- Email: [email protected]
Effect of cryopreservation on the immunogenicity of umbilical cord blood cells
Transfusion and Apheresis Science 30 (2004) 47–54 www.elsevier.com/locate/transci
Effect of cryopreservation on the immunogenicity of umbilical cord blood cells Natkunam Ketheesan a,b,*, Cass Whiteman a,b, Agnieszka B. Malczewski b, Robert G. Hirst a, Justin T. La Brooy b a
Microbiology and Immunology, James Cook University, Queensland, Australia 4811 b School of Medicine, James Cook University, Queensland, Australia 4811 Received in revised form 1 January 2003; accepted 1 May 2003
Abstract Cryopreservation is the accepted method for long-term storage of cord blood (CB) cells. We evaluated the effects of using different cooling rates (1, 5, 7.5 and 10 C/min) on CB cell allostimulatory and alloproliferative function, antigen expression and clonogenic potential. Significant decreases (P < 0:001–0:003) in viable cell recovery observed between fresh CB cells and CB cells cryopreserved at each cooling rate tested. Reductions in clonogenic potential of CB cells cryopreserved at cooling rates of 1, 5, 7.5 and 10 C/min were 44%, 76%, 88% and 93% respectively, compared to fresh controls. FACS analysis indicated no changes in percentages CD34+ cells or lymphocytes. Two sets of mixed lymphocyte reactions were carried out for each CB sample. It was observed that allostimulatory and alloproliferative function varied following cryopreservation at different cooling rates (1 and 5 C/min). Interestingly, there was a significant decrease (P < 0:001–0:04) in the alloproliferative function of six of the seven CB samples following cryopreservation using a cooling rate of 5 C/min. Cooling rates between 1 and 5 C/min may provide immunomodulation of CB with maintenance of haematopoietic progenitor cells function. 2003 Elsevier Ltd. All rights reserved. Keywords: Cord blood; Umbilical cord blood; Cryopreservation; Immunomodulation; Mixed lymphocyte reaction
1. Introduction It is accepted that there are definite advantages in the use of cord blood (CB) as a source of haematopoietic progenitor cells (HPC) in selected patients with malignant and non-malignant haematological conditions. Attractive features include
* Corresponding author. Tel.: +61-7-4781-6876; fax: +61-74779-1526. E-mail address: [email protected] (N. Ketheesan).
availability and a lower incidence of graft-versushost disease (GVHD) even with HLA mismatches [1–6]. However, most recipients of CB transplantation have been paediatric cases [2,3,6] and studies concentrating on adult recipients have only recently been undertaken [5,7,8]. Most current protocols for CB cryopreservation use a cooling rate of 1 C/min up to a set temperature, followed by faster cooling prior to storage in liquid nitrogen [9–12]. A concentration of 10% dimethyl sulfoxide (DMSO) has been used widely for cryopreservation [9–12]. However, studies that
1473-0502/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.transci.2003.05.002
48
N. Ketheesan et al. / Transfusion and Apheresis Science 30 (2004) 47–54
investigated optimal cooling rates did not assess possible changes in immunogenicity [10–12]. It is documented that freezing and thawing procedures have differential effects on particular cell populations and this fact has been previously exploited to immunomodulate allogeneic tissue other than bone marrow or CB [13–15]. Faster cooling rates have been shown to reduce the immunogenicity of allogeneic tissue by possibly selectively affecting the function of immunostimulatory cells [13–15]. It was the aim of this study to determine whether increasing the cooling rate during cryopreservation could promote immunomodulation of CB allostimulatory and alloproliferative function, while preserving clonogenic potential.
2. Materials and methods 2.1. Cord blood and peripheral blood mononuclear cell samples Informed consent was obtained from expecting mothers prior to collection of CB, as well as from healthy volunteers prior to collection of peripheral blood. Approval for this project was provided by the Townsville District Health Service Institutional Ethics Committee. CB samples (n ¼ 10) were procured using an ex vivo technique following placental delivery. Heparinised peripheral blood (40 ml) was collected from healthy volunteers. HLA typing was carried out on both CB and peripheral blood samples using sequence specific oligonucleotide probes (SSO). Acid citrate dextrose (10% ACD-A; Baxter Healthcare, Old Toonagabbie, Australia) was used as an anticoagulant in the collection of CB. Each CB sample was divided into two parts with one remaining as unfractionated whole CB at 25 C, while the other was cryopreserved subsequent to separation of mononuclear cells. CB mononuclear cells were separated by density gradient centrifugation (500g for 20 min) using Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden). Peripheral blood mononuclear cells (PBMC) were isolated from donor blood using the above technique. Viability of freshly isolated CB cells and PBMC was determined by trypan blue exclusion.
2.2. Cryopreservation of cord blood CB samples were subjected to cooling rates of either 1 and 5 C/min or 7.5 and 10 C/min. In the first series of experiments, CB mononuclear cells (n ¼ 7) were divided into two halves such that one half of all cell suspensions was cryopreserved using a cooling rate of 1 C/min, while the other half was cryopreserved using a 5 C/min cooling rate. A second set of experiments was carried out in which CB mononuclear cell suspensions (n ¼ 3) were cryopreserved using cooling rates of 7.5 and 10 C/ min. CB cells were cryopreserved in 5 ml cryotubes (NUNC International, Krastemballage, Denmark) with medium containing RPMI 1640 (Invitrogen, Mulgrave, Australia) supplemented with 20% pooled human serum and 10% DMSO (v/v) (Sigma, Sydney, Australia). Following the introduction of DMSO, CB cell suspensions were placed in a 4 C pre-cooled KRYO 10 (series III) controlled rate freezer (Planer Products, Sunbury-on-Thames, UK) and subsequently subjected to appropriately optimised cooling programs to obtain cooling rates of 1, 5, 7.5 and 10 C/min. Cooling rates of 1 or 5 C/min were used down to a temperature of )40 C and then a cooling rate of 5 C was used until a temperature of )160 C was attained. CB samples were finally stored in the gas phase of liquid nitrogen. Rapid thawing of CB samples was achieved by immersion in a 37 C waterbath until the appearance of small ice crystals. DMSO was removed from thawed cells, by gradual dilution with RPMI 1640 supplemented with 10% pooled human serum. CB cell suspensions were subsequently washed with RPMI 1640 (twice) and centrifuged at 500g for 10 min. Viability of CB cells was assessed using trypan blue exclusion. The number of viable cells recovered was expressed as a percentage of the total cell numbers of fresh samples. 2.3. Cord blood clonogenic potential Clonogenic potential of fresh and cryopreserved CB HPC was assessed using MethoCult GF medium (StemCell Technologies, Vancouver, Canada). Fresh and cryopreserved CB cells were
N. Ketheesan et al. / Transfusion and Apheresis Science 30 (2004) 47–54
plated at concentrations ranging from 0.05 · 105 to 0.2 · 105 cells/ml, with three replicates plated for each concentration. Following a 14-day incubation period at 37 C with 5% CO2 , CFU granulocyte macrophage were determined using an inverted microscope. 2.4. FACS analysis Antibodies from the Simultest IMK-Lymphocyte Kit (Becton Dickinson, San Jose, USA) were used for lymphocyte subset analysis of fresh and cryopreserved CB samples using a FACS Scane flow cytometer (Becton Dickinson). Following conventional gating of cells of interest (CD45+/ CD14)) the percentages of CD34+ cells, monocytes (CD14+/HLA-DR), memory T cells (CD3+/ CD45RO+), and NK cells (CD3)/CD16+CD56+) were determined. 2.5. Mixed lymphocyte cultures To assess allostimulatory function, mononuclear cells from fresh and cryopreserved CB samples were exposed to a dose of 3000 cGy c-radiation. Irradiated CB stimulator cells (2 · 105 cells/well) were co-cultured with donor PBMC (1 · 105 cells/well) in triplicate wells in 96 well round bottomed microtitre plates (NUNC International, Krastemballage, Denmark) in culture medium containing RPMI 1640 supplemented with Penicillin (100 000 units/l), Streptomycin (100 mg/l), L -Glutamine (2.0 mM, Sigma), HEPES Buffer (20 mM, Invitrogen), 2-mercaptoethanol (5 lM, Invitrogen) and pooled human serum (10%). Cells were incubated at 37 C in 5% CO2 with appropriate controls. Alloproliferative function was assessed using the above method by exposing donor PBMC to 3000 cGy c-radiation. Irradiated PBMC stimulator cells (2 · 105 cells/well) were cocultured with CB cells (1 · 105 cells/well) from fresh and cryopreserved samples in triplicate wells in 96 well microtitre plates in cell culture. Controls for stimulator and responder cells included unstimulated cells (cells with culture media) and cells stimulated with mitogen phytohaemagglutinin (10 lg/ml PHA; Sigma, Sydney, Australia). 3 H-thymidine (0.25 lCi/well, Amersham-Pharma-
49
cia Biotech, Sydney, Australia) was added to each well 4 h prior to harvesting. Cells were harvested at 120, 144 and 168 h of culture. These time points were determined as being optimal following preliminary studies. Cells were harvested onto glass fiber filtremats (Packard Biosciences, Mt Waverly, Australia). Results were recorded as counts per minute (cpm) using a TopCount microplate scintillation counter (Packard Biosciences). Stimulation indices (SI) were calculated by dividing the mean of the test cpm by the mean of the control cpm. 2.6. Statistical analysis QQ-plots were used to test dependent samples for normality and data were natural log transformed where necessary. Data for clonogenic potential of fresh and cryopreserved CB (1, 5, 7.5 and 10 C/min) and allostimulatory and alloproliferative function of fresh and cryopreserved (1 and 5 C/min) CB samples were analysed using two way analysis of variance (ANOVA) (SPSS version 10.0 software statistical package). The mean difference was determined to be significant if the probability of a type I error was less than 0.05% (P < 0:05). A least statistical difference test was used for separating the means when the ANOVA was significant. One way ANOVA was used to analyse data for clonogenic potential and allostimulatory and alloproliferative function of individual replicates of cryopreserved (1 and 5 C/ min) CB samples compared to fresh controls. Data for viability and antigen expression of fresh CB cells compared to cryopreserved (1, 5, 7.5 and 10 C/min) CB cells were also analysed using one way ANOVA.
3. Results 3.1. HLA typing Results of HLA typing for DRB1 for CB samples and peripheral blood used in the mixed lymphocyte reaction (MLRs) are presented in Table 1.
50
N. Ketheesan et al. / Transfusion and Apheresis Science 30 (2004) 47–54
Table 1 Cord blood and peripheral blood HLA Class II DRBI typing MLR No. 1 2 3 4 5 6 7
Cord blood 0404 03011 1301 15021 04011 04011 0701
Peripheral blood 1201/6 0408 15011 0401/7/22 1401 15021 15011
14011 03011 0101 0101 15011 03011 03011
0701 0701 03011 03011 0701 0801 0801
HLA-DRB1 typing of cord blood and peripheral blood indicated that the samples used in the mixed lymphocyte cultures (MLR) were not identical.
3.2. Effect of increasing cooling rate on CB cell recovery
cells cryopreserved at each of the cooling rates tested (1, 5, 7.5 and 10 C/min).
Assessment of fresh and cryopreserved CB cells recovered indicated that there was a reduction in viability with increasing cooling rates (Fig. 1). Significant decreases (P < 0:001–0:003) in CB cell viability were observed between fresh CB cells and
3.3. Effect of increasing cooling rate on CB clonogenic potential
Fresh 1°C/min
*
5°C/min 7.5°C/min 10°C/min 0
50 Recovery of viable cells (%)
100
Fig. 1. Effect of increasing cooling rate on cord blood cell recovery. Results are expressed as percent recovery of viable CB cells compared to fresh samples. Viability of fresh CB cells (n ¼ 10) is assigned a value of 100% and reduction of viable CB cells recovered following cryopreservation using cooling rates of 1 C/min (n ¼ 7), 5 C/min (n ¼ 7), 7.5 C/min (n ¼ 3) and 10 C (n ¼ 3) is expressed as a mean proportion of the corresponding fresh CB control. Compared to fresh controls, there were significant reductions in the recovery of viable CB cells following cryopreservation at cooling rates of 1 C/min (P < 0:003), 5 C/min (NP < 0:001), 7.5 C/min (NP < 0:001) and 10 C/min (NP < 0:001) (mean + SEM).
Analysis using two way ANOVA indicated that there was a significant decrease (P < 0:001) in the clonogenic potential of CB samples cryopreserved using cooling rates of 1, 5, 7.5 and 10 C/min compared to fresh controls (Fig. 2). There were progressive decreases in clonogenic potential following cryopreservation. The highest clonogenic potential was observed in fresh CB samples, while the lowest was observed in samples cryopreserved at a cooling rate of 10 C/min. At a cooling rate of 1 C/min, there was a significant reduction (P < 0:001–0:01) in the clonogenic potential of four of the seven CB samples tested compared to fresh controls. At a cooling rate of 5 C/min, a significant reduction (P < 0:001–0:01) in clonogenic potential was observed in all of the CB samples tested compared to fresh controls. The reduction in clonogenic potential compared to fresh controls in cells cryopreserved at cooling rates of 1, 5, 7.5 and 10 C/min was 44%, 76%, 88% and 93% respectively. 3.4. Effect of increasing cooling rate on antigen expression FACS analysis of CB cells expressing selected surface antigens indicated that there was no significant reduction in the proportion of CD34+ cells, monocytes (CD14+/HLA-DR) or memory T
N. Ketheesan et al. / Transfusion and Apheresis Science 30 (2004) 47–54
51
3.5. Effect of increasing cooling rate on CB allostimulatory function
Fresh 1°C/min
Overall a two way ANOVA indicated that there was a significant increase (P < 0:002) in the allostimulatory function of CB samples cryopreserved at a cooling rate of 1 C/min compared to fresh controls reaching significance in one of the seven CB samples tested. At a cooling rate of 5 C/min, there was a significant decrease (P < 0:01) in the allostimulatory function of one of the seven CB samples tested (data not shown).
*
5°C/min
* *
7.5°C/min *
10°C/min
50
0
100
Clonogenic Potential (%) Fig. 2. Effect of increasing cooling rate on cord blood clonogenic potential. Clonogenic potential of fresh CB cells (n ¼ 10) is assigned a value of 100% and reduction of clonogenic potential in samples cryopreserved using cooling rates of 1 C/min (n ¼ 7), 5 C/min (n ¼ 7), 7.5 C/min (n ¼ 3) and 10 C/min (n ¼ 3) is expressed as a mean proportion of the corresponding fresh CB control. Two way ANOVA indicated that there was a significant decrease (P < 0:001) between the clonogenic potential of samples cryopreserved at cooling rates of 1, 5, 7.5 and 10 C/min compared to fresh CB controls (mean + SEM).
cells (CD45+/CDRO+) following exposure to different cooling rates (1, 5, 7.5 and 10 C/min) during cryopreservation (Table 2). However, following cryopreservation at a cooling rate of 10 C/ min, there was a significant reduction (P < 0:02) in the proportion of NK cells (CD3)/CD16+/ CD56+) compared to fresh controls.
3.6. Effect of increasing cooling rate on CB alloproliferative function Overall a two way ANOVA indicated that there was a significant decrease (P < 0:001) in the alloproliferative function of CB samples cryopreserved at a cooling rate of 5 C/min compared to fresh controls. At a cooling rate of 5 C/min, there was a decrease in the alloproliferative function of each of the seven CB samples tested, reaching significance (P < 0:001–0:04) in six CB samples compared to fresh controls (Fig. 3). At a cooling rate of 1 C/ min, a significant increase (P < 0:03) in alloproliferative function was observed in one CB sample compared to fresh controls. The allostimulatory function and the alloproliferative function of CB samples cryopreserved at cooling rates of 7.5 and 10 C/min were not
Table 2 Effect of increasing cooling rate on antigen expression Treatment protocol
Fresh (n ¼ 10) 1 C/min (n ¼ 7) 5 C/min (n ¼ 7) 7.5 C/min (n ¼ 3) 10 C/min (n ¼ 3)
Cell type CD34+ cells
Monocytes (CD14+/HLA-DR)
Memory T cells (CD3+/45RO+)
NK cells (CD3)/CD16+CD56+)
0.74 ± 0.17 0.70 ± 0.11 0.73 ± 0.11 0.85 ± 0.55 1.01 ± 0.48
8.59 ± 2.60 5.98 ± 1.75 7.03 ± 1.94 14.78 ± 2.86 14.07 ± 1.95
2.06 ± 0.36 2.84 ± 0.29 2.14 ± 0.41 2.06 ± 0.95 2.33 ± 1.17
26.41 ± 3.61 23.65 ± 2.65 18.91 ± 2.73 19.76 ± 6.43 13.39 ± 5.79
The effect of increasing cooling rate on specific cell populations including CD34+ cells, monocytes (CD14+/HLA-DR), memory T cells (CD3+/45RO+) and NK cells (CD3)/CD16+CD56+) was determined. Data is expressed as mean percentages ± SEM of cells expressing specific surface antigens. Overall, there was no significant decrease in cell proportions (P > 0:05) compared to fresh controls. However, following cryopreservation at a cooling rate of 10 C/min, there was a significant decrease ( P < 0:02) in NK cell proportions compared to fresh controls.
52
N. Ketheesan et al. / Transfusion and Apheresis Science 30 (2004) 47–54
Fig. 3. Effect of increasing cooling rate on cord blood alloproliferative function. A significant decrease (P < 0:01, NP < 0:04, MP < 0:03, P < 0:007, P < 0:006, jP < 0:001) in the alloproliferative function was observed in six CB samples (No. 1–4 and No. 6–7) cryopreserved at a cooling rate of 5 C/ min compared to fresh CB controls. Following cryopreservation at a cooling rate of 1 C/min, there was a significant increase (MP < 0:03) in the alloproliferative function of one CB sample (No. 7) compared to fresh CB controls. Results are expressed as mean SI + SEM (n ¼ 3).
analysed due to significant decreases in the clonogenic potential observed at these cooling rates (Fig. 2).
4. Discussion Various studies have been undertaken to determine optimal techniques for the cryopreservation of CB cells [9,11,12]. A cooling rate of 1 C/ min and a concentration of DMSO of 10% are widely in use for cryopreservation of CB cells. However, to date there has not been a simultaneous analysis of the effect of increasing cooling rates on CB alloproliferative and allostimulatory function, surface antigen expression and clonogenic potential. Reduction of immunogenicity of allogeneic tissue other than bone marrow or CB following cryopreservation at faster cooling rates (>1 C/min) has been documented [13–15]. Studies investigating cryopreservation techniques have demonstrated the use of defined cooling rates for cryopreservation of particular cell types in mixed cell populations, with the possible selective ablation of immunostimulatory cells [13–15]. In con-
trast little is known about the immunogenicity of CB. The current study examined the effects of faster cooling rates during cryopreservation on CB allostimulatory and alloproliferative function and CB clonogenic potential. Recovery of viable CB cells decreased with increasing cooling rate (Fig. 1). Viability ranged from 100% for fresh CB samples to 75% for samples cryopreserved at a 10 C/min cooling rate. These findings are in agreement with observations made in a study by Donaldson et al. [11] in which increases in cooling rate (1, 5 and 10 C/min) were shown to be associated with significant decreases in CD34+ cell recovery rate. However, the propidium iodide exclusion method used by Donaldson et al. to assess viability may not give clear indications of functional damage sustained during cryopreservation and hence may not correlate with the repopulating potential of HPC. Significant differences were observed between the clonogenic potential of fresh CB samples and CB samples cryopreserved at cooling rates of 1, 5, 7.5 and 10 C/min (Fig. 2). In the current study, clonogenic potential was significantly reduced in four of the seven CB samples following cryopreservation at a 1 C/min cooling rate and in all samples following cryopreservation at a 5 C/min cooling rate. Similar observations were made by Nicol et al. [10] who found that CB cryopreserved at a cooling rate of 10 C/min had reduced clonogenic potential compared to fresh CB. A recent study has suggested that CB nucleated cell numbers before cryopreservation correlate with CD34+ cell numbers post thawing [7]. Decreased time to myeloid engraftment and event free survival were associated with high CD34+ numbers. Furthermore, large losses of clonogenic cells may lead to a decrease of the graft-versus-leukaemic effect. The reduced CB cell recovery and clonogenic potential observed in the current study following cryopreservation at faster cooling rates could be detrimental to CB transplant potential. FACS analysis confirmed that percentages of CD34+ cells, memory T cells (CD3+/CD45RO+) and NK cells (CD3)/CD16+56+) were similar to those found in other studies (Table 2) [16,17]. Although there was a significant reduction in the proportion of NK cell following cryopreservation
N. Ketheesan et al. / Transfusion and Apheresis Science 30 (2004) 47–54
at a cooling rate of 10 C/min, the use of faster cooling rates did not result in the complete ablation of any of the cell types assessed. HLA data (Table 1) confirmed that the paired CB and PBMC samples used in the MLR were different at HLA-DRB1. The disparity at HLA enabled the responder cell population to proliferate following the allostimulatory effect of the stimulator cell population. The allostimulatory function of CB was assessed in a MLR, as an in vitro system where CB cells were irradiated to arrest proliferation and facilitate the responder PBMC to proliferate. This assay was used as an in vitro correlate to simulate recipient response to the donor CB transplant (i.e. rejection or graft failure). An unexpected observation was that there was a significant increase (P < 0:002) observed in the allostimulatory function of CB samples cryopreserved at a cooling rate of 1 C/ min compared to fresh controls. The significant difference between fresh CB samples and samples cryopreserved at a cooling rate of 1 C/min cannot be explained by increases in HLA-DR expression as this was not observed in the monocyte population assessed (Table 2). Such a response in an in vivo situation may be detrimental to CB engraftment. The alloproliferative function of CB was assessed in an in vitro MLR system where CB cells were co-cultured with irradiated PBMC. Due to differences in MHC between PBMC and CB, the CB cells were able to proliferate. This enabled the assessment of the potential of CB to proliferate in response to allorecognition (i.e. GVHD). There was a significant decrease (P < 0:001) in the alloproliferative function of CB cells cryopreserved at a cooling rate of 5 C/min compared to fresh controls. Following cryopreservation at a cooling rate of 5 C/min there was a reduction in alloproliferative function of all CB samples, reaching significance in six of the seven CB samples compared to fresh controls (Fig. 3). Reduction of CB alloproliferative function may have implications in the prevention of GVHD. Thus, although the use of increased cooling rates during cryopreservation is able to decrease CB alloproliferative function, such cryopreservation protocols result in reduced clonogenic potential.
53
An assessment of the relative advantages gained by decreased alloproliferative function needs to be evaluated in light of the decreased recovery and clonogenic potential following cryopreservation. Compared to fresh controls, CB samples cryopreserved at a cooling rate of 1 C/ min had a clonogenic potential of 56%. This decreased to 24% in CB samples cryopreserved at a cooling rate of 5 C/min. Consideration must be given to the possibility of developing existing ex vivo expansion techniques to augment HPC numbers and thus compensate for the loss of viability following cryopreservation [18–20]. In the current study a second set of experiments conducted using cooling rates of 7.5 and 10 C/ min suggested that while cooling rates between 1 and 5 C/min may to some degree facilitate immunomodulation of CB, cooling rates greater than 5 C/min were sub-optimal for the clonogenic potential of CB HPC. In summary, the long-term aim of studies such as these is the development of a cryopreservation protocol that could be implemented as a donor pre-treatment regime prior to CB transplantation. Although in a proportion of the samples a degree of immunomodulation was observed using a faster cooling rate of 5 C/min, wider assessment of the functional viability of the HPC population should be undertaken.
Acknowledgements This work was supported in part by an Australian Research Council grant (ARCS204) and a grant from the Townsville Robert Towns Lions. We are appreciative of the assistance of Dr Sherif Farag, Pauline Pisters, Gordon McHugh and Andrew McCutchan of the Haematology Section, and the staff of the Tissue Typing Laboratory of the Queensland Health Pathology Services. We are grateful to the staff of the WomenÕs and ChildrenÕs Health Institute and the staff of Oncology Services, Townsville Hospital for their assistance with the processing of samples. We thank Dr Leigh Owens of James Cook University for his assistance with the statistical analysis and Dr Campbell Witt of Royal Perth Hospital, Australia and Dr John
54
N. Ketheesan et al. / Transfusion and Apheresis Science 30 (2004) 47–54
Kearney of The National Blood Services, UK for the critical review of the manuscript.
References [1] Wagner JE, Rosethal J, Sweetman R, Ou Shu X, Davies SM, Ramsey NKC, et al. Successful transplantation of HLA-matched and HLA-mismatched umbilical cord blood from unrelated donors: analysis of engraftment and acute graft-versus-host disease. Blood 1996;88:795–802. [2] Gluckman E, Rocha V, Boyer-Chammard A, Locatelli F, Arcese W, Pasquini R, et al. Outcome of cord-blood transplantation from related and unrelated donors. N Engl J Med 1997;337:373–81. [3] Rubinstein P, Carrier C, Scaradavou A, Kurtzberg J, Adamson J, Migliaccio AR, et al. Outcomes among 562 recipients of placental-blood transplants from unrelated donors. N Engl J Med 1998;339:1565–77. [4] Mogul MJ. Unrelated cord blood transplantation vs matched unrelated donor bone marrow transplantation: the risks and benefits of each choice. Bone Marrow Transplant 2000;25(Suppl 2):s58–60. [5] Laughlin MJ. Umbilical cord blood for allogeneic transplantation in children and adults. Bone Marrow Transplant 2001;27:1–6. [6] Ohnuma K, Isoyama K, Ikuta K, Toyoda Y, Nakamura J, Nakajima F, et al. Cord blood transplantation from HLAmismatched and unrelated donors as a treatment for children with haematological malignancies. Br J Haematol 2001;112:981–7. [7] Laughlin MJ, Barker J, Bambach B, Koc ON, Rizzieri DA, Wagner JE, et al. Hematopoietic engraftment and survival in adult recipients of umbilical cord-blood from unrelated donors. N Engl J Med 2001;344:1815–22. [8] Sanz GF, Saavedra S, Planelles D, Senent L, Cervera J, Barragan E, et al. Standardized, unrelated donor cord blood transplantation in adults with hematologic malignancies. Blood 2001;98:2332–8. [9] Campos L, Roubi N, Guyotat D. Definition of optimal conditions for collection and cryopreservation of umbilical cord hematopoietic cells. Cryobiology 1995;32:511–5.
[10] Nicol A, Nieda M, Donaldson C, Denning-Kendall P, Bradley B, Hows J. Analysis of cord blood CD 34+ cells purified after cryopreservation. Exp Hematol 1995;23: 1589–94. [11] Donaldson C, Armitage WJ, Denning-Kendall PA, Nicol AJ, Bradley BA, Hows JM. Optimal cryopreservation of human umbilical cord blood. Bone Marrow Transplant 1996;18:725–31. [12] Shlebak AA, Marley SB, Roberts IAG, Davidson RJ, Goldman JM, Goydon MY. Optimal timing for processing and cryopreservation of umbilial cord hemattopietic stem cells for clinical transplantation. Bone Marrow Transplant 1999;23:131–6. [13] Taylor MJ, London NJM, Thirdborough SM, Lake SP, James RFL. The cryobiology of rat and dendritic cells: preservation and destruction of membrane integrity by freezing. Cryobiology 1990;27:269–78. [14] Taylor MJ, Foreman J, Biwata Y, Tsukikawa S. Prolongation of islet allograft survival is facilitated by storage conditions using cryopreservation involving faster cooling and/or tissue culture. Transplant Proc 1992;24:2860–2. [15] Ketheesan N, Kearney JN, Ingham E. The effect of cryopreservation on the immunogenicity of allogeneic cardiac valves. Cryobiology 1996;33:41–53. [16] Hao Q, Shah AJ, Thiemann FT, Smogorzewska EM, Crooks GM. A functional comparison of CD 34+38) cells in cord blood and bone marrow. Blood 1995;86:3745–53. [17] DÕArena G, Musto P, Cascavilla N, Di Giorgio G, Fusilli S, Zendoli F, et al. Flow cytometric characterization of human umbilical cord blood lymphocytes: immunophenotypic features. Haematologica 1998;83:197–203. [18] Kogler G, Callejas J, Sorg RV, Wernet P. An eight-fold ex vivo expansion of long-term culture-initiating cells from umbilical cord blood in stirred suspension cultures. Bone Marrow Transplant 1998;21(Suppl 3):s48–53. [19] Lazzari L, Lucchi S, Montemurro T, Poretti L, Lopa R, Rebulla P, et al. Evaluation of the effect of cryopreservation on ex vivo expansion of hematopoietic progenitors from cord blood. Bone Marrow Transplant 2001;28:693–8. [20] Rice AM, Wood JA, Milross CG, Collins CJ, Case J, Nordon RE, et al. Prior cryopreservation of ex vivoexpanded cord blood cells is not detrimental to engraftment as measured in the NOD-SCID mouse model. J Hematotother 2001;10:157–65.
Effect of cryopreservation on the immunogenicity of umbilical cord blood cells Natkunam Ketheesan a,b,*, Cass Whiteman a,b, Agnieszka B. Malczewski b, Robert G. Hirst a, Justin T. La Brooy b a
Microbiology and Immunology, James Cook University, Queensland, Australia 4811 b School of Medicine, James Cook University, Queensland, Australia 4811 Received in revised form 1 January 2003; accepted 1 May 2003
Abstract Cryopreservation is the accepted method for long-term storage of cord blood (CB) cells. We evaluated the effects of using different cooling rates (1, 5, 7.5 and 10 C/min) on CB cell allostimulatory and alloproliferative function, antigen expression and clonogenic potential. Significant decreases (P < 0:001–0:003) in viable cell recovery observed between fresh CB cells and CB cells cryopreserved at each cooling rate tested. Reductions in clonogenic potential of CB cells cryopreserved at cooling rates of 1, 5, 7.5 and 10 C/min were 44%, 76%, 88% and 93% respectively, compared to fresh controls. FACS analysis indicated no changes in percentages CD34+ cells or lymphocytes. Two sets of mixed lymphocyte reactions were carried out for each CB sample. It was observed that allostimulatory and alloproliferative function varied following cryopreservation at different cooling rates (1 and 5 C/min). Interestingly, there was a significant decrease (P < 0:001–0:04) in the alloproliferative function of six of the seven CB samples following cryopreservation using a cooling rate of 5 C/min. Cooling rates between 1 and 5 C/min may provide immunomodulation of CB with maintenance of haematopoietic progenitor cells function. 2003 Elsevier Ltd. All rights reserved. Keywords: Cord blood; Umbilical cord blood; Cryopreservation; Immunomodulation; Mixed lymphocyte reaction
1. Introduction It is accepted that there are definite advantages in the use of cord blood (CB) as a source of haematopoietic progenitor cells (HPC) in selected patients with malignant and non-malignant haematological conditions. Attractive features include
* Corresponding author. Tel.: +61-7-4781-6876; fax: +61-74779-1526. E-mail address: [email protected] (N. Ketheesan).
availability and a lower incidence of graft-versushost disease (GVHD) even with HLA mismatches [1–6]. However, most recipients of CB transplantation have been paediatric cases [2,3,6] and studies concentrating on adult recipients have only recently been undertaken [5,7,8]. Most current protocols for CB cryopreservation use a cooling rate of 1 C/min up to a set temperature, followed by faster cooling prior to storage in liquid nitrogen [9–12]. A concentration of 10% dimethyl sulfoxide (DMSO) has been used widely for cryopreservation [9–12]. However, studies that
1473-0502/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.transci.2003.05.002
48
N. Ketheesan et al. / Transfusion and Apheresis Science 30 (2004) 47–54
investigated optimal cooling rates did not assess possible changes in immunogenicity [10–12]. It is documented that freezing and thawing procedures have differential effects on particular cell populations and this fact has been previously exploited to immunomodulate allogeneic tissue other than bone marrow or CB [13–15]. Faster cooling rates have been shown to reduce the immunogenicity of allogeneic tissue by possibly selectively affecting the function of immunostimulatory cells [13–15]. It was the aim of this study to determine whether increasing the cooling rate during cryopreservation could promote immunomodulation of CB allostimulatory and alloproliferative function, while preserving clonogenic potential.
2. Materials and methods 2.1. Cord blood and peripheral blood mononuclear cell samples Informed consent was obtained from expecting mothers prior to collection of CB, as well as from healthy volunteers prior to collection of peripheral blood. Approval for this project was provided by the Townsville District Health Service Institutional Ethics Committee. CB samples (n ¼ 10) were procured using an ex vivo technique following placental delivery. Heparinised peripheral blood (40 ml) was collected from healthy volunteers. HLA typing was carried out on both CB and peripheral blood samples using sequence specific oligonucleotide probes (SSO). Acid citrate dextrose (10% ACD-A; Baxter Healthcare, Old Toonagabbie, Australia) was used as an anticoagulant in the collection of CB. Each CB sample was divided into two parts with one remaining as unfractionated whole CB at 25 C, while the other was cryopreserved subsequent to separation of mononuclear cells. CB mononuclear cells were separated by density gradient centrifugation (500g for 20 min) using Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden). Peripheral blood mononuclear cells (PBMC) were isolated from donor blood using the above technique. Viability of freshly isolated CB cells and PBMC was determined by trypan blue exclusion.
2.2. Cryopreservation of cord blood CB samples were subjected to cooling rates of either 1 and 5 C/min or 7.5 and 10 C/min. In the first series of experiments, CB mononuclear cells (n ¼ 7) were divided into two halves such that one half of all cell suspensions was cryopreserved using a cooling rate of 1 C/min, while the other half was cryopreserved using a 5 C/min cooling rate. A second set of experiments was carried out in which CB mononuclear cell suspensions (n ¼ 3) were cryopreserved using cooling rates of 7.5 and 10 C/ min. CB cells were cryopreserved in 5 ml cryotubes (NUNC International, Krastemballage, Denmark) with medium containing RPMI 1640 (Invitrogen, Mulgrave, Australia) supplemented with 20% pooled human serum and 10% DMSO (v/v) (Sigma, Sydney, Australia). Following the introduction of DMSO, CB cell suspensions were placed in a 4 C pre-cooled KRYO 10 (series III) controlled rate freezer (Planer Products, Sunbury-on-Thames, UK) and subsequently subjected to appropriately optimised cooling programs to obtain cooling rates of 1, 5, 7.5 and 10 C/min. Cooling rates of 1 or 5 C/min were used down to a temperature of )40 C and then a cooling rate of 5 C was used until a temperature of )160 C was attained. CB samples were finally stored in the gas phase of liquid nitrogen. Rapid thawing of CB samples was achieved by immersion in a 37 C waterbath until the appearance of small ice crystals. DMSO was removed from thawed cells, by gradual dilution with RPMI 1640 supplemented with 10% pooled human serum. CB cell suspensions were subsequently washed with RPMI 1640 (twice) and centrifuged at 500g for 10 min. Viability of CB cells was assessed using trypan blue exclusion. The number of viable cells recovered was expressed as a percentage of the total cell numbers of fresh samples. 2.3. Cord blood clonogenic potential Clonogenic potential of fresh and cryopreserved CB HPC was assessed using MethoCult GF medium (StemCell Technologies, Vancouver, Canada). Fresh and cryopreserved CB cells were
N. Ketheesan et al. / Transfusion and Apheresis Science 30 (2004) 47–54
plated at concentrations ranging from 0.05 · 105 to 0.2 · 105 cells/ml, with three replicates plated for each concentration. Following a 14-day incubation period at 37 C with 5% CO2 , CFU granulocyte macrophage were determined using an inverted microscope. 2.4. FACS analysis Antibodies from the Simultest IMK-Lymphocyte Kit (Becton Dickinson, San Jose, USA) were used for lymphocyte subset analysis of fresh and cryopreserved CB samples using a FACS Scane flow cytometer (Becton Dickinson). Following conventional gating of cells of interest (CD45+/ CD14)) the percentages of CD34+ cells, monocytes (CD14+/HLA-DR), memory T cells (CD3+/ CD45RO+), and NK cells (CD3)/CD16+CD56+) were determined. 2.5. Mixed lymphocyte cultures To assess allostimulatory function, mononuclear cells from fresh and cryopreserved CB samples were exposed to a dose of 3000 cGy c-radiation. Irradiated CB stimulator cells (2 · 105 cells/well) were co-cultured with donor PBMC (1 · 105 cells/well) in triplicate wells in 96 well round bottomed microtitre plates (NUNC International, Krastemballage, Denmark) in culture medium containing RPMI 1640 supplemented with Penicillin (100 000 units/l), Streptomycin (100 mg/l), L -Glutamine (2.0 mM, Sigma), HEPES Buffer (20 mM, Invitrogen), 2-mercaptoethanol (5 lM, Invitrogen) and pooled human serum (10%). Cells were incubated at 37 C in 5% CO2 with appropriate controls. Alloproliferative function was assessed using the above method by exposing donor PBMC to 3000 cGy c-radiation. Irradiated PBMC stimulator cells (2 · 105 cells/well) were cocultured with CB cells (1 · 105 cells/well) from fresh and cryopreserved samples in triplicate wells in 96 well microtitre plates in cell culture. Controls for stimulator and responder cells included unstimulated cells (cells with culture media) and cells stimulated with mitogen phytohaemagglutinin (10 lg/ml PHA; Sigma, Sydney, Australia). 3 H-thymidine (0.25 lCi/well, Amersham-Pharma-
49
cia Biotech, Sydney, Australia) was added to each well 4 h prior to harvesting. Cells were harvested at 120, 144 and 168 h of culture. These time points were determined as being optimal following preliminary studies. Cells were harvested onto glass fiber filtremats (Packard Biosciences, Mt Waverly, Australia). Results were recorded as counts per minute (cpm) using a TopCount microplate scintillation counter (Packard Biosciences). Stimulation indices (SI) were calculated by dividing the mean of the test cpm by the mean of the control cpm. 2.6. Statistical analysis QQ-plots were used to test dependent samples for normality and data were natural log transformed where necessary. Data for clonogenic potential of fresh and cryopreserved CB (1, 5, 7.5 and 10 C/min) and allostimulatory and alloproliferative function of fresh and cryopreserved (1 and 5 C/min) CB samples were analysed using two way analysis of variance (ANOVA) (SPSS version 10.0 software statistical package). The mean difference was determined to be significant if the probability of a type I error was less than 0.05% (P < 0:05). A least statistical difference test was used for separating the means when the ANOVA was significant. One way ANOVA was used to analyse data for clonogenic potential and allostimulatory and alloproliferative function of individual replicates of cryopreserved (1 and 5 C/ min) CB samples compared to fresh controls. Data for viability and antigen expression of fresh CB cells compared to cryopreserved (1, 5, 7.5 and 10 C/min) CB cells were also analysed using one way ANOVA.
3. Results 3.1. HLA typing Results of HLA typing for DRB1 for CB samples and peripheral blood used in the mixed lymphocyte reaction (MLRs) are presented in Table 1.
50
N. Ketheesan et al. / Transfusion and Apheresis Science 30 (2004) 47–54
Table 1 Cord blood and peripheral blood HLA Class II DRBI typing MLR No. 1 2 3 4 5 6 7
Cord blood 0404 03011 1301 15021 04011 04011 0701
Peripheral blood 1201/6 0408 15011 0401/7/22 1401 15021 15011
14011 03011 0101 0101 15011 03011 03011
0701 0701 03011 03011 0701 0801 0801
HLA-DRB1 typing of cord blood and peripheral blood indicated that the samples used in the mixed lymphocyte cultures (MLR) were not identical.
3.2. Effect of increasing cooling rate on CB cell recovery
cells cryopreserved at each of the cooling rates tested (1, 5, 7.5 and 10 C/min).
Assessment of fresh and cryopreserved CB cells recovered indicated that there was a reduction in viability with increasing cooling rates (Fig. 1). Significant decreases (P < 0:001–0:003) in CB cell viability were observed between fresh CB cells and
3.3. Effect of increasing cooling rate on CB clonogenic potential
Fresh 1°C/min
*
5°C/min 7.5°C/min 10°C/min 0
50 Recovery of viable cells (%)
100
Fig. 1. Effect of increasing cooling rate on cord blood cell recovery. Results are expressed as percent recovery of viable CB cells compared to fresh samples. Viability of fresh CB cells (n ¼ 10) is assigned a value of 100% and reduction of viable CB cells recovered following cryopreservation using cooling rates of 1 C/min (n ¼ 7), 5 C/min (n ¼ 7), 7.5 C/min (n ¼ 3) and 10 C (n ¼ 3) is expressed as a mean proportion of the corresponding fresh CB control. Compared to fresh controls, there were significant reductions in the recovery of viable CB cells following cryopreservation at cooling rates of 1 C/min (P < 0:003), 5 C/min (NP < 0:001), 7.5 C/min (NP < 0:001) and 10 C/min (NP < 0:001) (mean + SEM).
Analysis using two way ANOVA indicated that there was a significant decrease (P < 0:001) in the clonogenic potential of CB samples cryopreserved using cooling rates of 1, 5, 7.5 and 10 C/min compared to fresh controls (Fig. 2). There were progressive decreases in clonogenic potential following cryopreservation. The highest clonogenic potential was observed in fresh CB samples, while the lowest was observed in samples cryopreserved at a cooling rate of 10 C/min. At a cooling rate of 1 C/min, there was a significant reduction (P < 0:001–0:01) in the clonogenic potential of four of the seven CB samples tested compared to fresh controls. At a cooling rate of 5 C/min, a significant reduction (P < 0:001–0:01) in clonogenic potential was observed in all of the CB samples tested compared to fresh controls. The reduction in clonogenic potential compared to fresh controls in cells cryopreserved at cooling rates of 1, 5, 7.5 and 10 C/min was 44%, 76%, 88% and 93% respectively. 3.4. Effect of increasing cooling rate on antigen expression FACS analysis of CB cells expressing selected surface antigens indicated that there was no significant reduction in the proportion of CD34+ cells, monocytes (CD14+/HLA-DR) or memory T
N. Ketheesan et al. / Transfusion and Apheresis Science 30 (2004) 47–54
51
3.5. Effect of increasing cooling rate on CB allostimulatory function
Fresh 1°C/min
Overall a two way ANOVA indicated that there was a significant increase (P < 0:002) in the allostimulatory function of CB samples cryopreserved at a cooling rate of 1 C/min compared to fresh controls reaching significance in one of the seven CB samples tested. At a cooling rate of 5 C/min, there was a significant decrease (P < 0:01) in the allostimulatory function of one of the seven CB samples tested (data not shown).
*
5°C/min
* *
7.5°C/min *
10°C/min
50
0
100
Clonogenic Potential (%) Fig. 2. Effect of increasing cooling rate on cord blood clonogenic potential. Clonogenic potential of fresh CB cells (n ¼ 10) is assigned a value of 100% and reduction of clonogenic potential in samples cryopreserved using cooling rates of 1 C/min (n ¼ 7), 5 C/min (n ¼ 7), 7.5 C/min (n ¼ 3) and 10 C/min (n ¼ 3) is expressed as a mean proportion of the corresponding fresh CB control. Two way ANOVA indicated that there was a significant decrease (P < 0:001) between the clonogenic potential of samples cryopreserved at cooling rates of 1, 5, 7.5 and 10 C/min compared to fresh CB controls (mean + SEM).
cells (CD45+/CDRO+) following exposure to different cooling rates (1, 5, 7.5 and 10 C/min) during cryopreservation (Table 2). However, following cryopreservation at a cooling rate of 10 C/ min, there was a significant reduction (P < 0:02) in the proportion of NK cells (CD3)/CD16+/ CD56+) compared to fresh controls.
3.6. Effect of increasing cooling rate on CB alloproliferative function Overall a two way ANOVA indicated that there was a significant decrease (P < 0:001) in the alloproliferative function of CB samples cryopreserved at a cooling rate of 5 C/min compared to fresh controls. At a cooling rate of 5 C/min, there was a decrease in the alloproliferative function of each of the seven CB samples tested, reaching significance (P < 0:001–0:04) in six CB samples compared to fresh controls (Fig. 3). At a cooling rate of 1 C/ min, a significant increase (P < 0:03) in alloproliferative function was observed in one CB sample compared to fresh controls. The allostimulatory function and the alloproliferative function of CB samples cryopreserved at cooling rates of 7.5 and 10 C/min were not
Table 2 Effect of increasing cooling rate on antigen expression Treatment protocol
Fresh (n ¼ 10) 1 C/min (n ¼ 7) 5 C/min (n ¼ 7) 7.5 C/min (n ¼ 3) 10 C/min (n ¼ 3)
Cell type CD34+ cells
Monocytes (CD14+/HLA-DR)
Memory T cells (CD3+/45RO+)
NK cells (CD3)/CD16+CD56+)
0.74 ± 0.17 0.70 ± 0.11 0.73 ± 0.11 0.85 ± 0.55 1.01 ± 0.48
8.59 ± 2.60 5.98 ± 1.75 7.03 ± 1.94 14.78 ± 2.86 14.07 ± 1.95
2.06 ± 0.36 2.84 ± 0.29 2.14 ± 0.41 2.06 ± 0.95 2.33 ± 1.17
26.41 ± 3.61 23.65 ± 2.65 18.91 ± 2.73 19.76 ± 6.43 13.39 ± 5.79
The effect of increasing cooling rate on specific cell populations including CD34+ cells, monocytes (CD14+/HLA-DR), memory T cells (CD3+/45RO+) and NK cells (CD3)/CD16+CD56+) was determined. Data is expressed as mean percentages ± SEM of cells expressing specific surface antigens. Overall, there was no significant decrease in cell proportions (P > 0:05) compared to fresh controls. However, following cryopreservation at a cooling rate of 10 C/min, there was a significant decrease ( P < 0:02) in NK cell proportions compared to fresh controls.
52
N. Ketheesan et al. / Transfusion and Apheresis Science 30 (2004) 47–54
Fig. 3. Effect of increasing cooling rate on cord blood alloproliferative function. A significant decrease (P < 0:01, NP < 0:04, MP < 0:03, P < 0:007, P < 0:006, jP < 0:001) in the alloproliferative function was observed in six CB samples (No. 1–4 and No. 6–7) cryopreserved at a cooling rate of 5 C/ min compared to fresh CB controls. Following cryopreservation at a cooling rate of 1 C/min, there was a significant increase (MP < 0:03) in the alloproliferative function of one CB sample (No. 7) compared to fresh CB controls. Results are expressed as mean SI + SEM (n ¼ 3).
analysed due to significant decreases in the clonogenic potential observed at these cooling rates (Fig. 2).
4. Discussion Various studies have been undertaken to determine optimal techniques for the cryopreservation of CB cells [9,11,12]. A cooling rate of 1 C/ min and a concentration of DMSO of 10% are widely in use for cryopreservation of CB cells. However, to date there has not been a simultaneous analysis of the effect of increasing cooling rates on CB alloproliferative and allostimulatory function, surface antigen expression and clonogenic potential. Reduction of immunogenicity of allogeneic tissue other than bone marrow or CB following cryopreservation at faster cooling rates (>1 C/min) has been documented [13–15]. Studies investigating cryopreservation techniques have demonstrated the use of defined cooling rates for cryopreservation of particular cell types in mixed cell populations, with the possible selective ablation of immunostimulatory cells [13–15]. In con-
trast little is known about the immunogenicity of CB. The current study examined the effects of faster cooling rates during cryopreservation on CB allostimulatory and alloproliferative function and CB clonogenic potential. Recovery of viable CB cells decreased with increasing cooling rate (Fig. 1). Viability ranged from 100% for fresh CB samples to 75% for samples cryopreserved at a 10 C/min cooling rate. These findings are in agreement with observations made in a study by Donaldson et al. [11] in which increases in cooling rate (1, 5 and 10 C/min) were shown to be associated with significant decreases in CD34+ cell recovery rate. However, the propidium iodide exclusion method used by Donaldson et al. to assess viability may not give clear indications of functional damage sustained during cryopreservation and hence may not correlate with the repopulating potential of HPC. Significant differences were observed between the clonogenic potential of fresh CB samples and CB samples cryopreserved at cooling rates of 1, 5, 7.5 and 10 C/min (Fig. 2). In the current study, clonogenic potential was significantly reduced in four of the seven CB samples following cryopreservation at a 1 C/min cooling rate and in all samples following cryopreservation at a 5 C/min cooling rate. Similar observations were made by Nicol et al. [10] who found that CB cryopreserved at a cooling rate of 10 C/min had reduced clonogenic potential compared to fresh CB. A recent study has suggested that CB nucleated cell numbers before cryopreservation correlate with CD34+ cell numbers post thawing [7]. Decreased time to myeloid engraftment and event free survival were associated with high CD34+ numbers. Furthermore, large losses of clonogenic cells may lead to a decrease of the graft-versus-leukaemic effect. The reduced CB cell recovery and clonogenic potential observed in the current study following cryopreservation at faster cooling rates could be detrimental to CB transplant potential. FACS analysis confirmed that percentages of CD34+ cells, memory T cells (CD3+/CD45RO+) and NK cells (CD3)/CD16+56+) were similar to those found in other studies (Table 2) [16,17]. Although there was a significant reduction in the proportion of NK cell following cryopreservation
N. Ketheesan et al. / Transfusion and Apheresis Science 30 (2004) 47–54
at a cooling rate of 10 C/min, the use of faster cooling rates did not result in the complete ablation of any of the cell types assessed. HLA data (Table 1) confirmed that the paired CB and PBMC samples used in the MLR were different at HLA-DRB1. The disparity at HLA enabled the responder cell population to proliferate following the allostimulatory effect of the stimulator cell population. The allostimulatory function of CB was assessed in a MLR, as an in vitro system where CB cells were irradiated to arrest proliferation and facilitate the responder PBMC to proliferate. This assay was used as an in vitro correlate to simulate recipient response to the donor CB transplant (i.e. rejection or graft failure). An unexpected observation was that there was a significant increase (P < 0:002) observed in the allostimulatory function of CB samples cryopreserved at a cooling rate of 1 C/ min compared to fresh controls. The significant difference between fresh CB samples and samples cryopreserved at a cooling rate of 1 C/min cannot be explained by increases in HLA-DR expression as this was not observed in the monocyte population assessed (Table 2). Such a response in an in vivo situation may be detrimental to CB engraftment. The alloproliferative function of CB was assessed in an in vitro MLR system where CB cells were co-cultured with irradiated PBMC. Due to differences in MHC between PBMC and CB, the CB cells were able to proliferate. This enabled the assessment of the potential of CB to proliferate in response to allorecognition (i.e. GVHD). There was a significant decrease (P < 0:001) in the alloproliferative function of CB cells cryopreserved at a cooling rate of 5 C/min compared to fresh controls. Following cryopreservation at a cooling rate of 5 C/min there was a reduction in alloproliferative function of all CB samples, reaching significance in six of the seven CB samples compared to fresh controls (Fig. 3). Reduction of CB alloproliferative function may have implications in the prevention of GVHD. Thus, although the use of increased cooling rates during cryopreservation is able to decrease CB alloproliferative function, such cryopreservation protocols result in reduced clonogenic potential.
53
An assessment of the relative advantages gained by decreased alloproliferative function needs to be evaluated in light of the decreased recovery and clonogenic potential following cryopreservation. Compared to fresh controls, CB samples cryopreserved at a cooling rate of 1 C/ min had a clonogenic potential of 56%. This decreased to 24% in CB samples cryopreserved at a cooling rate of 5 C/min. Consideration must be given to the possibility of developing existing ex vivo expansion techniques to augment HPC numbers and thus compensate for the loss of viability following cryopreservation [18–20]. In the current study a second set of experiments conducted using cooling rates of 7.5 and 10 C/ min suggested that while cooling rates between 1 and 5 C/min may to some degree facilitate immunomodulation of CB, cooling rates greater than 5 C/min were sub-optimal for the clonogenic potential of CB HPC. In summary, the long-term aim of studies such as these is the development of a cryopreservation protocol that could be implemented as a donor pre-treatment regime prior to CB transplantation. Although in a proportion of the samples a degree of immunomodulation was observed using a faster cooling rate of 5 C/min, wider assessment of the functional viability of the HPC population should be undertaken.
Acknowledgements This work was supported in part by an Australian Research Council grant (ARCS204) and a grant from the Townsville Robert Towns Lions. We are appreciative of the assistance of Dr Sherif Farag, Pauline Pisters, Gordon McHugh and Andrew McCutchan of the Haematology Section, and the staff of the Tissue Typing Laboratory of the Queensland Health Pathology Services. We are grateful to the staff of the WomenÕs and ChildrenÕs Health Institute and the staff of Oncology Services, Townsville Hospital for their assistance with the processing of samples. We thank Dr Leigh Owens of James Cook University for his assistance with the statistical analysis and Dr Campbell Witt of Royal Perth Hospital, Australia and Dr John
54
N. Ketheesan et al. / Transfusion and Apheresis Science 30 (2004) 47–54
Kearney of The National Blood Services, UK for the critical review of the manuscript.
References [1] Wagner JE, Rosethal J, Sweetman R, Ou Shu X, Davies SM, Ramsey NKC, et al. Successful transplantation of HLA-matched and HLA-mismatched umbilical cord blood from unrelated donors: analysis of engraftment and acute graft-versus-host disease. Blood 1996;88:795–802. [2] Gluckman E, Rocha V, Boyer-Chammard A, Locatelli F, Arcese W, Pasquini R, et al. Outcome of cord-blood transplantation from related and unrelated donors. N Engl J Med 1997;337:373–81. [3] Rubinstein P, Carrier C, Scaradavou A, Kurtzberg J, Adamson J, Migliaccio AR, et al. Outcomes among 562 recipients of placental-blood transplants from unrelated donors. N Engl J Med 1998;339:1565–77. [4] Mogul MJ. Unrelated cord blood transplantation vs matched unrelated donor bone marrow transplantation: the risks and benefits of each choice. Bone Marrow Transplant 2000;25(Suppl 2):s58–60. [5] Laughlin MJ. Umbilical cord blood for allogeneic transplantation in children and adults. Bone Marrow Transplant 2001;27:1–6. [6] Ohnuma K, Isoyama K, Ikuta K, Toyoda Y, Nakamura J, Nakajima F, et al. Cord blood transplantation from HLAmismatched and unrelated donors as a treatment for children with haematological malignancies. Br J Haematol 2001;112:981–7. [7] Laughlin MJ, Barker J, Bambach B, Koc ON, Rizzieri DA, Wagner JE, et al. Hematopoietic engraftment and survival in adult recipients of umbilical cord-blood from unrelated donors. N Engl J Med 2001;344:1815–22. [8] Sanz GF, Saavedra S, Planelles D, Senent L, Cervera J, Barragan E, et al. Standardized, unrelated donor cord blood transplantation in adults with hematologic malignancies. Blood 2001;98:2332–8. [9] Campos L, Roubi N, Guyotat D. Definition of optimal conditions for collection and cryopreservation of umbilical cord hematopoietic cells. Cryobiology 1995;32:511–5.
[10] Nicol A, Nieda M, Donaldson C, Denning-Kendall P, Bradley B, Hows J. Analysis of cord blood CD 34+ cells purified after cryopreservation. Exp Hematol 1995;23: 1589–94. [11] Donaldson C, Armitage WJ, Denning-Kendall PA, Nicol AJ, Bradley BA, Hows JM. Optimal cryopreservation of human umbilical cord blood. Bone Marrow Transplant 1996;18:725–31. [12] Shlebak AA, Marley SB, Roberts IAG, Davidson RJ, Goldman JM, Goydon MY. Optimal timing for processing and cryopreservation of umbilial cord hemattopietic stem cells for clinical transplantation. Bone Marrow Transplant 1999;23:131–6. [13] Taylor MJ, London NJM, Thirdborough SM, Lake SP, James RFL. The cryobiology of rat and dendritic cells: preservation and destruction of membrane integrity by freezing. Cryobiology 1990;27:269–78. [14] Taylor MJ, Foreman J, Biwata Y, Tsukikawa S. Prolongation of islet allograft survival is facilitated by storage conditions using cryopreservation involving faster cooling and/or tissue culture. Transplant Proc 1992;24:2860–2. [15] Ketheesan N, Kearney JN, Ingham E. The effect of cryopreservation on the immunogenicity of allogeneic cardiac valves. Cryobiology 1996;33:41–53. [16] Hao Q, Shah AJ, Thiemann FT, Smogorzewska EM, Crooks GM. A functional comparison of CD 34+38) cells in cord blood and bone marrow. Blood 1995;86:3745–53. [17] DÕArena G, Musto P, Cascavilla N, Di Giorgio G, Fusilli S, Zendoli F, et al. Flow cytometric characterization of human umbilical cord blood lymphocytes: immunophenotypic features. Haematologica 1998;83:197–203. [18] Kogler G, Callejas J, Sorg RV, Wernet P. An eight-fold ex vivo expansion of long-term culture-initiating cells from umbilical cord blood in stirred suspension cultures. Bone Marrow Transplant 1998;21(Suppl 3):s48–53. [19] Lazzari L, Lucchi S, Montemurro T, Poretti L, Lopa R, Rebulla P, et al. Evaluation of the effect of cryopreservation on ex vivo expansion of hematopoietic progenitors from cord blood. Bone Marrow Transplant 2001;28:693–8. [20] Rice AM, Wood JA, Milross CG, Collins CJ, Case J, Nordon RE, et al. Prior cryopreservation of ex vivoexpanded cord blood cells is not detrimental to engraftment as measured in the NOD-SCID mouse model. J Hematotother 2001;10:157–65.