Milano Cord Blood Bank, Centro Trasfusionale e di Immunologia dei Trapianti, IRCCS Ospedale Maggiore, Milan, Italy

Correspondence to: Dr L Lazzari, Cell Factory 'Franco Calori', Centro Trasfusionale e di Immunologia dei Trapianti, IRCCS Ospedale Maggiore, via Francesco Sforza 35, 20122 Milan, Italy

In previous studies, we identified a cytokine cocktail including thrombopoietin, Flt-3 ligand, interleukin (IL)-6 and IL-11 in serum-free medium, suitable to induce significant and sustained ex vivo expansion of primitive hematopoietic stem cells (HSCs) from cord blood (CB) for up to 10 weeks. The aim of the present study was to evaluate the effects of cryopreservation on ex vivo expansion of HSCs and their committed progenitors. CD34⁺ cells were purified from CB units, each of which was processed in part as such and in part as cryopreserved and thawed, then expanded for 5 weeks in serum-free medium with the cytokine cocktail described above. We determined the number of nucleated cells (NC), CD34⁺, CD34⁺/38^-/33^-, CD34⁺/61⁺, CD61⁺ cells and the clonogenic potential. After 2 weeks the median fold expansion of NC, CD34⁺ and CD34⁺/38^-/33^- cells was around two log both with fresh and cryopreserved CB and the expansion continued similarly until week 5. Our data suggest that this serum free protocol induces similar ex vivo expansion of HSCs and their committed progenitors from both fresh and cryopreserved CB. Our findings can be useful in view of clinical applications, since CB used for transplantation is stored in the cryopreserved state. Bone Marrow Transplantation (2001) 28, 693-698.

cryopreservation; transplantation; ex vivo expansion; cord blood

Cord blood (CB) is a valid alternative to bone marrow or mobilized peripheral blood as a source of hematopoietic stem cells (HSCs) for clinical use. So far, more than 1500 related and unrelated CB transplants have been performed world-wide in malignant and non-malignant diseases. Although clinical results are promising, approximately 80% of CB recipients are represented by pediatric patients.^1,2,3,4 In fact, the small number of HSCs present in CB prevents its use in most adult recipients. To overcome this limitation, in the last few years several protocols for ex vivo expansion of HSCs from CB have been developed^5,6,7,8 and tested successfully in an animal model.^9,10,11 Despite the positive findings so far collected, the clinical use of such protocols requires additional work. First, the expansion procedures must be scaled-up to fit the body size requirements of human recipients, with due consideration and strict adherence to regulatory issues. Second, the positive results obtained with fresh CB must be confirmed with CB stored in the cryopreserved state, which is the standard storage condition of CB units banked for transplantation purposes. In this regard, it was shown that the proliferative capacity of immature HSCs was not impaired by cryopreservation and storage in liquid nitrogen for up to 10 years.¹²

In the present work, we compared the expansion obtained with our protocol⁸ using in parallel fresh and cryopreserved aliquots of CB units. Moreover, as the prolonged time to platelet reconstitution remains a significant problem in CB transplantation, we evaluated the number of megakaryocyte (Mk) progenitors in cultures expanded from both fresh and cryopreserved CB units.

Materials and methods

Collection of CB

CB was collected after informed consent of the mother from full-term newborns. After delivery of the baby, the umbilical cord was clamped and disinfected and CB was recovered with the placenta in utero into sterile CB collection bags containing 29 ml of citrate-phosphate dextrose (CPD) as anticoagulant (Macopharma, Tourcoing, France).

Freezing and thawing of CB

CB samples were processed within 24 h of collection. Two-thirds of a CB unit were resuspended vol/vol in ice-cold freezing medium containing 70% RPMI-1640 w/o phenol red (Sigma, St Louis, MO, USA), 20% dimethylsulfoxide (DMSO; Cryoserv, Research Industries Corporation, Salt Lake City, UT, USA) and 10% human serum albumin (Farma Biagini, Lucca, Italy) and cryopreserved with a controlled-rate freezing procedure.

CB was thawed as described by Rubinstein et al.¹³ Briefly, the CB bags were placed in the gas phase of liquid nitrogen for 30 min, at room temperature for 5 min, and finally thawed in a 37°C water bath as rapidly as possible.

The thawed CB bags were immediately diluted with an equal volume of a solution containing 5% human serum albumin and 10% Dextran 40 (Solplex 40; Sifra, Verona, Italy) in 0.9% NaCl and subsequently centrifuged at 250 g for 10 min. The supernatant was removed and sedimented cells were resuspended in RPMI-1640 and 10% fetal calf serum (FCS; GIBCO BRL, Grand Island, NY, USA).

CD34⁺ cell purification

Mononuclear cells were isolated by centrifugation of the CB cells on density gradient Lympholyte-H (1.077 g/ml; Cedarlane, Hornby, Ontario, Canada) at 400 g for 30 min at room temperature. The low density cell fraction was collected and washed twice in Ca²⁺-, Mg²⁺-free Dulbecco's phosphate-buffered saline (PBS; GIBCO BRL) containing 1% bovine serum albumin (BSA; Sigma). The CD34⁺ cell population was isolated by paramagnetic microbeads selection procedure (CD34⁺ progenitor isolation kit, Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of the selected population was 75-95% both in fresh and cryopreserved CB samples, as assessed by flow cytometry with an anti-human CD34 antibody.

Flow cytometry

Cells were analyzed for phenotype expression of surface proteins specific for subpopulations of HSCs. Isolated or cultured CD34⁺ cells were incubated with 1 ml of PBS containing 4% FCS and 4% mouse immunoglobulins for 30 min at 4°C in order to block non-specific binding to Fc receptor. Cells were then washed and stained for 30 min at 4°C in the dark with the following monoclonal antibodies: mouse isotype control (1 fluorescein isothiocyanate (FITC)/1 phycoerythrin (PE)/1 Cy-Crome), anti-CD34 PE (HPCA-2) clone 8G12, anti-CD38 Cy-Crome, anti-CD33 FITC and anti-CD61 FITC. Antibodies were purchased from Becton Dickinson (San Jose, CA, USA). The cells were also incubated with 7-amino-actinomycin D (7-AAD, 1 g) for 20 min at 4°C in the dark for the evaluation of cell viability. At least 10 000 events were acquired with a FACSort flow cytometer (Becton Dickinson) and analyzed using the CellQuest software. For CD34⁺ cell analysis, consequential gates were used to consider only viable cells (7-AAD negative) which were anti-CD34 PE-positive and showed a scatter similar to that of lymphomonocytic cells. Specific subsets of CD34⁺ cells were evaluated in the dot plot FITC vs Cy-Crome.

Clonogenic assay of granulocyte/monocyte (GM) progenitors

The purified CD34⁺ cells and those obtained after liquid culture were plated in duplicate in 35 mm tissue culture plates at concentrations of 200 and 2000 cells/ml, respectively. The medium contained 0.9% methylcellulose, 30% FBS, 1% BSA, 10^-4 m 2-mercaptoethanol, 3 U/ml erythropoietin, 50 ng/ml SCF, 10 ng/ml GM-CSF, 10 ng/ml IL-3 (StemCell Technologies, Vancouver, Canada). Cultures were incubated for 14 days at 37°C in a 5% CO₂ fully humidified atmosphere and colony forming units (CFU)-GM, identified as colonies of 50 translucent cells, were scored by microscopy.

Clonogenic assay of Mk progenitors

One thousand purified CD34⁺ cells or 5000 CD34⁺ cells recovered from expansion cultures were seeded in 1.6 ml of serum-free collagen medium containing TPO (50 ng/ml), IL-6 (10 ng/ml), IL-3 (10 ng/ml) and a defined serum substitute (MegaCult-C system, StemCell Technologies), incubated in two double-chamber slides at 37°C in 5% CO₂ for 13 days and then fixed in 1:3 methanol:acetone. CFU-Mk colonies were stained using a primary antibody to the Mk-specific glycoprotein GPIIb/IIIa (CD41) linked to a secondary biotinylated antibody-alkaline phosphatase avidin- conjugated detection system and detected with an inverted microscope. Three categories of colonies were identified: pure Mk colonies (CFU-Mk), mixed Mk colonies (containing other lineages in addition to Mk), and non-Mk colonies.

Study design and expansion procedure

The study design is shown in Figure 1. Seven CB units were processed as follows: CD34⁺ cells were purified using MiniMacs immunomagnetic columns from one-third of the fresh unit while the remaining two-thirds were cryopreserved. The cryopreserved aliquots were thawed after 7 days of storage in liquid nitrogen. One ml of CD34⁺ cells isolated from fresh and cryopreserved aliquots was plated in serum-free medium (StemPro-34; GIBCO BRL) at 5 ´ 10³/ml in 24-well plates in triplicate. The cells were incubated at 37°C in a fully humidified atmosphere with 5% CO₂ for 5 weeks. A cytokine cocktail, including TPO (10 ng/ml), FL (50 ng/ml), IL-6 (10 ng/ml) and IL-11 (10 ng/ml) (Peprotech EC, London, UK), was added at the onset of culture and replaced twice a week. At the onset and from the second to the fifth weeks of culture we evaluated the number and fold expansion of nucleated cells (NC), CD34⁺ and CD34⁺/38^-/33^- cells and CFU-GM both from fresh and cryopreserved CB units. Furthermore, at the onset and after 2 weeks of culture we determined the number of CD34⁺/61⁺ and CD61⁺ cells and of Mk progenitors both from fresh and cryopreserved CB units.

Statistical analysis

The Wilcoxon signed-rank sum test was used to determine the significance of differences between paired groups. Values of P lower than 0.05 were considered as statistically significant. Results are reported as median and range.

Each week the total NC number was obtained by multiplying the number of NC counted by light microscopy in a Bürker chamber for 2ⁿ, where n represents the number of cell demipopulations performed. The fold expansion of NC, CD34⁺ cells and CFU-GM was determined as follows: fold expansion of NC = total NC at time T / time 0; fold expansion of CD34⁺ cells = total NC ´ % CD34⁺ cells at time T / time 0; fold expansion of CFU-GM = total NC ´ CFU-GM counted at time T/time 0.

Results

Ex vivo expansion of NC, CD34⁺ cells, CD34⁺/38^-/33^- cells and CFU-GM

The purified fresh and thawed CD34⁺ cells median and range viability at time 0 was 86 (78-95) and 86 (75-94), respectively. The median and range number of CD34⁺ cells recovered per 1 ´ 10⁸ NC input was 180 ´ 10⁵ (42-320) when fresh cord blood was processed, and 103 ´ 10⁵ (56-274) when frozen CB was used (n = 7).

After 2 weeks of culture in serum-free medium the median fold expansion of NC and CFU-GM (Figure 2), CD34⁺ cells and CD34⁺/38^-/33^- cells (Figure 3) was around two log both with fresh and cryopreserved CB. After 5 weeks, the ex vivo expansion reached the highest level. The median (and range) fold expansions of CFU-GM from fresh and cryopreserved CB were 72 (44-262) and 72 (24-164) at 2 weeks and 469 (356-1111) and 467 (442-592) at 5 weeks (Figure 2). The median (and range) fold expansions of CD34⁺/38^-/33^- cells from fresh and cryopreserved CB were 181 (75-759) and 176 (68-388) at 2 weeks and 389 (74-2597) and 832 (204-1894) at 5 weeks, respectively (Figure 3). The differences were not statistically significant. The number of CD34⁺ cells during a 5 week expansion is reported in Table 1. Also for this parameter, there were no statistically significant differences between fresh and cryopreserved/thawed samples.

Maintenance and expansion of CFU-Mk progenitors after cryopreservation and thawing

To evaluate the effects of cryopreservation and thawing on CFU-Mk progenitors, we compared the number of CFU-Mk, CD34⁺/61⁺ and CD61⁺ cells per 10³ cells initially seeded in the culture wells before expansion, in both fresh and cryopreserved cells. To this aim, we used a defined serum-free kit for the evaluation of CFU-Mk and flow cytometry for the analysis of CD34⁺/61⁺ and CD61⁺ cells. The results are shown in Figure 4.

The median (and range) number of CFU-Mk was 45 (6-108) and 40 (27-96) in fresh and cryopreserved CB, respectively. The median (and range) number of CD34⁺/61⁺ cells was 64 (23-464) and 75 (35-117) in fresh and cryopreserved CB, respectively. The differences were not statistically significant.

After 2 weeks of culture the median (and range) number of CFU-Mk was 439 (60-1354) and 411 (84-1274) in fresh and cryopreserved CB respectively (Figure 4). The median (and range) number of CD34⁺/61⁺ cells was 14881 (13 070-31 106) and 3675 (648-20 925) in fresh and cryopreserved CB, respectively (Figure 4). All differences were not statistically significant.

Moreover, after 2 weeks expansion the percentage of CD61⁺ cells was 28 (11-38) in fresh CB and 17 (11-40) in cryopreserved CB. This difference was also not statistically significant.

Discussion

The outcome of CB transplantation is significantly influenced by the number of cells infused into the recipient.^4,13 The current evidence shows that the clinical outcome of CB transplant is better in patients receiving more than 37 ´ 10⁶ nucleated cells per kg body weight.^3,14 To overcome this limitation, a number of protocols for ex vivo expansion of CB-HSCs have been developed.^{5,6,7,8,10,12,15,16} The validation of such protocols for pre-clinical studies and their ultimate clinical application requires the demonstration that cryopreservation, the current storage condition of CB prospectively banked for clinical use, does not impact negatively on the expansion potential of HSCs from CB.

Our previous studies indicated that a serum-free medium and a cocktail including TPO, FL, IL-6 and IL-11 are able to induce significant and sustained ex vivo expansion of long-term culture-initiating cells (LTC-IC) from CB⁸ and that the expanded cells are able to engraft into NOD/SCID mice.¹⁷ The studies reported in this article were undertaken to compare the performance of our expansion protocol when applied to fresh and cryopreserved aliquots of the same CB units. We selected this experimental design to remove potential bias generated by the use of two independent series of fresh and cryopreserved units. Our current data show that TPO, FL, IL-6 and IL-11 in serum-free medium support the amplification and the self-renewal of early HSCs similarly in fresh and cryopreserved CB. Although with a different expansion protocol, Gilmore et al¹⁸ recently reported similar findings.

In view of clinical applications, an additional objective of our in vitro study was to evaluate if cryopreservation and thawing impair Mk progenitors and their clonogenic capacity. In this regard, it is possible that thrombocytopenia after CB transplantation is due to a reduced speed in the process of CB-Mk maturation into platelets.¹⁹ Therefore, we investigated if our ex vivo expansion protocol, that includes early-acting growth factors able to impact on the development of the Mk lineage, could induce some Mk maturation. As far as the components of our expansion cytokine cocktail are concerned, TPO is considered the most important regulator of megakaryopoiesis,^20,21,22 while IL-6 and IL-11 enhance human megakaryopoiesis.^23,24 Moreover, Li et al²⁵ recently demonstrated that FL, in combination with TPO, is able to augment the expansion of CFU-Mk in liquid culture. After 2 weeks we observed a three to five log expansion of Mk precursors both in fresh and cryopreserved CB samples. Therefore, our data suggest that cryopreservation does not significantly modify the function of Mk progenitors. Our observations corroborate the report by Lu et al,²⁶ who showed that CD34⁺ cells recovered from cryopreserved CB could be efficiently transduced and expanded ex vivo.

Other studies on ex vivo expansion systems for clinical purposes have been recently published.^{27,28,29,30,31} In this regard, Querol et al²⁷ determined optimal conditions for CB thawing and expansion with a system using Teflon bags. These investigators evaluated the impact of thawing and CD34⁺ cell selection on the expansion of frozen CB cells. Using a combination of four cytokines (TPO, stem cell factor (SCF), IL-3 and FL) and an expansion interval of 6 days, they observed similar clonogenic efficiencies of CD34⁺ cells from both cryopreserved and fresh samples. They also reported that the functionality of the HSCs, as determined by a clonogenic assessment in semisolid media and ex vivo expansion cultures, was maintained after cryopreservation and thawing.²⁷

Another group of investigators studied the effect of different thawing methods on the ex vivo expansion of primitive and committed progenitors from CB.²⁸ These investigators thawed CB samples using a solution containing dextran and albumin as described by Rubinstein et al¹³ or FCS and RPMI-1640 and found that the former protocol significantly enhances the recovery of CD34⁺ cells, CFC and LTC-IC. Moreover, they expanded the thawed CD34⁺ cells in the presence of IL-3, IL-6, SCF, epo, granulocyte-colony stimulating factor (G-CSF) and showed a 20- to 40-fold CFC and a two-fold LTC-IC expansion after 7 to 14 days.

The clinical efficacy of CB expansion has been recently evaluated by Shpall et al²⁹ and McNiece et al.³⁰ These investigators transplanted 40 cancer patients receiving high-dose chemotherapy with CB cells expanded in the presence of SCF, G-CSF and megakaryocyte growth and development factor. Their results suggest that expanded CB cells may reduce the engraftment failure rate that affects transplantation of unexpanded CB.²⁹ Moreover, these investigators recently developed a two-step culture system able to increase the expansion of NC and the maturation of neutrophil precursors, as compared to the one-step procedure previously developed.³¹

As regards our protocol, based on the fold expansion observed after 2 weeks, it is possible to compute the number of cells that would be available for a recipient of expanded CB as compared to non-expanded CB. This computation should be performed considering that, for ethical reasons related to the safety of the procedure, the expanded cells will be transplanted into an initial set of recipients together with non-expanded cells. In our system, we chose to use one-third of the available cells for the expansion protocol, while the remaining two-thirds would be used unexpanded. Therefore, from our data we determined that the number of NC cells transplantable into a recipient of 2/3 non-expanded and 1/3 expanded cells will be approximately 30 times higher than the number of cells present in the same entirely non-expanded CB unit.

In conclusion, the evidence so far collected supports the possibility of expanding cryopreserved CB cells with the same efficacy as fresh cells. Moreover, our system has the ability to expand Mk progenitors, thus providing a potential tool to shorten the prolonged time to platelet reconstitution currently affecting most CB recipients. Clinical studies of adequate size are necessary to determine the impact of ex vivo expansion on short- and long-term outcomes of CB transplantation.

Acknowledgements

This work was supported by grants from Cariplo 1997, 1998, 1999 and from Ricerca Corrente 1996 (515/01), Ricerca Corrente 1997 (515/02), Ricerca Finalizzata 1997, MURST 1998 (prot. 9806156213 006), Eurocord BIOMED II - QLRT-1999-00380 (Cord blood as a source of stem cells for clinical use).

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Figure 1 Study design. Each CB unit was divided into two parts: 1/3 of the unit was processed as such and the remaining 2/3 were cryopreserved and thawed. CD34⁺ cells were expanded for 5 weeks and at the onset and from the second to the fifth weeks of culture nucleated cells, CD34⁺, CD34⁺/38^-/33^- and CD61⁺ cells, clonogenic potential of GM were determined. At the onset and after 2 weeks of culture, we also determined the clonogenic potential of MK progenitors. All the parameters were analyzed both from fresh and cryopreserved CB.

Figure 2 Expansion of total nucleated cells (NC) and CFU-GM observed during 5 weeks culture of CD34⁺ CB cells in serum-free medium including TPO, FL, IL- 6 and IL-11. The white bars show the results obtained using fresh samples and the grey bars using cryopreserved samples. Data are shown as median and range of the fold expansion of seven experiments.

Figure 3 Expansion of CD34⁺ and CD34⁺/38^-/33^- cells observed during 5 weeks culture of CD34⁺ CB cells in serum-free medium including TPO, FL, IL- 6 and IL-11. The white bars show the results obtained using fresh samples and the grey bars using cryopreserved samples. Data are shown as median and range of the fold expansion of seven experiments.

Figure 4 Number of CFU-Mk, CD34⁺/61⁺ and CD61⁺ cells obtained from fresh or cryopreserved CB samples before (columns A and B) and after (columns C and D) ex vivo expansion in the presence of TPO, FL, IL-6 and IL-11. Columns A and C represent fresh CB. Columns B and D cryopreserved, thawed, unexpanded CB. Data are shown as median and range (n = 7).

Table 1 Number of CD34⁺ cells ´ 10³ during expansion of CD34⁺ cells purified from fresh and cryopreserved CB