长期冻存脐带血细胞方案评价

1087 Received May 25, 2011; final acceptance May 15, 2012. Online prepub date: October 3, 2012. 1These authors provided equal contribution to this work. Address correspondence to Mario Mairhofer, Department of Obstetrics and Gynecology, Medical University of Vienna, Vienna, Austria. Tel: +43 1 40400-7692; Fax: +43 1 40400-7842; E-mail: mario.mairhofer@meduniwien.ac.at Cell Transplantation, Vol. 22, pp. 1087–1099, 2013 0963-6897/13 $90.00 + .00 Printed in the USA. All rights reserved. DOI: http://dx.doi.org/10.3727/096368912X657396 Copyright  2013 Cognizant Comm. Corp. E-ISSN 1555-3892 www.cognizantcommunication.com Evaluation of a Xeno-Free Protocol for Long-Term Cryopreservation of Cord Blood Cells M. Mairhofer,*1 J. C. Schulz,†1 M. Parth,* U. Beer,* H. Zimmermann,†‡1 and A. Kolbus*1 *Department of Obstetrics and Gynecology, Medical University of Vienna, Vienna, Austria †Fraunhofer Institute for Biomedical Engineering (IBMT), St. Ingbert, Germany ‡University of Saarland, Chair of Molecular and Cellular Biotechnology/Nanotechnology, Saarbrücken, Germany Cord blood is regarded as a powerful source for adult stem cells. Cord blood transplants have been used success- fully to treat children and adults in autologous and allogeneic settings. Nevertheless, in many cases, the clinically relevant cell number (CD34+ cells and total leukocytes) is a limiting factor. To enable standardized cell banking and future in vitro expansion of adult stem/progenitor cells, elimination of serum, which inevitably differs from lot to lot and donor to donor, is highly desirable. Here, we demonstrate the feasibility of a xeno-free, chemically defined cryopreservation procedure for cord blood-derived cells over a period of 1 year. Cell recoveries with respect to retrieval of clinically relevant CD34+ cells, colony-forming units, and in vitro cultures of erythroid pro- genitor cells under standardized conditions were analyzed after 1 week or 1 year of cryopreservation and found to be very high and similar to the samples before freezing. The established xeno-free procedure is an important step toward using the full potential of adult stem cells from cord blood, enabling the elimination of serum-derived factors negatively influencing proliferation, differentiation, and survival of hematopoietic stem cells. Key s: Cord blood; Optimization of cryopreservation; Hematopoietic stem/progenitor cells; Xeno-free cryomedium; Erythroid progenitor cells (EPCs) for cryopreservation and storage (8). Nowadays, most cord blood banks store red cell-depleted, volume-reduced sam- ples (leukocyte-rich plasma, LRP) that are cryopreserved in a cocktail of dimethyl sulfoxide (DMSO) and dextran-40 (8,23). The stored products are thawed and subsequently washed in order to reduce the DMSO concentration before infusion. Total leukocyte recoveries after cryopreservation typically are close to 80%, which means that a substantial amount of dead/apoptotic cells is transfused into the recipi- ent. Therapy success, however, always correlates with the transfused cell dose (total CD34+ cells) (6,21,22). Recently, several cases of cardiovascular toxicity have been reported (12,16), and Ma et al. argued that dextran-40 could be responsible for this adverse effect (16). These reports indi- cate that the cryopreservation of cord blood cells should be improved to maximize the number of transfused cells and to avoid transplant-induced complications in the diseased recipients. One solution for the persistent problem of inad- equate cell numbers in cord blood transplants could be the in vitro expansion of hematopoietic stem cells (HSCs) and INTRODUCTION The allogeneic transplantation of umbilical cord blood is widely accepted as a therapeutic treatment of hematolog- ical disorders and malignant diseases in children and adults (2,6,9–11,13,21,22). Umbilical cord blood transplantation (UCBT) has several advantages to bone marrow transplan- tation (BMT) (15): (i) the fast procurement due to thou- sands of stored and human leukocyte antigen (HLA) typed cord blood units worldwide; (ii) lower incidence of severe graft-versus-host disease (GVHD); and (iii) less stringent requirements for HLA matching. However, there are also major disadvantages: (i) delayed engraftment and increased risk of graft failure compared to BMT; (ii) delayed T-cell immune reconstitution; and (iii) increased costs of hospi- talization. Due to the continued shortage of HLA-matched bone marrow donors, UCBT is successfully used to treat a broad variety of malignant and nonmalignant diseases when no HLA-matched BMT is available. An adequate supply of high-quality cord blood cells can only be guaran- teed upon establishing standardized and efficient protocols Administrator 高亮 1088 MAIRHOFER ET AL. progenitor cells. Successful ex vivo expansion of HSCs has already been demonstrated in the mouse system (19,30). This approach has already been envisaged in the year 1996 as “the next generation of cellular therapeutics” (7), but its transformation to the clinic has proven to be difficult. To enable standardized expansion protocols for cord blood cells, it is important to avoid human plasma, which is con- tained in most stored cord blood samples. Cytokines like tumor necrosis factor-a (TNF-a) or transforming growth factor-b (TGF-b) in the plasma may negatively influence in vitro cell expansion even at very low concentrations. Therefore, a fully defined cryopreservation medium would facilitate standardized conditions for subsequent cell expansion by avoiding additional cytokines contained in most cryomedia. Cells derived from cord blood are gen- erally not used directly for therapy, but have to be stored for several years, demanding high standards for the cryo- preservation protocol and the storage conditions. Efficient recovery of hematopoietic progenitor cells after long-term cryopreservation has been reported (4). Recently, long- term cryopreserved cord blood cells have been used for competitive repopulation experiments in mice, thereby demonstrating that true HSCs are still functional after more than 20 years of cryopreservation (3). In addition, the same group also used the cryopreserved cord blood as a starting material for reprogramming into induced pluri- potent stem (iPS) cells and for the isolation of endothelial progenitors, demonstrating the therapeutic potential of the stored cord blood units (3). In a recent study, we have char- acterized a xeno-free, fully defined cryomedium, which gave excellent recoveries of different stem/progenitor cell types after cryopreservation (29). This cryomedium, which is chemically fully defined, was tested with three different primary cell types: umbilical cord blood (UCB)- derived erythroid progenitor cells (EPCs), UCB-derived endothelial colony-forming cells, and adipose tissue-derived mesenchymal stromal cells. For all three cell types, cell recovery after cryopreservation was equal to or better than a reference medium consisting of 90% fetal bovine serum (FBS) and 10% DMSO (29). The aim of the present study was to evaluate the long- term storage of cord blood cells in this xeno-free and chemically fully defined cryomedium. The efficiency of the cryopreservation procedure was assessed by mea- suring the viability and recovery of total mononuclear cells (MNCs) and CD34+ cells, as well as the amount of colony-forming units (CFUs). Standardized EPC cultures were performed to characterize the potential for in vitro expansion cultures. EPC cultures were chosen as a com- monly used and well-characterized model for expansion cultures from cord blood because of their high prolifera- tive potential, and the ability to perform cultures from small aliquots of cryopreserved cord blood cells, which was a prerequisite for our study. MATERIALS AND METHODS Cord Blood Collection Cord blood samples were collected after obtaining informed consent from the mother according to the insti- tutional guidelines of the General Hospital of Vienna. Cord blood was collected by venipuncture after cesarean section or spontaneous birth into 250-ml cord blood bags containing 35 ml of citrate phosphate dextrose adenine (CPDA-1) anticoagulant solution (Baxter Healthcare, Vienna, Austria). Samples with cord blood volumes below 50 ml were excluded. The collected cord blood was stored at room temperature until the isolation of the mononuclear cells (MNCs) was started. Isolation of MNCs, determina- tion of cell viability, quantification of CD34+ cells, estab- lishing prefreezing cultures and CFU assays, and freezing of cells were completed within 12 h after isolation of the cord blood. Isolation of MNCs From Cord Blood The cord blood was dispensed into 50-ml Falcon tubes (BD Biosciences, Franklin Lakes, NJ, USA) under sterile conditions and diluted 1:2 with Dulbecco’s PBS (Gibco/ Invitrogen, Carlsbad, CA, USA). The diluted cord blood was carefully layered on top of a Ficoll cushion (Biocoll Separating Solution, Biochrom, Berlin, Germany). The tubes were centrifuged at room temperature (RT) for 40 min at 600 ´ g. Next, the blood/Ficoll interphase was collected, diluted with PBS, and the cells were pelleted (300 ´ g, 10 min, RT) and resuspended in 50 ml of hypotonic lysis buffer (8.99 g of ammonium chloride, 1 g of KHCO3, 0.037 g of EDTA per liter, pH to 7.3; all from Sigma-Aldrich, St. Louis, MO, USA). Aliquots were removed for cell count- ing (CASY counter; Roche-Innovatis, Indianapolis, IN, USA) and determination of CD34+ cells. The MNCs were then resuspended in Fraunhofer Institute for Biomedical Engineering (IBMT) medium (available from Fischer Procryotect, Ruedlingen, Switzerland) at a concentration of 1 ´ 107 cells/ml. The medium contains a protein-free basal medium, an ethylene oxide/propylene oxide block copolymer (Pluronic F-68), and DMSO as cryoprotec- tant, and does not contain any additional proteins. For cell viability determination in triplicates and CD34 analytics, one aliquot was further diluted 1:10 with cryomedium and dispensed into 10 aliquots containing 1 ´ 106 cells/ ml. For prefreeze analysis, one aliquot of 1 ´ 107 cells and three aliquots of 1 ´ 106 cells were processed for CFU assays and in vitro cultivation and cell viability analysis, respectively. The other samples were frozen as outlined in Figure 1 and in the section below. XENO-FREE CRYOPRESERVATION OF CORD BLOOD 1089 Freezing and Thawing of Cells Isolated mononuclear cells were placed on ice and transferred to a temperature-controlled freezer (SY-LAB, Purkersdorf, Austria). The cells were frozen with the fol- lowing protocol: 4°C for 30 min (precooling), tempera- ture gradient −1°C/min from 4°C to −80°C, −80°C for 2 h (hold). Frozen cells were transferred to the gas phase of a N2 storage tank (Taylor-Wharton K series equipped with CryoCon AFT-3L module for temperature monitor- ing and automatic N2 refill; Taylor-Wharton Cryogenics, Theodore, AL, USA) and stored for the indicated periods. For thawing, cells were transferred from the storage tank to a 37°C water bath and swirled continuously until thawed. The cryovials were immersed in 70% EtOH, dried, and transferred to a laminar flow cell culture hood. After thaw- ing, the cells were washed once with 10 ml of prewarmed Roswell Park Memorial Institute (RPMI) medium (Lonza, Basel, Switzerland), pelleted (300 ´ g, 5 min, RT), and resuspended in StemSpan medium (STEMCELL Tech- nologies, Vancouver, Canada) for cultivation. Cell Viability Analysis With the 1 ´ 106 MNC Aliquots To enumerate the viable cells, triplicate samples from each cord blood sample were analyzed at each time point (prefreeze, 1 week cryo and 1 year cryo). Two hundred microliters of the cell suspension was transferred to fluores- cence-activated cell sorting (FACS) tubes (BD Biosciences) and diluted with 300 µl of PBS. Dead cells were stained by addition of 5 µl of propidium iodide (PI) staining solution [50 µg PI/ml in PBS (pH 7.4)], and the fraction of viable cells (PI negative) was determined by flow cytometry on a FACScan (BD Biosciences). Nucleated cell counts were determined with the CASY counter (60-µm capillary, size range 5–15 µm). The number of viable MNCs was calcu- lated by multiplication of the percentage of viable cells with the total number. Cultivation of EPCs From 1 ´ 107 MNC Aliquots The mononuclear cells resuspended in cryomedium were washed once with 10 ml of RPMI medium and pel- leted (300 ´ g, 5 min, RT), and the supernatant was aspi- rated carefully. The cell pellets were resuspended in 1 ml of StemSpan medium (STEMCELL Technologies, Vancouver, Canada), and an aliquot was removed for the CFU assays. Then, the cells were supplemented with 2 U/ml erythro- poietin (Erypo, 10,000 U/ml, Jannsen-Cilag, Vienna, Austria), 100 ng/ml stem cell factor (SCF), 1 ´ 10−6 M dex- amethasone (Dex), 40 ng/ml insulin-like growth factor-1 Figure 1. Schematic representation of the experimental setup and of the analyses performed with the 11 cord blood (CB) samples. After isolation of mononuclear cells (MNCs), aliquots of the cells were directly assayed for total cell viability and colony-forming unit (CFU) capability in triplicates. Viable CD34+ cells were quantified and endothelial progenitor cell (EPC) cultures were initiated. The other aliquots were frozen as described in Materials and Methods. After 1 week or 1 year of storage, cells were thawed and the indi- cated tests were performed with the cryopreserved samples. 1090 MAIRHOFER ET AL. (IGF-1), and 20 µg/ml of a cholesterol-rich lipid mix (all from Sigma-Aldrich) and cultivated as described (5,14). Briefly, partial medium changes were performed daily, and adherent cells were removed by daily transfer into fresh culture dishes. Outgrowth of erythroid progenitor cells (EPCs) was monitored by cytospin preparations and stainings according to May–Gruenwald–Giemsa (Biomed Labordiagnostik GmbH, Oberschleißheim, Germany) and by analyzing the CASY cell counter profiles at days 4, 8, 12, and 14. As soon as erythroid cells start to domi- nate the culture (days 6–10), the cell concentration was adjusted to 2 ´ 106 cells/ml and maintained at this value by daily partial medium replacements. At day 14, more than 95% of the cells show the characteristics of EPCs in hematological staining, cell diameter, and flow cytometry analysis (>95% of the cells are positive for CD71 and CD36). Flow cytometry for surface marker analysis was performed with live cells freshly harvested from the cul- tures and washed once with PBS. Approximately 2 ´ 105 cells were stained per approach. Antibodies were diluted according to the manufacturer’s instructions. Stainings were performed in approximately 50 µl of buffer (PBS supplemented with 0.2% BSA and 1 mM EDTA) for 30 min at 4°C. After staining, the cells were washed once with 1 ml of buffer and resuspended in approximately 400 µl of buffer for measurement. The corresponding iso- type controls were used to control for unspecific antibody binding. A minimum of 10,000 events were collected for every staining. The following antibodies were used: anti- CD3 (clone UCHT1), anti-CD34 (clone 581), anti-CD15 (clone 80H5), anti-CD19 (clone J3-119), and anti-CD56 (clone N901) from Beckman-Coulter; anti-CD14 (clone MjP9), anti-CD36 (clone CB38), anti-CD45 (clone HI30), anti-CD71 (clone M-A712), anti-CD117 (clone YB5.B8), anti-CD235A (clone GA-R2/HIR2), and anti-CD42b (clone HIP1) from BD Biosciences. Colony-Forming Assays (CFU) For CFU assays, approximately 100,000 MNCs were diluted to 500 µl with Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 2% bovine serum albumin (BSA; both from STEMCELL Technologies, Vancouver, Canada). Four hundred microliters of this dilution was mixed with 3.6 ml of methylcellulose medium supporting eryth- roid burst-forming unit (BFU-E), erythroid colony-forming unit (CFU-E), granulocyte macrophage colony-forming unit (CFU-GM), and granulocyte, erythrocyte, monocyte/ macrophage, megakaryocyte (CFU-GEMM) colony growth (STEMCELL Technologies). With a 5-ml syringe and a 16-gauge needle (both BD Biosciences), aliquots of 1.1 ml methylcellulose were seeded in triplicates into 35-mm cell culture dishes (Nunc, Roskilde, Denmark). Approximately 20,000 MNCs were seeded per dish. Cells in methylcellulose were cultivated for 14 days, and colonies were then scored using an inverted microscope (Olympus, Tokyo, Japan) and 4´ and 10´ objectives. Determination of Viable CD34+ Cells From Fresh Cord Blood Units and Mononuclear Cells For quantification of viable CD34+ cells from cord blood, an aliquot of 50 µl was mixed with 20 µl of CD45/ CD34 staining solution (BD Biosciences, Franklin Lakes, NJ, USA) in TruCount tubes (BD Biosciences) according to the manufacturer’s instructions. Cells were stained for 15 min at RT in the dark, and red blood cells (RBCs) were then lysed by addition of 1 ml of ammonium chloride lys- ing buffer (BD Biosciences). After RBC lysis, 20 µl of 7-aminoactinomycin D (7-AAD; BD Biosciences) dye was added to exclude dead cells. Flow cytometry analysis was performed on a FACScan, and the viable CD34+ cells were quantified using a modified International Society of Hematotherapy and Graft Engineering (ISHAGE) pro- tocol (1,26). Gating for the different cell types (granu- locytes, lymphocytes, monocytes) was performed. The staining protocol was calibrated with the BD Stem Cell Control Kit (BD Biosciences). For determination of CD34+ cells from isolated MNCs, the cells were diluted with 1 ml of PBS with 0.1% BSA after staining (RBC lysis not necessary), mixed with 7-AAD, and analyzed as described above. Statistical Analysis The paired Student’s t test was performed to assess the significance of differences between the measurements at different time points. Correlations between independent variables were analyzed using Pearson’s product moment correlation. The analysis was performed with SigmaPlot 12 (Systat Software, San Jose, CA, USA). RESULTS To examine whether xeno-free cryopreservation of cord blood stem/progenitor cells is an alternative to cur- rent protocols using either human or bovine serum, we decided to thoroughly evaluate the performance of the serum-free cryopreservation protocol on 11 cord blood units by four different tests (Fig. 1). First, we directly measured the clinically relevant numbers of viable CD34+ cells before and after cryopreservation. Second, we ana- lyzed total MNC numbers and cell viability before and after cryopreservation. Third, we tested the multilineage differentiation properties of the cord blood-derived cells by performing CFU assays before and after cryopreserva- tion. Fourth, we performed well-characterized erythroid progenitor cell (EPC) expansion cultures before and after cryopreservation. Eleven cord blood units were collected, and an aliquot of 50 ml of each cord blood was removed for determination of cells expressing CD34. MNCs were isolated and resuspended in IBMT medium as described XENO-FREE CRYOPRESERVATION OF CORD BLOOD 1091 in Materials and Methods. Cells were frozen and stored for either 1 week or 1 year. Reference cells were directly assayed for CD34 expression and total cell viability, and CFU assays and expansion cultures of EPCs were star - ted as shown in Figure 1. The corresponding experiments were performed with the cryopreserved cells after 1 week and 1 year of storage, respectively. Viable CD34+ cells in whole cord blood were quantified using a single-platform method and a modified ISHAGE gating strategy (1) and the numbers of cells expressing CD34 in whole blood and after isolation of MNCs were determined (Table 1). As expected, there is