- Email: [email protected]
Influence of volume reduction and cryopreservation methodologies on quality of thawed umbilical cord blood units for transplantation
Available online at www.sciencedirect.com
Cryobiology 56 (2008) 152–158 www.elsevier.com/locate/ycryo
Influence of volume reduction and cryopreservation methodologies on quality of thawed umbilical cord blood units for transplantation Pilar Solves *, Vicente Mirabet, Dolores Planelles, Francisco Carbonell-Uberos, Roberto Roig Umbilical Cord Blood Bank, Valencia Transfusion Centre, Avda del Cid, 65-A, 46014 Valencia, Spain Received 27 August 2007; accepted 6 February 2008 Available online 13 February 2008
Abstract Introduction: Although there is considerable variability in methodology among umbilical cord blood banks, their common goal is to achieve optimal product quality for transplantation. Cryopreservation is a critical issue for a long-term maintenance of cord blood viability and colony-forming capacities. Materials and methods: We designed a prospective study to compare controlled (CRF) vs. non-controlled freezing (URF) of volumereduced cord blood units. In addition, the influence of hydroxy ethyl starch (HES) on cryopreservation was also assayed. To assess the efficiency of protocols used, cell recoveries were measured and the presence of hematopoietic colony-forming units was quantified. Results: In the study phase, we observed similar CB haematopoietc recoveries for CRF and URF strategies, except for TNC recovery that was better for HES volume reduced CB units in the URF group. When we analysed the data of routine processed CB units in samples from satellite cryovials, we found better BFU-E, CFU-GM, CFU-GEMM and CFU recoveries for those units processed with HES than without HES, in an URF manner. Conclusions: URF of CB units is a cryopreservation procedure that allows similar hematopoietic progenitor recoveries than CRF with programmed devices. However, our study suggests that those banks that cryopreserve CB units in a URF manner should use HES for volume reduction. On the other hand, for CRF cryopreservation methodology volume reduction with and without HES are equally useful. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Cord blood; Cryopreservation; Volume reduction
Umbilical cord blood (CB) has become an established source of haematopoietic progenitors for paediatric and young patients without a suitable donor [4]. As a result, many CB banks have been created throughout the world. Although there is considerable variability in methodology among banks, the quality of CB stored is an important part of the success of CB transplants [15]. Cryopreservation is a critical issue for a long-term maintenance of CB viability and colony-forming capacities [5]. Cryopreservation methods for CB have been based on techniques established for haematopoietic progenitor cells from bone marrow or peripheral blood [27]. Currently, the most widely used system for CB cryopreservation is controlled rate freezing *
Corresponding author. Fax: +00 96 350 24 69. E-mail address: [email protected] (P. Solves).
0011-2240/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2008.02.002
(CRF) of 1 °C/min in programmed devices, but this technique is time-consuming and requires sophisticated equipment, unavailable in many centres [7]. This procedure also increases the global costs of transplantation. Thus, uncontrolled-rate freezing (URF) in 80 °C mechanical freezers is an attractive alternative aimed at reducing costs and ease of routine freezing procedures. Volume reduction of CB units before cryopreservation not only technically maximizes storage space, but also clinically alleviates the toxicity resulting from the infusion of larger volumes of dimethylsulfoxide (Me2SO) and hemolyzed products in the unfractionated infusates [19]. Many techniques have been tested for the volume reduction of CB units purpose but to date, most transplants have involved CB units processed by the hydroxy ethyl starch (HES)-technique developed by the New York Cord Blood
P. Solves et al. / Cryobiology 56 (2008) 152–158
Center [21,25,3]. This method adds HES to the cord blood as red blood cells sedimentant agent. In addition to this, HES is a well-known extracellular cryoprotectant, used for haematopoietic stem cell cryopreservation [14]. However, other cord blood banks reduce the volume using semi-automated systems as top and bottom fractionation with different devices [2,8]. In this case, cryoprotectant solution consists only of Me2SO and dextran, without HES. We designed a prospective study to compare: controlled (CRF) vs non-controlled (URF) rate cryopreservation and HES vs non-HES addition. To validate the volume reduction and cryopreservation methods fully, the CB units were volume-reduced, cryopreserved, thawed and washed, as for transplantation in the study group. In the routine group, cell recoveries were analysed from satellite cryovials. Materials and methods Cord blood collection CB donors must sign informed consent before delivery, during last months of pregnancy. CB samples were obtained from normal full-term deliveries in the obstetric departments of collaborating Hospitals. In most cases, the collections were made after placental delivery as previously reported [24]. Briefly, after child delivery, umbilical cord was clamped immediately and cleaned with 70% alcohol and an iodine swab. CB was collected from the umbilical vein by gravity in a closed sterile collection 350 ml double bag (R 1315, Baxter Healthcare, USA) containing 23 ml citrate phosphate dextrose (CPD)-adenine solution anticoagulant, or in a triple bag system (R MQT 2205PU, Maco Pharma, 59338 Tourcoing Cedex, France) containing 21 ml CPD anticoagulant. Fig. 1 shows the general scheme of CB units processing. Red cell depletion using HES sedimentation Within 48 h after collection, CB units were processed. More than 10 108 TNC content was required to accept cord blood units for the bank. Volume reduction was performed by the method developed by Rubinstein et al. [21]. After removal of the aliquots for routine testing, 6% hydroxy-ethyl-starch (HES, Grifols, Spain) solution was mixed with the umbilical cord blood in a ratio of one HES volume to four volumes of blood in the collection bag (1.2% HES final concentration). Then, the bag was
153
centrifuged at 45g for seven minutes at 10 °C (Varifuge 3.ORS, Heraeus Instruments). The removed supernatant leucocyte-rich plasma and 12 g of red blood cells were centrifuged again at 600g for 15 min at 10 °C. Finally, the sedimented WBC concentrate was resuspended in supernatant plasma to a total volume of 42 ml. Red cell depletion using a top and bottom system with two different devices The cord blood units collected in triple bag system were centrifuged in oval buckets at 3000g for 12 min at 22 °C, ensuring that the bags were well supported to prevent disruption of the buffy coat layer. For top and bottom fractionation of CB units two different devices were used: Optipress II (Baxter Healthcare, Deerfield, IL 60015, USA) and Compomat G4 (Fresenius Hemocare Inc., Redmond, WA). A standard protocol programmed into the Optipress II, together with the standard backplate for buffy coat preparation was used to process the CB units. The programme was set with the following parameters: buffy coat volume of 42 ml, a buffy coat level of 5.5 and a force of 25. Different programs were used in Compomat G4, as previously described [22]. Cryopreservation of cord blood units Cryopreservation of CB units was performed by adding 50% Me2SO solution with dextran 10% (Rheomacrodex, Ibyss Fresenius Kabi, Barcelona, Spain) to reach a 10% Me2SO final concentration. For the study group, after removal 2 ml of sample for cells counts, 10 ml of freezing solution were added with syringe for 15 min to the cord blood bags. Cord blood was separated into two 50 ml cryopreservation bags (Cryocyte R4R9951, Nexwell Therapeutics Inc., Irvine, CA 92618, USA) containing 25 ml each one. The cryocyte bags were placed in aluminium cassettes for immediate freezing. One of the bags was cryopreserved in a CRF manner and the other one in a URF way. For URF, cord blood units were placed horizontally on the surface of a styrene-foam box, previously introduced and cooled in the 80 °C mechanical freezer (Koxka). After 24 h, the units were transferred to and stored in the vapour phase of a liquid nitrogen tank. The temperature during cooling was monitored by introducing a thermometer (Fluke S1 K/J, John Fluke MFG, Co, Inc. Everett, Washington) in the 80 °C freezer and recorded each 30 s with a thermosensor, which was introduced in a cryopreservation
Fig. 1. General scheme of CB units processing.
154
P. Solves et al. / Cryobiology 56 (2008) 152–158
bag containing 25 ml of cord blood. For programmed freezing, Planer Biomed, Kryo 10 device was used with a cooling-rate of 1.5 °C/min. At the end of the procedure, the bags were transferred into the vapour phase of liquid nitrogen tank. For the routine group, cord blood was cryopreserved separately in two 50 ml cryopreservation bags containing 29 (60%) and 19 (40%) ml each one, in order to reserve a part of CB for future expansion protocols. The cryocyte bags were placed in aluminium cassettes (1 cm depth 9.5 cm width 16 cm height) for immediate freezing. Units were usually frozen by CRF and stored in the vapour phase of liquid nitrogen. When the programmed freezer was not available, cord blood units were introduced and cryopreserved in the 80 °C mechanical freezer as previously described. A cryovial from each CB units was also cryopreserved for quality control of the process, and stored in liquid nitrogen. Thawing of cord blood units and cryovials After 7 days, cord blood units cryopreserved in the study group were thawed as described previously [21]. Bags were removed from the liquid nitrogen tank and exposed to room temperature for 5 min. Then, the bags were submerged in a 37 °C water bath for thawing and the cells were diluted by adding a volume equal to the CB volume, of a solution containing dextran and human albumin (2.5% final concentration). After centrifugation at 400g for 10 min, the supernatant was removed and the sedimented cells were carefully resuspended in the thawing solution. Cryovials were submerged in a 37 °C water bath and samples for cell counts were drawn directly without washing the cells. The temperature during the cooling was registered in the same way as for the bags. Biological controls In the study group, before processing, cord blood volume was calculated and samples were drawn for cell counts. Immediately before cryopreservation and after thawing, samples were drawn directly from the bags for cell counts, CD34+ cells analysis, viability and clonogenic assays. Cell recoveries were calculated. In the routine group, a cryovial of each cryopreserved CB unit was thawed for quality control. Cell count, viability and clonogenic assays were performed. TNC and CFU recoveries of the CB unit were calculated from this sample.
fluorescein (FITC) and anti-CD34 phycoerythrin (PE; Becton–Dickinson, San Jose, CA, USA) for 10 min at room temperature and then residual red blood cells were lysed with FACS lysing solution (Becton–Dickinson). The cells were fixed in 0.5% paraformaldehyde and stored at 4 °C until analysis. Fifty thousands live events per tube were acquired in the FACScan with Procount software (Becton–Dickinson). Clonogenic assays Clonogenic assays were performed using a commercially prepared complete methylcellulose medium (Methocult GF H4434), supporting growth of CFUGM, BFU-E and CFU-GEMM. CFUs were calculated as the sum of the three kinds of colonies. For the study group, cultures were plated at 2 104 cells/plate, in duplicate 35 mm diameter Petri dishes and incubated for 14 days at 37 °C with 5% CO2 in a humidified atmosphere. Colonies defined as aggregates of more than 40 cells were counted under an inverted microscope. Samples for routine clonogenic assays were drawn from a thawed cryovial, taking a volume previously calculated (between 0.010 and 0.015 ml/plate) according to the total nucleated cell and CD34+ cells content of cord blood units before cryopreservation. Viability Cell viability was assessed using ethidium bromide and acridine orange. The non-viable cells stained orange. Acridine orange is a membrane-permeable cationic dye that binds to nucleic acids of viable cells and causes green fluorescence, while ethidium bromide penetrates the membranes of non-viable cells causing orange fluorescence. Statistical analysis Statistical Package for Social Sciences (SPSS) v. 10 was used to perform the statistical analysis. Results of variables are expressed as means ± standard deviation. The Kolmogorov–Smirnov goodness-of-fit test was used to evaluate whether the variables were normally distributed. Since most variables were not normally distributed, the nonparametric tests Kruskal–Wallis and Mann–Whitney U test were used for comparing the different groups. A p < 0.05 was considered to be significant. Results Results of the study group
Cell counts Cells and differential counts were made with an autoanalyser Sysmex K800 (Toa Medical Electronics, Kobe, Japan). Total nucleated cells content (TNC) was calculated. CD34 assay CD34+ cells were quantified by flow cytometry. Briefly, cord blood cells (5 105) were incubated with anti-CD45
Before freezing TNC, CD34+ cells and CFUs content per bag were 4.5 ± 1.7 108, 2.0 ± 1.6 106 and 66.6 ± 39.9 104, respectively, for HES methodology, and 4.3 ± 2.0 108, 1.6 ± 1.5 106 and 58.6 ± 35.6 104, respectively, for top and bottom volume reduction. Differences between both groups were not statistically significant, except for CD34+ cells (p = 0.014). Cell viability was similar among
P. Solves et al. / Cryobiology 56 (2008) 152–158 10 0
Temperature (ºC)
groups (data not shown). Cell recoveries of the study phase are shown in the Table 1. In the group of URF, TNC recovery was significantly better for those CB units processed with HES. Curves of freezing temperature representing the median values for cryopreservation in a mechanical freezer and in a programmed freezer are shown in Figs. 2 and 3. These freezing curves showed a median pre-transition freezing rate of 2.3 °C/min and 1.1 °C/min, and post-transition freezing rate of 1.2 °C/min and 1.8 °C/min for CB bags cryopreserved in mechanical freezer (Fig. 2) and programmed device (Fig. 3), respectively. Warming rate was 196 °C/min for the bags.
155
-10 -20 -30 -40 -50 -60 -70 -80 0
10
20
30
40
50
60
Time (min) Fig. 2. Freezing curve of CB cells cryopreserved in bags and metallic canisters, and introduced in double styrene-foam boxes inside a 80 °C electric freezer.
For a 7 year-period a total of 6250 cord blood units were collected and 1182 were finally cryopreserved containing TNC P 10 108. Most of non-cryopreserved units were refused by low TNC count. Results of the routine group are shown in the Table 2. TNC recovery, lymphocyte recovery and RBC depletion of CB units after volume reduction were 78.6 ± 8.1%, 79.5 ± 10.9% and 56.5 ± 14.8% for Optipress and 79.3 ± 8.1%, 78.5 ± 7.9% and 52.6 ± 19.4% for Compomat G4 (p = 0.013, p = 0.004 and p < 0.001, respectively). There were no statistical differences for CD34+ cell content before cryopreservation between Optipress II and Compomat (44.8 ± 32.6 105 and 43.6 ± 26.6 105, respectively; p = 0.537). In addition to this, CD34+ cell content before cryopreservation was similar for all groups (HES: URF: 43.6 ± 32.6 105 and CRF: 41.4 ± 27.7 105; (p = 0.650); Top and bottom: URF: 40.2 ± 29.0 105 and CRF: 39.4 ± 29.6 105; (p = 0.674). In the URF group, in spite of better TNC recovery after thawing for top and bottom volume-reduced CB, clonogenic assays were significantly higher for those CB units volume-reduced
Table 1 Study group: Results of CRF and URF cryopreservation of volume reduced CB units with and without HES Mode of cryopreservation URF Means ± SD
CRF Means ± SD
P
TNC recovery (%)
A B
87.1 ± 11.8 78.3 ± 8.2 p = 0.003
83.4 ± 10 78.8 ± 12.2 p = 0.145
0.136 0.680
CD34+ cells recovery (%)
A B
83 ± 21.1 92.6 ± 20.5 p = 0.248
83 ± 19 94.6 ± 31.3 p = 0.529
0.679 0.586
CFUs recovery (%)
A B
65 ± 31.1 57.7 ± 24.9 p = 0.478
53 ± 32 74.2 ± 11.3 p = 0.212
0.198 0.442
Results are expressed as means ± SD. A: volume reduction with HES (n = 31). B: volume reduction by top and bottom methodology (n = 49, 29 Optipress II, 20 Compomat G4). URF: uncontrolled rate freezing. CRF: controlled rate freezing.
10 0 -10
Temperature (ºC)
Results of the routine group
-20 -30 -40 -50 -60 -70 -80 0
10
20
30
40
50
60
Time (min) Fig. 3. Freezing curve of CB cells cryopreserved in bags and metallic canisters and introduced in a programmed device.
with HES. When comparing URF and CRF, for those CB units volume reduced by top and bottom system, clonogenic assays were significantly higher for CRF method. All these statistical differences were maintained when analysing separately the two different devices in top and bottom group with the HES group, while there no were differences between the two different top and bottom devices for CB units freezed by CRF method and URF, respectively (data not shown). However, there no was any difference between CRF and URF method for CB units volume-reduced with HES. Red blood cell (RBC) depletion is shown in Table 3. RBC content before cryopreservation and after thawing was significantly higher for those CB units volume reduced by top and bottom methodology. Fig. 4 shows the curve of freezing for cryovials introduced in a 80 °C mechanical freezer. The pre-transition freezing rate was 3.3 °C/min, while there were two clearly different freezing rates in post-transition phase: a first part with a freezing rate of 0.6 °C/min and a second part with a freezing rate of 1.6 °C/min. Warming rate of cryovials was approximately 204 °C/min.
156
P. Solves et al. / Cryobiology 56 (2008) 152–158
Table 2 Routine group: Results of CRF and URF of volume reduced CB cells in cryovials with and without HES Mode of cryopreservation URF Means ± SD
CRF Means ± SD
A B
150 220
162 650
Volume (ml)
A B
113.1 ± 24.8 107.2 ± 21.0 p = 0.016
112.5 ± 24.8 110.9 ± 44.5 p = 0.340
TNC 107
A B
14.3 ± 4.1 14.3 ± 3.8 p = 0.971
RBC 109/ml
A B
TNC 108 After volume reduction
P
Mode of cryopreservation URF Means ± SD
CRF Means ± SD
P
n
A B
150 220
162 650
0.522 0.345
RBC 109 After volume reduction
A B
75.0 ± 26.8 180.5 ± 45.1 p < 0.001
75.2 ± 27.1 175.2 ± 43.3 p < 0.001
0.372 0.045
14.6 ± 4.8 14.7 ± 4.4 p = 0.449
0.930 0.469
RBC 109 After thawing
A B
57.7 ± 22.5 133.6 ± 32.5 p < 0.001
55.3 ± 16.2 142.5 ± 35.5 p < 0.001
0.851 0.874
3.4 ± 0.4 3.6 ± 0.3 p = 0.186
3.5 ± 0.4 3.6 ± 0.4 p = 0.225
0.225 0.742
Results are expressed as means ± SD. A: volume reduction with HES. B: volume reduction by top and bottom methodology. URF: uncontrolled rate freezing. CRF: controlled rate freezing.
A B
10.7 ± 3.3 11.1 ± 3.3 p = 0.176
10.9 ± 3.7 11.0 ± 3.4 p = 0.380
0.910 0.491
TNC 106/ml After volume reduction
A B
22.4 ± 7.5 23.2 ± 6.9 p = 0.176
22.8 ± 7.8 23.0 ± 7.0 p = 0.380
0.910 0.491
TNC recovery (%) After volume reduction
A B
74.3 ± 7.8 77.3 ± 8.5 p = 0.054
74.6 ± 8.2 75.3 ± 8.4 p = 0.611
0.903 <0.001
TNC 108 After thawing
A B
10.6 ± 3 11.3 ± 2.8 p = 0.594
10.8 ± 3.5 11.4 ± 3.9 p = 0.474
0.690 0.860
74.0 ± 8.0 78.8 ± 8.0 p < 0.001
75.1 ± 8.6 78.2 ± 7.4 p < 0.001
0.692 0.490
TNC recovery (%) After thawing
A B
0 -10 -20 -30 -40 -50 -60 -70 -80 0
Viability (%) After thawing
A B
77.9 ± 9.9 77.3 ± 9.3 p = 0.481
80.1 ± 8.6 76.8 ± 10.6 p = 0.056
0.154 0.690
BFU-E 104 After thawing
A B
55.2 ± 37.3 41.8 ± 26.4 p = 0.001
58.1 ± 41.0 53.3 ± 30.2 p = 0.531
0.464 <0.001
CFU-GM 104 After thawing
A B
46.1 ± 35.0 22.3 ± 17.2 p< 0.001
47.3 ± 45.5 28.1 ± 21.7 p < 0.001
0.990 <0.001
CFU-GEMM 10 After thawing
A B
6.1 ± 8.8 2.4 ± 3.2 p < 0.001
6.1 ± 7.5 4.0 ± 4.6 p < 0.001
0.066 <0.001
CFU 104 After thawing
A B
107.5 ± 74.3 67.1 ± 42.9 p< 0.001
108.3 ± 80.0 84.8 ± 51.1 p = 0.027
0.862 <0.001
Length of cryovial storage (days)
A B
33.8 ± 17.9 48.4 ± 18.7 p < 0.001
42.6 ± 17.0 59.7 ± 36.8 p = 0.002
4
10
Temperature (ºC)
n
Table 3 Routine group: Results of RBC depletion of CRF and URF of volumereduced CB cells in cryovials with and without HES
0.016 0.020
Results are expressed as means ± SD. A: volume reduction with HES. B: volume reduction by top and bottom methodology. URF: uncontrolled rate freezing. CRF: controlled rate freezing. In the top and bottom group and CRF, 160 CB were volume reduced with Optipress and 60 with Compomat G4, while in URF 338 were processed with Optipress and 312 with Compomat G4.
Length of cryovial storage is shown in the Table 2 and was statistically different among groups.
10
20
30
40
50
60
Time (min) Fig. 4. Freezing curve of CB cells cryopreserved in cryovials and introduced in double styrene-foam boxes inside a 80 °C mechanical freezer.
Comparison between bags and cryovials TNC recovery was significantly better for samples drawn directly from the bags (86.1 ± 11.5%) than samples drawn from cryovials (75.5 ± 8.4%) without taking into account volume reduction and cryopreservation methods (p = 0.025). When considering volume reduction and cryopreservation. TNC recoveries were also significantly better for samples drawn from the bags (data not shown). However there were not differences for CFUs recoveries between bag and cryovial samples. Discussion Haematological reconstitution after cord blood transplantation requires efficient cryopreservation and storage techniques to reduce the loss and the functional damage of pluripotent haematopoietic progenitor cells. The particular characteristics of CB collections as the limited and many times scarce number of progenitors contained makes
P. Solves et al. / Cryobiology 56 (2008) 152–158
mandatory to optimize processing and cryopreservation techniques. The objective of this study was to research the influence of volume reduction and cryopreservation methods in thawed cord blood quality. In addition, we decided to investigate the feasibility of URF, in order to save time and reduce transplantation costs. There are only a few studies concerning a consistent number of patients transplanted with haematopoietic progenitors from bone marrow or peripheral blood cryopreserved by URF [26,10,9,1,13]. These studies have demonstrated similar cell recovery and short-term haematopoietic recovery for CRF and URF cryopreserved haematopoietic progenitors. Other authors have evaluated the long-term haematopoietic reconstitution after autologous peripheral blood progenitor cell transplantation cryopreserved with URF [18]. These authors conclude that, URF is safe and allows sustained long-term engraftment without increasing the risk of transplantation, even though the early engraftment after URF is slower. Optimal cryopreservation of CB haematopoietic cells has been achieved with 5–10% Me2SO at a controlled cooling rate of 1 °C/min [7,11,16]. However, URF methods could represent an alternative to CRF cryopreservation procedures which are more expensive, time consuming and require high level techniques. In our study, we observed similar CB haematopoietic progenitor recoveries for CRF and URF strategies, except for TNC recovery that was better for HES volume-reduced CB units in the URF group. These results are similar to those published by Itoh et al [12], that is to our knowledge, the only study evaluating the feasibility of large-scale URF of CB units. These authors conclude the results of haematopoietic cell recoveries were comparable to the method using a controlled-rate programmed freezer. Previous volume reduction methodology was not evaluated. We have previously shown that, although there are some differences for TNC and RBC recovery among different volume reduction techniques, final CB units CD34+ and CFU content were similar in all [22,23]. Surprisingly, when we analysed the data of routine processed CB units in samples from satellite cryovials (Table 2), we found better BFU-E, CFU-GM, CFU-GEMM and CFU recoveries for those units processed with HES than without HES, in an URF manner. For those CB units volume-reduced by top and bottom methodology, better BFU-E, CFU-GM, CFU-GEMM and CFUs recoveries were found for CRF than by URF method. These results are important because some authors have demonstrated that times for neutrophil and platelet engraftment were significantly correlated with both the doses of infused TNC and haematopoietic colony-forming progenitor cells [17]. Our hypothesis is that the presence of HES as extracellular non-penetrating cryoprotector, although present at low final concentration (less than 1%), may be the cause of better results in volume reduced CB units with HES and cryopreserved in URF manner than without HES. In the study by Itoh et al. [12], volume reduction of CB units is per-
157
formed by sedimentation with HES that can explain the similar results for both methods of cryopreservation. Other authors have compared blood stem cell cryopreservation using Me2SO vs Me2SO plus HES, showing a faster white blood cell recovery in those patients receiving stem cells cryopreserved with Me2SO and HES [20]. One possible explanation is that cells committed to the granulocyte lineage are more sensitive to possible toxic effects of the Me2SO-only cryopreservation than cells committed to platelet differentiation, and that the addition of HES provides better cryopreservation of these precursors. In addition to this, several studies have confirmed the benefit of extracellular cryoprotectants as HES for haematopoietic progenitor cell cryopreservation in 80 °C electrical freezers [26,28]. For CB haematopoietic progenitors, there are no previous studies comparing the cryopreservation with and without HES. Only Donaldson et al. [7] showed that varying HES concentration in solutions containing 5% Me2SO had no significant effect on CD34+ cell recovery. We have no explanation for better TNC recovery of volume reduced CB units by top and bottom methodology. The two devices used in top and bottom arm have shown some differences in the volume reduction process: Optipress II produces higher TNC and lymphocyte recovery and RBC depletion than Compomat G4. Anyway, haematopoietic content after volume reduction quantified as CD34+ cell content was similar for both machines. Excluding the addition of HES, the main difference between HES and top and bottom volume reduction methods is the RBC reduction that is significantly higher for HES sedimentation [23]. Other authors, also using a direct plating method, have shown a negative influence of RBC content on CFUGM growth if a concentration of >0.02 109/ml was present in the CFU medium [6]. The higher residual RBC content in top and bottom volume reduced CB units could explain the lower CFU counts as compared to HES volume reduced CB, and also the differences between CRF and URF in those units processed without HES. However, we have not determined the concentration of RBC in culture medium for clonogenic assays. On the other hand, the length of cryovial storage could also explain the differences among groups. Cryovials are stored in liquid nitrogen tanks of 40 l capacity approximately, and the racks are taken out of liquid nitrogen for a few minutes several times a week to remove samples for biological assays. These oscillations in temperature could impair the viability of haematopoietic progenitors. However, time of storage has not shown influence on clonogenic assays of cord blood units processed with HES. Anyway, this time (2–3 months) is very short for a sample stored in liquid nitrogen. When analysing the freezing curves, we observed that the freezing rates for URF and CRF were similar and acceptable. However, we observed for cryovials a suboptimal freezing rate (3 °C/min), although in similar cryopreservation conditions, results of clonogenic assays were significantly better for those cells cryopreserved with HES
158
P. Solves et al. / Cryobiology 56 (2008) 152–158
than without HES in URF manner. A possible explanation for these results is again the protective effect of HES. In conclusion, URF of CB is a cryopreservation procedure that allows similar haematopoietic progenitor recoveries than CRF with programmed devices. However, our study suggests that those banks that cryopreserve CB units in an URF manner should use HES for previous volume reduction. On the other hand, for CRF methodology, volume reduction with and without HES is equally useful. Acknowledgments We thank all the members of Valencia Cord Blood Bank their support and assistance. References [1] C. Almici, P. Ferremi, A. Lanfranchi, et al., Uncontrolled-rate freezing of peripheral blood progenitor cells allows successful engraftment by sparing primitive and committed hematopoietic progenitors, Haematologica 88 (2003) 1390–1395. [2] S. Armitage, D. Fehily, A. Dickinson, C. Chapman, C. Navarrete, M. Contreras, Cord blood banking: volume reduction of cord blood units using a semi-automated closed system, Bone Marrow Transplantation 23 (1999) 505–509. [3] P. Aroviita, K. Teramo, P. Westman, V. Hiilesmaa, R. Kekomaki, Associations among nucleated cell, CD34+ cell and colony-forming cell contents in cord blood units obtained through a standardized banking process, Vox Sanguinis 84 (2003) 219–227. [4] K.K. Ballen, New trends in umbilical cord blood transplantation, Blood 105 (2005) 3786–3792. [5] H.E. Broxmeyer, E.F. Srour, G. Hangoc, S. Cooper, S.A. Anderson, D. Bodine, High-efficiency recovery of functional hematopoietic progenitor and stem cells from human cord blood cryopreserved for 15 years, Proc. Natl. Acad. Sci. USA 100 (2003) 645–650. [6] A.M. de Kreuk, A. Zevenbergen, J.W. van Oostveen, G.F. Schuurhuis, P.C. Huijgens, A.R. Jenkhoff, A single-step colony-forming unit assay for unseparated mobilized peripheral blood, cord blood, and bone marrow, J. Hematother. Stem Cell Res. 10 (2001) 795–806. [7] C. Donaldson, W.J. Armitage, P.A. Denning-Kendall, A.J. Nicol, B.A. Bradley, J.M. Hows, Optimal cryopreservation of human umbilical cord blood, Bone Marrow Transplant 18 (1996) 725–731. [8] M.I. Godinho, M.E. de Sousa, A. Carvalhais, I.L. Barbosa, Umbilical cord blood processing with the Optipress II blood extractor, Cytotherapy 2 (2000) 439–444. [9] P. Halle, O. Tournilhac, W. Knopinska-Poluszny, et al., Uncontrolled-rate freezing and storage at 80 °C, with only 3.5-percent DMSO in cryoprotective solution for 109 autologous peripheral blood progenitor cell transplantations, Transfusion 41 (2001) 667– 673. [10] F. Hernandez-Navarro, E. Ojeda, R. Arrieta, et al., with nonprogrammed freezing by immersion in a methanol bath: results in 213 cases, Bone Marrow Transplant 21 (1998) 511–517. [11] C.J. Hunt, S.E. Armitage, D.E. Pegg, Cryopreservation of umbilical cord blood: 2. Tolerance of CD34+ cells to multimolar dimethyl sulphoxide and the effect of cooling rate on recovery after freezing and thawing, Cryobiology 46 (2003) 76–87.
[12] T. Itoh, M. Minegishi, J. Fushimi, et al., A simple controlled-rate freezing method without a rate-controlled programmed freezer provides optimal conditions for both large-scale and small-scale cryopreservation of umbilical cord blood cells, Transfusion 43 (2003) 1303–1308. [13] Y. Kudo, M. Minegishi, T. Itoh, et al., Evaluation of hematological reconstitution potential of autologous peripheral blood progenitor cells cryopreserved by a simple controlled-rate freezing method, Tohoku J. Exp. Med. 205 (2005) 37–43. [14] K. Luo, G. Wu, Q. Wang, Y. Sun, H. Liu, Effect of dimethylsulfoxide and hydroxyethyl starch in the preservation of fractionated human marrow cells, Cryobiology 31 (1994) 349–354. [15] J. McCullough, D. McKenna, D. Kadidlo, T. Schierman, J. Wagner, Issues in the quality of umbilical cord blood stem cells for transplantation, Transfusion 45 (2005) 832–841. [16] T.P.H. Meyer, B. Hofmann, J. Zaisserer, et al., Analysis and cryopreservation of hematopoietic stem and progenitor cells from umbilical cord blood, Cytotherapy 8 (2006) 265–276. [17] A.R. Migliaccio, J.W. Adamson, C.E. Stevens, et al., Cell dose and speed of engraftment in placental/umbilical cord blood transplantation: graft progenitor cell content is a better predictor than nucleated cell quantity, Blood 96 (2000) 2717–2722. [18] M. Montanari, D. Capelli, A. Poloni, et al., Long-term hematologic reconstitution after autologous peripheral blood progenitor cell transplantation: a comparison between controlled-rate freezing and uncontrolled-rate freezing at 80 °C, Transfusion 43 (2003) 42–49. [19] T. Nagamura-Inoue, M. Shioya, M. Sugo, et al., Wash-out of DMSO does not improve the speed of engraftment of cord blood transplantation: follow-up of 46 adults patients with units shipped from a single cord blood bank, Transfusion 43 (2003) 1285–1294. [20] S.D. Rowley, Z. Feng, L. Chen, et al., A randomized phase III clinical trial of autologous blood stem cell transplantation comparing cryopreservation using dimethylsulfoxide vs dimethylsulfoxide with hydroxyethylstarch, Bone Marrow Transplant 31 (2003) 1043–1051. [21] P. Rubinstein, P.E. Taylor, A. Scaradavou, et al., Unrelated placental blood for bone marrow reconstitution: organization of the placental blood program, Blood Cells 20 (1994) 587–596. [22] P. Solves, V. Mirabet, F. Carbonell-Uberos, M.A. Soler, R. Roig, Automated separation of cord blood units in top and bottom bags using the Compomat G4, Clin. Lab. Haematol. 28 (2006) 202–207. [23] P. Solves, V. Mirabet, D. Planelles, I. Blasco, A. Perales, F. Carbonell-Uberos, M.A. Soler, R. Roig, Red blood cell depletion with a semiautomated system or hydroxyethyl starch sedimentation for routine cord blood banking: a comparative study, Transfusion 45 (2005) 867–873. [24] P. Solves, R. Moraga, E. Saucedo, et al., Comparison between two strategies for umbilical cord blood collection, Bone Marrow Transplantation 31 (2003) 269–273. [25] S. Stanworth, R. Warwick, D. Fehily, et al., An international survey of unrelated umbilical cord blood banking, Vox Sang 80 (2001) 236– 243. [26] P.J. Stiff, A.R. Koester, M.K. Weidner, et al., Autologous bone marrow transplantation using unfractionated cells cryopreserved in dimethylsulfoxide and hydroxyethyl starch without controlled-rate freezing, Blood 70 (1987) 974–978. [27] J.E. Wagner, H.E. Broxmeyer, S. Cooper, Umbilical cord and placental blood hematopoietic stem cells: collection, cryopreservation and storage, J. Hematother. 1 (1992) 167–173. [28] S.Y. Wang, C.K. Ho, P.M. Chen, et al., Comparison of stem cell viability of bone marrow cryopreserved by two different methods, Cryobiology 24 (1987) 229–237.
Cryobiology 56 (2008) 152–158 www.elsevier.com/locate/ycryo
Influence of volume reduction and cryopreservation methodologies on quality of thawed umbilical cord blood units for transplantation Pilar Solves *, Vicente Mirabet, Dolores Planelles, Francisco Carbonell-Uberos, Roberto Roig Umbilical Cord Blood Bank, Valencia Transfusion Centre, Avda del Cid, 65-A, 46014 Valencia, Spain Received 27 August 2007; accepted 6 February 2008 Available online 13 February 2008
Abstract Introduction: Although there is considerable variability in methodology among umbilical cord blood banks, their common goal is to achieve optimal product quality for transplantation. Cryopreservation is a critical issue for a long-term maintenance of cord blood viability and colony-forming capacities. Materials and methods: We designed a prospective study to compare controlled (CRF) vs. non-controlled freezing (URF) of volumereduced cord blood units. In addition, the influence of hydroxy ethyl starch (HES) on cryopreservation was also assayed. To assess the efficiency of protocols used, cell recoveries were measured and the presence of hematopoietic colony-forming units was quantified. Results: In the study phase, we observed similar CB haematopoietc recoveries for CRF and URF strategies, except for TNC recovery that was better for HES volume reduced CB units in the URF group. When we analysed the data of routine processed CB units in samples from satellite cryovials, we found better BFU-E, CFU-GM, CFU-GEMM and CFU recoveries for those units processed with HES than without HES, in an URF manner. Conclusions: URF of CB units is a cryopreservation procedure that allows similar hematopoietic progenitor recoveries than CRF with programmed devices. However, our study suggests that those banks that cryopreserve CB units in a URF manner should use HES for volume reduction. On the other hand, for CRF cryopreservation methodology volume reduction with and without HES are equally useful. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Cord blood; Cryopreservation; Volume reduction
Umbilical cord blood (CB) has become an established source of haematopoietic progenitors for paediatric and young patients without a suitable donor [4]. As a result, many CB banks have been created throughout the world. Although there is considerable variability in methodology among banks, the quality of CB stored is an important part of the success of CB transplants [15]. Cryopreservation is a critical issue for a long-term maintenance of CB viability and colony-forming capacities [5]. Cryopreservation methods for CB have been based on techniques established for haematopoietic progenitor cells from bone marrow or peripheral blood [27]. Currently, the most widely used system for CB cryopreservation is controlled rate freezing *
Corresponding author. Fax: +00 96 350 24 69. E-mail address: [email protected] (P. Solves).
0011-2240/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2008.02.002
(CRF) of 1 °C/min in programmed devices, but this technique is time-consuming and requires sophisticated equipment, unavailable in many centres [7]. This procedure also increases the global costs of transplantation. Thus, uncontrolled-rate freezing (URF) in 80 °C mechanical freezers is an attractive alternative aimed at reducing costs and ease of routine freezing procedures. Volume reduction of CB units before cryopreservation not only technically maximizes storage space, but also clinically alleviates the toxicity resulting from the infusion of larger volumes of dimethylsulfoxide (Me2SO) and hemolyzed products in the unfractionated infusates [19]. Many techniques have been tested for the volume reduction of CB units purpose but to date, most transplants have involved CB units processed by the hydroxy ethyl starch (HES)-technique developed by the New York Cord Blood
P. Solves et al. / Cryobiology 56 (2008) 152–158
Center [21,25,3]. This method adds HES to the cord blood as red blood cells sedimentant agent. In addition to this, HES is a well-known extracellular cryoprotectant, used for haematopoietic stem cell cryopreservation [14]. However, other cord blood banks reduce the volume using semi-automated systems as top and bottom fractionation with different devices [2,8]. In this case, cryoprotectant solution consists only of Me2SO and dextran, without HES. We designed a prospective study to compare: controlled (CRF) vs non-controlled (URF) rate cryopreservation and HES vs non-HES addition. To validate the volume reduction and cryopreservation methods fully, the CB units were volume-reduced, cryopreserved, thawed and washed, as for transplantation in the study group. In the routine group, cell recoveries were analysed from satellite cryovials. Materials and methods Cord blood collection CB donors must sign informed consent before delivery, during last months of pregnancy. CB samples were obtained from normal full-term deliveries in the obstetric departments of collaborating Hospitals. In most cases, the collections were made after placental delivery as previously reported [24]. Briefly, after child delivery, umbilical cord was clamped immediately and cleaned with 70% alcohol and an iodine swab. CB was collected from the umbilical vein by gravity in a closed sterile collection 350 ml double bag (R 1315, Baxter Healthcare, USA) containing 23 ml citrate phosphate dextrose (CPD)-adenine solution anticoagulant, or in a triple bag system (R MQT 2205PU, Maco Pharma, 59338 Tourcoing Cedex, France) containing 21 ml CPD anticoagulant. Fig. 1 shows the general scheme of CB units processing. Red cell depletion using HES sedimentation Within 48 h after collection, CB units were processed. More than 10 108 TNC content was required to accept cord blood units for the bank. Volume reduction was performed by the method developed by Rubinstein et al. [21]. After removal of the aliquots for routine testing, 6% hydroxy-ethyl-starch (HES, Grifols, Spain) solution was mixed with the umbilical cord blood in a ratio of one HES volume to four volumes of blood in the collection bag (1.2% HES final concentration). Then, the bag was
153
centrifuged at 45g for seven minutes at 10 °C (Varifuge 3.ORS, Heraeus Instruments). The removed supernatant leucocyte-rich plasma and 12 g of red blood cells were centrifuged again at 600g for 15 min at 10 °C. Finally, the sedimented WBC concentrate was resuspended in supernatant plasma to a total volume of 42 ml. Red cell depletion using a top and bottom system with two different devices The cord blood units collected in triple bag system were centrifuged in oval buckets at 3000g for 12 min at 22 °C, ensuring that the bags were well supported to prevent disruption of the buffy coat layer. For top and bottom fractionation of CB units two different devices were used: Optipress II (Baxter Healthcare, Deerfield, IL 60015, USA) and Compomat G4 (Fresenius Hemocare Inc., Redmond, WA). A standard protocol programmed into the Optipress II, together with the standard backplate for buffy coat preparation was used to process the CB units. The programme was set with the following parameters: buffy coat volume of 42 ml, a buffy coat level of 5.5 and a force of 25. Different programs were used in Compomat G4, as previously described [22]. Cryopreservation of cord blood units Cryopreservation of CB units was performed by adding 50% Me2SO solution with dextran 10% (Rheomacrodex, Ibyss Fresenius Kabi, Barcelona, Spain) to reach a 10% Me2SO final concentration. For the study group, after removal 2 ml of sample for cells counts, 10 ml of freezing solution were added with syringe for 15 min to the cord blood bags. Cord blood was separated into two 50 ml cryopreservation bags (Cryocyte R4R9951, Nexwell Therapeutics Inc., Irvine, CA 92618, USA) containing 25 ml each one. The cryocyte bags were placed in aluminium cassettes for immediate freezing. One of the bags was cryopreserved in a CRF manner and the other one in a URF way. For URF, cord blood units were placed horizontally on the surface of a styrene-foam box, previously introduced and cooled in the 80 °C mechanical freezer (Koxka). After 24 h, the units were transferred to and stored in the vapour phase of a liquid nitrogen tank. The temperature during cooling was monitored by introducing a thermometer (Fluke S1 K/J, John Fluke MFG, Co, Inc. Everett, Washington) in the 80 °C freezer and recorded each 30 s with a thermosensor, which was introduced in a cryopreservation
Fig. 1. General scheme of CB units processing.
154
P. Solves et al. / Cryobiology 56 (2008) 152–158
bag containing 25 ml of cord blood. For programmed freezing, Planer Biomed, Kryo 10 device was used with a cooling-rate of 1.5 °C/min. At the end of the procedure, the bags were transferred into the vapour phase of liquid nitrogen tank. For the routine group, cord blood was cryopreserved separately in two 50 ml cryopreservation bags containing 29 (60%) and 19 (40%) ml each one, in order to reserve a part of CB for future expansion protocols. The cryocyte bags were placed in aluminium cassettes (1 cm depth 9.5 cm width 16 cm height) for immediate freezing. Units were usually frozen by CRF and stored in the vapour phase of liquid nitrogen. When the programmed freezer was not available, cord blood units were introduced and cryopreserved in the 80 °C mechanical freezer as previously described. A cryovial from each CB units was also cryopreserved for quality control of the process, and stored in liquid nitrogen. Thawing of cord blood units and cryovials After 7 days, cord blood units cryopreserved in the study group were thawed as described previously [21]. Bags were removed from the liquid nitrogen tank and exposed to room temperature for 5 min. Then, the bags were submerged in a 37 °C water bath for thawing and the cells were diluted by adding a volume equal to the CB volume, of a solution containing dextran and human albumin (2.5% final concentration). After centrifugation at 400g for 10 min, the supernatant was removed and the sedimented cells were carefully resuspended in the thawing solution. Cryovials were submerged in a 37 °C water bath and samples for cell counts were drawn directly without washing the cells. The temperature during the cooling was registered in the same way as for the bags. Biological controls In the study group, before processing, cord blood volume was calculated and samples were drawn for cell counts. Immediately before cryopreservation and after thawing, samples were drawn directly from the bags for cell counts, CD34+ cells analysis, viability and clonogenic assays. Cell recoveries were calculated. In the routine group, a cryovial of each cryopreserved CB unit was thawed for quality control. Cell count, viability and clonogenic assays were performed. TNC and CFU recoveries of the CB unit were calculated from this sample.
fluorescein (FITC) and anti-CD34 phycoerythrin (PE; Becton–Dickinson, San Jose, CA, USA) for 10 min at room temperature and then residual red blood cells were lysed with FACS lysing solution (Becton–Dickinson). The cells were fixed in 0.5% paraformaldehyde and stored at 4 °C until analysis. Fifty thousands live events per tube were acquired in the FACScan with Procount software (Becton–Dickinson). Clonogenic assays Clonogenic assays were performed using a commercially prepared complete methylcellulose medium (Methocult GF H4434), supporting growth of CFUGM, BFU-E and CFU-GEMM. CFUs were calculated as the sum of the three kinds of colonies. For the study group, cultures were plated at 2 104 cells/plate, in duplicate 35 mm diameter Petri dishes and incubated for 14 days at 37 °C with 5% CO2 in a humidified atmosphere. Colonies defined as aggregates of more than 40 cells were counted under an inverted microscope. Samples for routine clonogenic assays were drawn from a thawed cryovial, taking a volume previously calculated (between 0.010 and 0.015 ml/plate) according to the total nucleated cell and CD34+ cells content of cord blood units before cryopreservation. Viability Cell viability was assessed using ethidium bromide and acridine orange. The non-viable cells stained orange. Acridine orange is a membrane-permeable cationic dye that binds to nucleic acids of viable cells and causes green fluorescence, while ethidium bromide penetrates the membranes of non-viable cells causing orange fluorescence. Statistical analysis Statistical Package for Social Sciences (SPSS) v. 10 was used to perform the statistical analysis. Results of variables are expressed as means ± standard deviation. The Kolmogorov–Smirnov goodness-of-fit test was used to evaluate whether the variables were normally distributed. Since most variables were not normally distributed, the nonparametric tests Kruskal–Wallis and Mann–Whitney U test were used for comparing the different groups. A p < 0.05 was considered to be significant. Results Results of the study group
Cell counts Cells and differential counts were made with an autoanalyser Sysmex K800 (Toa Medical Electronics, Kobe, Japan). Total nucleated cells content (TNC) was calculated. CD34 assay CD34+ cells were quantified by flow cytometry. Briefly, cord blood cells (5 105) were incubated with anti-CD45
Before freezing TNC, CD34+ cells and CFUs content per bag were 4.5 ± 1.7 108, 2.0 ± 1.6 106 and 66.6 ± 39.9 104, respectively, for HES methodology, and 4.3 ± 2.0 108, 1.6 ± 1.5 106 and 58.6 ± 35.6 104, respectively, for top and bottom volume reduction. Differences between both groups were not statistically significant, except for CD34+ cells (p = 0.014). Cell viability was similar among
P. Solves et al. / Cryobiology 56 (2008) 152–158 10 0
Temperature (ºC)
groups (data not shown). Cell recoveries of the study phase are shown in the Table 1. In the group of URF, TNC recovery was significantly better for those CB units processed with HES. Curves of freezing temperature representing the median values for cryopreservation in a mechanical freezer and in a programmed freezer are shown in Figs. 2 and 3. These freezing curves showed a median pre-transition freezing rate of 2.3 °C/min and 1.1 °C/min, and post-transition freezing rate of 1.2 °C/min and 1.8 °C/min for CB bags cryopreserved in mechanical freezer (Fig. 2) and programmed device (Fig. 3), respectively. Warming rate was 196 °C/min for the bags.
155
-10 -20 -30 -40 -50 -60 -70 -80 0
10
20
30
40
50
60
Time (min) Fig. 2. Freezing curve of CB cells cryopreserved in bags and metallic canisters, and introduced in double styrene-foam boxes inside a 80 °C electric freezer.
For a 7 year-period a total of 6250 cord blood units were collected and 1182 were finally cryopreserved containing TNC P 10 108. Most of non-cryopreserved units were refused by low TNC count. Results of the routine group are shown in the Table 2. TNC recovery, lymphocyte recovery and RBC depletion of CB units after volume reduction were 78.6 ± 8.1%, 79.5 ± 10.9% and 56.5 ± 14.8% for Optipress and 79.3 ± 8.1%, 78.5 ± 7.9% and 52.6 ± 19.4% for Compomat G4 (p = 0.013, p = 0.004 and p < 0.001, respectively). There were no statistical differences for CD34+ cell content before cryopreservation between Optipress II and Compomat (44.8 ± 32.6 105 and 43.6 ± 26.6 105, respectively; p = 0.537). In addition to this, CD34+ cell content before cryopreservation was similar for all groups (HES: URF: 43.6 ± 32.6 105 and CRF: 41.4 ± 27.7 105; (p = 0.650); Top and bottom: URF: 40.2 ± 29.0 105 and CRF: 39.4 ± 29.6 105; (p = 0.674). In the URF group, in spite of better TNC recovery after thawing for top and bottom volume-reduced CB, clonogenic assays were significantly higher for those CB units volume-reduced
Table 1 Study group: Results of CRF and URF cryopreservation of volume reduced CB units with and without HES Mode of cryopreservation URF Means ± SD
CRF Means ± SD
P
TNC recovery (%)
A B
87.1 ± 11.8 78.3 ± 8.2 p = 0.003
83.4 ± 10 78.8 ± 12.2 p = 0.145
0.136 0.680
CD34+ cells recovery (%)
A B
83 ± 21.1 92.6 ± 20.5 p = 0.248
83 ± 19 94.6 ± 31.3 p = 0.529
0.679 0.586
CFUs recovery (%)
A B
65 ± 31.1 57.7 ± 24.9 p = 0.478
53 ± 32 74.2 ± 11.3 p = 0.212
0.198 0.442
Results are expressed as means ± SD. A: volume reduction with HES (n = 31). B: volume reduction by top and bottom methodology (n = 49, 29 Optipress II, 20 Compomat G4). URF: uncontrolled rate freezing. CRF: controlled rate freezing.
10 0 -10
Temperature (ºC)
Results of the routine group
-20 -30 -40 -50 -60 -70 -80 0
10
20
30
40
50
60
Time (min) Fig. 3. Freezing curve of CB cells cryopreserved in bags and metallic canisters and introduced in a programmed device.
with HES. When comparing URF and CRF, for those CB units volume reduced by top and bottom system, clonogenic assays were significantly higher for CRF method. All these statistical differences were maintained when analysing separately the two different devices in top and bottom group with the HES group, while there no were differences between the two different top and bottom devices for CB units freezed by CRF method and URF, respectively (data not shown). However, there no was any difference between CRF and URF method for CB units volume-reduced with HES. Red blood cell (RBC) depletion is shown in Table 3. RBC content before cryopreservation and after thawing was significantly higher for those CB units volume reduced by top and bottom methodology. Fig. 4 shows the curve of freezing for cryovials introduced in a 80 °C mechanical freezer. The pre-transition freezing rate was 3.3 °C/min, while there were two clearly different freezing rates in post-transition phase: a first part with a freezing rate of 0.6 °C/min and a second part with a freezing rate of 1.6 °C/min. Warming rate of cryovials was approximately 204 °C/min.
156
P. Solves et al. / Cryobiology 56 (2008) 152–158
Table 2 Routine group: Results of CRF and URF of volume reduced CB cells in cryovials with and without HES Mode of cryopreservation URF Means ± SD
CRF Means ± SD
A B
150 220
162 650
Volume (ml)
A B
113.1 ± 24.8 107.2 ± 21.0 p = 0.016
112.5 ± 24.8 110.9 ± 44.5 p = 0.340
TNC 107
A B
14.3 ± 4.1 14.3 ± 3.8 p = 0.971
RBC 109/ml
A B
TNC 108 After volume reduction
P
Mode of cryopreservation URF Means ± SD
CRF Means ± SD
P
n
A B
150 220
162 650
0.522 0.345
RBC 109 After volume reduction
A B
75.0 ± 26.8 180.5 ± 45.1 p < 0.001
75.2 ± 27.1 175.2 ± 43.3 p < 0.001
0.372 0.045
14.6 ± 4.8 14.7 ± 4.4 p = 0.449
0.930 0.469
RBC 109 After thawing
A B
57.7 ± 22.5 133.6 ± 32.5 p < 0.001
55.3 ± 16.2 142.5 ± 35.5 p < 0.001
0.851 0.874
3.4 ± 0.4 3.6 ± 0.3 p = 0.186
3.5 ± 0.4 3.6 ± 0.4 p = 0.225
0.225 0.742
Results are expressed as means ± SD. A: volume reduction with HES. B: volume reduction by top and bottom methodology. URF: uncontrolled rate freezing. CRF: controlled rate freezing.
A B
10.7 ± 3.3 11.1 ± 3.3 p = 0.176
10.9 ± 3.7 11.0 ± 3.4 p = 0.380
0.910 0.491
TNC 106/ml After volume reduction
A B
22.4 ± 7.5 23.2 ± 6.9 p = 0.176
22.8 ± 7.8 23.0 ± 7.0 p = 0.380
0.910 0.491
TNC recovery (%) After volume reduction
A B
74.3 ± 7.8 77.3 ± 8.5 p = 0.054
74.6 ± 8.2 75.3 ± 8.4 p = 0.611
0.903 <0.001
TNC 108 After thawing
A B
10.6 ± 3 11.3 ± 2.8 p = 0.594
10.8 ± 3.5 11.4 ± 3.9 p = 0.474
0.690 0.860
74.0 ± 8.0 78.8 ± 8.0 p < 0.001
75.1 ± 8.6 78.2 ± 7.4 p < 0.001
0.692 0.490
TNC recovery (%) After thawing
A B
0 -10 -20 -30 -40 -50 -60 -70 -80 0
Viability (%) After thawing
A B
77.9 ± 9.9 77.3 ± 9.3 p = 0.481
80.1 ± 8.6 76.8 ± 10.6 p = 0.056
0.154 0.690
BFU-E 104 After thawing
A B
55.2 ± 37.3 41.8 ± 26.4 p = 0.001
58.1 ± 41.0 53.3 ± 30.2 p = 0.531
0.464 <0.001
CFU-GM 104 After thawing
A B
46.1 ± 35.0 22.3 ± 17.2 p< 0.001
47.3 ± 45.5 28.1 ± 21.7 p < 0.001
0.990 <0.001
CFU-GEMM 10 After thawing
A B
6.1 ± 8.8 2.4 ± 3.2 p < 0.001
6.1 ± 7.5 4.0 ± 4.6 p < 0.001
0.066 <0.001
CFU 104 After thawing
A B
107.5 ± 74.3 67.1 ± 42.9 p< 0.001
108.3 ± 80.0 84.8 ± 51.1 p = 0.027
0.862 <0.001
Length of cryovial storage (days)
A B
33.8 ± 17.9 48.4 ± 18.7 p < 0.001
42.6 ± 17.0 59.7 ± 36.8 p = 0.002
4
10
Temperature (ºC)
n
Table 3 Routine group: Results of RBC depletion of CRF and URF of volumereduced CB cells in cryovials with and without HES
0.016 0.020
Results are expressed as means ± SD. A: volume reduction with HES. B: volume reduction by top and bottom methodology. URF: uncontrolled rate freezing. CRF: controlled rate freezing. In the top and bottom group and CRF, 160 CB were volume reduced with Optipress and 60 with Compomat G4, while in URF 338 were processed with Optipress and 312 with Compomat G4.
Length of cryovial storage is shown in the Table 2 and was statistically different among groups.
10
20
30
40
50
60
Time (min) Fig. 4. Freezing curve of CB cells cryopreserved in cryovials and introduced in double styrene-foam boxes inside a 80 °C mechanical freezer.
Comparison between bags and cryovials TNC recovery was significantly better for samples drawn directly from the bags (86.1 ± 11.5%) than samples drawn from cryovials (75.5 ± 8.4%) without taking into account volume reduction and cryopreservation methods (p = 0.025). When considering volume reduction and cryopreservation. TNC recoveries were also significantly better for samples drawn from the bags (data not shown). However there were not differences for CFUs recoveries between bag and cryovial samples. Discussion Haematological reconstitution after cord blood transplantation requires efficient cryopreservation and storage techniques to reduce the loss and the functional damage of pluripotent haematopoietic progenitor cells. The particular characteristics of CB collections as the limited and many times scarce number of progenitors contained makes
P. Solves et al. / Cryobiology 56 (2008) 152–158
mandatory to optimize processing and cryopreservation techniques. The objective of this study was to research the influence of volume reduction and cryopreservation methods in thawed cord blood quality. In addition, we decided to investigate the feasibility of URF, in order to save time and reduce transplantation costs. There are only a few studies concerning a consistent number of patients transplanted with haematopoietic progenitors from bone marrow or peripheral blood cryopreserved by URF [26,10,9,1,13]. These studies have demonstrated similar cell recovery and short-term haematopoietic recovery for CRF and URF cryopreserved haematopoietic progenitors. Other authors have evaluated the long-term haematopoietic reconstitution after autologous peripheral blood progenitor cell transplantation cryopreserved with URF [18]. These authors conclude that, URF is safe and allows sustained long-term engraftment without increasing the risk of transplantation, even though the early engraftment after URF is slower. Optimal cryopreservation of CB haematopoietic cells has been achieved with 5–10% Me2SO at a controlled cooling rate of 1 °C/min [7,11,16]. However, URF methods could represent an alternative to CRF cryopreservation procedures which are more expensive, time consuming and require high level techniques. In our study, we observed similar CB haematopoietic progenitor recoveries for CRF and URF strategies, except for TNC recovery that was better for HES volume-reduced CB units in the URF group. These results are similar to those published by Itoh et al [12], that is to our knowledge, the only study evaluating the feasibility of large-scale URF of CB units. These authors conclude the results of haematopoietic cell recoveries were comparable to the method using a controlled-rate programmed freezer. Previous volume reduction methodology was not evaluated. We have previously shown that, although there are some differences for TNC and RBC recovery among different volume reduction techniques, final CB units CD34+ and CFU content were similar in all [22,23]. Surprisingly, when we analysed the data of routine processed CB units in samples from satellite cryovials (Table 2), we found better BFU-E, CFU-GM, CFU-GEMM and CFU recoveries for those units processed with HES than without HES, in an URF manner. For those CB units volume-reduced by top and bottom methodology, better BFU-E, CFU-GM, CFU-GEMM and CFUs recoveries were found for CRF than by URF method. These results are important because some authors have demonstrated that times for neutrophil and platelet engraftment were significantly correlated with both the doses of infused TNC and haematopoietic colony-forming progenitor cells [17]. Our hypothesis is that the presence of HES as extracellular non-penetrating cryoprotector, although present at low final concentration (less than 1%), may be the cause of better results in volume reduced CB units with HES and cryopreserved in URF manner than without HES. In the study by Itoh et al. [12], volume reduction of CB units is per-
157
formed by sedimentation with HES that can explain the similar results for both methods of cryopreservation. Other authors have compared blood stem cell cryopreservation using Me2SO vs Me2SO plus HES, showing a faster white blood cell recovery in those patients receiving stem cells cryopreserved with Me2SO and HES [20]. One possible explanation is that cells committed to the granulocyte lineage are more sensitive to possible toxic effects of the Me2SO-only cryopreservation than cells committed to platelet differentiation, and that the addition of HES provides better cryopreservation of these precursors. In addition to this, several studies have confirmed the benefit of extracellular cryoprotectants as HES for haematopoietic progenitor cell cryopreservation in 80 °C electrical freezers [26,28]. For CB haematopoietic progenitors, there are no previous studies comparing the cryopreservation with and without HES. Only Donaldson et al. [7] showed that varying HES concentration in solutions containing 5% Me2SO had no significant effect on CD34+ cell recovery. We have no explanation for better TNC recovery of volume reduced CB units by top and bottom methodology. The two devices used in top and bottom arm have shown some differences in the volume reduction process: Optipress II produces higher TNC and lymphocyte recovery and RBC depletion than Compomat G4. Anyway, haematopoietic content after volume reduction quantified as CD34+ cell content was similar for both machines. Excluding the addition of HES, the main difference between HES and top and bottom volume reduction methods is the RBC reduction that is significantly higher for HES sedimentation [23]. Other authors, also using a direct plating method, have shown a negative influence of RBC content on CFUGM growth if a concentration of >0.02 109/ml was present in the CFU medium [6]. The higher residual RBC content in top and bottom volume reduced CB units could explain the lower CFU counts as compared to HES volume reduced CB, and also the differences between CRF and URF in those units processed without HES. However, we have not determined the concentration of RBC in culture medium for clonogenic assays. On the other hand, the length of cryovial storage could also explain the differences among groups. Cryovials are stored in liquid nitrogen tanks of 40 l capacity approximately, and the racks are taken out of liquid nitrogen for a few minutes several times a week to remove samples for biological assays. These oscillations in temperature could impair the viability of haematopoietic progenitors. However, time of storage has not shown influence on clonogenic assays of cord blood units processed with HES. Anyway, this time (2–3 months) is very short for a sample stored in liquid nitrogen. When analysing the freezing curves, we observed that the freezing rates for URF and CRF were similar and acceptable. However, we observed for cryovials a suboptimal freezing rate (3 °C/min), although in similar cryopreservation conditions, results of clonogenic assays were significantly better for those cells cryopreserved with HES
158
P. Solves et al. / Cryobiology 56 (2008) 152–158
than without HES in URF manner. A possible explanation for these results is again the protective effect of HES. In conclusion, URF of CB is a cryopreservation procedure that allows similar haematopoietic progenitor recoveries than CRF with programmed devices. However, our study suggests that those banks that cryopreserve CB units in an URF manner should use HES for previous volume reduction. On the other hand, for CRF methodology, volume reduction with and without HES is equally useful. Acknowledgments We thank all the members of Valencia Cord Blood Bank their support and assistance. References [1] C. Almici, P. Ferremi, A. Lanfranchi, et al., Uncontrolled-rate freezing of peripheral blood progenitor cells allows successful engraftment by sparing primitive and committed hematopoietic progenitors, Haematologica 88 (2003) 1390–1395. [2] S. Armitage, D. Fehily, A. Dickinson, C. Chapman, C. Navarrete, M. Contreras, Cord blood banking: volume reduction of cord blood units using a semi-automated closed system, Bone Marrow Transplantation 23 (1999) 505–509. [3] P. Aroviita, K. Teramo, P. Westman, V. Hiilesmaa, R. Kekomaki, Associations among nucleated cell, CD34+ cell and colony-forming cell contents in cord blood units obtained through a standardized banking process, Vox Sanguinis 84 (2003) 219–227. [4] K.K. Ballen, New trends in umbilical cord blood transplantation, Blood 105 (2005) 3786–3792. [5] H.E. Broxmeyer, E.F. Srour, G. Hangoc, S. Cooper, S.A. Anderson, D. Bodine, High-efficiency recovery of functional hematopoietic progenitor and stem cells from human cord blood cryopreserved for 15 years, Proc. Natl. Acad. Sci. USA 100 (2003) 645–650. [6] A.M. de Kreuk, A. Zevenbergen, J.W. van Oostveen, G.F. Schuurhuis, P.C. Huijgens, A.R. Jenkhoff, A single-step colony-forming unit assay for unseparated mobilized peripheral blood, cord blood, and bone marrow, J. Hematother. Stem Cell Res. 10 (2001) 795–806. [7] C. Donaldson, W.J. Armitage, P.A. Denning-Kendall, A.J. Nicol, B.A. Bradley, J.M. Hows, Optimal cryopreservation of human umbilical cord blood, Bone Marrow Transplant 18 (1996) 725–731. [8] M.I. Godinho, M.E. de Sousa, A. Carvalhais, I.L. Barbosa, Umbilical cord blood processing with the Optipress II blood extractor, Cytotherapy 2 (2000) 439–444. [9] P. Halle, O. Tournilhac, W. Knopinska-Poluszny, et al., Uncontrolled-rate freezing and storage at 80 °C, with only 3.5-percent DMSO in cryoprotective solution for 109 autologous peripheral blood progenitor cell transplantations, Transfusion 41 (2001) 667– 673. [10] F. Hernandez-Navarro, E. Ojeda, R. Arrieta, et al., with nonprogrammed freezing by immersion in a methanol bath: results in 213 cases, Bone Marrow Transplant 21 (1998) 511–517. [11] C.J. Hunt, S.E. Armitage, D.E. Pegg, Cryopreservation of umbilical cord blood: 2. Tolerance of CD34+ cells to multimolar dimethyl sulphoxide and the effect of cooling rate on recovery after freezing and thawing, Cryobiology 46 (2003) 76–87.
[12] T. Itoh, M. Minegishi, J. Fushimi, et al., A simple controlled-rate freezing method without a rate-controlled programmed freezer provides optimal conditions for both large-scale and small-scale cryopreservation of umbilical cord blood cells, Transfusion 43 (2003) 1303–1308. [13] Y. Kudo, M. Minegishi, T. Itoh, et al., Evaluation of hematological reconstitution potential of autologous peripheral blood progenitor cells cryopreserved by a simple controlled-rate freezing method, Tohoku J. Exp. Med. 205 (2005) 37–43. [14] K. Luo, G. Wu, Q. Wang, Y. Sun, H. Liu, Effect of dimethylsulfoxide and hydroxyethyl starch in the preservation of fractionated human marrow cells, Cryobiology 31 (1994) 349–354. [15] J. McCullough, D. McKenna, D. Kadidlo, T. Schierman, J. Wagner, Issues in the quality of umbilical cord blood stem cells for transplantation, Transfusion 45 (2005) 832–841. [16] T.P.H. Meyer, B. Hofmann, J. Zaisserer, et al., Analysis and cryopreservation of hematopoietic stem and progenitor cells from umbilical cord blood, Cytotherapy 8 (2006) 265–276. [17] A.R. Migliaccio, J.W. Adamson, C.E. Stevens, et al., Cell dose and speed of engraftment in placental/umbilical cord blood transplantation: graft progenitor cell content is a better predictor than nucleated cell quantity, Blood 96 (2000) 2717–2722. [18] M. Montanari, D. Capelli, A. Poloni, et al., Long-term hematologic reconstitution after autologous peripheral blood progenitor cell transplantation: a comparison between controlled-rate freezing and uncontrolled-rate freezing at 80 °C, Transfusion 43 (2003) 42–49. [19] T. Nagamura-Inoue, M. Shioya, M. Sugo, et al., Wash-out of DMSO does not improve the speed of engraftment of cord blood transplantation: follow-up of 46 adults patients with units shipped from a single cord blood bank, Transfusion 43 (2003) 1285–1294. [20] S.D. Rowley, Z. Feng, L. Chen, et al., A randomized phase III clinical trial of autologous blood stem cell transplantation comparing cryopreservation using dimethylsulfoxide vs dimethylsulfoxide with hydroxyethylstarch, Bone Marrow Transplant 31 (2003) 1043–1051. [21] P. Rubinstein, P.E. Taylor, A. Scaradavou, et al., Unrelated placental blood for bone marrow reconstitution: organization of the placental blood program, Blood Cells 20 (1994) 587–596. [22] P. Solves, V. Mirabet, F. Carbonell-Uberos, M.A. Soler, R. Roig, Automated separation of cord blood units in top and bottom bags using the Compomat G4, Clin. Lab. Haematol. 28 (2006) 202–207. [23] P. Solves, V. Mirabet, D. Planelles, I. Blasco, A. Perales, F. Carbonell-Uberos, M.A. Soler, R. Roig, Red blood cell depletion with a semiautomated system or hydroxyethyl starch sedimentation for routine cord blood banking: a comparative study, Transfusion 45 (2005) 867–873. [24] P. Solves, R. Moraga, E. Saucedo, et al., Comparison between two strategies for umbilical cord blood collection, Bone Marrow Transplantation 31 (2003) 269–273. [25] S. Stanworth, R. Warwick, D. Fehily, et al., An international survey of unrelated umbilical cord blood banking, Vox Sang 80 (2001) 236– 243. [26] P.J. Stiff, A.R. Koester, M.K. Weidner, et al., Autologous bone marrow transplantation using unfractionated cells cryopreserved in dimethylsulfoxide and hydroxyethyl starch without controlled-rate freezing, Blood 70 (1987) 974–978. [27] J.E. Wagner, H.E. Broxmeyer, S. Cooper, Umbilical cord and placental blood hematopoietic stem cells: collection, cryopreservation and storage, J. Hematother. 1 (1992) 167–173. [28] S.Y. Wang, C.K. Ho, P.M. Chen, et al., Comparison of stem cell viability of bone marrow cryopreserved by two different methods, Cryobiology 24 (1987) 229–237.