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Keywords:

  • Cord blood processing;
  • Filtration;
  • Umbilical cord blood;
  • Stem cell transplantation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. UCB Processing by BC Preparation
  6. UCB Processing by Filtration
  7. Cryopreservation Procedure of WB, BC, and Filter Samples
  8. Thawing of the Samples
  9. Cell Counting and Analysis by Flow Cytometry
  10. Hematopoietic Progenitor Cell Assays and Cell Viability
  11. Paired NOD/SCID Mice Transplantation Experiments
  12. Statistical Analysis
  13. Results
  14. Discussion
  15. Conclusion
  16. Acknowledgements
  17. References

Umbilical cord blood (UCB) preparation needs to be optimized in order to develop more simplified procedures for volume reduction, as well as to reduce the amount of contaminating cells within the final stem cell transplant. We evaluated a novel filter device (StemQuickE) and compared it with our routine buffy coat (BC) preparation procedure for the enrichment of hematopoietic progenitor cells (HPCs). Two groups of single or pooled UCB units were filtered (each n = 6), or equally divided in two halves and processed by filtration and BC preparation in parallel (n = 10). The engraftment capacity of UCB samples processed by whole blood (WB) preparation was compared with paired samples processed by filtration in the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse animal model. Filtration of UCB units in the two groups with a mean volume of 87.8 and 120.7 ml, respectively, and nucleated cell (NC) content of 9.7 and 23.8 × 108 resulted in a sufficient mean cell recovery for mononucleated cells ([MNCs] 74.2%-77.5%), CD34+ cells (76.3%-79.0%), and colony-forming cells (64.1%-86.3%). Moreover, we detected a relevant depletion of the transplants for RBCs (89.2%-90.0%) and platelets ([PLTs] 77.5%-86.1%). In contrast, the mean depletion rate using BC processing proved to be significantly different for PLTs (10%, p = 0.03) and RBCs (39.6%, p < 0.01). The NC composition showed a highly significant increase in MNCs and a decrease in granulocytes after filtration (p < 0.01), compared with a less significant MNC increase in the BC group (p < 0.05). For mice transplanted with WB-derived progenitors, we observed a mean of 15.3% ± 15.5% of human CD45+ cells within the BM compared with 19.9% ± 16.8% for mice transplanted with filter samples (p = 0.03). The mean percentage of human CD34+ cells was 4.2% ± 3.1% for WB samples and 4.5% ± 3.2% for filter samples (p = 0.68). As the data of NOD/SCID mice transplantation demonstrated a significant engraftment capacity of HPCs processed by filtration, no negative effect on the engraftment potential of filtered UCB cells versus non-volume-reduced cells from WB transplants was found. The StemQuickE filter devices proved to be a useful tool for Good Manufacturing Practices conform enrichment of HPCs and MNCs out of UCB. Filtration enables a quick and standardized preparation of a volume-reduced UCB transplant, including a partial depletion of granulocytes, RBCs, and PLTs without the need for centrifugation. Therefore, it seems very probable that filter-processed UCB transplants will also result in sufficient hematopoietic reconstitution in humans.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. UCB Processing by BC Preparation
  6. UCB Processing by Filtration
  7. Cryopreservation Procedure of WB, BC, and Filter Samples
  8. Thawing of the Samples
  9. Cell Counting and Analysis by Flow Cytometry
  10. Hematopoietic Progenitor Cell Assays and Cell Viability
  11. Paired NOD/SCID Mice Transplantation Experiments
  12. Statistical Analysis
  13. Results
  14. Discussion
  15. Conclusion
  16. Acknowledgements
  17. References

The transplantation of umbilical cord blood (UCB) is increasingly recognized as an alternative for allogeneic transplantation with curative intent [1, 2]. To build up large-scale banks of unrelated UCB transplants for broader clinical use, techniques had to be established to minimize the volume of the collected samples in order to reduce the need for storage space in liquid nitrogen [3]. Because clinical data have shown that a low infused cell dose hurt the patient's outcome after transplantation [1, 2], investigators have focused on the problem of how to reduce the volume of UCB transplants without causing major loss of hematopoietic progenitor cells (HPCs), such as techniques for RBC depletion [47], hydroxyethyl starch (HES) sedimentation followed by centrifugation [8, 9], or separation by buffy coat (BC) preparation [1012]. At the Mannheim Cord Blood Bank, we established volume reduction by using a BC separation protocol [13]. Nevertheless, most of these recently described techniques require special equipment such as blood bag centrifuges or blood component extractors because, otherwise, the units cannot be processed under Good Manufacturing Practices (GMP) conditions in sterile closed systems. Therefore, methods for UCB volume reduction still need to be optimized in order to develop a simplified and less time- and equipment-consuming procedure. Finally, the reduction of contaminating cells within the frozen transplant, i.e., granulocytes and platelets (PLTs), is important to achieve a high quality final transplant.

We evaluated a novel white blood cell collection filter device for the processing of UCB (StemQuickE, Asahi Medical; Tokyo, Japan; http://www.asahi-kasei.co.jp/medical/en) [14, 15]. The study compared filtration with an established BC processing protocol to evaluate its feasibility for volume reduction and for the enrichment of HPCs. Additionally, the engraftment capacity of UCB progenitors processed either by whole blood (WB) processing or by StemQuickE filtration was investigated in a paired study using the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse transplantation model.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. UCB Processing by BC Preparation
  6. UCB Processing by Filtration
  7. Cryopreservation Procedure of WB, BC, and Filter Samples
  8. Thawing of the Samples
  9. Cell Counting and Analysis by Flow Cytometry
  10. Hematopoietic Progenitor Cell Assays and Cell Viability
  11. Paired NOD/SCID Mice Transplantation Experiments
  12. Statistical Analysis
  13. Results
  14. Discussion
  15. Conclusion
  16. Acknowledgements
  17. References

Study Design

In the first set of experiments, the feasibility of the StemQuickE filter system for processing of low-volume (n = 6) and high-volume pooled UCB units (n = 6) was determined by measuring the operation time for filtration and by calculating postprocessing cell recovery. Furthermore, we compared both BC and filter processing in parallel by using equally divided nonpooled UCB samples (n = 10). Additionally, the pre- and postprocessing nucleated cell (NC) composition was measured on 15 samples for both preparation methods.

Finally, 10 UCB units were divided, and each half of the split samples was processed either by WB preparation or by filtration. After freezing and thawing, CD34+ cells of each group were transplanted to NOD/SCID mice, and the animals were analyzed for human engraftment within their bone marrow (BM).

Collection of UCB

After informed consent of the mother was obtained, placental blood was collected from the unborn placenta by a well-trained team of gynecologists and midwives from the Department of Gynecology and Obstetrics at the Mannheim University Hospital. For collection and processing of the UCB, a triple-bag system with top/bottom configuration was used (Cord Blood Collection System, Eltest; Bonn, Germany; http://www.sarstedt.com), containing 17 ml of citrate/phosphate/dextrose in the collection bag. The units were stored and processed at room temperature within 48 hours of blood collection.

UCB Processing by BC Preparation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. UCB Processing by BC Preparation
  6. UCB Processing by Filtration
  7. Cryopreservation Procedure of WB, BC, and Filter Samples
  8. Thawing of the Samples
  9. Cell Counting and Analysis by Flow Cytometry
  10. Hematopoietic Progenitor Cell Assays and Cell Viability
  11. Paired NOD/SCID Mice Transplantation Experiments
  12. Statistical Analysis
  13. Results
  14. Discussion
  15. Conclusion
  16. Acknowledgements
  17. References

To reduce the volume of the UCB, we centrifuged the units at 3,500 × g for 15 minutes as described previously [13]. After centrifugation, the units were processed using a semiautomated separator (Compomat G4, Fresenius Hemocare; Bad Homburg, Germany; http://www.fresenius-hemocare.com) to transfer the RBCs and the plasma into the respective satellite bags, resulting in a final UCB volume within the collection bag of 25 ml [13].

UCB Processing by Filtration

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. UCB Processing by BC Preparation
  6. UCB Processing by Filtration
  7. Cryopreservation Procedure of WB, BC, and Filter Samples
  8. Thawing of the Samples
  9. Cell Counting and Analysis by Flow Cytometry
  10. Hematopoietic Progenitor Cell Assays and Cell Viability
  11. Paired NOD/SCID Mice Transplantation Experiments
  12. Statistical Analysis
  13. Results
  14. Discussion
  15. Conclusion
  16. Acknowledgements
  17. References

To process the UCB units by filtration, we used the novel StemQuickE filter device, a two-bag system with an integrated leukocyte filter for the enrichment of HPCs from UCB [14, 15]. In principle, the NCs are trapped within the filter during the filtration of the anticoagulated blood, while the RBCs and PLTs flow through the filter pores into a drain bag. In a second step, the trapped cells are eluted from the filter by flushing the fibers with an inverted flow, and the cells are washed out into the recovery bag of the system.

The system consists of a spike needle for connection to the primary bag containing the collected UCB, followed by a mesh chamber and the leukocyte filter (Fig. 1). One bag is used to collect the drained cells and the second bag contains the recovered NCs. After connection of the filter system with the primary UCB bag, the bag is hung 45 cm over the surface of the clean bench, and the UCB flows down through the filter fibers to the drain bag. After filtration, the cells within the filter are recovered by flushing 19 ml solution consisting of 3 ml human serum albumin (HSA) 20% (DRK-Blutspendedienst; Baden-Württemberg, Germany; http://www.blutspende.de) and 16 ml Dextran 40 (Longasteril, Fresenius Kabi; Friedberg, Germany; http://www.fresenius-kabi.com), along with 18 ml of sterile air rapidly through a port at the outlet of the filter to the recovery bag. For the elution procedure, a syringe filled with the recovery solution is pressed out at constant speed and force by a special recovery device.

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Figure Figure 1.. Diagram of the StemQuickE filter device, a two-bag system with an integrated leukocyte filter.It consists of a spike needle for connection to the UCB bag (A), an air vent filter (B) at the mesh chamber (C), filter (D), port for the application of flushing solution by the recovery device of the system (E), recovery bag (F), and drain bag (G). In addition, two clamps (1, 2) and one roller clamp (3) are attached to the tubes in order to open and close the lines during processing.

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Cryopreservation Procedure of WB, BC, and Filter Samples

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. UCB Processing by BC Preparation
  6. UCB Processing by Filtration
  7. Cryopreservation Procedure of WB, BC, and Filter Samples
  8. Thawing of the Samples
  9. Cell Counting and Analysis by Flow Cytometry
  10. Hematopoietic Progenitor Cell Assays and Cell Viability
  11. Paired NOD/SCID Mice Transplantation Experiments
  12. Statistical Analysis
  13. Results
  14. Discussion
  15. Conclusion
  16. Acknowledgements
  17. References

The cryoprotective solution for freezing the non-volume-reduced WB samples was prepared by mixing 32 ml of saline solution (Jonosteril; Fresenius Kabi), 8 ml of HSA 20%, and 10 ml of dimethyl sulfoxide ([DMSO] Cryoserv; WAK-Chemie, Bad Soden, Germany; http://www.wak-chemie.de) within a sterile transfer blood bag; the cryoprotective solution for BC samples was prepared out of autologous UCB plasma and DMSO. For both WB and BC samples, an aliquot of the specific cryoprotective solution was added to various sample volume, resulting in a concentration of 5.5% DMSO within the final cell supension.

After transferring the filtered cells to the freezing bag, the samples were cryopreserved by adding 3 ml of a 50% solution of DMSO (1.5 ml DMSO plus 1.5 ml saline solution) to the cell suspension that showed a mean volume of 22 ml. This resulted in a final concentration of 6% DMSO within the cryopreserved cell suspension. All the samples were frozen using a microprocessor-controlled cell freezer (Kryo 10/Serie III, Planer; Sunbury, UK; http://www.planer.co.uk) and were then stored in the vapor phase of liquid nitrogen for at least 48 hours before thawing.

Thawing of the Samples

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. UCB Processing by BC Preparation
  6. UCB Processing by Filtration
  7. Cryopreservation Procedure of WB, BC, and Filter Samples
  8. Thawing of the Samples
  9. Cell Counting and Analysis by Flow Cytometry
  10. Hematopoietic Progenitor Cell Assays and Cell Viability
  11. Paired NOD/SCID Mice Transplantation Experiments
  12. Statistical Analysis
  13. Results
  14. Discussion
  15. Conclusion
  16. Acknowledgements
  17. References

Both the BC and filter samples were thawed according to a modified technique described by Rubinstein et al. [8]. The samples were thawed quickly within a water bath at 37°C and diluted in an equal volume of washing solution (12.5% [vol/vol] HSA 20% plus 50% [vol/vol] Dextran 40 within saline solution). After centrifugation, the supernatant was removed and the cell pellet was resuspended in washing solution and analyzed immediately.

On the day of NOD/SCID mouse transplantation, both the WB and the filter-processed parts of the paired samples were thawed quickly at 37°C. To eliminate DMSO and dead cells sufficiently, the thawed samples were washed using the solution described above. Additionally, recombinant human DNase (Pulmozyme, Roche Pharmaceuticals; Grenzach-Wyhlen, Germany; http://www.roche.com) was added in a final concentration of 1% to avoid cell clotting [16].

Cell Counting and Analysis by Flow Cytometry

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. UCB Processing by BC Preparation
  6. UCB Processing by Filtration
  7. Cryopreservation Procedure of WB, BC, and Filter Samples
  8. Thawing of the Samples
  9. Cell Counting and Analysis by Flow Cytometry
  10. Hematopoietic Progenitor Cell Assays and Cell Viability
  11. Paired NOD/SCID Mice Transplantation Experiments
  12. Statistical Analysis
  13. Results
  14. Discussion
  15. Conclusion
  16. Acknowledgements
  17. References

Cell counts were performed with an automated cell analyzer (Cell-Dyn 3200, Abbott; Wiesbaden, Germany; http://abbott.stellenprofile.de). The volume of the samples was determined by weighing the blood bags. For NC subset analysis, the percentage of granulocytes, monocytes, and lymphocytes was quantified by flow cytometry with a FACSCalibur cytometer using Cell Quest software (Becton Dickinson; Heidelberg, Germany; http://www.bd.com). The NC subsets were analyzed either by their forward scatter (FSC) and side scatter (SSC) signal, or after staining by fluorescein isothiocyanate (FITC)-labeled anti-CD4, anti-CD8, anti-CD14, anti-CD19, anti-CD33, and peridin chlorophyll-a protein-conjugated anti-CD45, or mouse IgG isotype control antibodies. For CD34+ cell enumeration, the NCs were stained simultaneously with FITC-labeled anti-CD45 and phycoerythrin (PE)-conjugated anti-CD34 (HPCA-2, Becton Dickinson). CD34+ cells were determined by their FSC/SSC signals, followed by analysis of the CD45+ cells for their expression of the second PE-labeled signal.

For NOD/SCID mouse engraftment analysis, the BM samples were first incubated with 5% (vol/vol) mouse gamma globulin (Dianova; Hamburg, Germany; http:// www.dianova.org) to block the murine Fc receptor, followed by incubation with anti-human (ahu) CD45-PE and costaining with FITC-conjugated ahu CD34, CD33, CD19, CD3, and CD61, respectively (Dianova). Isotypic controls were performed with FITC- and PE-conjugated anti-mouse gamma globulin (anti-γ1-FITC and anti-γ1-PE; Becton Dickinson). NC subsets were determined by analyzing the cells for ahu CD45 expression together with the second FITC-labeled signal. Cells were considered positive if they were both positive for ahu CD45 and the corresponding second signal.

Hematopoietic Progenitor Cell Assays and Cell Viability

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. UCB Processing by BC Preparation
  6. UCB Processing by Filtration
  7. Cryopreservation Procedure of WB, BC, and Filter Samples
  8. Thawing of the Samples
  9. Cell Counting and Analysis by Flow Cytometry
  10. Hematopoietic Progenitor Cell Assays and Cell Viability
  11. Paired NOD/SCID Mice Transplantation Experiments
  12. Statistical Analysis
  13. Results
  14. Discussion
  15. Conclusion
  16. Acknowledgements
  17. References

For the identification and quantification of colony-forming units (CFUs), we performed progenitor cell assays using a commercial semisolid medium containing various recombinant human growth factors (MethoCult, StemCell Technologies; Vancouver, Canada; http://www.stemcell.com). Briefly, 1-10 × 105 NCs, or a fixed volume of mouse BM cell suspension, were incubated for 14 days on sterile plastic dishes in a 5% CO2 fully humidified atmosphere. The slides were scored by inverted microscopic examination for morphological quantification of total CFU. In the cases of testing mouse BM, we analyzed samples on the existence of visible colonies after the incubation period. In some experiments, cell viability assay was performed by tryphan-blue staining.

Paired NOD/SCID Mice Transplantation Experiments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. UCB Processing by BC Preparation
  6. UCB Processing by Filtration
  7. Cryopreservation Procedure of WB, BC, and Filter Samples
  8. Thawing of the Samples
  9. Cell Counting and Analysis by Flow Cytometry
  10. Hematopoietic Progenitor Cell Assays and Cell Viability
  11. Paired NOD/SCID Mice Transplantation Experiments
  12. Statistical Analysis
  13. Results
  14. Discussion
  15. Conclusion
  16. Acknowledgements
  17. References

In order to study the influence of the filtration procedure on the hematopoietic engraftment potential, paired NOD/SCID mice transplantation experiments were performed. Ten UCB units with a mean volume of 110.0 ± 10.5 ml containing 11.0 ± 2.4 × 108 NCs and 3.8 ± 1.6 × 106 CD34+ cells were divided into two samples of equal volume prior to processing, followed by WB preparation or by StemQuickE filtration. Both paired samples were cryopreserved as described above. After thawing and washing, the cells of the paired samples were resuspended in Iscove's modified Dulbecco's medium ([IMDM] Sigma Chemical; Irvine, UK; http://www.sigmaaldrich.com) and transplanted into NOD/SCID mice in parallel. The animals aged 4-8 weeks were maintained in a specialized animal facility; the care of the animals was in accordance with institutional guidelines. For one set of parallel transplantation experiments, each of five mice were injected with 2 × 104 CD34+ cells collected by filtration, and another five mice were transplanted with 2 × 104 CD34+ cells from the WB sample. One mouse per experiment was injected only with IMDM and served as negative control. Prior to transplantation, the mice were irradiated sublethally with 100 cGy, and genetically modified rat fibroblasts were cotransplanted in order to enhance the engraftment by producing human interleukin-3 [17]. Forty-nine days after transplantation, the mice were sacrificed and BM cells were obtained by flushing both femora with saline solution plus 5% (vol/vol) fetal calf serum for flow cytometric analysis.

Engraftment potential was calculated as the mean value of five animals transplanted with cells from the same sample. Engraftment was assumed if the percentage of human CD45+ cells in murine BM proved to be higher than in the corresponding control animal.

Statistical Analysis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. UCB Processing by BC Preparation
  6. UCB Processing by Filtration
  7. Cryopreservation Procedure of WB, BC, and Filter Samples
  8. Thawing of the Samples
  9. Cell Counting and Analysis by Flow Cytometry
  10. Hematopoietic Progenitor Cell Assays and Cell Viability
  11. Paired NOD/SCID Mice Transplantation Experiments
  12. Statistical Analysis
  13. Results
  14. Discussion
  15. Conclusion
  16. Acknowledgements
  17. References

The values represent the mean ± standard deviation (SD). Different groups of samples were compared using the t tests for paired samples by running computer software (SPSS; Munich, Germany; http://www.spss.com). To compare the cell recovery rates of paired BC and filter-processed samples, we performed the nonparametric Wilcoxon matched-pair rank test, and p values of <0.05 were considered to be statistically significant.

For each group of five mice transplanted with the same sample (WB or filter), the mean values of engraftment rate were calculated, represented by the percentage of human CD45+ cells in the mouse BM. Because the data of the engraftment rate showed to be normally distributed when tested by the Kolmogorov-Smirnow test, we compared the mean values of the paired groups by the parametric t tests for paired samples. The data were also evaluated by descriptive analysis.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. UCB Processing by BC Preparation
  6. UCB Processing by Filtration
  7. Cryopreservation Procedure of WB, BC, and Filter Samples
  8. Thawing of the Samples
  9. Cell Counting and Analysis by Flow Cytometry
  10. Hematopoietic Progenitor Cell Assays and Cell Viability
  11. Paired NOD/SCID Mice Transplantation Experiments
  12. Statistical Analysis
  13. Results
  14. Discussion
  15. Conclusion
  16. Acknowledgements
  17. References

UCB Processing with the StemQuickE Filter

The filter system was evaluated by comparison of six single UCB units (mean NC content, 9.7 × 108) and six pooled units (mean NC content, 23.8 × 108). The mean volumes of the single samples and pooled samples were 87.8 ± 24.0 ml and 120.7 ± 26.9 ml, respectively (Table 1). The processing time for the single units showed to be 476 ± 132 seconds (240-590 seconds), and 652 ± 147 seconds (465-870 seconds) for the pooled units. Thus, an identical mean filtration rate of 1 ml per 5.4 seconds was observed for lower-volume as well as higher-volume units.

Table Table 1.. Comparison of cell recovery rates in % (mean ± SD) of 12 UCB units after processing by using the StemQuickE filter device
  1. a

    Six samples were processed as single units (mean NC content, 9.7 × 108), and the other six as pooled units (NC content, 23.8 × 108).

 n samplesMean volume (ml)NCMNCCD34*cellCFURBCPLT
Single samples687.859.6 ± 10.377.5 ± 5.976.3 ± 10.486.3 ± 9.310.0 ± 9.313.9 ± 8.3
Pooled samples6120.763.0 ± 5.074.2 ± 6.479.0 ± 16.764.1 ± 15.210.8 ± 2.822.5 ± 5.5

The NC recovery was calculated as 59.6% ± 10.3% and 63.0% ± 5.0%, whereas the mean mononuclear cell (MNC) and CD34+ cell recovery ranged from 74.2%-77.5% and 76.3%-79.0%, respectively. With respect to CFU recovery, slightly poorer results were obtained in the pooled sample group, showing 86.3% ± 9.3% for single samples in contrast to 64.1% ± 15.2% for pooled samples. Contaminating cells such as RBCs and PLTs could be eliminated very effectively from the UCB units, resulting in a mean recovery rate of only 10.0%-10.8% for RBCs and 13.9%-22.5% for PLTs.

StemQuickE Filtration versus BC Processing

The standard BC technique was compared to the filtration method by processing 10 splitted UCB units in parallel (Table 2, Figs. 2A and 2B). The mean operation time for the filter procedure was 9.6 ± 3.1 minutes, whereas the overall BC preparation took 45 minutes, including centrifugation and processing at the blood separator.

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Figure Figure 2.. Comparison of mean MNC (A) and CD34+cell (B) recovery rates of nine paired UCB samples after filtration () or BC processing ().Pair 4 was excluded from analysis because BC recovery >120%. BC preparation showed higher values in eight out of nine samples.

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Table Table 2.. Comparison of cell recovery rates in % (mean ± SD) of 10 UCB units after StemQuickE filtration or BC processing
  1. a

    The samples were divided in two equal-sized aliquots with a mean volume of 62.4 ml prior to parallel processing by both techniques. The Wilcoxon-matched pair rank test was used to calculate significant differences between the two groups. NS = not significant.

 NCMNCCD34*cellCFURBCPLT
Filter62.3 ± 11.680.7 ± 12.678.9 ± 10.574.8 ± 39.820.1 ± 10.232.1 ± 10.9
Buffy coat115.3 ± 51.8125.5 ± 67.592.4 ± 32.9125.0 ± 68.860.4 ± 30.590.0 ± 87.1
p value<0.01<0.01NS<0.05<0.010.03

Calculating the recovery rates of the different cell types, we observed a higher variability between the samples within the BC group compared with the filter group. Therefore, the StemQuickE filtration procedure results in a more standardized final stem cell product. Nevertheless, in performing the nonparametric Wilcoxon test, significantly higher recovery rates within the LBC group compared with the filter group could be observed for NCs, MNCs, and RBCs (each p < 0.01), as well as for PLTs (p = 0.03) and CFUs (p < 0.05). In contrast, no difference for CD34+ cell recovery was calculated.

To determine the viability in the post-thawing cell suspension, the 10 splitted samples processed by both techniques were frozen. After thawing, filter samples showed a better mean viability of 99.4% compared with 93.1% for BC samples, although there was no difference between the two groups postprocessing (>98%).

NC Composition of BC and Filter Samples Preprocessing and Postprocessing

We addressed the question of whether a different NC composition could be observed before and after BC or filter processing. Therefore, seven units with a median volume of 98 ml were processed by BC preparation and eight units with a median volume of 91 ml by StemQuickE filtration (Table 3). The absolute median cell numbers for BC versus filter samples before processing were 20.5 × 108 versus 6.9 × 108 for NCs, 14.4 × 108 versus 3.6 × 108 for granulocytes, 4.6 × 108 versus 2.6 × 108 for lymphocytes, and 1.1 × 108 versus 0.6 × 108 for monocytes, respectively. After processing, the median cell count of BC versus filter units changed to 12.0 × 108 versus 4.3 × 108 for NCs, 7.2 × 108 versus 1.7 × 108 for granulocytes, 3.0 × 108 versus 2.0 × 108 for lymphocytes, and 0.9 × 108 versus 0.3 × 108 for monocytes, respectively.

Table Table 3.. NC composition (%) of preprocessing and postprocessing samples, processed either by BC preparation or by StemQuickE filtration (mean ± SD) of 7 to 8 experiments
  1. a

    Abbreviation: NS = not significant.

 Buffy coat (n = 7)StemQuickE(n = 8)
 PreprocessingPostprocessingp valuePreprocessingPostprocessingp value
Granulocytes65.2 ± 7.458.9 ± 6.7<0.0557.4 ± 8.646.6 ± 10.7<0.01
MNCs33.6 ± 7.340.2 ± 7.2<0.0541.5 ± 8.752.2 ± 11.0<0.01
CD4+ cells14.4 ± 6.516.1 ± 5.3NS11.5 ± 2.919.3 ± 4.5<0.01
CD8+ cells5.0 ± 2.26.1 ± 2.3NS6.5 ± 2.38.6 ± 3.0<0.05
CD14+ cells6.2 ± 3.06.8 ± 1.9NS6.7 ± 1.47.4 ± 1.2<0.05
CD19+ cells4.0 ± 1.74.8 ± 1.8NS5.2 ± 2.76.0 ± 2.8<0.05
CD33+ cells6.8 ± 2.97.7 ± 2.1NS5.6 ± 4.45.9 ± 4.9NS

A significant drop in the relative number of granulocytes (p < 0.01) in combination with a significant increase in the percentage of MNC, CD4+, CD8+, CD14+, and CD19+ cells (p < 0.01 to 0.05) was observed in the filter group. In contrast, the percentage of the CD33+ cell subset proved not to be significantly changed by filtration. The MNC subsets remained unchanged within the BC group, although a slightly significant increase for total MNCs and a decrease for granulocytes was observed (p < 0.05).

NOD/SCID Mice Engraftment Analysis of WB and Filter Units

Paired transplantation experiments (n = 10) were analyzed with respect to the rate of NOD/SCID mouse engraftment with human cells. Overall, 8 of 110 mice (7.3%) died before day 49 and could not be analyzed, three within the WB group and five within the filter group.

Figures 3A and 3B show the engraftment results for the two different groups of mice transplanted with the paired samples. For mice transplanted with WB-derived progenitors, we calculated a mean of 15.3% ± 15.5% of human CD45+ cells within the mouse BM compared with 19.9% ± 16.8% for mice transplanted with filter samples (p = 0.03; WB/filter pairs: 57.6%/60.1%, 18.4%/36.6%, 10.6%/17.6%, 6.9%/7.8%, 13.7%/12.8%, 4.3%/3.0%, 7.4%/10.1%, 14.0%/18.9%, 7.6%/12.1%, and 12.7%/20.2%). The mean percentage of human CD19+ cells showed to be 10.0% ± 10.9% for WB samples and 13.8% ± 12.8% for filter samples (p = 0.09), whereas the proportion of human CD34+ cells was calculated to be 4.2% ± 3.1% for WB samples and 4.5% ± 3.2% for filter samples (p = 0.68, Fig. 3B).

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Figure Figure 3.. Mean value and SD of 10 experiments for the engraftment rate (%) of human cells within mouse BM.A) Expressing markers CD45+and CD19+. The values for CD45+cells in the filter group and in the WB group were 19.9% ± 16.8% and 15.3% ± 15.5%, respectively (p = 0.03). The value for CD19+cells in the filter group was 13.8% ± 12.8% and 10.0% ± 10.9% in the WB group. B) Expressing markers CD34+, CD33+, CD3+, and CD61+. No significant difference between the filter group and the WB group could be calculated for the percentage of cells expressing these markers.

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Therefore, in 8 out of 10 experiments, a higher mean engraftment rate represented by the percentage of human CD45+ cells was detected for the group of mice transplanted with cells processed by the StemQuickE filter. Analyzing all 10 experiments, a slightly significant difference (p = 0.03) was observed between the two groups with respect to the overall engraftment rate. In contrast, no significant difference was detected for the percentage of human CD34+ cells or cells expressing CD19, CD33, CD3, or CD61.

We also analyzed the mouse BM for the presence of human CFUs, and all control animals showed a negative growth of human CFUs. In 5 out of 10 paired transplantation experiments, CFUs were grown in parallel from mice transplanted with WB and filter-derived cells, whereas in the other five experiments, no human CFUs could be demonstrated. There was no relation between the rate of bone marrow engraftment and CFU detection.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. UCB Processing by BC Preparation
  6. UCB Processing by Filtration
  7. Cryopreservation Procedure of WB, BC, and Filter Samples
  8. Thawing of the Samples
  9. Cell Counting and Analysis by Flow Cytometry
  10. Hematopoietic Progenitor Cell Assays and Cell Viability
  11. Paired NOD/SCID Mice Transplantation Experiments
  12. Statistical Analysis
  13. Results
  14. Discussion
  15. Conclusion
  16. Acknowledgements
  17. References

Clinical evidence indicates that CD34+ cells from UCB can be used as an alternative source of HPCs for transplantation [18]. Previously, a variety of methods for volume reduction of placental blood samples and for enrichment of HPCs have been developed in order to store large numbers of UCB specimens in liquid nitrogen containers [3]. Techniques using Percoll or Ficoll [6, 1923], gelatin [7], or immunoaffinity separation [24, 25] gave high rates of NC recovery, but they did not meet GMP standards because of open system processing.

Recently, methods for BC separation by centrifugation have been published that meet the criteria for large-scale preparation of UCB samples within a closed system [713]. These techniques involve either centrifugation with or without the addition of HES or gelatin as exogenous material for RBC sedimentation [7, 8, 10], or volume reduction by differential centrifugation followed by automated expression of RBCs and plasma [1113]. BC separation of UCB samples is very similar to well-established methods of WB processing in transfusion services and can easily be integrated into routine work of a blood bank [13]. However, in contrast to open system techniques, it requires specialized equipment such as multiple bag collection systems, blood bag centrifuges, and component separators. In addition, the single-step BC separation primarily results in a concentration of all NCs, including granulocytes, within a very small final freezing volume of 31-50 ml [11]. This could conceivably lead to negative effects after thawing of the cells, such as cell clotting by damaged granulocytes followed by the release of DNA, thus making DNase-containing processing solutions necessary [16, 26].

As an alternative for volume reduction, our study results clearly demonstrate that UCB units with a volume ranging from 62-121 ml can be easily processed by a new filtration technology using the StemQuickE filter device. The filter proved to be easy in handling, and the processing was performed by a technician inside a clean bench under GMP conditions. UCB processing by filtration proved to be less time consuming, with an operation time of only 4-14.5 minutes compared with a BC processing time of 45 minutes. The postprocessing volume for filter samples proved to be the same as for the BC samples. Sufficient recovery rates of MNCs, CD34+ cells, and CFUs were demonstrated in each single and pooled units, comparable to other methods of UCB volume reduction [8, 10]. Furthermore, the filtration method resulted in a highly significant increase in MNCs and a decrease in granulocytes, whereas BC processing resulted in a less significant enrichment of MNCs and reduction of granulocytic cells. Compared with BC preparation, a significant difference in the RBC and PLT depletion rate could be observed, with 67.9%-79.9% for filtered samples but only 10.0%-39.6% for BC-processed samples. The RBC depletion rate for BC samples was found to be in line with previously published data [12]. In addition, cell recovery values of the filter group showed much smaller variability than those for the BC group, reflecting a higher reproducibility and a more standardized blood component preparation. Nevertheless, NC, MNC, and CFU recovery proved to be significantly reduced for the filter group.

Similar recovery results testing the same filter system for processing a total of 12 UCB units were published by Yasutake et al. [14]. They reported recovery rates of lymphocytes, monocytes, and granulocytes of 79.5%, 79.8%, and 39.0%, with the rate of granulocytes being significantly lower than that of monocytes and lymphocytes (p ≤0.0001). The reduction of granulocytic cells and decreased contamination with PLTs and RBCs results in higher quality of the final transplant, and may lead to reduced occurrence of clumping of the thawed cell suspension [16].

Although results of cell recovery studies are quite valuable, the evaluation of cell function is of paramount interest. The NOD/SCID mouse transplant model offers one of the best approaches for investigating the in vivo repopulating ability of human HPCs [27]. Taking this into consideration, we performed paired NOD/SCID mice transplantation experiments with 10 divided UCB units, injecting the same dosage of CD34+ progenitors in each mouse in order to compare the engraftment capacity of both the WB and the filter-processing methods. For mice transplanted with WB-derived progenitors, we observed a mean of 15.3% human cells within the BM compared with 19.9% for mice transplanted with filter samples. In 8 out of 10 experiments, the engraftment rate of filter samples proved to be enhanced compared with the corresponding WB samples (p = 0.03). Therefore, a higher engraftment capacity of progenitors processed by StemQuickE filtration compared with WB preparation could be observed. This result might indicate an increased biological availability of filtered cells after transplantation because of lower content of contaminating cells within the transplant and, therefore, a smaller amount of released DNA after thawing [16]. In contrast, no significant difference was found in the percentage of human CD34+ cells or cells expressing CD19, CD3, CD33, or CD61. In conclusion, at least no negative effect on the biological potential of filtered cells versus non-volume-reduced cells from WB transplants could be observed. Tokushima et al. also reported results from paired NOD/SCID mice transplantation of three divided UCB units that were either processed by HES separation or by filtration [15]. A total of 1 × 106 or 5 × 106 NCs from thawed and washed units were transplanted. Although the interpretation of the data proved to be difficult because 22 of 42 mice (52.4%) died before analysis, no significant difference in the hematopoietic reconstitution pattern could be observed between the two groups of mice. The results of this and our study clearly indicate that filter-processed UCB samples have at least the same engraftment capacity as samples processed by WB preparation or by the standard HES method.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. UCB Processing by BC Preparation
  6. UCB Processing by Filtration
  7. Cryopreservation Procedure of WB, BC, and Filter Samples
  8. Thawing of the Samples
  9. Cell Counting and Analysis by Flow Cytometry
  10. Hematopoietic Progenitor Cell Assays and Cell Viability
  11. Paired NOD/SCID Mice Transplantation Experiments
  12. Statistical Analysis
  13. Results
  14. Discussion
  15. Conclusion
  16. Acknowledgements
  17. References

In summary, the processing of UCB by the StemQuickE filter device proved to be a useful technique resulting in high recovery rates for MNCs and HPCs and a significant depletion of contaminating cells. This closed system method enables quick and standardized preparation of volume-reduced UCB preparations without the need for centrifugation. Furthermore, data of a paired transplantation study using a NOD/SCID mouse animal model demonstrated a significant engraftment capacity of hematopoietic progenitors processed by filtration. No negative effect on the engraftment potential of filtered UCB cells versus non-volume-reduced UCB cells from WB transplants was observed. Therefore, it seems very probable that filter-processed UCB transplants will also result in sufficient hematopoietic reconstitution in humans.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. UCB Processing by BC Preparation
  6. UCB Processing by Filtration
  7. Cryopreservation Procedure of WB, BC, and Filter Samples
  8. Thawing of the Samples
  9. Cell Counting and Analysis by Flow Cytometry
  10. Hematopoietic Progenitor Cell Assays and Cell Viability
  11. Paired NOD/SCID Mice Transplantation Experiments
  12. Statistical Analysis
  13. Results
  14. Discussion
  15. Conclusion
  16. Acknowledgements
  17. References
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