Ex vivo expansion of umbilical cord blood CD34+ cells in a closed system: a multicentric study

Vox Sanguinis (2006) 90, 183–190 © 2006 Blackwell Publishing DOI: 10.1111/j.1423-0410.2006.00751.x

ORIGINAL PAPER

Ex vivo expansion of umbilical cord blood CD34+ cells in a closed system: a multicentric study

Blackwell Publishing Ltd

G. Astori,1 J. Larghero,4 T. Bonfini,3 R. Giancola,3 M. Di Riti,3 L. Rodriguez,2 M. Rodriguez,2 G. Mambrini,1 L. Bigi,1 A. Iacone,3 J. P. Marolleau,4 I. Panzani,1 J. Garcia2 & S. Querol2 1

DIDECO srl Mirandola (MO), Italy Barcelona Cord Blood Bank, Blood and Tissue Bank, Barcelona, Spain 3 Department of Transfusion Medicine, General Hospital, Pescara, Italy 4 Cell Therapy Unit, Saint-Louis Hospital, Paris, France 2

Background and Objectives The Dideco ‘Pluricell System’ is a CE-marked medical device allowing haematopoietic stem cell (HSC) expansion. It comprises a kit of cGMP cytokines and reagents, a closed-cell expansion chamber and a cell-washing set. We tested the system in a multicentric study by expanding CD34+ cells from eight fresh umbilical cord blood (UCB) samples. Materials and Methods During culture, the mean nucleated cell (NC) count, the mean CD34+ cell count, fold expansion, viability and apoptosis were measured. Clonogenic assays and immunophenotypical characterization were performed on days 0, 7 and 12. On the expanded cellular product, in three cases cell genotyping, endotoxin level and mycoplasma detection (by polymerase chain reaction) were performed. Results The mean CD34+ cell expansion on days 7 and 12 was sevenfold and 12-fold respectively and the mean NC expansion was 69-fold and 180-fold. The mean NC viability on day 12 was 96·9% (94·4–99·1). After 12 days, granulocyte–macrophage colony-forming units (GM-CFU) showed a 20-fold increase: a slight increase in CD34+ cell apoptosis was observed during culture. In all of three cases neither chromosomal alterations nor mycoplasma contamination was detected. No significant endotoxin levels were detected after expansion.

Received: 13 October 2005, revised 26 November 2005, accepted 20 December 2005, published online 16 February 2006

Conclusions The device allows the ex vivo expansion of NC and CD34+ cells in a closed system. The expanded cellular product is a mixture of progenitors (CD34+ cells) and differentiated (mainly myeloid and megakaryocytic) cells. To reduce cell apoptosis, more frequent cell feeding during culture should be tested. Key words: bioreactors, disposable equipment, haematopoietic stem cell transplantation, umbilical cord blood.

Introduction Over the past 15 years, a number of clinical protocols have been developed based on the isolation and manipulation of haematopoietic stem cells (HSC). The availability of clinical-grade Statement of disclosure: G.A., G.M., L.B. and I.P. were employed by Dideco srl, which manufactures the Pluricell System studied in this work. Correspondence: Giovanni Mambrini, DIDECO srl, Via Statale 12 nord, 86, 41037 Mirandola (MO), Italy E-mail: [email protected]

devices for the selection of HSC from peripheral blood (PB) or bone marrow (BM) has facilitated the application of cell selection techniques for clinical use. To date, many patients have been transplanted with ex vivo-expanded cells from umbilical cord blood (UCB), PB or BM, and clinical trials have demonstrated the safety and feasibility of the expansion procedures [1–11]. The latter were performed in commercial tissue culture flasks, culture bags, bioreactors or dedicated devices with different growth factor combinations, culture conditions, and in the presence or absence of serum from several animal sources, using either unselected cells or selected CD34+ cells. 183

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The term ‘cell therapies’, defines ‘the use of manipulated or modified cells for therapeutic purposes’; recently, a number of official indications published by regulatory bodies from several countries have been released to address legal, ethical and quality control issues [12–15]. Collaborative research and development programmes have involved biologists, engineers and physicians in integrating engineering technology with the life sciences. This has resulted in the combination of cell culture and biomaterial technologies, allowing the production and use of several therapeutic products. To comply with the regulatory indications, all necessary control measures had to be considered in order to ensure appropriate sourcing and control of all materials used in the manufacture of the cell therapy product (CTP). In particular, the integrity and functionality of the cells after ex vivo expansion, in compliance with the highest quality and safety standards of the procedures involved in cell manipulation should be controlled. Moreover, prior to release, the CTP must be evaluated for safety (sterility, endotoxin level, mycoplasma contamination) and identity (genotyping). The Dideco ‘Pluricell System’ is a CE-marked commercially available medical device (patent pending) that allows HSC expansion in the absence of stroma layer in a closed and defined system comprising an expansion chamber, a system of bags for the final washing of the expanded cellular product and a kit of certified reagent. In the present study, this device has been independently tested by three institutions and the data obtained were compared with the expected performances declared by the manufacturer.

Materials and methods Study design The institutions involved in this study were the Barcelona Cord Blood Bank, Blood and Tissue Bank (Barcelona, Spain), the Department of Transfusion Medicine, General Hospital (Pescara, Italy) and the Cell Therapy Unit, Hopital Saint-Louis (Paris, France). A protocol was designed and approved by the participating institutions to ensure harmonization of data collection and the laboratory methods used. The following data were collected: mean UCB volume; nucleated cells (NC); CD34+ cell content and percentage in the unfractionated sample; purity of CD34+ cells after cell selection; mean number of cells and volume of medium seeded in the device. During culture, the following data were collected: mean NC and CD34+ cell count and fold expansion; cell viability and apoptosis. Cell genotyping was performed and the endotoxin level measured on the expanded product. Clonogenic assay was performed on days 0, 7 and 12 of culture. On day 12, immunophenotypical characterization of the expanded cells was performed by staining the cells with anti-CD3, antiCD13, anti-CD14, anti-CD15, anti-CD19, anti-CD38, anti-

Fig. 1 The expansion chamber and the washing set of the cell expansion device.

CD41 and anti-HLA-DR fluorescent immunoglobulin followed by flow cytometric analyses.

Short description of the device The device comprises a 175-cm2 polystyrene expansion chamber equipped with sterilizing filters for the injection of culture media, a vented filter for gas exchange, two sites for injection of cell inoculum and sampling, and a system of bags and filters for the final washing and recovery of the expanded cellular product (Fig. 1). The expansion chamber and the bags are certified sterile and pyrogen-free. The device includes a ready-to-use kit of reagents comprising two vials of serumfree media (Med-A and Med-B vials) and two vials containing a balanced mixture of Flt3/Flk2 ligand (Flt-3L), thrombopoietin (TPO), interleukin-3 (IL-3) and stem cell factor (SCF), cytokines (CK Mix-A and CK Mix-B vials) known to induce expansion of HSC in vitro [16–21]. The cytokines were produced under cGMP. The reagents do not contain animal derivatives and are certified sterile, mycoplasma and endotoxin-free.

UCB collection Fresh samples were collected from normal full-term delivery placentas: blood was drained into collection bags (MacoPharma, Tourcoin, France) containing 29 ml of citrate–phosphate– dextrose (CPD). After collection, UCB samples were stored at 4 °C and processed within 24 h. Prior to collection, informed consent was obtained. The protocol was approved by the local ethical committees of the institutions involved in this study.

Purification of CD34+ cells The bags were transferred to a laminar flow hood and NC were isolated from UCB samples by density-gradient centrifugation © 2006 Blackwell Publishing Ltd. Vox Sanguinis (2006) 90, 183–190

Ex vivo expansion of UCB cells 185

(d = 1·077 g/ml). The CD34+ cells were purified using the direct CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany) following the manufacturer’s protocol. Briefly, mononuclear cells (MNC) were incubated at 4 °C for 30 min with the FcR blocking reagent and with magnetic microbeads coated with the anti-CD34 primary antibody (QBEND/10). After washing, the CD34+ cells were isolated by slow flow of the cell suspension through a separation column placed in a magnetic field. Magnetically retained cells were eluted by removing the column from the magnetic field. The separation step was repeated twice.

Ex vivo expansion of CD34 cells CD34+ purified cells were expanded in the device following the manufacturer’s instructions. Briefly, ‘complete medium’ [the ‘Med-A’ vial added together with 5% (v/v) human AB plasma and the ‘CK Mix-A’ vial] was injected into the expansion chamber through a port equipped with a 0·2-µm sterilizing filter. The CD34+ purified cells were then injected into the chamber through the dedicated port at a final concentration of 20 000 cells/ml in the ‘complete medium’. Cultures were incubated at 37 °C in a fully humidified atmosphere of 5% (v/v) CO2 for 12 days and fed, after 7 days, through a dedicated port (equipped with a 0·2-µm sterilizing filter) by the addition of fresh ‘complete medium’ [all of the ‘Med-B’ vial together with 5% (v/v) human AB plasma and the ‘CK Mix-B’ vial]. On days 4, 7, 10 and 12 of culture, cells were sampled for NC count, phenotypic analysis and clonogenic assays. Cell viability was evaluated by either the Trypan blue exclusion test or by flow cytometry using 7-aminoactinomycin D (7-AAD) staining. The expansion values represent the fold increase over the number of CD34+ cells initially purified.

Flow cytometric analysis of the cells CD34+ cell staining was performed following the ISHAGE protocol [22]. Cells were double stained with anti-CD45 fluorescein isothiocyanate (FITC) and anti-CD34 phycoerythrin (PE)-conjugated antibodies (2D1 and anti-HPCA-2 clones respectively; Becton Dickinson, San Jose, CA, USA). Expression of other haematopoietic lineage-associated surface antigens was evaluated by staining the cells with anti-human monoclonal antibodies (mAbs) against CD3, CD13, CD14, CD15, CD19, CD34, CD41 (PE labelled), CD38 and HLA-DR [allophicocyanin (APC) labelled], and CD45 (FITC labelled). For the cell viability test, samples were incubated with 7-AAD. Analyses were performed using a FACScan flow cytometer (Becton Dickinson) acquiring at least 100 000 events. Isotype-matched murine PE-, FITC- and APC-conjugated immunoglobulin were used as controls. All the reagents were purchased from Becton Dickinson. © 2006 Blackwell Publishing Ltd. Vox Sanguinis (2006) 90, 183–190

Apoptosis assay The induction of apoptosis was evaluated on CD34+ expanded cells on days 0, 4, 7, 10 and 12 of culture and on CD34+ cells after the washing procedure. Cells were collected and incubated immediately with FITC-Annexin V (Bender MedSystems, Vienna, Austria) combined with CD34 and CD45 antibodies. The cells (5 × 105) were incubated for 15 min with Annexin V, following the manufacturer’s instructions, and then washed in phosphate-buffered saline (PBS) containing 0·1% bovine serum albumin (BSA). As an internal control, a subset of ‘living’ lymphocytes was taken as a threshold for viability. For the calculation of apoptotic cells, the patterns of CD45 fluorescence and scatter were used for all subsets analysed, except CD34 cells where CD45/CD34 fluorescence characteristics were used.

Clonogenic assay The clonogenic assay was performed on selected CD34+ cells and on expanded cells on days 7 and 12 of culture. Cells were cultured in Methocult GF H4434 (Stemcell Technologies Inc., Vancouver, BC, Canada) containing 1% methylcellulose in Iscove’s MDM, 30% fetal bovine serum, 1% BSA, 10 × 10−4 M 2-mercaptoethanol, 2 mM L-glutamine, 3 U/ml recombinant human erythropoietin, 50 ng/ml recombinant human (rh) SCF, 10 ng/ml rh granulocye–macrophage colony-stimulating factor (GM-CSF) and 10 ng/ml rh IL-3. Briefly, cells were plated in duplicate in tissue culture plates (Costar, Acton, MA, USA). Plates were incubated at 37 °C in a fully humidified atmosphere of 5% CO2. On day 14, each culture well was examined under an inverted microscope. Granulocyte– macrophage colony-forming units (GM-CFU) and blastforming units erythroid/mix (BFU-E/mix) were identified and counted using standard criteria. Total CFU, defined as the total count of colonies, regardless of their lineage, were used for analysing the recovery of colonies. Colonies were considered as aggregates of more than 50 cells.

Cell washing Cell washing after expansion was performed, following the manufacturer’s instructions, in a three-bag set provided with the kit. The washing procedure is particularly stringent and is designed to remove the culture medium and the growth factors [below the detection level measured by enzyme-linked immunosorbent assay (ELISA)] after the cell expansion. Briefly, the washing set was connected to the expansion chamber using a sterile connection device (SCD), and the expanded cells were transferred in the ‘collection bag’. The expansion flask was then disconnected from the washing set. A volume of washing solution (PBS pH 7·4) was introduced, through tubing equipped with a 0·22-µm sterilizing filter,

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into the bag containing the cells, until a final volume of 400 ml was reached. The washing set was then centrifuged at 600 g for 15 min at 4 °C with moderate deceleration, placed on a manual plasma extractor and the supernatant extracted and collected in the preconnected ‘waste bag’. The procedure was repeated with a further 400 ml of washing solution. Cells were then resuspended in washing solution, counted and stained for viability and apoptosis, as described above.

Fig. 2 Fold expansion of CD34+ cells during culture. Results are expressed as mean ± standard error of the mean (SEM) (n = 8).

Endotoxin test The limulus amebocyte lysate (LAL) test was used to detect the presence of Gram-negative bacterial endotoxin in the expanded cell product [23]. Endotoxin testing was performed on three samples after cell washing using the Cambrex Pyrogent gel clot assay, sensitivity 0·06 endotoxin units (EU) /ml (Cambrex Biosciences, Walkersville, MD, USA), following the manufacturer’s instructions.

Mycoplasma detection Mycoplasma detection was performed on three samples by polymerase chain reaction (PCR) testing the boiled extracts of the cell culture supernatants after 12 days of expansion. The testing was performed by using the MycoSensor PCR Assay kit (Stratagene, La Jolla, CA, USA) following the manufacturer’s instructions. The kit detects eight Mycoplasma and Acholeaplasma species, which make up the most commonly encountered agents of tissue culture infections. To confirm the size of the mycoplasma PCR product and to validate that the PCR reaction had occurred, a positive control template was included.

Genotyping The presence of transformed cells after ex vivo expansion was tested by G-banding and karyotyping samples of three cell types following standard techniques and according to the International Nomenclature [24].

Results The results obtained following the manufacturer’s instructions for use were as follows: the average volume of the eight UCB units collected was 107 ± 26 ml (range 84·7–142·0) and the mean NC content was 1·2 ± 0.04 × 109 (range 1·08–1·32). The mean percentage and the number of CD34+ cells were 0·43 ± 0·06% (range 0·23–0·58) and 4·39 ± 0·57 × 106 (range 2·49–5·77) respectively. After immunomagnetical separation, purity was 87 ± 11% (range 78–95). The mean number of inoculated cells was 752·000 (range 630·000–878·000) and the mean volume of ‘complete medium’ was 38 ml (range 32–

Fig. 3 Fold expansion of nucleated cells (NC) during culture. Results are expressed as mean ± standard error of the mean (SEM) (n = 8).

Table 1 Viability of CD34+ cells (7-aminoactinomycin D staining) during culture (n = 6) CD34+ cell viability during culture

Mean SEM

0 days

4 days

7 days

10 days

12 days

98·8 0·3

98·8 0·1

97·6 1·1

97·3 1·0

96·9 0·8

SEM, standard error of the mean.

44 ml). After 12 days in culture, the mean CD34+ fold expansion was 12 ± 2 (Fig. 2), and the mean NC fold expansion was 180 ± 29 (Fig. 3). The cell viability showed no great variations during culture, varying from 98·8 ± 0·3% on day 0 to 97·0 ± 0·8% on day 12. Cell apoptosis within the CD34+ cell population varied during culture from 4·3 ± 0·8% on day 0 to 10·8 ± 3·1% on day 12. Complete data are reported in Table 1 and Fig. 4 respectively. Expression of differentiation antigens on expanded cells at 12 days of culture is reported in Fig. 5. Neither T- nor B-cell subpopulations were present in the expanded cells. The percentage of CD34+ cells on day 12 of culture was 5·7 ± 0·7%: phenotypic characterization of the CD34+ cell subpopulation revealed that 92·7 ± 2% of the cells were CD34+ CD38+, 5·3 ± 2·3% were CD34+ CD38– and 6·3 ± 2·5% of the cells were CD34+ HLA-DR–. © 2006 Blackwell Publishing Ltd. Vox Sanguinis (2006) 90, 183–190

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Fig. 4 Mean cell apoptosis (annexin-V) within the CD34+ cell population during culture. Results are expressed as mean ± standard error of the mean (SEM) (n = 5).

with a mean cell viability of 94·9 ± 0·5% and cell apoptosis of 10·0 ± 1·5%. Three samples were used to test the expanded cellular product for chromosomal alterations, endotoxin level, bacterial (both aerobic and anaerobic), fungal and mycoplasma contaminations. Chromosome analyses were performed by G-banding and karyotyping: no structural or numerical chromosome alterations were observed. The presence of pyrogens was examined using the LAL test: two samples were negative, whilst one showed an endotoxin concentration between 0·24 EU/ml and 0·48 EU/ml (a concentration of < 0·5 EU/ml represents the threshold for cell culture products). Cultures, tested weekly for bacterial (both aerobic and anaerobic) or fungal contamination, remained negative until the end of the expansion period. No contamination with mycoplasma was detected by PCR. All results are expressed as mean ± standard error of the mean (SEM).

Discussion

Fig. 5 Expression of differentiation antigens of expanded cells on day 12 of culture. Results are expressed as mean ± standard error of the mean (SEM) [n = 5, except for CD41 (n = 3)].

Fig. 6 Fold increase of total colony-forming units (CFU), granulocye– macrophage CFU (GM-CFU) and blast-forming units erythroid/mix (BFU-E/ mix) of expanded cells on days 7 and 12 of culture. Results are expressed as mean ± standard error of the mean (SEM) (n = 8).

The total CFU, GM-CFU and BFU-E/mix fold expansion on day 12 was 13 ± 4, 20 ± 7 and 5 ± 1 respectively. Complete results of CFU assay at day 7 and 12 are reported in Fig. 6. In three cases, we quantified the NC and CD34+ cell recovery after the washing procedure: the percentage of NC recovery was 78·1 ± 3·7%. The recovery of CD34+ cells was 70·7 ± 2·5%, © 2006 Blackwell Publishing Ltd. Vox Sanguinis (2006) 90, 183–190

The use of UCB as a source of haematopoietic progenitor cells as an alternative for bone marrow transplantation has rapidly increased during the last 10 years. However, the time to neutrophil and platelet engraftment in UCB recipients is delayed when compared with BM [25]. One possible explanation is the low number of nucleated and CD34+ cells infused compared with BM or mobilized PB. Furthermore, even studies of mobilized PB transplantation report a period of severe thrombocytopenia and obligate neutropenia [25]. The ex vivo expansion of haematopoietic progenitors has been suggested in order to ameliorate the short-term engraftment and thus the speed of engraftment [26]. Once infused, haematopoietic progenitor cells should home to the BM and complete their maturation in vivo to produce mature functional cells. Recently, Prince et al. [27] assessed the efficacy of expanded autologous CD34+ cells from mobilized PB in patients with breast cancer, evidencing that expanded cells substantially improve both neutrophil and platelet recovery. The clinical evidence acquired so far may indicate that in order to sustain both short- and long-term cell engraftment, an expanded cell population could comprise both uncommitted CD34+ as well as more committed myeloid and megakaryocytic cells. In the expanded product investigated here, megakaryocytic cells (defined as CD45+ CD41+ cells) represent 15·8 ± 5·1% (n = 3) of the population (Fig. 7). Considering that for UCB, it is still under debate whether time for platelet and neutrophil engraftment is related to the number of CD34+ cells in the reinfused cell population or to the presence of committed myeloid and megakaryocytic cells, it is difficult to draw conclusions. A 12-fold expansion of CD34+ cells was observed at 12 days of culture. The fold expansion is comparable with the data obtained on day 10 (11-fold expansion), perhaps indicating

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Fig. 7 Light scatter and CD45+/CD41a+ megakaryocytic cells after 12 days of culture. PE, phycoerythrin.

that cell growth and differentiation reached a plateau on day 12. This observation seems to be confirmed when the NC fold expansion trend is investigated: NC showed a 173-fold expansion on day 10 compared with a slight increment (180fold) on day 12. The manufacturer stated a fivefold expansion of CD34+ cells at day 12: in seven of eight (88%) of the experiments we obtained a maximum CD34+ expansion of 23-fold, well above the value stated by the manufacturer. The result was also higher than the value of 100-fold stated for TNC in seven of eight (88%) of the cases, which reached a maximum value of 329-fold. The NC cell viability remained high during culture, ranging from 98.8% on day 0 to 96·9% on day 12. The percentage of apoptotic cells, assessed by annexin V staining, varied from 4·3% on day 0 to 10·8% on day 12 (n = 6). The data indicate that a percentage of cells are in an early stage of apoptosis. As CD34+ cell selection is required before expansion, we cannot exclude that cell apoptosis could be partially induced by the selection procedure itself (density-gradient centrifugation, cell separation, frequent cell washing). Alternatively, cell apoptosis could reflect an inadequate timing in cell feeding during culture: more frequent feeding (i.e. on days 4 and 8) could be tested. In any case, data suggest that it is mandatory to collect the cells not later than day 12. A major concern in UCB ex vivo expansion is the reproducibility of the procedure. We have observed variations both in terms of NC and CD34+ fold increase, as well as in the percentage of the cell phenotypes obtained in the different expansion experiments. The CD34+ fold expansion varied (at the same Institution) from 4·06 to 23·01. In addition, the fold

expansion of NC cells varied, ranging from 81 to 329 at 12 days. The phenotype of the expanded cells showed less variation (evidenced by SEM) for the B and T cells subset and for the CD34+. A major range of variation was observed for CD45+ CD13+ (range 35·08–95·2) and CD45+ CD15+ (range 24·2–49·6) cells, perhaps reflecting the asynchronous expression of these markers during myeloid maturation, thus evidencing different stages of cell commitment/maturation during ex vivo expansion of UCB cells. In contrast, the CD34+ and TNC fold expansion in Pluricell of mobilized PB CD34+ cells from adult patients, albeit reduced, showed only slight variations in the different experiments (G. Astori, personal communication). This could be a result of the more mature progenitor cell compartment in the adult BM, or of cell commitment caused by the growth factors used during BM cell mobilization. This observation leads to the conclusion that UCB cells responds in a different extent to the cytokines in the media, possibly reflecting different stages of commitment of the naïve cells. The ability to obtain an expanded CD34+ cell population that is still capable of reconstituting a myeloablated bone marrow after ex vivo expansion is a major concern. After ex vivo expansion, the CD34+ cells underwent a differentiation process that can be evidenced by fluorescence-activated cell sorter (FACS) analysis. Figure 8 shows the logical gating of the CD45+ CD34+ CD38+ cells on day 0 (purified cells) and on day 12 of expansion. Cell maturation is evidenced by observing the increment in the light scatter properties of expanded CD34+ cells and by acquisition of the CD38 cell marker. Nevertheless, this cell population has been demonstrated to be able to successfully engraft the bone marrow of myeloablated non-obese diabetic/severe combined immunodeficient (NOD-SCID) mice [28]. The ability of the cells to engraft an immunoablated patient must, in any case, be proved in a controlled trial. The results of the colony assays after 12 days in culture evidenced an expansion of total-CFU, BFU-E/mix and GM-CFU, indicating maintenance of the more immature cell subset. In particular, the GM-CFU expansion (20-fold) is comparable with the data obtained by Verfaillie et al. in stroma-free cultures [29]. As discussed, to comply with the regulatory requirements, the washing procedure of the cells was very stringent to ensure the complete removal of cytokines from the CTP. The mean result of three experiments indicates that 21·9% of NC cells and 29·3% of CD34+ cells were lost after washing (loss of the latter perhaps caused by the lower cell density of the more immature CD34+ cells). In this device, a reduced number of cells (ranging around 150 × 106 in our experience) must be washed in cell bags in a great volume of buffer: to reduce cell loss, alternative close washing methods in compliance with regulatory requirements and able to ensure high cell recoveries and depletion of residual cytokines and medium, © 2006 Blackwell Publishing Ltd. Vox Sanguinis (2006) 90, 183–190

Ex vivo expansion of UCB cells 189

Fig. 8 Maturation of CD34+ cells during culture. Logical gates of CD34+ cells following the ISHAGE protocol: (a–e) CD45 cells; (b–f) CD34+ cells gated in R1. (c–g) Light scatter of cells gated in R2. (d–h) CD34/CD38 cells gated in R3. Cell maturation is evidenced by the progressive increasing of the dimension (FSC) and complexity (SSC) of CD34+ cells and by acquisition of the CD38+ antigen (arrow). FITC, fluorescein isothiocyanate; PE, phycoerythrin.

could be tested [30]. No CD34+ cell viability decrements (measured by 7-AAD), or increments in NC apoptosis (measured by annexin V), were observed after cell washing. Before release of the CTP, consideration should be given to the transformation potential of cells in response to growth factors, as transformed cells may gain a growth advantage over non-transformed cells under defined in vitro culture conditions: we tested for the presence of transformed cells after ex vivo expansion by G-banding and karyotyping three samples of cells, and no alterations were observed. We measured the endotoxin level on three CTP; one sample was positive, even though the concentration was below the threshold for cell culture products. The CTP were free of any bacterial, fungal or mycoplasma contamination. Recently [31,32], the transplantation of two partially HLAmatched UCB units to enhance engraftment in adults with haematological malignancies has been suggested. In the clinical report by Barker et al. because of the availability of a double UCB unit, 88% of the patients eligible in that study received a transplant with an adequate cell dose. Prior to double-unit transplantation, the percentage was only 30%. Enhanced short-term donor engraftment derived from both donors was demonstrated, although long-term haematopoiesis was derived from a single donor. The hypothesis is that the graft-vs.-graft reaction could be responsible for single-donor predominance. As B and T cells are represented in ex vivoexpanded cell products at low percentages, we speculate that transplantation of an unfractionated UCB, together with an expanded unit, could result in sustained double-unit engraftment, as also recently reported in immunodeficient mice [33]. © 2006 Blackwell Publishing Ltd. Vox Sanguinis (2006) 90, 183–190

Acknowledgements We are grateful to Dr Massimo Dominici (Oncology & Haematology Department, University of Modena and Reggio Emilia, Italy), for the critical reading of the manuscript.

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