programs* Variation Fast-rate, fast-rate I II freezing program; D/P (%): 20 20 Slow-rate, slow-rate Temp D/P (°C): 20-24 2-6 freezing program Temp WBC-enriched product (°C): 20-24 20-24 Total WBCs, ×109/unit WBC-enriched product After adding D/P Fast-rate 3.63 ± 0.13 3.64 ± 0.15 Slow-rate 3.57 ± 0.33 3.67 ± 0.25a After thawing Fast-rate 3.56 ± 0.14 3.57 ± 0.17 Slow-rate 3.39 ± 0.24 3.46 ± 0.22 Total MNCs, ×109/unit WBC-enriched product After adding D/P 1.71 ± 0.20b Fast-rate 1.69 ± 0.19a Slow-rate 1.71 ± 0.20 1.77 ± 0.19c After thawing Fast-rate 1.60 ± 0.22 1.61 ± 0.24 Slow-rate 1.62 ± 0.24 1.66 ± 0.24d Total lymphocytes, ×109/unit WBC-enriched product After adding D/P Fast-rate 1.33 ± 0.22 1.33 ± 0.23 Slow-rate 1.37 ± 0.23 1.41 ± 0.22a After thawing Fast-rate 1.27 ± 0.24 1.30 ± 0.27 Slow-rate 1.26 ± 0.17 1.27 ± 0.17b WBC viability, % After adding D/P Fast-rate 89.0 ± 4.1 89.8 ± 3.5 Slow-rate 86.9 ± 7.6 86.25 ± 7.1a,l After thawing Fast-rate 54.3 ± 3.6 52.7 ± 3.5e Slow-rate 60.2 ± 7.0 62.9 ± 5.6e WBC viability, ×109/unit WBC-enriched product After adding D/P Fast-rate 3.34 ± 0.16 3.43 ± 0.24 Slow-rate 3.07 ± 0.43 3.21 ± 0.52 After thawing Fast-rate 1.89 ± 0.16 1.88 ± 0.12 Slow-rate 2.00 ± 0.33 2.22 ± 0.43
III 20 2-6 2-6
IV 50 20-24 20-24
V 50 2-6 20-24
VI 50 2-6 2-6
3.57 ± 0.18 3.59 ± 0.25
3.54 ± 0.18 3.64 ± 0.28
3.60 ± 0.13 3.61 ± 0.14 3.58 ± 0.21a 3.66 ± 0.12
3.53 ± 0.16 3.40 ± 0.23
3.47 ± 0.19 3.42 ± 0.13
3.56 ± 0.17 3.38 ± 0.19
1.69 ± 0.22 1.74 ± 0.21
1.57 ± 0.18a 1.66 ± 0.20b 1.69 ± 0.21 1.73 ± 0.18 1.70 ± 0.19c 1.77 ± 0.15
1.59 ± 0.23 1.64 ± 0.25
1.51 ± 0.18 1.65 ± 0.20
1.57 ± 0.21 1.58 ± 0.22 1.62 ± 0.22d 1.65 ± 0.22
1.32 ± 0.25 1.40 ± 0.24
1.25 ± 0.20 1.39 ± 0.20
1.31 ± 0.23 1.33 ± 0.24 1.36 ± 0.21a 1.40 ± 0.16
1.26 ± 0.24 1.27 ± 0.19
1.21 ± 0.19 1.26 ± 0.14
1.26 ± 0.23 1.25 ± 0.24 1.25 ± 0.16b 1.28 ± 0.16
90.1 ± 4.9 88.8 ± 6.8l
88.1 ± 5.4j 85.8 ± 7.1m
88.5 ± 5.0k 87.2 ± 6.6a
91.3 ± 2.5j,k 87.9 ± 7.3m
54.0 ± 3.5f 64.6 ± 5.6f
56.1 ± 3.2g 65.1 ± 8.2g
55.3 ± 1.3h 64.6 ± 9.1h
56.9 ± 4.7i 68.3 ± 5.7i
3.41 ± 0.10 3.21 ± 0.42
3.23 ± 0.34 3.18 ± 0.65
3.30 ± 0.15 3.17 ± 0.44
3.49 ± 0.16 3.21 ± 0.52
1.80 ± 0.06e 1.89 ± 0.21 2.16 ± 0.32e 2.19 ± 0.49
1.87 ± 0.09 2.17 ± 0.50
1.93 ± 0.16f 2.30 ± 0.31f
3.55 ± 0.15 3.45 ± 0.16
* Results are shown as mean ± SD. a, b, c, dp < 0.05 paired t test, 20% versus 50% D/P. e, f, g, h, ip < 0.05 unpaired t test, fast-rate versus slow-rate freezing program. j, k, l, mp < 0.05 ANOVA for repeated measurements, Variations I to III or Variations IV to VI.
increase was approximately 6°C for adding precooled D/P to precooled WBC-enriched product, which is comparable with that found by Nicoud and colleagues.11 The temperature increase was less when WBC-enriched product was at RT. An explanation for this might be the faster penetration of DMSO into the cell when the absolute temperature is higher,26 resulting in a lower increase in temperature of the product. Without precooling, the WBC-enriched product had a mean maximum temperature between 25 and 26°C, which appears to still be acceptable for a good cell recovery and viability after thawing. In the literature, it is suggested that addition of D/P to HPC causes a local overheating that causes protein denaturation and will be deleterious for the cells.2 However, in this study, this was not confirmed by results of total WBC recovery and WBC viability.
The temperature course of adding DMSO to plasma was also measured. For this a difference in maximum temperature of approximately 10°C was seen between 20 and 50% D/P. For adding 50% DMSO to plasma at RT, the maximum temperature was more than 40°C and the D/P became cloudy, indicating denaturation of proteins. Nicoud and colleagues11 described appearance of a “mushroom cloud” when adding DMSO solution quickly and this is probably the same phenomenon. However, in our study addition of a cloudy D/P did not affect total WBC recovery or viability after thawing, while Nicoud and colleagues11 found inferior results for fast compared to slow adding of DMSO. Although 20% D/P is most commonly used for freezing HPCs, 50% D/P did not show inferior results after thawing. These results are confirmed by Rubinstein and Volume **, ** **
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colleagues,10 although they used a 50% DMSO solution in 5% dextran 40. Therefore, it might be preferable to use higher concentrations D/P so that less volume is needed to be frozen. And even the volume of DMSO added to the patient will be a bit lower, which might reduce some adverse events caused by DMSO.7-9 In this study it was shown that the freezing curve influenced the postthaw results. A slow-rate freezing including a steep cooling rate to achieve vitrification and avoid nucleation had superior results above fast-rate freezing. Sputtek and coworkers13 showed that the optimum cooling rate was from 1 to 5°C/min from end of plateau phase until −45°C. Both tested programs in our study fall within these cooling rates. Tijssen and colleagues14 described a further optimization of the freezing curve, which is dependent of the final DMSO concentration. However, Tijssen and coworkers14 performed their experiments in cryotubes, while in our study cryobags were used. We did this, because in a previous study we showed that there might be a difference in thawing results between freezing in bags and tubes.23 Except for the steep cooling rate to achieve vitrification, the slow-rate freezing program is quite comparable to the optimized freezing program of Tijssen and colleagues.14 Further, freezing in a −80°C freezer instead of controlled-rate freezing has been described. It appeared that products in this case also have freezing rates in the 1 to 5°C/min range and result in good thawing results.15,24,27 From these studies, including our study, it appeared that a “slow-freezing” rate is preferred over a “fast-freezing” rate, but this depends on the final DMSO concentration. Furthermore, testing in tubes should be confirmed by testing in bags. One remarkable point is the contradiction in total WBC recovery and WBC viability. Total WBC recovery is optimal for fast-rate freezing (only shown for the experiments in which effect of precooling, initial DMSO concentration, and freezing program was studied) while WBC viability is optimal for the slow-rate freezing program. Total WBC recovery is calculated from WBC counts measured using the impedance technique of an automatic cell analyzer, while WBC viability is measured using a flow cytometric 7-aminoactinomycin D method. A possible explanation is that for the fast-rate freezing program more nonviable WBCs are present and for the slow-rate program these nonviable cells get lost. Since WBC viability is more important than total WBC recovery, slow-rate freezing is preferred over fast-rate freezing. Finally, the effect of storage in vapor-phase nitrogen versus liquid nitrogen was studied. Rowley and Byrne22 described this effect for temperature change during storage depending on the material of the storage racking system, distance from the liquid nitrogen, and opening of the lid of the tank. Their findings show that temperature of samples in an aluminum racking system barely increases at greater distance from liquid nitrogen in con8
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trast to samples in steel racking systems and that temperature of liquid nitrogen is more stable than that of vapor-phase nitrogen. Temperature of vapor-phase nitrogen remained for aluminum racking systems well below −150°C. Valeri and Pivacek15 described a comparison of storage of HPCs in provials in vapor-phase and liquid nitrogen and found no significant differences in recovery and viability and in culture assay. Our results of WBCenriched product in bags confirm these results as no difference in recovery and viability between both storage methods was found. Thus, for safety of the patient, because of the lower chance of cross-contamination, and for safety of the technician, because of the lower chance of spoiling liquid nitrogen, storage of HPCs in vaporphase nitrogen might be preferred over storage of HPCs in liquid nitrogen. In conclusion, precooling of HPCs or D/P seems not to be necessary, but 50% D/P showed for some variations a higher recovery of WBC viability after thawing than 20% D/P. The slow-rate freezing program is preferred above the fast-rate freezing program. Further, based on safety, storage in vapor-phase nitrogen is preferred over storage in liquid nitrogen. However, additional testing using real HPCs, and thus containing CD34+ and other immature cells, might be necessary to confirm these results. ACKNOWLEDGMENT We thank Paul Thijssen, head of the stem cell laboratory of Sanquin Blood Supply in Groningen, for critical reviewing of the manuscript.
CONFLICT OF INTEREST The authors have disclosed no conflicts of interest.
REFERENCES 1. Rowley SD. Hematopoietic stem cell processing and cryopreservation. J Clin Apher 1992;7:132-4. 2. Smagur A, Mitrus I, Giebel S, et al. Impact of different dimethyl sulphoxide concentrations on cell recovery, viability and clonogenic potential of cryopreserved peripheral blood hematopoietic stem and progenitor cells. Vox Sang 2013;104:240-7. 3. Baust JG, Gao D, Baust JM. Cryopreservation: an emerging paradigm change. Organogenesis 2009;5:90-6. 4. Rodriguez L, Velasco B, Garcia J, et al. Evaluation of an automated cell processing device to reduce the dimethyl sulfoxide from hematopoietic grafts after thawing. Transfusion 2005;45:1391-7. 5. Akkok CA, Holte MR, Tangen JM, et al. Hematopoietic engraftment of dimethyl sulfoxide-depleted autologous peripheral blood progenitor cells. Transfusion 2009;49:35461.
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6. Keung YK, Lau S, Elkayam U, et al. Cardiac arrhythmia after infusion of cryopreserved stem cells. Bone Marrow Transplant 1994;14:363-7. 7. Stroncek DF, Fautsch SK, Lasky LC, et al. Adverse reactions in patients transfused with cryopreserved marrow. Transfusion 1991;31:521-6. 8. Windrum P, Morris TC, Drake MB, et al. Variation in dimethyl sulfoxide use in stem cell transplantation: a survey of EBMT centres. Bone Marrow Transplant 2005;36: 601-3. 9. Zambelli A, Poggi G, Da PG, et al. Clinical toxicity of cryopreserved circulating progenitor cells infusion. Anticancer Res 1998;18(6B):4705-8. 10. Rubinstein P, Dobrila L, Rosenfield RE, et al. Processing and cryopreservation of placental/umbilical cord blood for unrelated bone marrow reconstitution. Proc Natl Acad Sci U S A 1995;92:10119-22. 11. Nicoud IB, Clarke DM, Taber G, et al. Cryopreservation of umbilical cord blood with a novel freezing solution that mimics intracellular ionic composition. Transfusion 2012; 52:2055-62. 12. McCullough J, Haley R, Clay M, et al. Long-term storage of peripheral blood stem cells frozen and stored with a conventional liquid nitrogen technique compared with cells frozen and stored in a mechanical freezer. Transfusion 2010;50:808-19. 13. Sputtek A, Jetter S, Hummel K, et al. Cryopreservation of peripheral blood progenitor cells: characteristics of suitable techniques. Beitr Infusionsther Transfusionsmed 1997; 34:79-83. 14. Tijssen MR, Woelders H, de Vries-van RA, et al. Improved postthaw viability and in vitro functionality of peripheral blood hematopoietic progenitor cells after cryopreservation with a theoretically optimized freezing curve. Transfusion 2008;48:893-901.
17. Foundation for the Accreditation of Cellular Therapy (FACT) and Joint Accreditation Committee ISCT and EBMT (JACIE). International standards for cellular therapy product collection, processing, and administration accreditation manual. 5.3 ed. Omaha (NE): FACT/JACIE; 2012. 18. Tedder RS, Zuckerman MA, Goldstone AH, et al. Hepatitis B transmission from contaminated cryopreservation tank. Lancet 1995;346:137-40. 19. Fountain D, Ralston M, Higgins N, et al. Liquid nitrogen freezers: a potential source of microbial contamination of hematopoietic stem cell components. Transfusion 1997;37: 585-91. 20. Grout BW, Morris GJ. Contaminated liquid nitrogen vapour as a risk factor in pathogen transfer. Theriogenology 2009; 71:1079-82. 21. Mirabet V, Alvarez M, Solves P, et al. Use of liquid nitrogen during storage in a cell and tissue bank: contamination risk and effect on the detectability of potential viral contaminants. Cryobiology 2012;64:121-3. 22. Rowley SD, Byrne DV. Low-temperature storage of bone marrow in nitrogen vapor-phase refrigerators: decreased temperature gradients with an aluminum racking system. Transfusion 1992;32:750-4. 23. Dijkstra-Tiekstra MJ, Setroikromo AC, de Wildt-Eggen J. Freezing “stem cells” in a bag and tube under various freezing conditions? Vox Sang 2012;102:273. 24. Almici C, Ferremi P, Lanfranchi A, et al. Uncontrolled-rate freezing of peripheral blood progenitor cells allows successful engraftment by sparing primitive and committed hematopoietic progenitors. Haematologica 2003;88:1390-5. 25. Humpe A, Beck C, Schoch R, et al. Establishment and optimization of a flow cytometric method for evaluation of viability of CD34+ cells after cryopreservation and comparison with trypan blue exclusion staining. Transfusion
15. Valeri CR, Pivacek LE. Effects of the temperature, the duration of frozen storage, and the freezing container on in
2005;45:1208-13. 26. Meryman HT. Cryopreservation of living cells: principles
vitro measurements in human peripheral blood mononuclear cells. Transfusion 1996;36:303-8. 16. Mazur P. The role of intracellular freezing in the death of cells cooled at supraoptimal rates. Cryobiology 1977;14: 251-72.
and practice. Transfusion 2007;47:935-45. 27. Solves P, Mirabet V, Planelles D, et al. Influence of volume reduction and cryopreservation methodologies on quality of thawed umbilical cord blood units for transplantation. Cryobiology 2008;56:152-8.
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