Evaluating A Novel Method For The Cryopreservation Of Human Haematopoietic Stem Cells

Introduction. Haematopoietic stem cells (HSCs) are multipotent cells widely used for the treatment of haematological diseases. Due to the prolonged time between their extraction and use, cryopreservation is necessary. The currently used cryoprotectant dimethyl sulfoxide (DMSO) leads to low cell viability and severe adverse effects in patients (1). A recent study showed that trehalose (T), a non-toxic disaccharide, in combination with PP50 (P), an amphipathic polymer, is effective in the cryopreservation of human nucleated cell lines (2).

Hypothesis and aims. I hypothesized that trehalose in combination with PP50 (TP) is more effective and less toxic than DMSO for the cryopreservation of HSCs. My aims were to: 1) Assess the efficacy of TP, and 2) Optimize the conditions for the cryopreservation of HSCs with TP.

Methods and Results. HSCs were isolated from the bone marrow and spleen of deceased human organ donors with informed consent and cryopreserved with DMSO, T or TP. The post-thaw viability and percentages of HSCs was assessed using flow cytometry and showed no significant difference between the conditions. Two parameters were optimized: the incubation time with the cryoprotectants and the cell concentration. An incubation time of 1 h was associated with an increase in the viability of splenocytes cryopreserved with TP, whereas the 3 h incubation was toxic. With TP, using 108 cells/ml was associated with greater viability than 106 and 107 cells/ml. Colony forming unit (CFU) assays were performed to analyse the function of HSCs in vitro. A similar number of colonies were formed with TP and DMSO. The number of colonies was higher when using HSCs isolated from younger donors compared to older donors.

Conclusions. TP might be as effective as DMSO in the cryopreservation of HSCs. The optimal conditions for the cryopreservation with TP appear to be 1 h incubation time and 108 cells/ml. HSCs cryopreserved with TP can form colonies of all the blood cell lineages and donor age may have an impact on the function of HSCs.

References:

1. Shu Z, Heimfeld S and Gao D. (2014). Haematopoietic SCT with cryopreserved grafts: adverse reactions after transplantation and cryoprotectant removal before infusion. Bone Marrow Transplantation. 49: 469-476.

2. Sharp DM et al. (2013). Amphipathic polymer-mediated uptake of trehalose for dimethyl sulfoxide-free human cell cryopreservation. Cryobiology. 67:305-11.

Key words: HSC, DMSO, trehalose, PP50, cryopreservation