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Automated washing of long-term cryopreserved peripheral blood stem cells promotes cell viability and preserves CD34+ cell numbers

Peripheral blood stem cell (PBSC) transplantation has become an established treatment option for a range of malignant and inherited diseases. PBSCs are usually cryopreserved in the presence of dimethyl sulfoxide (DMSO) and stored in liquid nitrogen. However, cryopreservation and thawing of PBSCs may affect cell viability, resulting in delayed engraftment and other risks related to low stem cell numbers [1]. DMSO is commonly used at a concentration of 10% during freezing of PBSCs. However, DMSO itself is toxic and lowering doses of DMSO may have a favorable effect on hematopoietic recovery after transplantation [2, 3]. Generally, PBSCs are thawed and infused without removal of DMSO. This has been associated with a wide range of adverse effects (AEs), ranging from minor to severe life-threatening events [4, 5]. Although not all toxic events can be contributed to DMSO, grafts containing lower concentrations of DMSO typically display a reduced incidence of AEs [5, 6]. To decrease AEs and improve graft quality after thawing, it has been suggested to remove DMSO prior to transplantation [4, 7] using different washing systems [8, 9]. Here, we aimed to remove DMSO from long-term cryopreserved PBSCs using an automated, fully closed system and assessed effects on CD34+ stem cells, viability, and colony-forming capacity.

PBSCs were collected from patients and healthy donors (n = 12), aged 46.8 ± 12.9 years, according to the standardized procedures. Ethical permission no. GO15/352-I8 was obtained from the internal Ethical Review Board of the Hacettepe University. PBSCs were collected using Fenwal Amicus (Fresenius Kabi, IL, USA) in a total volume of 60 mL, consisting of 30% cells and 70% plasma and Heparin (H3393, Sigma-Aldrich). Cells were frozen in 10% DMSO and stored for 3.9 ± 2.0 years in liquid nitrogen. Stem cell collection bags (OriGen Biomedical, Cryostore CS500CN, TX, USA) were rapidly thawed and placed on ice. To remove DMSO, the cell suspensions were washed with a fully automated, closed system (Biosafe, Sepax2, Switzerland) using 500 mL Voluven™ (Pfizer, USA), while maintaining the volume at 60 mL.

Directly after thawing of the PBSCs and after washing, cells were counted and cell viability was assessed using 0.4% Trypan Blue and Acridine Orange. Cells were stained with anti-CD34 (clone 561, BioLegend) and CD45-PE (clone HI30, BioLegend). To assess apoptosis, cells were stained with Annexin-V-FITC and propidium iodide (BioLegend). Events were acquired using a FACSARIA (BD Biosciences, San Jose, CA, USA) and analyzed using FACSDIVA v8.0.1 software. Colony-forming capacity was tested using MethoCultTM H4434 Classic (Stem Cell Technologies, Vancouver, Canada). Statistical analyses were done using Microsoft® Excel® for Mac 2011, version 14.7.3. For paired analysis of groups Student’s t-test was used.

Total cell counts before freezing of PBSCs was 1.38 × 1010 ± 0.32 × 1010 cells (n = 12), after thawing 0.99 × 1010 ± 0.32 × 1010 cells, and after washing 0.78 × 1010 ± 0.23 × 1010 cells. Washing of PBSCs resulted in minimal cell loss, with recovery of 80.7% of total cells in comparison to pre-washing cell numbers (Fig. 1a). Cell viability was significantly increased (p < 0.01) after washing with 61.0 ± 13.9% viable cells before and 75.2 ± 9.7% after washing (Fig. 1b). Apoptosis was significantly decreased (p < 0.05) after washing of PBSCs (Fig. 1c). Therefore, removal of DMSO and dead cells using automated washing resulted in minimal cell loss and increased cellular viability.

Fig. 1

Effect of thawing and washing of PBSCs. a Effect of washing on number of white blood cells (WBCs) per apheresis bag. b Cell viability after thawing and washing of PBSCs. c Percentage of apoptotic cells before and after washing of PBSCs. Average ± standard deviations (n = 12) are depicted. *p < 0.05; **p < 0.01; ***p < 0.001

The absolute number of CD34+ cells directly after thawing was 162.7 × 106 ± 81.8 × 106 and after washing 162.5 × 106 ± 87.2 × 106, showing that washing resulted in depletion of non-viable CD34-negative cells and maximal recovery of viable CD34+ cells. The frequency of CD34high cells was relatively increased (Figs. 2a,b), whereas colony numbers (both BFU-E and CFU-GM) per 104 plated cells (p < 0.01) and per 103 CD34+ cells (p < 0.01) were relatively low after washing (Fig. 2c). This is consistent with the fact that CD34high cells have a higher long-term repopulation potential, are less differentiated, and therefore form fewer colonies in culture.

Fig. 2

Frequency of CD34+ cells and CFUs before and after washing. a Representative FACS plots from samples 09 and 11 before and after washing with on the x-axis CD45 and on the y-axis CD34. b Relative frequency of CD34high cells is increased after washing. c Colony-forming units before and after washing of PBSC grafts (n = 12). CFU-GEMM colony-forming unit—granulocyte/erythroid/monocyte/macrophage, CFU-GM colony-forming unit—granulocyte/monocyte, BFU-E burst-forming unit—erythroblast. *p < 0.01

Here, the Sepax-2 system was used to remove DMSO and increase cell viability of the PBSC grafts. Washed cells showed increased viability in comparison to unprocessed PBSCs and contained increased levels of CD34high cells. Manual washing with a simple saline/citrate solution can be used to deplete DMSO without significant loss of CD34+ cells, but with sustained engraftment potential, the downsides of manual washing include cell clumping, cell activation, and contamination, in addition to being time-consuming and labor-intensive [7]. For automated washing, the Sepax S-100 (Biosafe) [8, 10], Sepax-2 (Biosafe) [9], CytomateTM (Baxter Oncology) [11], and Cobe 2991 Cell Processor (Terumo Medical Corporation) [12] have all shown to allow rapid processing and good recovery of viable CD34+ cells. Importantly, transplantation of washed cells resulted in similar engraftment characteristics, although with considerably less and lower-grade AEs [8, 10]. The Sepax-2 system was previously shown to eliminate >95% of the DMSO in the PBSC products and support a post-wash recovery of 80% [9]. Using this system, we found that post-wash cell viability was significantly enhanced, and virtually all CD34 cells present in the grafts were recovered.

In conclusion, washing of long-term frozen PBSC grafts using a fully automated, closed system results in high recovery of viable CD34+ cells. However, the effect on AEs after infusion and hematopoietic recovery, long-term repopulation potential of washed PBSCs, and the cost effectiveness of the procedure remain to be explored in a clinical setting.


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This study was supported by a grant from the Hacettepe University Scientific Research Program Coordination Unit, project number EOZ_2016_7263.

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Correspondence to Fatima Aerts-Kaya.

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Aerts-Kaya, F., Koca, G., Sharafi, P. et al. Automated washing of long-term cryopreserved peripheral blood stem cells promotes cell viability and preserves CD34+ cell numbers. Bone Marrow Transplant 53, 1225–1227 (2018).

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