Abstract
Background aims. Previous data have shown that the addition of docosahexanoic acid (DHA)/arachidonic acid (AA) has a beneficial effect on cytokine-mediated in vitro generation of megakaryocytes (MK) from umbilical cord blood (UCB).Cryopreservation forms an inherent part of UCB banking and MK progenitors are known to be very sensitive to the stresses of freezing. It is therefore imperative to generate functional cells from cryopreserved cells, and the generated cells need to be cryopreserved until used. In the present study, cryopreservation of ex vivo-expanded MK as well as MK generation from cryopreserved UCB samples was investigated. Methods. MK generated with or without DHA/AA were cryopreserved in freezing medium containing 10% dimethyl sulfoxide (DMSO). Freezing efficacy was tested by quantitating MK after revival. Cryopreserved CD34+ cells were cultured with stem cell factor (SCF) and thrombopoietin (TPO), in the presence and absence of DHA/AA for 10 days, and then quantitated for MK. Results. We observed a 1.5–3-fold increase in MK numbers, their progenitor content and their expression of phenotypic markers and MK-related transcription factors. DHA/AA sets showed a 2–5-fold improved engraftment in NOD/SCID mice. These data showed that the beneficial effect of DHA/AA obtained during MK expansion was not altered after freezing stress. The enhancement in MK generation obtained from fresh cord blood (CB) cells was reproduced with comparable efficiency when we used cryopreserved CB samples. Conclusions. Taken together, our data suggest that in vitro-generated DHA/AA MK survive cryoinjuries in a functionally better state. DHA/AA support a more efficient generation of MK from cryopreserved UCB.
Acknowledgments
The authors thank Drs Prakash Daithankar, Arvind Sangamnerkar Girish Godbole and Veena Todkar for providing CB samples. We thank all the core facilities of NCCS, such as the flow cytometry, confocal microscopy and animal experimentation sections.
This work was supported by a project grant from Department of Biotechnology (DBT), Government of India. Namrata Shabrani is a Senior Research Fellow of Indian Council of Medical Research (ICMR), India.
Author contributions: Namrata Shabrani, collection of data, data analysis and interpretation, manuscript writing; Nikhat Firdaus Q. Khan, collection and analysis of murine data; Vaijayanti P. Kale, data analysis and interpretation, manuscript writing and co-investigator in the project; Lalita S. Limaye, conception and design, data analysis and interpretation, manuscript writing and principal investigator in the project.
Authorship and disclosures: The authors reported no potential conflicts of interest.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.