Cryopreservation of umbilical cord blood: 1. Osmotically inactive volume, hydraulic conductivity and permeability of CD34+ cells to dimethyl sulphoxide

Cryobiology 46 (2003) 61–75 www.elsevier.com/locate/ycryo

Cryopreservation of umbilical cord blood: 1. Osmotically inactive volume, hydraulic conductivity and permeability of CD34þ cells to dimethyl sulphoxideq Charles J. Hunt,a,* Susan E. Armitage,b and David E. Peggc a

NBS Tissue Services R&D, East Anglian Blood Centre, Long Road, Cambridge CB2 2PT, UK b NBS London Cord Blood Bank, Deansbrook Road, Edgeware, Middlesex HA8 9BD, UK c Department of Biology, Medical Cryobiology Unit, University of York, York YO1 5DD, UK Received 30 July 2002; accepted 20 November 2002

Abstract Umbilical cord blood (UCB) is an accepted treatment for the reconstitution of bone marrow function following myeloablative treatment predominantly in children and juveniles. Current cryopreservation protocols use methods established for bone marrow and peripheral blood progenitors cells that have largely been developed empirically. Such protocols can result in losses of up to 50% of the nucleated cell population: losses unacceptable for cord blood. The design of optimal cryopreservation regimes requires the development of addition and elution protocols for the chosen cryoprotectant; protocols that minimise damaging osmotic transients. The biophysical parameters necessary to model the addition and elution of dimethyl sulphoxide to and from cord blood CD34þ cells have been established. An electronic particle counting method was used to establish the volumetric response of CD34þ cells to changes in osmolality of the suspending medium. The non-osmotic volume of the cell was 0.27 of the cells isotonic volume. The permeation kinetics of CD34þ cells to water and dimethyl sulphoxide were investigated at two temperatures, +1.5 and +20 °C. Values for the hydraulic conductivity were 3:2  108 and 2:8  107 cm/atm/s, respectively. Values for the permeability of dimethyl sulphoxide at these temperatures were 4:2  107 and 7:4  106 cm/s, respectively. Clonogenic assays indicated that the ability of CD34þ cells to grow and differentiate was significantly impaired outside the limits 0.6–4 isotonic. Based on the Boyle vanÕt Hoff plot, the tolerable limits for cell volume excursion were therefore 45– 140% of isotonic volume. The addition and elution of cryoprotectant was modelled using a two-parameter model. Current protocols for the addition of cryoprotectant based on exposure at +4 °C would require additional time for complete equilibration of the cryoprotectant. During the elution phase current protocols are likely to cause CD34þ cells to exceed tolerable limits. The addition of a short holding period during elution reduces the likelihood of this occurring. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Cord blood; Haematopoietic stem cells; Cryopreservation; CD34; Solute permeability; Hydraulic conductivity

q

This work was funded by the Joely Bear Appeal. Corresponding author. Fax: +44-1223-548072. E-mail address: [email protected] (C.J. Hunt). *

0011-2240/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0011-2240(02)00180-3

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Transplantation of haematopoietic progenitor cells (HPCs) for a range of haematological malignancies and non-malignant disorders is an established treatment. The sources of HPCs for reconstitution of haematopoiesis are bone marrow (BM), peripheral blood and more recently, UCB [15,43]. These transplants can be from autologous, syngeneic or allogeneic donors. Allogeneic donors may be related or unrelated; however the proportion of patients for whom HLA-matched related donors are available is relatively small. The chance of finding an allogeneic donor from amongst living unrelated donors recorded on the bone marrow registries has been reported to be between 60 and 75%, falling as low as 20–30% in the case of patients from ethic minority backgrounds [7]. Thus banking of UCB, as a source of HLA-matched allogeneic HPCs has become common over the past decade with UCB banks being established worldwide. Such banks require an efficient method of long-term storage and cryopreservation has become the accepted, indeed the only, method of preservation. Though cryopreservation of human haematopoietic stem cells has been practised for over three decades, there is still no concensus on an optimal method for cryopreservation [41]. Current techniques for preservation of bone marrow and peripheral blood progenitor cells (PBSC) were derived empirically. They generally involve the use of dimethyl sulphoxide (Me2 SO), slow cooling and storage below )120 °C with varying degrees of success; recoveries ranging from 10 to 120% have been reported [35,38]. Whilst the current cryopreservation protocols are effective from a clinical perspective, there is little evidence that they are optimal either in the case of BM and PBSC or for UCB. The design of an optimum cryopreservation protocol should take into account the sequence of events occurring during the cryopreservation process for the target cell population. Cells are first equilibrated with cryoprotectant (CPA) and damage may occur either from temperature dependent toxicity or from osmotic excursions as the cells respond to the osmotic gradient produced by the high osmolality of the external environment. With time the cells return to normal

volume as the CPA penetrates the cell. During cooling, cells are exposed to detrimental effects of ice formation, either directly during rapid cooling and intracellular ice formation or, indirectly during excessively slow cooling, through extracellular ice formation and the damaging effects of the resulting hypertonic solution. Finally, the cells must be returned to isotonicity following thawing and will experience further osmotic transients during the elution of the CPA. In this and the companion paper [20], these events are investigated for CD34þ cells from UCB since this is the most frequently used marker for isolation and quantitation of haematopoietic stem and progenitor cells [23]. The permeability properties of the membrane to both water and CPA and the tolerable limits for cell volume excursions are important factors in the design of an effective cryopreservation protocol. Changes in cell volume may be determined experimentally, but a number of other parameters are required: cell surface area, isotonic volume, osmotically inactive cell volume and permeability of the membrane to water and CPA. In the first part of this study, the osmotic response of CD34þ cells from UCB to a range of anisotonic solutions was investigated using an electronic particle counting method. The data were used to construct a Boyle vanÕt Hoff plot from which was determined the non-osmotic volume. The same method was also used to chart the time course for volumetric response to the addition of the chosen cryoprotectant. Me2 SO was chosen, as it appears to yield the best results empirically [36]. The experiments were conducted in 10% w/w Me2 SO (1.225 M), a concentration commonly used in current cryopreservation protocols. The effect of cell volume excursions on membrane integrity and functional capacity were investigated and used to establish safe limits for cell shrinkage and swelling. The kinetic data were used to determine the permeability of the membrane to water and cryoprotectant at two temperatures commonly used to introduce and remove Me2 SO from UCB. The parameters defined by the study have been used to model current addition and elution protocols and to propose a method that will minimise harmful swelling.

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Materials and methods Collection of UCB donations Cells were isolated from low-volume, UCB donations that would otherwise have been discarded and for which consent for research had been obtained. Each cord blood sample was collected from a single placenta into CPD according to London Cord Blood Bank (LCBB) protocols and stored at room temperature [24]. Storage times varied up to a maximum of 24 h post-donation. Six UCB donations were used for each set of experiments. Cell isolation Depending on the assay method used, either a mononuclear cell fraction containing CD34þ cells or a purified CD34þ cell population was obtained. Platelet removal For determination of osmotically inactive cell volume and permeability coefficients it was necessary to deplete cord blood of platelets. This was carried out using one of two methods: clot formation (using CaCl2 /thrombin) or differential centrifugation and CD61þ cell depletion. Clot formation. This was carried out prior to preparation of the mononuclear cell fraction by adding 20 ll of 1 M CaCl2 and 2 U of bovine thrombin per ml of UCB and inducing clot formation and retraction onto a splayed wooden applicator. Low speed centrifugation and CD61þ cell depletion. This was conducted following preparation of the mononuclear cell fraction. The cells were suspended in cold PBS and centrifuged (twice) at 200g for 10 min to lower the platelet concentration. Further reduction in platelet numbers was carried out by magnetic activated cell separation (MACS) using super paramagnetic beads coupled to an anti-human CD61þ antibody (www.MiltenyiBiotech.com) according to manufacturerÕs protocol. The beads bind to megakaryocytes and platelets expressing the CD61 antigen. Depletion was effected by magnetic adsorption of cells, coated with the beads, to a separation column held within a

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powerful magnetic field. Cells expressing the CD61 antigen were retained and the negative mononuclear cell fraction containing CD34þ cells was collected after passage through the column [37]. Preparation of a mononuclear cell fraction UCB was treated with a polyclonal panagglutinin to agglutinate red cells, and diluted 1:4 in Ca2þ =Mg2þ -free PBS (DulbeccoÕs, containing 2 mM EDTA and 0.5% BSA—designated PBSþþ ). A mononuclear cell population was obtained by density gradient centrifugation over Ficoll at 1300g for 35 min [17]. The interface layer was collected and washed twice in cold PBS. Cells were maintained on ice during subsequent manipulation. CD34þ purification CD34þ cells were isolated and enriched by MACS separation technology [9] using the CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec) according to manufactureÕs protocol. Briefly, mononuclear cells were incubated with human IgG (to prevent non-specific binding) and primary anti-CD34 antibody for 15 min at 4–8 °C. After washing, the cells were resuspended in a colloidal suspension of magnetic beads capable of binding to the CD34 antibody and incubated for a further 15 min at 4–8 °C. Positive selection of CD34þ cells was carried out using a VarioMACS column held within a strong magnetic field. CD34þ cells were retained on the column and eluted with PBS by positive pressure after removing the column from the magnetic field. Purity in excess of 80% was obtained using single column filtration. Passage through a second column increased the purity to >92%. Cell counts Total nucleated cell counts were performed on whole blood and mononuclear cell fractions using a Coulter counter. Platelet counts, both pre-and post-removal, were performed on a Sysmex cell counter. CD34þ cell counts were performed on whole blood, the mononuclear cell fraction and CD34þ -purified cell suspensions using flow cytometry with anti-CD45 FITC (fluorescein isothiocyanate) and anti-CD34 PE (phycoerythrin)

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labelled antibodies using a dual platform method [40]. Cell viability assays and cell culture Survival of CD34þ cells was determined after exposure to anisotonic conditions using both membrane integrity tests and cell culture. Exposure to anisotonic conditions Cells were exposed to PBS solutions made anisotonic by adjusting only the NaCl concentration. Cells were centrifuged and resuspended in isotonic Ca2þ =Mg2þ -free PBS containing 0.5% BSA but no EDTA (PBSþ ) and equilibrated in this solution for 10 min at 20 °C. The osmolality of each anisotonic solution was measured using a freezing point osmometer. The measured osmolality of isotonic PBSþ was 295 mOsmol/kg. Cells were exposed to anisotonic conditions (70, 160, 225, 600, 900, 1200, 1500, and 1800 mOsmol/ kg respectively) for 10 min at 20 °C, by adding an aliquot of cell suspension (500 ll) to a similar volume of an appropriate stock test solution. The stock test solutions were designed so as to provide the required osmolality after dilution with the cell suspension. Following exposure, cells were returned to isotonicity by dilution in an appropriate stock recovery solution. The NaCl concentration of the stock recovery solution was adjusted so that the final osmolality was equivalent to isotonic PBSþ (295 mOsmol/kg) after addition of 140 ll of the anisotonic cell suspension to 840 ll of the appropriate recovery solution. Cells exposed to anisotonic conditions and returned to isotonicity were compared with controls in which equal numbers of cells were exposed only to isotonic PBSþ in the same manner. Membrane integrity tests Two membrane integrity tests were used to determine the effects of anisotonic exposure: fluorescein diacetate/propidium iodide (FDA/PI) staining by confocal microscopy and flow cytometry with 7-amino-actinomycin D (7-AAD). On entering the cell, non-fluorescent FDA is cleaved to fluorescein by non-specific esterases. This is retained if the cell membrane is intact. PI enters

through damaged membranes, binding to the nucleus. For flow cytometric studies 7-AAD offers advantages over PI in that there is minimum spectral overlap with FITC and PE [16]. FDA/PI staining. CD34þ cells purified by singlecolumn MACS separation were used for this test. Following exposure to anisotonic conditions and equilibration in isotonic PBSþ , aliquots of purified CD34þ cell suspension were centrifuged (1200g for 10 min) and stained with 20 lM FDA + 40 lM PI for 5 min at 37 °C. The cells were then spread over a known area under a glass coverslip and photographed using a dual wavelength (488/514 nm) confocal microscope. Six fields were examined per sample using a 10 objective, permitting analysis of 200–500 cells per field. Two B&W images were obtained for each field using red and green filters respectively and combined into a colour-encoded image for digital analysis using Metamorph software. The percentage of non-PI stained cells was calculated for each anisotonic condition and the data normalised to the isotonic control. Thus: Recovery ¼

ðcellsTC  cellsPIþ Þ

Anisot

ðcellsTC  cellsPIþ ÞIsot

;

ð1Þ

where cellsTC is the total number of FDA and PI staining cells in each sample. Flow cytometry with 7AAD. The Ficoll-separated mononuclear cell fraction was used for this assay. Following exposure to anisotonic PBS and return to isotonicity, aliquots of the mononuclear cell fraction were incubated with anti-CD45 FITC and anti-CD34 PE conjugated antibodies (BD Biosciences) for 30 min at 20 °C. IgG PE was used for determination of non-specific fluorescence. Following incubation and lysis of contaminating red cells, the cells were centrifuged, and the cell pellet resuspended in PBSþ to which was added 7AAD (final concentration: 1 lg/ml) and incubated for 15 min. Analysis of the CD34þ cell population was carried out on a dual laser FACScan flow cytometer using CELLQuest software. In total, 100,000 events were acquired. Sequential gating of the CD45þ cell population was used to identify the CD34þ sub-population according to the dual platform ISHAGE guidelines [40]. The fluores-

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cence cut-off for 7-AAD negativity (i.e., cells with intact membranes that excluded the stain) was established by preliminary experiments and was set so as to exclude <2% of cells in a fresh untreated sample of UCB. The percentage of 7-AAD negative cells was calculated for each anisotonic condition. Thus: Recovery ¼

ðcellsTC  cells7-AADþ Þ ðcellsTC Þ

Anisot

Anisot

;

ð2Þ

where cellsTC is the total number of cells within the CD34þ cell region. The data were normalised to the isotonic control. Cell culture The mononuclear cell fraction was used for all clonogenic assays. Progenitor cell viability was assayed by culture in a semi-solid methylcellulose medium (Methocult GF H4434, Stem Cell Technologies) supplemented with IscoveÕs modified DulbeccoÕs medium (IMDM). The medium is capable of supporting differentiation of progenitor cells into three lineages: CFU-GM, BFU-E, and CFU-GEMM. Following anisotonic exposure and return to isotonic PBSþ , cells were mixed with the growth medium and plated at three concentrations in triplicate wells of a 24 well plate. The cells were incubated at 37 °C in 95% air/5% CO2 and scored for growth of all three lineages at day 14 based on standard criteria [39]. The volume of cell suspension added to the medium was adjusted to give final cell concentrations of 0.25, 0.5 and 1  105 nucleated cells/ml of growth medium. The volume required was determined from the total nucleated cell count of the isotonic control. The number of CFU for all three lineages was added together and the results expressed as: Total CFUAnisotonic =103 Nucleated cellsIsotonic :

ð3Þ

The results were normalised to the isotonic control.

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for determination of Vb . Hypertonic and hypotonic solutions were prepared by the addition of sucrose or pure water to PBSþ (295 mOsmol/kg). The osmolality was adjusted to 140, 175, 200, 225, 600, 900, 1200, 1500, and 1800 mOsmol/kg respectively. The volumetric response of CD34þ cells to abrupt exposure to anisotonic conditions was measured at 20 °C using an electronic particle counting method [26,32]. Cells in isotonic PBSþ (200 ll) were injected into 20 ml of anisotonic test solution and allowed to equilibrate for 10 min. For each experiment (n ¼ 6), a minimum of 1000 cells were analysed per run and a minimum of 6 runs were conducted at each osmolality. Cell volume, expressed as peak voltage, was measured for each cell passing through the 100 lm orifice of a Coulter counter (model ZM) attached to a PC. The data were captured using an ADwin 1 MHz AD converter running under a purposedesigned Testpoint interface (Capitol Equipment, USA) to Windows NT and stored for subsequent processing by PASCAL programs. The modal voltage for each run was calculated and the average value at each osmolality determined. Cell volumes relative to the volume at isotonicity were plotted against the reciprocal of osmolality, normalised to the isotonic value (Boyle vanÕt Hoff plot). The data were fitted to a straight line by linear least square methods and extrapolated by linear regression to determine the cell volume at infinite concentration (Vb ). The Boyle vanÕt Hoff equation is defined by   V Miso Vb Vb 1 ¼ ; ð4Þ þ Viso M Viso Viso where V is the volume of the cell at osmolality M, Viso is the isotonic volume at Miso . The data were forced through the point 1,1 corresponding to the isotonic values of V and M permitting Vb to be defined as the intercept of the regression line on the y-axis. Determination of permeability parameters

Determination of osmotically inactive cell volume ðVb Þ Platelet-depleted CD34þ cell suspensions, purified by two-column MACS separation, were used

Platelet-depleted, CD34þ cells purified by twocolumn MACS separation were used in this part of the study. The volume response of CD34þ cells to abrupt (single step) exposure to Me2 SO was

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investigated at two temperatures, +1.5 and +20 °C, using the Coulter counter method. Solution temperatures were held to within 0.5 °C using a water or water/ice jacketed sample holder. Purified CD34þ cells in PBS2þ were injected into a stirred Me2 SO solution close to the orifice of the Coulter tube. The injection mechanism included an electronic switch that triggered data acquisition; thus kinetic changes in cell volume for the cell population could be recorded. An aliquot (1000 ll) of cell suspension was injected into 19 ml of 10% w/w Me2 SO–PBSþ to give a final concentration of 9.5% w/w Me2 SO and the change in volume represented by change in voltage recorded over time. For the time course experiments, sampling intervals varied according to the temperature of exposure. At +20 °C the cell suspension was sampled after 1 s and at 2-s intervals thereafter up to 30 s. Data acquisition then continued up to 3 min postinjection by which time the cells were approaching their equilibrium Me2 SO concentration. At +1.5 °C sampling times of 5 s up to 30 s were employed followed by sampling at increasing intervals up to 15 min. Further data were acquired at 30, 60, and 120 min post-injection at both temperatures. At least 1000 cells were acquired at each time interval. Duplicate runs were carried out in each experiment. Six cord donations were used for each exposure temperature. Calculation of permeability parameters The data acquired from the time course experiments was used to calculate the modal voltage for the cell population at each time interval following exposure to the cryoprotectant. This was expressed as volume relative to that in isotonic PBSþþ . The relative volumes measured over the time course at each temperature were fitted to a two-parameter model defined by the equations below [22]:

Jms ¼ Vs  Ps ðC

Modelling Me2 SO addition and elution protocols The effect on cell volume excursion of various addition and elution protocols for Me2 SO was modelled using the PASCAL program with the values of Ps and Lp established by the time course experiments. The protocols were tested against the tolerable limits for cell volume excursions established by the viability studies.

Results Osmotically inactive volume

Jmw ¼ Lp ðpe  pi Þ; e

non-ideal behaviour of the intracellular solutes— see [6,33]; Jms is the volumetric flux of Me2 SO; Vs is the partial molar volume of Me2 SO; Ps the permeability coefficient for Me2 SO and C e and C i are the external and internal concentrations of Me2 SO, respectively. The solute concentration was expressed in mol L1 [21] and the partial molar volume for Me2 SO obtained by dividing the molecular weight of Me2 SO by its density at 20 °C giving a Vs ¼ 71 cm3 . No correction was made for change in density with temperature. The model assumes that there is no coupled transport between water and cryoprotectant. The physiological volume of the cell was calculated from the mean voltage at isotonicity using a latex bead calibration method to determine the Coulter offset voltage. Cellular water volume was assumed to be 0.87 of the cellÕs physiological volume [11]. The value for Ponders R was calculated using the non-osmotic volume provided by the Boyle vanÕt Hoff plot. Curve fitting was performed using a purposewritten PASCAL program running on a standard PC. A weighted least squares best fit was obtained by manual iteration to minimise the value of v2 [44] and provided the values for Lp and Ps at the two temperatures.

i

 C Þ;

where Jmw is the volumetric water flux; Lp is the hydraulic conductivity; pe and pi are the external and internal osmolality respectively (assuming

CD34þ cells purified from UCB responded to anisotonic solutions by exhibiting an ideal osmometric response over the range 160–1800 mOsmol/ kg (Fig. 1). Cell volume after equilibration in a 140-milliosmolal solution appeared to deviate slightly from ideal behaviour, however there was

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Fig. 1. Boyle vanÕt Hoff plot for human CD34þ cells from cord blood (n ¼ 6). Mean values (SEM).

no significant difference between the intercept on the y-axis with and without the inclusion of this data point. Extrapolation of the regression line to infinite osmolality gave an osmotically inactive volume of 0:27  0:01 (mean  SEM). Osmotic tolerance The limits of tolerance to shrinkage and swelling in response to exposure to anisotonic conditions was investigated using a colony forming assay and two membrane integrity tests. Cells were exposed to hypertonic and hypotonic conditions and stained with FDA/PI (Fig. 2A) following return to isotonicity. A decrease in the number of FDA staining cells with respect to isotonic controls was observed over a range of osmolalities, with CD34þ cells appearing to tolerate exposure to hypotonic conditions less well than hypertonic exposure. In hypotonic solution, a statistically significant reduction in FDA positive cells was observed at 225 mOsmol/ kg and below. However, this was not repeated in the flow cytometric, membrane integrity assay at that osmolality even though 7-AAD is a fluorescent nucleic acid dye that enters through damaged membranes and intercalates with DNA in much the same way as PI. After exposure to hypotonic solutions a statistically significant response was seen only at 160 mOsmol/kg and below. Under hypertonic conditions both methods demonstrated a statistically significant increase in membrane

Fig. 2. Osmotic tolerance of CD34þ cells as determined by membrane integrity tests. (A) FDA/PI staining; (B) 7-AAD dual-platform FACS analysis. The cells were exposed (20 °C, 10 min) to solutions made anisotonic by altering the NaCl concentration to give the osmolalities shown. All values are expressed as mean  SEM. Statistical analysis was carried out using Tukey–Kramer ANOVA (*p ¼ 0:05, **p ¼ 0:01, ***p ¼ 0:001).

damage only after exposure to the highest osmolality chosen (1800 mOsmol/kg). The effect of exposure to anisotonic conditions on the ability of CD34þ cells to form one of three cell lineages; CFU-GM, BFU-E, or CFU-GEMM together with total colony formation is shown in Fig. 3. This is a far more stringent test of osmotic damage since it requires both cell division and differentiation. Colony forming capacity was expressed as a proportion of the isotonic control and indicated statistically significant damage below 160 mOsmol/kg and at 1500 mOsmol/kg and above (Fig. 3A). An effect of exposure to the anisotonic solutions was seen in all three lineages (Figs. 3B– D). The level of statistical significance seen in recovery of total-CFU (Fig. 3A) is mirrored in the

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Fig. 3. Osmotic tolerance of CD34þ cells as determined by clonogenic assay. (A) total CFU; (B) CFU-GM; (C) BFU- E; (D) CFUGEMM colony formation. The cells were exposed (20 °C, 10 min) to solutions made anisotonic by altering the NaCl concentration to give the osmolalities shown. All values are expressed as mean  SEM. Statistical analysis was carried out using Tukey–Kramer ANOVA (*p ¼ 0:05, **p ¼ 0:01, ***p ¼ 0:001).

recovery of CFU-GM and reflects the weighting in the population for this lineage. Permeability parameters for CD34þ cells Isotonic cell volume measurements were made using the Coulter counter. The modal voltage was calculated for purified CD34þ cell populations equilibrated in PBSþ at 20 °C, adjusted to take account of the Coulter counter offset voltage and a value for the isotonic volume obtained by comparison with latex particles of known volume. The calculations assumed a spherical shape to the cell. The physiological volume was calculated to be 2:74  1010 cm3  0:13 (mean  SEM). Other basic cell parameters such as the cell water volume and the osmotic water fraction, either calculated or assumed, are shown in Table 1.

The dynamic response of cell volume to the abrupt exposure of UCB CD34þ cells to a 10% w/w Me2 SO–PBSþ solution is shown in Fig. 4. Cells were exposed at two temperatures, +20 and +1.5 °C. At +20 °C, data on the volumetric response was collected on 1000 cells every 2 s during the first 30 s and at increasing time intervals over the next 2 min. The data from six separate cord donations was normalised to isotonic control values and pooled to obtain the mean relative volume at each time interval. The mean values obtained during this period were modelled using the assumed parameters to give a best-fit curve (Fig. 4A). This indicated that CD34þ cells shrink to approximately 60% of their isotonic volume within 20 s of exposure to the cryoprotectant, as water flows out of the cell along the osmotic gradient. Isotonic volume was regained within 5– 10 min (data not shown).

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Table 1 Calculated permeability properties and other cell parameters for CD34þ cells from umbilical cord blood Cell properties Hydraulic conductivity (Lp ) Solute permeability (Ps ) Mean cell volume [SEM] Cell water volume Non-osmotic volume (Vb ) [SEM] Osmotic water fraction (R) Safe limits for volume excursion

+20 °C

+1.5 °C 7

2:8  10 cm/atm/s 7:4  106 cm/s 2:74  1010 cm3 ½0:13] 2:38  1010 cm3 0.27 [0.01] 0.96 45–140% of isotonic volume

At +1.5 °C data were collected at 5–10 s intervals over the first 2 min and at 50 and 100 s intervals thereafter up to 15 min. Data was again normalised to isotonic controls and the mean values modelled as before (Fig. 4B). Cells shrank to approximately 55% of isotonic volume within 2 min of exposure to Me2 SO. The cells re-expanded reaching approximately 90% of isotonic volume within 80 min (data not shown). In both

3:2  108 cm/atm/s 4:2  107 cm/s

cases the model fitted the observed pattern of shrinkage and swelling well. v2 values were 0.01 and 0.05, respectively. The hydraulic conductivity (Lp ) and permeability of the membrane to Me2 SO (Ps ) were calculated from the model for both temperatures and are shown in Table 1. Both Lp and Ps were decreased by approximately one order of magnitude at the lower temperature. Modelling of UCB bank Me2 SO addition and elution protocols The calculated permeability parameters were used to model the addition and elution of Me2 SO based on protocols developed by the New York Blood Centre [36] and adapted for use by the LCBB (Figs. 5 and 6).

Fig. 4. The best-fit curves using the two parameter model, for kinetic response of CD34þ cells to a single step exposure to 1.225 M Me2 SO in PBSþ solution at (A) +20 °C; (B) +1.5 °C. All values are mean  SEM (n ¼ 6). The v2 vales are shown on the graphs.

Addition protocols The standard method (Fig. 5A) involved the addition of a 20% v/v solution of Me2 SO in isotonic solution to an equal volume of UCB over 15 min on ice using a constant velocity syringe pump delivering cryoprotectant at 1.66 ml/min. The second addition protocol (data not shown) used for volume reduced cords involved the addition of 6 ml of a 50% Me2 SO solution to 25 ml of UCB buffy coat at a constant rate of 0.4 ml/min under similar conditions. In both cases, the model indicated that cell volume decreased to approximately 60% of isotonic volume by halfway through the addition period, remaining at this level to the end of the addition phase. The PASCAL program also modelled the intracellular concentration of Me2 SO as a function of time and indicated that at the end of the addition period, Me2 SO had not reached its equilibrium concentration. The effect of

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Fig. 5. Calculated osmotically induced excursions in cell volume (dotted line) and intracellular Me2 SO concentration (solid line) for CD34þ cells during the addition of cryoprotectant. (A) Slow addition of 1.4 M Me2 SO using the standard LCBB protocol. (B) Effect of introducing a 15-min equilibration period. Cells reach a minimum volume of approximately 60% of isotonic volume and Me2 SO is equilibrated by the end of the holding period.

introducing a defined holding period (15 min, on ice) was also modelled (Fig. 5B). In both cases this permitted the intracellular Me2 SO concentration to attain near-equilibrium values with the cells still remaining below their isotonic volume at the end of the equilibration period. Elution protocols Methods for elution of 10% v/v Me2 SO were compared for their effect on cell volume. The elution protocol used was dependent on the method of addition as this governed the final volume of the stored UCB unit. Me2 SO was eluted on ice using a 5% dextran–2.25% human albumin solution in a twostep dilution process that included a centrifugation step. The centrifugation step was modelled in both cases as an extended equilibration period between the dilution steps. The model indicated that the safe limits for volume excursion were exceeded in both

Fig. 6. Calculated osmotically induced excursions in cell volume (dotted line) and intracellular Me2 SO concentration (solid line) for CD34þ cells during the elution of cryoprotectant. Dilution was with a 5% dextran–2.25% human albumin solution. (A) A standard two step dilution incorporating centrifugation. The centrifugation step itself involved 20-min centrifugation sandwiched between two short equilibration periods of 5 min each. Cell volume excursions exceed a ‘‘safe’’ level. (B) A three-step process incorporating a two step, post-centrifugation dilution phase. This scheme would restrict cell swelling to a ‘‘safe’’ level.

elution protocols after centrifugation, during the final dilution step (Fig. 6A). Cell volume exceeded 150% of isotonic volume. A post-centrifugation, two-stage addition of diluent was modelled, in which approximately 20% of the remaining volume of diluent was added to the residual supernatant. This reduced cell swelling to approximately 130% of isotonic volume, well within the safe limit for volume expansion (Fig. 6B).

Discussion The development of cryopreservation protocols for UCB stem cells has relied largely on an empirical approach utilising methods adapted from

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bone marrow and peripheral blood stem cells [35]. Studies of UCB stem cells have largely been directed to establishing that such methods provide an adequate level of post-thaw survival rather than attempting to derive optimal cryopreservation protocols from first principles [12,27,34]. A methodological approach has been advocated for cord blood and the steps required to achieve it described elsewhere [30]. This approach requires as a first step, the determination of a number of biophysical parameters that describe the kinetic response of the cell to changes in solute concentration together with information regarding the tolerated limits for cell volume excursion imposed by such changes. The membrane permeability coefficients to water (Lp ) and the chosen cryoprotectant (Ps ) derived from such experiments can then be used to model both the maximum decrease in cell volume caused by exposure to any cryoprotectant solution of known composition, as well as the maximum increase in volume occurring with the elution of the cryoprotectant. Subsequent steps require an investigation of cryoprotectant toxicity, maximum tolerable cryoprotectant concentration and optimal cooling rate. These subsequent steps are the subject of a companion paper [20]. This approach has been applied to a variety of cell types [29,33,46] but is made more difficult with haematopoietic cells due to the lack of a specific marker for the pluripotent stem cell. However, a number of surrogate markers exist which are used to predict the haematopoietic potential of stem cell grafts including the cell surface antigen CD34 and the number of haematopoietic progenitor cells as determined by culture in semi-solid medium (clonogenic assay). The CD34þ count has been shown to correlate closely with clonogenic potential [10], and both it and the colony-forming ability of hematopoietic stem cells are used as prognostic indicators of engraftment and estimators of viability after processing or cryopreservation [2,13]. Though there is some dispute as to whether the pluripotent stem cell resides solely within this sub-population of nucleated cells (see [28]), determination of biophysical parameters, osmotic tolerance and subsequent modelling of the addition and elution protocols for Me2 SO were based on the behaviour of the CD34þ cell population.

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Measurement of the volumetric response of CD34þ cells was carried out using an electronic particle counting method. Cells moving through the orifice of the counter tube cause perturbations in the applied electric field. The volume of the cell is proportional to the voltage across the orifice through which a constant current is flowing. Such a method compares favourably with optical methods but changes in the composition of the suspending medium, as in the case of extreme hypotonic solutions, can affect the electronic signal and lead to anomalous results [1]. This can be avoided by measuring the peak voltage of osmotically inactive latex particles of known volume over a range of osmolalities to determine the limits of proportionality. In this study, there was no effect on the peak voltage of latex particles over the range 140–1800 mOsmol/kg when using sucrose as the osmoticant. Changes in cell volume were calculated using the modal voltage for the cell population rather than the mean. The CD34þ cell suspension, though a purified population still contained significant numbers of platelets. Unlike the mean, the mode is unaffected by outlying data and has been shown to be more representative of the population than the mean when there is a broad distribution in cell volume [5,32]. The results showed that CD34þ cells behaved as ideal osmometers over the range 140–1800 mOsmol/kg and indicated that the osmotically inactive volume was 0.27. This is within the range observed for many different cell types including lymphocytes [18], granulocytes [42], and platelets [4]. It agrees closely with that recently derived for CD34þ cells isolated from UBC [45] and mobilised peripheral blood from healthy donors [19] but is significantly higher than that reported for CD34þ =CD33 cells isolated from bone marrow [14]. The damaging effect of excessive excursions in cell volume, in response to abrupt changes in solution osmolality, was investigated using two membrane integrity tests together with functional assessment by clonogenic assay. The ability of membrane integrity tests such as FDA/PI to accurately reflect certain types of injury has been critically reviewed [31] and a clonogenic assay was included to provide a more rigorous test of cell incapacity, since it examines both cellular differ-

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entiation and division. Comparison with the results of the clonogenic assay indicated that both membrane integrity tests tended to overestimate the effect of hypotonic exposure and underestimate the effect of hypertonicity. In the case of 7-AAD, this poor correlation may have been compounded by using a two-platform FACS analysis where absolute cell number is determined using a nonflow cytometric technique leading to an inevitable increase in variability. The clonogenic assay indicated that CD34þ cells tolerated cell volume excursions over a range of osmolalities from 160 to 1200 mOsmol/kg with no statistically significant loss of colony forming capacity. Although not statistically significant, a slight reduction in total-CFU was seen at the limits of this range. All cell lineages tested by the colonyforming assay showed similar responses to both hypotonic and hypertonic exposure which is consistent with similar published observations [25,45]. To enable addition and elution protocols to be evaluated, some permissible limits for shrinkage and swelling are required. These were based predominantly on the results of the clonogenic assays. An arbitrarily defined cut-off of 85% of the clonogenic capacity for the isotonic controls gave a ‘‘safe’’ limit for anisotonicity of between 180 and 1200 mOsmol/kg. The critical limits for cell volume excursions were based on this ‘‘safe’’ range and were calculated from the Boyle vanÕt Hoff plot to be 45–140% of the isotonic volume. The limit for hypotonic exposure is consistent with that from other published studies [45], whereas the limit for exposure to hypertonic solutions is lower than that published by Woods et al for similar cells exposed under identical conditions (cf. 600 vs 1200 mOsmol/kg). The parameters that describe water and solute permeability are derived from experimental observations of the rate of change in cell volume, in response to an abrupt change in osmolality. Modelling of these changes is most commonly conducted using the Kedem and Katchalsky formalism (1958). However, Kleinhans [22] has proposed a two-parameter formalism that does not assume the coupled transport of water and cryoprotectant through the same membrane channels: an assumption that is rarely secure. This simpler

two-parameter formalism was used to describe the experimental curve and generate the values for hydraulic conductivity (Lp ) and solute permeability (Ps ). The best-fit curve for each temperature was generated by minimising the squares of the difference between the mean observation and the value predicted by the model, weighted by the variance associated with each data point. The v2 statistic was used to indicate the goodness-offit in each case. The v2 values were 0.009 and 0.05 for exposure at +20 and 1.5 °C respectively indicating in both cases that there was a close fit to the data. A value for Lp of 2:8  107 cm/atm/s was determined for CD34þ cells at +20 °C in the presence of 1.225 mol L1 Me2 SO. There is no consensus on the units used to express hydraulic conductivity but this equates to a value of 0.17 lm/atm/min or 2:82  1014 m3 =N=s. This value is identical to that reported by Woods et al. [45] for CD34þ cells from UBC though slightly lower than that reported for bone marrow stem cells [25] at +20 °C. At +1.5 °C the hydraulic conductivity decreased 10-fold to 3:2  108 cm/atm/s. This is close to that determined at this temperature for mobilised peripheral blood CD34þ cells from healthy donors [19], though significantly lower than that reported for bone marrow [25]. The solute permeability of the membrane to Me2 SO at +20 °C estimated to be 7:4  106 cm/s (0:44  103 cm/min), is close to that calculated by Woods et al. [45] from their experimental observations. We also determined the permeability to Me2 SO at +1.5 °C. This gave a value of 4:2  107 cm/s; an approximate 5-fold decrease in permeability. The estimates for Lp and Ps were used to model the introduction and removal of Me2 SO in a clinical situation. The protocols for addition and elution of cryoprotectant vary between cord blood banks, but generally Me2 SO is added slowly to a final concentration of 10%, with many banks employ a volume reduction step to UCB prior to addition (for review see [3]). Following cryopreservation, thawed units are eluted of cryoprotectant and washed prior to transplant. The elution phase usually consists of a two-step dilution process separated by a centrifugation step [8,36]. These

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empirically derived protocols are conducted on ice, with each step lasting from 5 to 10 min. Modelling the LCBB addition protocols using the estimates for Lp and Ps obtained at +1.5 °C showed that the reduction in cell volume did not exceed the tolerable limits imposed when Me2 SO was added slowly over a 15-min period. The cells shrank to approximately 60% of their isotonic volume within the first 5 min. However, intracellular cryoprotectant concentration rose to only 90% of the equilibrium concentration by the end of the addition phase. Protocols that immediately transfer cells to the cooling phase of the cryopreservation process are thus unlikely to reach the desired cryoprotectant concentration before freezing is initiated. A short equilibration period permits equilibrium concentrations of cryoprotectant to be attained. Elution of Me2 SO in the clinical setting imposes particular problems since cells have to be treated within closed systems to minimise contamination and the final volumes for re-infusion are kept to a minimum. Elution of Me2 SO is generally carried out by serial dilution, the first step being to dilute the Me2 SO concentration by 50% at which point the cells are centrifuged and the supernatant partially removed, reducing the solution volume by approximately 80%. Sufficient isotonic diluent is then added to reconstitute the solution to its original volume, further diluting the cryoprotectant concentration. The LCBB employs a 20-min centrifugation step, though shorter centrifugation times are used elsewhere. For the purposes of modelling this was treated as an equilibration step (total equilibration time with handling was 30 min). The model showed that this common elution protocol imposed osmotic transients in excess of tolerated limits, with cells reaching over 150% of isotonic volume during post-centrifugation re-suspension. Shorter centrifugation times would result in an even greater volume expansion since there would be less time for the cells to shrink back towards their isotonic volume before addition of the diluent. The model indicated that phased addition of a cryoprotectant-free diluent would reduce cell swelling. The protocol required the addition of a volume of diluent equivalent to the volume of the original cell suspension using a constant velocity

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syringe pump. The protocol was as follows: slow addition of 50% of the diluent volume, followed by centrifugation and removal of 80% of the supernatant; slow addition of approximately 20% of the remaining diluent followed by a 5-min equilibration period; slow addition of the remaining diluent followed by an equilibration period at +1.5 °C. This protocol reduced swelling to below 130% of isotonic volume—well within ‘‘safe’’ limits. These theoretical simulations indicate that the empirically derived protocols are likely to be sub-optimal. Minor adjustments to current addition and elution protocols may well improve post-thaw survival without imposing technical or time constraints on processing of donations within the cord blood bank. Acknowledgments We would like to thank the donor co-ordinators and processing staff of the London Cord Blood Bank for their help in providing cord blood. We are also indebted to the staff of the H&I Department NBS Colindale for their help and advice. References [1] J.P. Acker, J. Pasch, I. Heschel, G. Rau, L.E. McGann, Comparison of optical measurement and electrical measurement techniques for the study of osmotic responses of cell suspensions, Cryo-Lett. 20 (1999) 315–324. [2] T.A. Amos, M.Y. Gordon, Sources of human haematopoietic stem cells for transplantation—a review, Cell Transplant. 4 (1995) 547–569. [3] S. Armitage, Collection and processing of cord blood units, in: S.B.A. Cohen, E. Gluckman, P. Rubinstein, J.A. Madrigal (Eds.), Cord Blood Characteristics: Role in Stem Cell Transplantation, Martin Dunitz, London, 2000, pp. 129–148. [4] W.J. Armitage, N. Parmar, C.J. Hunt, The effects of osmotic stress on human platelets, J. Cell. Physiol. 123 (1985) 241–249. [5] W.J. Armitage, B.K. Juss, Osmotic response of mammalian cells: effects of permeating cryoprotectants on nonsolvent volume, J. Cell. Physiol. 168 (1996) 532–538. [6] F.G. Arnaud, D.E. Pegg, Permeation of glycerol and propane-1,2-diol into human platelets, Cryobiology 27 (1990) 107–118. [7] P.G. Beatty, M. Mori, E. Milford, Impact of racial genetic polymorphism on the probability of finding an HLAmatched donor, Transplantation 60 (1985) 778–783.

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