Cell separation and cryopreservation of cord blood fractions for immunotherapeutic applications

In 2008 Anthony Nolan opened the UK’s first public umbilical cord blood (CB) bank (Anthony Nolan Cell Therapy Centre, ANCTC) in which CB is stored for haematopoietic stem cell (HSC) transplantation. Due to strict quality thresholds, the majority of units are not suitable for transplantation. Therefore, ANCTC aims to create a Biobank allowing these units to be stored for other purposes. To ensure cell products retained high levels of viability and potency, this study optimised banking processes starting with assessing the effects of transport conditions. It is essential that units are of the highest possible quality upon arrival at ANCTC, yet there is no consensus as to the optimal transport conditions of HSCs. Therefore, different fresh storage temperatures and the effect of delaying cryopreservation was assessed on three sources of HSCs. Cells were found to be better maintained at refrigerated temperatures and to avoid significant losses in potency, delays in cryopreservation should be minimised to <24 hours for bone marrow and <48 hours for CB and mobilised peripheral blood stem cells. Based upon these observations, ANCTC maintains all fresh samples at refrigerated temperatures and aims to cryopreserve them within 24 hours of collection. Banking cells involves cryopreservation and potentially long term storage of samples, however, suboptimal cryopreservation conditions can result in reduced cell viability. Cryoprotectants are used to reduce damage during the freeze and thaw stages of cryopreservation but they have been linked to toxic effects observed in cells. Dimethyl sulphoxide (DMSO) was found to exhibit a dose-dependent toxic effect on CB and optimal concentrations were found to be between 7.5% and 10% (v/v). This study also highlights the importance of minimising exposure to DMSO to <1hour prior to freezing and <30 minutes post-thaw. In addition, the presence of 1% (w/v) dextran-40 in the cryoprotective solution was found to be crucial to maintaining cell potency. The Biobank would require the storage of specific pure cell populations. Tregs are vital for the homeostasis of the immune system and have the potential to be used therapeutically in autoimmunity or transplantation, thus making these cells an ideal candidate for the Biobank. CB Tregs were found to have reduced suppressive abilities compared to their adult counterparts, due to lower levels of FoxP3 intensity and CD39 expression. However, their higher frequency and a more defined CD25+ population facilitates the isolation process. Two banking strategies were assessed, using both research and GMP grade methods. Firstly banking an isolated pure Treg population which, post-thaw, the cells maintained their phenotype but viability and suppressive ability was reduced. Alternatively, mononuclear cells were banked and Tregs isolated post-thaw. This strategy resulted in comparable isolation yields and purities compared to fresh cells, however, improved viabilities and higher suppressive abilities were observed compared to the cryopreserved pure Treg samples (p=0.0012). Therefore, this banking strategy was found to be more efficient. Overall, this study has optimised banking procedures from the transport of fresh samples to isolation and cryopreservation of pure cell populations. Therefore, this study has laid the foundations for the creation of a Biobank within the ANCTC allowing the distribution of cell products not only for research purposes, but also potentially for their use in immunotherapeutic interventions.