Optimization of the freezing process for hematopoietic progenitor cells: effect of precooling, initial dimethyl sulfoxide concentration, freezing program, and storage in vapor-phase

O R I G I N A L A R T I C L E

Optimization of the freezing process for hematopoieticprogenitor cells: effect of precooling, initial dimethyl sulfoxide

concentration, freezing program, and storage in vapor-phase orliquid 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) tohematopoietic progenitor cells (HPCs) causes an exo-thermic reaction, potentially affecting their viability. Thefreezing method might also influence this. The aim wasto investigate the effect of 1) precooling of DMSO andplasma (D/P) and white blood cell (WBC)-enrichedproduct, 2) DMSO concentration of D/P, 3) freezingprogram, and 4) storage method on WBC quality.STUDY DESIGN AND METHODS: WBC-enrichedproduct without CD34+ cells was used instead of HPCs.This was divided into six or eight portions. D/P (20 or50%; precooled or room temperature [RT]) was addedto the WBC-enriched product (precooled or RT), result-ing in 10% DMSO, while monitoring temperature. Theproduct was frozen using controlled-rate freezing (“fast-rate” or “slow-rate”) and placed in vapor-phase or liquidnitrogen. After thawing, WBC recovery and viabilitywere determined.RESULTS: Temperature increased most for precooledD/P to precooled WBC-enriched product, without influ-ence of 20 or 50% D/P, but remained for all variationsbelow 30°C. WBC recovery for both freezing programswas more than 95%. Recovery of WBC viability washigher for slow-rate freezing compared to fast-ratefreezing (74% vs. 61%; p < 0.05) and also for 50%compared to 20% D/P (two test variations). Effect ofprecooling D/P or WBC-enriched product and of storagein vapor-phase or liquid nitrogen was marginal.CONCLUSION: Based on these results, precooling isnot necessary. Fifty percent D/P is preferred over 20%D/P. Slow-rate freezing is preferred over fast-rate freez-ing. For safety reasons storage in vapor-phase nitrogenis preferred over storage in liquid nitrogen. Additionaltesting using real HPCs might be necessary.

To protect hematopoietic progenitor cells (HPCs)during cryopreservation, a cryoprotectant likedimethyl sulfoxide (DMSO) is added. The DMSOpenetrates cells and binds water molecules in

solution thereby preventing cellular dehydration.1,2

However, a disadvantage of DMSO is that it may cause cellinjury before freezing and after thawing.2-4 Other disad-vantages are that by adding DMSO the final volume frozenis increased and sometimes DMSO needs to be washedaway before transplantation of the HPCs, causing adelayed platelet engraftment.5 If DMSO is transplantedtogether with the HPCs it may cause mild reactions likenausea, vomiting, chills, and fever or the more severe butless 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, mostly20%, before addition to HPCs. To reduce storage volumeand absolute amount of DMSO, a higher preconcentrationDMSO can be used, for example, 50%.10 Because additionof DMSO to HPCs causes an exothermic reaction, whichcan 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

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often precooled. Furthermore, the used DMSO concentra-tion may be of importance in cell survival after thawing,since it is expected that a higher concentration or fasteraddition of DMSO causes more local overheating resultingin protein denaturation.2,11

Another point of attention is the freezing rate. Cellscan be frozen using controlled-rate freezing as well asuncontrolled-rate freezing in a −80 or −135°C mechanicalfreezer.12-15 The freezing program is important. Duringtoo-fast cooling, intracellular crystallization may occur,which could result in cell membrane rupture. During too-slow cooling, extracellular ice formation can occur, whichcould result in dehydration of the cell due to osmosis.1,16

Storage of HPCs can be done at temperatures between −80and −196°C.12-15 Until about 10 years ago, HPCs werestored in liquid nitrogen. Because of the possibility ofcross-contamination of microbial pathogens when HPCsare stored in liquid nitrogen, it is advised to store HPCs inthe vapor phase of nitrogen,17,18 although the vapor phaseof nitrogen might also be a potential source of pathogencontamination.19-21 However, literature for studying differ-ences between storage in vapor-phase or liquid nitrogen isscarce. One study described the effect on temperature,22

and another described the effect on quality of HPCs inprovials,15 which might deviate from results of HPCs inbags.15,23 Because it is difficult to obtain HPC products forresearch purposes, in this study a white blood cell (WBC)-enriched product with comparable cell counts and hema-tocrit (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-enrichedproduct compared to room temperature (RT), 2) theDMSO concentration of D/P (20 or 50%), and 3) the freez-ing program (“fast-rate” or “slow-rate”), on temperaturecourse, total cell recovery (WBCs, mononuclear cells[MNCs], and lymphocytes) and WBC viability. Besidesthis, the effect of storage of WBC-enriched product ineither liquid nitrogen or vapor-phase nitrogen on totalWBC recovery and viability was studied.

MATERIALS AND METHODS

The WBC-enriched productBecause HPCs are not readily available for research pur-poses, a WBC-enriched product, with comparable cellcounts and Hct, but absence of CD34+ and immaturecells, was used in this study. The WBC-enriched productwas prepared from buffy coats and plasma. In short,plasma was added to a residual buffy coat pool via a usedleukoreduction filter (Compostop, Fresenius Hemocare,Emmer Compascuum, the Netherlands) in the oppositedirection to increase the WBC count. The used plasma andresidual buffy coat pool derived from whole blood collec-tion of 1 day previously. Six of these plasma-remaining

buffy coat pool units were pooled and diluted usingplasma to a WBC concentration of approximately10 × 109/L and a Hct of about 10%. This was divided overseveral 400-mL bags and allowed to sediment by gravityfor 105 minutes. The supernatant including the buffy coatlayer was pressed to a satellite bag, sampled, and centri-fuged at 3900 × g for 14 minutes. From the sample theWBC count was determined. The supernatant was pressedfrom the pellets. The pellets were resuspended in super-natant plasma and pooled resulting in approximately150 mL of WBC-enriched product with a WBC concentra-tion of approximately 200 × 109/L.

Preparing D/PFor preparation of 20% D/P, four volume parts of plasmawere placed into in two bags. Subsequently the probe of adata logger (Escort Junior, −40 to 70°C, Escort DataLogging Systems LTD, New Lynn, Auckland, New Zealand)was placed in each bag and was activated (one measure-ment/5 sec). One bag was stored at RT and the other wasplaced between two precooled gel packs (Cool Gel, SCACool Logistics, Leighton Buzzard, UK) at 2 to 6°C for 15minutes. 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) wasadded slowly under continuous agitation to each bag.After DMSO was added, the bags were stored for at least15 minutes again at RT or at 2 to 6°C between precooledgel packs. For 50% D/P the procedure for 20% D/P asdescribed was followed but with equal volumes of plasmaand DMSO.

Study effect of precooling, initial DMSOconcentration, and freezing programThe processing of the WBC-enriched product with thevarious study variations is shown in a flow chart (Fig. 1).After overnight storage at 2 to 6°C, the WBC-enrichedproduct was divided into six portions of 20 mL in 150-mLtransfer bags (Fresenius Hemocare), in which the probe ofa data logger (Escort Junior, −40 to 70°C) was placed andactivated (one measurement/5 sec). To three bags 12 mLplasma was added. Two bags with only WBC-enrichedproduct and two bags with additional plasma were kept atRT (20-24°C) and the remaining two bags were precooledbetween precooled gel packs at 2 to 6°C for at least 15minutes. The 20 and 50% D/P at RT were sterile connectedto the WBC-enriched product stored at RT and the pre-cooled 20 and 50% D/P were sterile connected to theremaining two bags with WBC-enriched product stored atRT and also to two bags with precooled WBC-enrichedproduct. The 50% D/P was docked on the bags containingthe additional plasma, to keep final volumes equal.Twenty milliliters of 20% D/P or 8 mL of 50% D/P was

DIJKSTRA-TIEKSTRA ET AL.

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added slowly under continuous agitation to the WBC-enriched product. The WBC-enriched product had a finalDMSO concentration of 10% and a final WBC concentra-tion of 100 × 109/L. At the moment the temperaturedid not increase further, the products were sampled(1 mL) and transferred to 250-mL freezing containers(CryoMACS, Miltenyi Biotec, Bergisch Gladbach,Germany) and weighed.

Reference bagThe reference bag was used to monitor the temperatureduring the freezing of the cells. Therefore, the probe of a

data logger (Escort Precision with PT300 adaptor, -200 to +300°C, Escort DataLogging Systems LTD) was placed in a250-mL freezing bag. Subsequently forhalf of the experiments 20 mL of WBC-enriched product and 20 mL 20% D/Pwere added slowly under continuousagitation. For the other half of theexperiments 20 mL of WBC-enrichedproduct, 12 mL of plasma, and 8 mL50% D/P were added slowly under con-tinuous agitation. The data logger wasactivated (one measurement/sec).

Freezing and thawing of theWBC-enriched productAll freezing bags (six study variationsand a reference) were frozen usingcontrolled-rate freezing with either afast-rate or slow-rate freezing programin a controlled-rate freezer (Kryo 560-16, Planer, Middlesex, UK). The fast-rateprogram was as follows: start tempera-ture, 10°C; 5 minutes at 10°C; −4°C/minuntil 0°C; 2 minutes at 0°C; −1°C/minuntil −15°C, −4°C/min until −25°C;−5°C/min until −35°C; −8°C/min until−100°C; −15°C/min until −160°C; and10 minutes at −160°C. The slow-rateprogram was as follows: start tempera-ture, 10°C; 1 minute at 10°C; −1°C/minuntil −8°C; −20°C/min until −60°C; 2minutes at −60°C; 15°C/min until −35°C;−0.5°C/min until −45°C; and −5.0°C/minuntil −140°C (Fig. 2).

Subsequently the products wereplaced in an aluminum racking systeminto the vapor phase of nitrogen. Afterstorage for at least 1 week, the productswere thawed in a 37°C water bath and

immediately fivefold diluted using phosphate-bufferedsaline.

Study effect of storage in vapor-phase orliquid nitrogenFor this, eight variations (VII-XIV) were compared withvariations for 20 or 50% D/P, freezing using either the fast-rate or slow-rate freezing program, and storage in vapor-phase nitrogen or liquid nitrogen (see Table 1). For allvariations precooled D/P was added to precooled WBC-enriched product. The followed procedure was asdescribed above.

± 150 mL WBC-enriched product (cells)

Var III20 mLcells +0 mL

plasma(6-10°C)

Var I20 mLcells +0 mL

plasma(20-24°C)

Var V20 mLcells +12 mL

plasma(20-24°C)

Var IV20 mLcells +12 mL

plasma(20-24°C)

Var II20 mLcells +0 mL

plasma(20-24°C)

Var VI20 mLcells + 12 mL

plasma(6-10°C)

20 mL20% D/P (20-24°C)

20 mL20% D/P(6-10°C)

20 mL20% D/P(6-10°C)

8 mL50% D/P(20-24°C)

8 mL50% D/P (6-10°C)

8 mL50% D/P (6-10°C)

cells +D/P in

freezing bag

cells +D/P in

freezing bag

cells +D/P in

freezing bag

cells +D/P in

freezing bag

cells +D/P in

freezing bag

Add D/P to cells and transferto freezing bag

Reference20 mLcells

in freezing

bag

20 mL20% D/P (20-24°C)

Add D/P to cells

Add D/P to cells and transferto freezing bag

Thawing

cells +D/P in

freezing bag

Freezing using either the fast-rate or the slow-rate program

Referencecells +D/P in

freezing bag

Fig. 1. Flow chart for freezing and thawing of WBC-enriched product in six variations

(Var). Experiments were performed separately, thus nonpaired, for fast-rate and

slow-rate freezing programs. The reference bag was prepared in half of the experi-

ments using 20 mL of WBC-enriched product and 20 mL of 20% D/P (as is shown in

the flow chart) and in the other half of the experiments using 20 mL of WBC-

enriched product, 12 mL of plasma, and 8 mL of 50% D/P. Temperature was

monitored for Variations I to VI for preparation of D/P and for adding D/P to

WBC-enriched product. The temperature of the reference bag was monitored while

freezing the WBC-enriched product with D/P.

OPTIMIZATION OF THE HPC FREEZING PROCESS


TestsTests were performed before adding D/P, after adding D/P,and after thawing. WBC count (including differentiation)was determined using a hematology analyzer (ModelXT1800i, Sysmex, Kobe, Japan). The MNCs consisted ofmonocytes and lymphocytes. WBC viability was measuredusing a flow cytometric 7-aminoactinomycin D methodaccording to the prescriptions of the manufacturer (BDstem cell enumeration kit, BD Biosciences, San Jose, CA)on a flow cytometer (FACSCalibur, BD Biosciences). Thepercentages of recovery after thawing compared to afteradding D/P for total WBCs, total MNCs, total lymphocytes,and WBC viability were calculated.

Statistical analysisExperiments for studying the effect of precooling, initialDMSO concentration, and freezing program using thefast-rate freezing program were independently per-formed from experiments using the slow-rate freezingprogram. The other variations were performed in apaired design. All experiments for studying the effect ofstorage in vapor-phase or liquid nitrogen were per-formed in a paired design. Experiments were performedwith n = 6.

For statistical analysis computer software (MicrosoftExcel 2002, Microsoft Corp., Redmond, WA; and Instat,GraphPad, 2005, San Diego, CA) was used. Results areshown as mean (±SD). For comparison of data either at test (paired or unpaired) or an analysis of variance(ANOVA), regular or for repeated measurements, was usedfollowed by Tukey posttest as is indicated in the legends of

the figures or tables. A p value of lessthan 0.05 was used to indicate a signifi-cant difference.

RESULTS

Effect of precooling, initialDMSO concentration, andfreezing program

Temperature courseTemperature increase was lower foradding 20% DMSO to plasma comparedto 50% DMSO (p < 0.05; Table 2). Asexpected, the maximum temperaturereached was lower when DMSO wasadded to precooled plasma (p < 0.05).

For adding D/P to the WBC-enriched product, it was found thathighest temperature was reached whenD/P and/or WBC-enriched productswere at RT (p < 0.05; Table 2). However,the increase in temperature was highest

when both D/P and WBC-enriched product were pre-cooled (p < 0.05).

WBCsFor all variations except for Variation I (20% D/P andWBC-enriched product at RT) total WBC recovery washigher for WBC-enriched product frozen using the fast-rate program than when using the slow-rate program(p < 0.05; Fig. 3A). Total WBC recovery was approximately98% for freezing the fast-rate program and approximately95% for the slow-rate freezing program. No influence ontotal WBC recovery of precooling and 20 or 50% D/P wasfound.

MNCsTotal MNC recovery was between 93 and 95% for the fast-rate freezing program and between 90 and 92% for theslow-rate freezing program (Fig. 3B, p > 0.05). The abso-lute amounts of total MNCs per unit after adding D/P werehigher for 20% D/P than for 50% D/P, Variation II versusVariation V (precooled D/P and WBC-enriched product atRT) for both freezing programs and for the fast-rate freez-ing program also for Variation I versus Variation IV (D/Pand WBC-enriched product at RT; Table 3, p < 0.05).However, no influence on total MNC recovery of precool-ing and 20% or 50% D/P was found.

LymphocytesTotal lymphocyte recovery was between 94 and 98% forthe fast-rate freezing program and between 90 and 92% forthe slow-rate freezing program, with only significant dif-ferences for Variations II and III (Fig. 3C, p < 0.05).Amounts of total lymphocytes after adding D/P were

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Fig. 2. Temperature course of the fast-rate (gray line) and slow-rate (black line)

freezing programs. Temperature was measured in the reference bag that was frozen

simultaneously with the bags of the experiments (continuous lines). The curve as

programmed is shown by the dotted lines.



different between 20 and 50% only for experiments withthe slow-rate freezing program, with higher amounts oflymphocytes per unit for 20% D/P, Variation II versusVariation V (Table 3, p < 0.05). However, no influence ontotal lymphocyte recovery of precooling and 20 or 50%D/P was found.

WBC viabilityThe WBC viability was approximately 93% before addingD/P and decreased to 88% to 91% and 86% to 88% afteradding D/P for experiments with the fast-rate and slow-rate freezing programs, respectively. After thawing, WBCviability was further decreased to 53% to 57% for the fast-rate freezing program and 60% to 68% for the slow-ratefreezing program (Table 3). The recovery of the WBCviability showed even more pronounced differencesbetween both freezing programs, which was significant forall variations except for Variation I (Fig. 3D, p < 0.05).Comparing 20 and 50% D/P, a lower recovery of the WBCviability was found for 20% D/P (p < 0.05), for Variation II

(59%) versus Variation V (63%) when using the fast-ratefreezing program and for Variation III (73%) versusVariation VI (78%) when using the slow-rate freezingprogram.

The absolute number of viable WBCs only shows asignificant difference between both freezing programs forVariations III and VI (D/P and WBC-enriched product areprecooled), with a lower number of viable WBCs for thefast-rate freezing program compared to the slow-ratefreezing program, 1.80 × 109 versus 2.16 × 109 for VariationIII 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 recoveryFor these experiments no differences in total WBC recov-ery were found between storage in vapor phase or liquidnitrogen (Table 1). Neither differences were foundbetween both freezing programs or between 20% D/P and

TABLE 1. Results for testing effect of storage WBC-enriched product in vapor-phase or liquid nitrogenVariation D/P (%) Freezing program Storage Total WBC recovery* WBC viability*

VII 20 Fast-rate Vapor phase 95.8 ± 2.0 63.8 ± 4.3a

VIII 20 Fast-rate Liquid nitrogen 95.8 ± 1.3 61.7 ± 2.8b,e

IX 20 Slow-rate Vapor phase 96.0 ± 1.9 73.9 ± 3.6a

X 20 Slow-rate Liquid nitrogen 95.0 ± 2.9 71.4 ± 7.0b

XI 50 Fast-rate Vapor phase 96.5 ± 1.7 65.2 ± 5.4c

XII 50 Fast-rate Liquid nitrogen 96.3 ± 1.8 65.0 ± 4.0d,e

XIII 50 Slow-rate Vapor phase 96.2 ± 1.9 73.5 ± 4.3c

XIV 50 Slow-rate Liquid nitrogen 94.9 ± 2.9 71.7 ± 5.8d

* Results show recovery after thawing compared to after adding D/P; results (%) are shown as mean ± SD.a,b,c,dp < 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 (%) Temp DMSO (°C) Start temp plasma (°C) Max temp (°C) Max increase (°C)

I 20 20-24 22.7 ± 0.8a 32.8 ± 0.9a,c 10.1 ± 0.5a,c

II/III 20 20-24 8.5 ± 1.1a 21.4 ± 1.1a,d 12.9 ± 1.2a,d

IV 50 20-24 22.7 ± 0.6b 43.3 ± 0.8b,c 20.6 ± 0.4b,c

V/VI 50 20-24 8.3 ± 0.6b 34.7 ± 1.7b,d 26.3 ± 1.6b,d

Adding D/P to WBC-enriched product

Variation D/P (%) Temp D/P (°C) Start temp WBC-enriched product (°C) Max temp (°C) Max increase (°C)

I 20 20-24 23.0 ± 0.8e,f 25.5 ± 0.9e,f 2.5 ± 0.8e

II 20 6-10 20.7 ± 1.7e,g,j 23.8 ± 1.3e,g,j 3.1 ± 1.4f

III 20 6-10 9.2 ± 1.0f,g 15.1 ± 1.2f,g 5.9 ± 1.3e,f

IV 50 20-24 23.0 ± 1.2h 25.6 ± 0.7h 2.7 ± 1.2h

V 50 6-10 23.1 ± 0.8i,j 25.9 ± 1.3i,j 2.8 ± 1.6i

VI 50 6-10 9.1 ± 1.0h,i 15.6 ± 1.8h,i 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 intemperature. n = 12, except for adding DMSO to Plasma Variation IV with n = 9. Adding DMSO to plasma: ap < 0.05, paired t test betweenVariations 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 toWBC-enriched product: e, f,gp < 0.05, ANOVA for repeated measurements between Variations I to III; h, ip < 0.05, ANOVA betweenVariations IV to VI; jp < 0.05, paired t test between 20 and 50% D/P.



50% D/P. For all tested variations the mean total WBCrecovery was between 95 and 96%.

WBC viabilityRecovery of WBC viability also did not differ betweenstorage in vapor-phase or liquid nitrogen. Differenceswere found between both freezing programs, with highervalues for the slow-rate freezing program (p < 0.05). A dif-ference was also found between 20 and 50% D/P for Varia-tion VIII versus Variation XII (fast-rate freezing program,liquid nitrogen; 62% vs. 65%; p < 0.05). For other varia-tions no differences between 20% D/P and 50% D/P werefound.

DISCUSSION

In this article it was discussed that pre-cooling of eitherD/P or WBC-enriched product minimally affected recov-ery of total WBCs, total MNCs, total lymphocytes, andWBC viability after thawing. However, 20% D/P showed a

lower WBC viability compared to 50% D/P (2%-5% differ-ence, p < 0.05 for two of six variations tested), and slow-rate freezing showed better viability results after thawingthan fast-rate freezing (8%-15% difference; p < 0.05 for fiveof six variations tested).

For this study a WBC-enriched product was usedsince real HPC products are not readily available forresearch purposes, due to ethical and practical consider-ations. Although this WBC-enriched product does notcontain CD34+ cells or other immature cells we think thisproduct can be used to optimize the freezing process ofHPCs. Since WBCs are less stable than CD34+ cells,24,25 wethink that when a method results in good WBC recoveryand viability, it will be also the case for CD34+ cells.However, confirmation using real HPC product might stillbe desired.

It can be expected that when products are precooledthis will positively influence the results after thawing,because an exothermic reaction takes place when DMSOis added to plasma or HPCs. Our maximum temperature

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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.



increase was approximately 6°C for adding precooled D/Pto precooled WBC-enriched product, which is comparablewith that found by Nicoud and colleagues.11 The tempera-ture increase was less when WBC-enriched product was atRT. An explanation for this might be the faster penetrationof DMSO into the cell when the absolute temperature ishigher,26 resulting in a lower increase in temperature of theproduct. Without precooling, the WBC-enriched producthad a mean maximum temperature between 25 and 26°C,which appears to still be acceptable for a good cell recov-ery and viability after thawing. In the literature, it is sug-gested that addition of D/P to HPC causes a localoverheating that causes protein denaturation and will bedeleterious for the cells.2 However, in this study, this wasnot confirmed by results of total WBC recovery and WBCviability.

The temperature course of adding DMSO to plasmawas also measured. For this a difference in maximum tem-perature of approximately 10°C was seen between 20 and50% D/P. For adding 50% DMSO to plasma at RT, themaximum temperature was more than 40°C and the D/Pbecame cloudy, indicating denaturation of proteins.Nicoud and colleagues11 described appearance of a“mushroom cloud” when adding DMSO solution quicklyand this is probably the same phenomenon. However, inour study addition of a cloudy D/P did not affect total WBCrecovery or viability after thawing, while Nicoud and col-leagues11 found inferior results for fast compared to slowadding of DMSO.

Although 20% D/P is most commonly used for freez-ing HPCs, 50% D/P did not show inferior results afterthawing. These results are confirmed by Rubinstein and

TABLE 3. Results for six variations of precooling D/P and WBC-enriched product using two freezing programs*

Fast-rate, fast-ratefreezing program;Slow-rate, slow-ratefreezing program

D/P (%):

Variation

I II III IV V VI20 20 20 50 50 50

Temp D/P (°C): 20-24 2-6 2-6 20-24 2-6 2-6Temp WBC-enriched product (°C): 20-24 20-24 2-6 20-24 20-24 2-6

Total WBCs, ×109/unit WBC-enriched productAfter adding D/P

Fast-rate 3.63 ± 0.13 3.64 ± 0.15 3.57 ± 0.18 3.54 ± 0.18 3.60 ± 0.13 3.61 ± 0.14Slow-rate 3.57 ± 0.33 3.67 ± 0.25a 3.59 ± 0.25 3.64 ± 0.28 3.58 ± 0.21a 3.66 ± 0.12

After thawingFast-rate 3.56 ± 0.14 3.57 ± 0.17 3.53 ± 0.16 3.47 ± 0.19 3.56 ± 0.17 3.55 ± 0.15Slow-rate 3.39 ± 0.24 3.46 ± 0.22 3.40 ± 0.23 3.42 ± 0.13 3.38 ± 0.19 3.45 ± 0.16

Total MNCs, ×109/unit WBC-enriched productAfter adding D/P

Fast-rate 1.69 ± 0.19a 1.71 ± 0.20b 1.69 ± 0.22 1.57 ± 0.18a 1.66 ± 0.20b 1.69 ± 0.21Slow-rate 1.71 ± 0.20 1.77 ± 0.19c 1.74 ± 0.21 1.73 ± 0.18 1.70 ± 0.19c 1.77 ± 0.15

After thawingFast-rate 1.60 ± 0.22 1.61 ± 0.24 1.59 ± 0.23 1.51 ± 0.18 1.57 ± 0.21 1.58 ± 0.22Slow-rate 1.62 ± 0.24 1.66 ± 0.24d 1.64 ± 0.25 1.65 ± 0.20 1.62 ± 0.22d 1.65 ± 0.22

Total lymphocytes, ×109/unit WBC-enrichedproduct

After adding D/PFast-rate 1.33 ± 0.22 1.33 ± 0.23 1.32 ± 0.25 1.25 ± 0.20 1.31 ± 0.23 1.33 ± 0.24Slow-rate 1.37 ± 0.23 1.41 ± 0.22a 1.40 ± 0.24 1.39 ± 0.20 1.36 ± 0.21a 1.40 ± 0.16

After thawingFast-rate 1.27 ± 0.24 1.30 ± 0.27 1.26 ± 0.24 1.21 ± 0.19 1.26 ± 0.23 1.25 ± 0.24Slow-rate 1.26 ± 0.17 1.27 ± 0.17b 1.27 ± 0.19 1.26 ± 0.14 1.25 ± 0.16b 1.28 ± 0.16

WBC viability, %After adding D/P

Fast-rate 89.0 ± 4.1 89.8 ± 3.5 90.1 ± 4.9 88.1 ± 5.4j 88.5 ± 5.0k 91.3 ± 2.5j,k

Slow-rate 86.9 ± 7.6 86.25 ± 7.1a,l 88.8 ± 6.8l 85.8 ± 7.1m 87.2 ± 6.6a 87.9 ± 7.3m

After thawingFast-rate 54.3 ± 3.6 52.7 ± 3.5e 54.0 ± 3.5f 56.1 ± 3.2g 55.3 ± 1.3h 56.9 ± 4.7i

Slow-rate 60.2 ± 7.0 62.9 ± 5.6e 64.6 ± 5.6f 65.1 ± 8.2g 64.6 ± 9.1h 68.3 ± 5.7i

WBC viability, ×109/unit WBC-enrichedproduct

After adding D/PFast-rate 3.34 ± 0.16 3.43 ± 0.24 3.41 ± 0.10 3.23 ± 0.34 3.30 ± 0.15 3.49 ± 0.16Slow-rate 3.07 ± 0.43 3.21 ± 0.52 3.21 ± 0.42 3.18 ± 0.65 3.17 ± 0.44 3.21 ± 0.52

After thawingFast-rate 1.89 ± 0.16 1.88 ± 0.12 1.80 ± 0.06e 1.89 ± 0.21 1.87 ± 0.09 1.93 ± 0.16f

Slow-rate 2.00 ± 0.33 2.22 ± 0.43 2.16 ± 0.32e 2.19 ± 0.49 2.17 ± 0.50 2.30 ± 0.31f

* 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-ratefreezing program. j,k, l,mp < 0.05 ANOVA for repeated measurements, Variations I to III or Variations IV to VI.



colleagues,10 although they used a 50% DMSO solution in5% dextran 40. Therefore, it might be preferable to usehigher concentrations D/P so that less volume is neededto be frozen. And even the volume of DMSO added to thepatient will be a bit lower, which might reduce someadverse events caused by DMSO.7-9

In this study it was shown that the freezing curveinfluenced the postthaw results. A slow-rate freezingincluding a steep cooling rate to achieve vitrification andavoid nucleation had superior results above fast-ratefreezing. Sputtek and coworkers13 showed that theoptimum cooling rate was from 1 to 5°C/min from end ofplateau phase until −45°C. Both tested programs in ourstudy fall within these cooling rates. Tijssen and col-leagues14 described a further optimization of the freezingcurve, which is dependent of the final DMSO concentra-tion. However, Tijssen and coworkers14 performed theirexperiments in cryotubes, while in our study cryobagswere used. We did this, because in a previous study weshowed that there might be a difference in thawing resultsbetween freezing in bags and tubes.23 Except for the steepcooling rate to achieve vitrification, the slow-rate freezingprogram is quite comparable to the optimized freezingprogram of Tijssen and colleagues.14 Further, freezing in a−80°C freezer instead of controlled-rate freezing has beendescribed. It appeared that products in this case also havefreezing rates in the 1 to 5°C/min range and result in goodthawing results.15,24,27 From these studies, including ourstudy, it appeared that a “slow-freezing” rate is preferredover a “fast-freezing” rate, but this depends on the finalDMSO concentration. Furthermore, testing in tubesshould be confirmed by testing in bags.

One remarkable point is the contradiction in totalWBC recovery and WBC viability. Total WBC recovery isoptimal for fast-rate freezing (only shown for the experi-ments in which effect of precooling, initial DMSO concen-tration, and freezing program was studied) while WBCviability is optimal for the slow-rate freezing program.Total WBC recovery is calculated from WBC counts mea-sured using the impedance technique of an automatic cellanalyzer, while WBC viability is measured using a flowcytometric 7-aminoactinomycin D method. A possibleexplanation is that for the fast-rate freezing program morenonviable WBCs are present and for the slow-rate programthese nonviable cells get lost. Since WBC viability is moreimportant than total WBC recovery, slow-rate freezing ispreferred over fast-rate freezing.

Finally, the effect of storage in vapor-phase nitrogenversus liquid nitrogen was studied. Rowley and Byrne22

described this effect for temperature change duringstorage depending on the material of the storage rackingsystem, distance from the liquid nitrogen, and openingof the lid of the tank. Their findings show that tempera-ture of samples in an aluminum racking system barelyincreases at greater distance from liquid nitrogen in con-

trast to samples in steel racking systems and that tem-perature of liquid nitrogen is more stable than that ofvapor-phase nitrogen. Temperature of vapor-phase nitro-gen remained for aluminum racking systems well below−150°C. Valeri and Pivacek15 described a comparison ofstorage of HPCs in provials in vapor-phase and liquidnitrogen and found no significant differences in recoveryand viability and in culture assay. Our results of WBC-enriched product in bags confirm these results as no dif-ference in recovery and viability between both storagemethods was found. Thus, for safety of the patient,because of the lower chance of cross-contamination, andfor safety of the technician, because of the lower chanceof spoiling liquid nitrogen, storage of HPCs in vapor-phase nitrogen might be preferred over storage of HPCsin liquid nitrogen.

In conclusion, precooling of HPCs or D/P seems notto be necessary, but 50% D/P showed for some variationsa higher recovery of WBC viability after thawing than 20%D/P. The slow-rate freezing program is preferred above thefast-rate freezing program. Further, based on safety,storage in vapor-phase nitrogen is preferred over storagein liquid nitrogen. However, additional testing using realHPCs, and thus containing CD34+ and other immaturecells, 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 periph-

eral 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. Transfu-

sion 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;49:354-

61.



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. Trans-

fusion 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. Anti-

cancer 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 con-

ventional 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 suit-

able 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.

15. Valeri CR, Pivacek LE. Effects of the temperature, the dura-

tion of frozen storage, and the freezing container on in

vitro measurements in human peripheral blood mono-

nuclear 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.

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 con-

taminants. 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 suc-

cessful engraftment by sparing primitive and committed

hematopoietic progenitors. Haematologica 2003;88:1390-5.

25. Humpe A, Beck C, Schoch R, et al. Establishment and opti-

mization of a flow cytometric method for evaluation of

viability of CD34+ cells after cryopreservation and com-

parison with trypan blue exclusion staining. Transfusion

2005;45:1208-13.

26. Meryman HT. Cryopreservation of living cells: principles

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.



Documents

Optimization of the freezing process for hematopoietic progenitor cells: effect of precooling, initial dimethyl sulfoxide concentration, freezing program, and storage in vapor-phase