1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Conflict of Interest
  8. References


Cord blood (CB) is a valuable source of hematopoietic stem cells (HSCs). Extended storage of CB is possible provided that validated cryopreservation procedures are used. The study objective was to determine optimal methods of CB cryopreservation.

Study Design and Methods

In the study we 1) compared the effect of two-step cryopreservation and controlled-rate freezing method on the postthaw quality of CB (Study A) and 2) evaluated the postthaw quality of HSC fractions isolated from CB with various methods and frozen with controlled-rate freezing method (Study B). The same cryoprotectant mixture was used for 20 CB units (Study A) and 122 CB units (Study B).


In Study A, 13.79 × 108 and 13.29 × 108 initial white blood cell (WBC) counts decreased to 6.38 × 108 and 6.02 × 108 after thaw for the two methods, respectively. The mononuclear cell (MNC) counts decreased from 5.90 × 108 to 3.71 × 108 and from 5.64 × 108 to 3.47 × 108 dependent on the method. MNC viability decreased from 99.0% to 97.4% for the former and from 98.5% to 97.2% for the latter method. The differences were insignificant. In Study B, postthaw WBC recovery in HSC fractions was 74.4% to 103.5%, MNC recovery 106.4% to 118.5%, CD34+ cell recovery 102.5% to 150.2%, and MNC viability 94.1% to 97.4%.


Neither the cryopreservation procedure nor the freezing of isolated HSCs affected product quality, which may indicate that various freezing methods can be used for cell banking provided the they follow recommendations of good manufacturing practice and Directive 2004/33/EC.


cord blood


hematopoietic stem cell(s)

Hematopoietic stem cell (HSC) transplantation is the method of choice for hematopoiesis reconstitution in malignant and nonmalignant disorders as well as in genetic diseases.1-4 It relies on three main sources of HSCs: marrow, peripheral blood, and cord blood (CB). The latter is a unique source of allogenic HSCs designated for no particular recipient and can undergo long-term storage.

In October 1988, E. Gluckman and colleagues[5] were the first to use CB for sibling transplantation in a child-patient with Fanconi anemia. The first unrelated CB stem cell transplantation was performed by J. Kurtzberg and coworkers in 1993.[6]

It is estimated that for approximately 30% of patients there is the chance of sibling marrow donors; unrelated donors can be found for 40% to 50% but for the remaining 10%-11% donor matching is often unsuccessful[7, 8] and stem cells from CB are the only option.

CB, however, requires very special handling; before issue for clinical use it must be frozen following precise, validated procedures and stored in appropriate conditions.

All available methods of CB freezing are based on cryoprotective properties of dimethyl sulfoxide (DMSO; Merck, Darmstadt, Germany) mixed with albumin, dextran, hydroxyethyl starch (HES), or other components.9-11 The cryoprotective effect of DMSO on white blood cells (WBCs) and platelets (PLTs) has been known for years.

The choice of cryoprotectant as well as proper freezing rate serve to protect cells from adverse effects of small ice crystal accumulation that may cause permanent cell damage.[12, 13]

CB freezing techniques follow the well-known freezing procedures for marrow and peripheral blood stem cells. To suppress cellular metabolism blood is stored in special containers in vapor nitrogen. Initially, the whole volume of CB was frozen but the requirements of extended storage called for more effective storage conditions. Currently only isolated stem cell fractions are stored, their volume being much reduced compared to that of whole CB units. This resulted in substantial reduction of storage costs.[14, 15] No time limits for CB storage have been determined.

The study objective was to determine optimal methods of CB preparation for long-term storage in the stem cell bank of the Institute of Hematology and Transfusion Medicine. The tested CB units were collected solely for the purpose of research but they met the national criteria for public banking. Our study was divided into two parts. In Study A we assessed the impact of the freezing technique on the quality variables of CB units stored in vapor nitrogen. To this effect we compared two methods of CB freezing: the two-step method and the controlled-rate freezing method (Fig. 1). We then evaluated CB cell quality before freezing and immediately after thawing. In Study B we evaluated the postthaw quality of isolated HSC fractions. To this effect we compared the quality of CB units previously isolated with various methods16-18 and then subjected to controlled-rate freezing and extended storage (Fig. 2).


Figure 1. Flow diagram of Study A (comparison of two freezing methods: Methods 1 and 2).

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Figure 2. Flow diagram of Study B (evaluation of postthaw quality of HSC fractions).

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Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Conflict of Interest
  8. References

Collection of CB units

For the purpose of the whole study we used a total of 142 CB units (mean volume, 86.7 mL; range, 47-175 mL): 20 units for Study A and 122 units for Study B. Immediately after child delivery CB was collected into 150-mL triple bags with 21 plus 10 mL CPD (Macopharma, Tourcoing, France) or “top-bottom” system bags with (21 + 10) mL of CPD (Opti, Baxter, Deerfield, IL) placed on a blood mixer. Before preparation blood was stored at +4°C in a temperature-controlled refrigerator. We used only units stored up to 24 hours of collection. During preparation a sterile connection device (TSCD, Terumo, Tokyo, Japan) was used.

Two methods of CB freezing: Study A

We compared the effect of two alternate freezing methods on the quality of 20 CB units with the same cryoprotectant mixture added (Fig. 1). Each unit was divided into two portions; 20 portions were then frozen with the two-step freezing method (Method 1) and the other 20 portions with the controlled-rate freezing method (Method 2). A mixture of 20% vol. DMSO and 80% vol. of 5% human albumin (Biomed, Lublin, Poland) was used for whole blood freezing. This cryoprotectant mixture was cooled to 4°C and then added to CB at a 1:1 volume ratio in sterile conditions with constant mixing.

Freezing of HSC fractions isolated from CB: Study B

Following the preparation method described in our institute studies,16-18 we froze 122 units of HSC fractions according to the controlled-rate freezing method (Method 2). We relied on such CB isolation processes as 1% hetastarch (Plasmasteril, Fresenius, Bad Homburg, Germany), HES (6% HES, Fresenius), gelatin (Braun, Melsungen, Germany), TF-4 filters (Terumo), and buffy coat (Fig. 2).

The two-step freezing method (Method 1)

The CB portions with cryoprotectant mixture were transferred into cryogenic Teflon bags DF-200 (NPBI, Fresenius) with special plate protection, placed for 1 to 16 hours at −80°C and transferred to vapor nitrogen for further storage.

Controlled freezing method (Method 2)

CB portions in Teflon bags DF-200 (NPBI) with special plate protection were placed in a control freezer (IceCube 1810, SY-LAB, Neupurkersdorf, Austria), frozen from initial 6°C to final −50°C at a cooling rate of 1°C/min and then to −90°C at a cooling rate of 3°C/min. They were then placed in vapor nitrogen for further storage.

CB thawing

The frozen CB was removed from nitrogen vapors, placed in a water bath at 37°C until liquid state, weighed, and then sampled.

Cell variable analysis

Study samples (Study A and Study B) were collected before freezing and immediately after thawing. White blood cell (WBC) count was determined using a hematology analyzer (Sysmex K-4500; Toa Medical Electronics Co. Ltd., Kobe, Japan). Mononuclear cell (MNC) count was determined as percentage of WBCs using a flow cytometry analyzer. The gate was placed on a RT-SC versus FW-SC cytogram for cells morphologically corresponding to MNCs and cell percentage was read from the WBC total within the gate. The percentage of CD34+ cells was assayed using a flow cytometry analyzer (Cytoron Absolute, Ortho Diagnostic Systems, Raritan, NJ) with monoclonal antibody anti-CD34PE (anti HPCA-2 clone; Becton Dickinson, Franklin Lakes, NJ). WBC and MNC viability was determined with propidium iodide using a flow cytometry analyzer (Cytoron Absolute, Ortho Diagnostic Systems).

Statistical analysis

Statistical analysis was performed with analysis of variance (ANOVA). The results are presented as mean ± SD. The p values were calculated with ANOVA and a value of less than 0.05 was considered significant.


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Conflict of Interest
  8. References

Evaluation of the two CB freezing methods: Study A

The freezing effect was determined by comparing the variables of 20 CB portions frozen with the two-step method (Method 1) and those of the 20 CB portions frozen with controlled-rate freezing (Method 2). Mean volume with cryoprotectant was 94.9 mL per portion for the former and 91.4 mL for the latter method. Postthaw WBC recoveries were 48.0% (for Method 1) and 46.2% (for Method 2). MNC recoveries were 64.5 and 63.3% for Methods 1 and 2, respectively; for CD34+ cells they were 56.7 and 53.0% for the two methods, respectively. Postthaw viable WBC percentage was 70.7 and 79.6%, respectively, while the percentages of MNCs were 98.8 and 98.6%. The only differences were observed for lymphocyte recovery: 38.6% for the two-step freezing method and 49.6% for control freezing with special equipment. The differences, however, were not significant. Other results are presented in Table 1 and Fig. 3. No significant differences were observed in the recovery of variables in Study A.


Figure 3. Variable recovery (%) for CB frozen with two freezing methods: (□) two-step method (Method 1) and (image) controlled-rate freezing method (Method 2); comparison (Study A).

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Table 1. Quality variables for CB frozen with two-step and control freezing methods (Study A)*
ParametrsTwo-step freezing (Method 1)Control freezing (Method 2)
Before freezingAfter thawingBefore freezingAfter thawing
  1. * Data are reported as mean ± SD.

Volume (mL)94.0 ± 32.591.8 ± 31.891.4 ± 22.189.4 ± 21.7
WBCs × 108/unit13.79 ± 9.56.38 ± 3.9713.29 ± 7.906.02 ± 3.36
MNCs × 108/unit5.90 ± 3.713.71 ± 2.235.64 ± 2.833.47 ± 1.51
CD34+ × 106/unit6.76 ± 11.203.35 ± 4.785.96 ± 8.982.01 ± 1.62
WBC viability (%)96.0 ± 2.467.8 ± 8.896.0 ± 2.476.5 ± 9.8
MNC viability (%)99.0 ± 1.397.4 ± 1.698.5 ± 1.397.2 ± 1.7

Evaluation of the postthaw quality of HSC fractions isolated with various techniques: Study B

For isolation of HSC fractions we used sedimentation with 6% HES, sedimentation with 1% HES (Plasmasteril), sedimentation with 3% gelatin, buffy coat isolation, and isolation with TF-4 filter for WBC removal.

We determined the postthaw HSC variables. WBC recovery ranged from 74.4% (1% Plasmasteril) to 103.5% (3% gelatin), MNC recovery from 110.2% (3% gelatin) to 118.5% (TF-4 filters), and CD34+ recovery from 102.5% (6% HES) to 150.2% (1% Plasmasteril). Viable WBC recovery ranged from 62.5% (TF-4 filters) to 79.1% (6% HES) and viable MNC was between 95.0% (TF-4 filters) and 97.9% (3% gelatin). The other results are presented in Table 2 and Fig. 4. Statistical analysis is presented in Table 3.


Figure 4. Cell and viability recovery (%) in thawed HSCs isolated with different methods (Study B): (□) 6% HES, (image) 1% Plasmasteril (image), 3% gelatin, (image) buffy coat, and (image) TF-4 filter.

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Table 2. Cell count and viability in CB material acquired by various isolation methods immediately after thawing (Study B)*
MethodNumber of unitsVolume (mL) (without cryoprotectant)Variables
Cell content/unitViability (%)
WBCs × 108MNCs × 108CD34+ × 106WBCsMNCs
  1. * Data are reported as mean ± SD.

6% HES2724.3 ± 5.26.80 ± 3.024.30 ± 2.404.8 ± 5.478.3 ± 7.295.0 ± 4.7
1% Plasmasteril2723.8 ± 3.16.70 ± 2.615.18 ± 2.266.69 ± 5.6277.6 ± 11.596.1 ± 2.1
3% gelatin2720.0 ± 0.04.85 ± 2.02.62 ± 0.562.30 ± 0.8570.9 ± 7.797.4 ± 1.3
Buffy coat2625.3 ± 0.55.72 ± 1.124.71 ± 1.044.65 ± 3.9472.6 ± 8.594.5 ± 1.8
TF-4 filter1522.9 ± 2.55.57 ± 2.483.17 ± 1.323.76 ± 3.7164.4 ± 10.894.1 ± 3.2
Table 3. Statistical analysis of p values for Study B
Cell parameters6% HES:1% Plasmasteril6% HES: gelatin6% HES: buffy coat6% HES: TF filter1% Plasmasteril: gelatin1% Plasmasteril: buffy coat1% Plasmasteril: TF-4 filterGelatin: buffy coatGelatin: TF-4 filterBuffy coat: TF-4 filter
WBCs0.0015NS (0.3159)NS (0.1204)NS (0.6763)0.0001NS (0.2586)0.00020.0016NS (0.9742)0.0092
MNCsNS (0.9895)NS (0.9998)NS (0.9998)NS (0.9780)NS (0.9704)NS (0.9054)NS (0.9999)NS (0.9989)NS (0.9469)NS (0.8548)
CD34+NS (0.2752)NS (0.3934)NS (0.1838)NS (0.4816)NS (0.9977)NS (0.9998)NS (0.9906)NS (0.9998)NS (0.9998)NS (0.9976)
WBC viabilityNS (0.9021)0.0075NS (0.3212)0.00180.0010NS (0.1118)0.0003NS (0.3526)NS (0.9882)NS (0.1473)
MNC viabilityNS (0.9465)NS (0.9989)NS (0.3840)NS (0.2623)NS (0.8579)NS (0.9407)NS (0.7228)NS (0.3589)NS (0.4003)0.0060


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Conflict of Interest
  8. References

The process of unrelated adult donor search, medical qualification, and HLA testing usually takes 3 to 6 months.[19] This waiting period can be reduced to 1 to 2 weeks if CB is used as transplant material. Moreover, the CB proliferation potential is approximately 10-fold higher than that of marrow; therefore, only 3 × 107 nuclear cells/kg recipient body mass are required for CB transplantation[20] compared to the 35 × 107/kg recipient body mass for marrow. Another advantage of CB grafts is the higher HLA disparity. CB can be collected from sibling donors and may be used for other family members provided that the relevant criteria are met. CB is considered excellent transplantation material. Recent data21-27 confirm that the CB cell dose restores all marrow functions of the recipient.

Development of uniform standards for CB collection, preparation, and storage would render CB stocks available to a larger number of transplantation centers. CB banks are expected to store as many CB units as possible; therefore, CB volume reduction and economizing storage space has become first priority.28-33 The global inventory of CB units has already exceeded 550,000 and is steadily growing.[34]

Freezing of HSCs is one of the crucial stages of CB preparation.[10, 35, 36] In our study we relied on the experience with freezing of stem cells isolated from both marrow and peripheral blood.37-40 In Study A we compared the controlled-rate freezing method and the two-step freezing method to see whether both were equally effective for preparation of transplant material for emergency situations or when equipment for controlled-rate freezing is unavailable. We focused on the evaluation of MNC count, CD34+ cell count, and viability as the postthaw variables used for routine quality control of CB transplant material. We observed no significant differences for results obtained with either method. The mononuclear and CD34+ cell count recovery suggest that both methods can be used interchangeably which is confirmed by no significance between results (p = 0.63 and p = 0.78, respectively). The results may also suggest that the freezing of whole CB units with no prior isolation of stem cell fractions may not serve the preservation of quality variables of transplantation material. One reason is that DMSO is no proper cryoprotectant for red blood cells (RBCs); therefore, postthaw RBC hemolysis occurs, which may also affect other cells. The results of our study are consistent with those reported by Shlebak and colleagues[35] who present results for MNCs isolated with medium (Lymphoprep, Nycomed Pharma AS, Oslo, Norway) and frozen in tubes according to either of the two freezing methods. MNC recoveries were 90% (the two-step method) and 97% (controlled-rate freezing method); MNC viabilities were 91 and 93%, respectively. Their results were higher than those reported in our study, most likely because they added DNase after thawing. Cell clusters in thawed products may contain WBCs, which might be the explanation for the lower MNC count.

Postthaw CD34+ cell recovery depends on the freezing technique.9,41-44 The optimal freezing rate of 1°C/min can be achieved only with special equipment (control freezers) that allows full control of the freezing process and the relevant documentation. In emergency it may be necessary to use an alternative two-step freezing method based on uncontrolled freezing procedure (i.e., standard freezer). During the first step the product is placed at −80°C for at least 1 hour and then transferred into a container with vapor nitrogen (second step).

In Study B we evaluated the effect of freezing on the quality of the isolated HSC fractions.16-18 These fractions obtained with selected methods of WBC isolation were thawed and their postthaw qualitative and quantitative variables were determined. Recovery variables, particularly for MNCs (from 110.2% to 118.5%) and CD34+ (from 102.5% to 150.2%) imply that all the cell isolation techniques and the freezing method used can readily be applied in routine practice. The results indicate that the quality variables are maintained regardless of the isolation techniques used for obtaining HSC fractions from CB units.

Postthaw WBC count is not an optimal variable for evaluation of the transplantation material because it is usually affected by granulocyte disintegration. Our results were significant for WBC recovery in case of 1% Plasmasteril compared to the other methods, except for buffy coat (p = 0.2586). Significance was also observed between gelatin method and buffy coat (p = 0.0016) as well as between buffy coat and TF-4 filter (p = 0.0092). However, no significance was found between methods for recovery of MNC (p = 0.8548 to p = 0.9999) and CD34+ cells (p = 0.1838 to p = 0.9998). Differences in WBC viability were observed between the 1% Plasmasteril and TF-4 filter (p = 0.0003) methods as well as in the 1% Plasmasteril and gelatin (p = 0.0010), 6% HES and gelatin (p = 0.0075), and 6% HES and TF-4 filter (p = 0.0018) methods. The MNC viability differences were, however, significant only for buffy coat and TF-4 filter method (p = 0.0060). Since the number of tested units was rather limited it might be expected that the results could differ if the number of units were bigger. At the same time, the obtained results imply that some of the variables that are relevant to the quality of transplantation material will be maintained regardless of the isolation method used. Depending on the isolation method used the functions of cells might be affected by various other factors. The differences in WBC recovery and viability may result from the effect of the isolation medium used. This was particularly noticeable in case of granulocytes that disintegrate more readily than other nuclear cells.

CB units for transplantation are usually stored frozen for many years. It is therefore extremely important to select a technique for WBC isolation and freezing that can be approved and accepted by all transplantation centers.

With the uncontrolled freezing procedure, no loss in cell count, viability, or clonogenic properties was observed for units frozen in double-compartment bags, while a slight loss of these variables was observed for samples frozen in tubes.[44] Almici and colleagues[45] report a 75% postthaw MNC recovery in cells previously isolated with various other media such as Ficoll and Hypaque. These cells lost none of their clonogenic properties; the media, however, were not approved for clinical use. In his article, McCullough and colleagues[46] state that freezing of peripheral blood stem cells to −80°C is comparable to the method of control freezing and suggests the possibility of using 5% DMSO in 6% HES. At the same time he draws attention to the fact that the evaluation of colony-forming ability varies in respect to both the person who performs the tests and the testing center. With the results of his study in mind we can predict that the same methods can be successfully used in case of CB. This seems to be supported by the results of our study.

In our study we focused on methods that could be put to routine use in cell banks that have to follow cell-banking requirements according to Directive 2004/23/EU[47] and good manufacturing practice. It should be noted, however, that equally important is the fulfillment of the highest possible quality standards, elimination of human errors, and documentation of each procedure step. To this effect it is recommended to rely on automation as much as possible. Several factors affect the quality of transplantation material. In their report, Barker and colleagues[48] state that there are major differences between the quality of CB units from different CB banks. The quality of CB units correlates with successful engraftment; therefore, Barker and colleagues suggest that the use of two CB units may prove clinically more effective because there is a better chance that at least 1 of the 2 units will be of superior potency. On the other hand, Wagner and coworkers[49] demonstrate no improvement in survival of children as a result of double CB transplantation.

Successful engraftment and promptness of posttransplant individual cell line recovery is the ultimate proof that the preparation method used guarantees high-quality transplantation material. In our study we focused only on the basic quality variables of postthaw CBs that are the usual criteria for CB unit selection. The blood in our study was not intended for clinical use and therefore we were in no position to assess its transplantation success. The results of our study may therefore be merely suggestive of the techniques to be used in CB banking. In her study Page and workers[27] report a strong correlation between postthaw CFUs and neutrophil and PLT recovery. In another study Page and colleagues[50] present an interesting retrospective analysis of 435 CB units and their prefreeze and postthaw variables. She developed a novel scoring system that determines quality standards for all variables that affect transplantation success. This system is predictive for effective engraftment provided a uniform set of testing and evaluation procedures is implemented in cell banks. This is also confirmed in a study by Brand and coworkers[51] that demonstrates differences in CD34+ and viability variables for the same CB units tested in nine different centers.

On the other hand, Rosenau and colleagues[52] evaluated autologous CB units for transplantation from different cell banks and prepared with various methods. The CB units were stored both in standard bags and in vials. Interestingly, the authors found no differences in total nucleated cell recovery related to the type of container; the number of samples however was rather limited (six bags and 11 vials). The samples were washed during thawing, which did not take place in our study. We evaluated only unwashed CB units because 5 mL of DMSO added to transplantation material (CB units prepared according to our institution procedures) is such a small volume that it practically puts the recipient at no risk.


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Conflict of Interest
  8. References

The authors thank Ms Krystyna Dudziak for her contribution and assistance.

Conflict of Interest

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Conflict of Interest
  8. References

The authors declare no conflict of interest relevant to the manuscript submitted to TRANSFUSION.


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Conflict of Interest
  8. References
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