JARB Journal of Animal Reproduction and Biotehnology

OPEN ACCESS pISSN: 2671-4639
eISSN: 2671-4663

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Journal of Embryo Transfer 2012; 27(3): 183-187

Published online September 30, 2012

Copyright © The Korean Society of Animal Reproduction and Biotechnology.

Activation of Caspase-3 and -7 on Porcine Bone Marrow Derived Mesenchymal Stem Cells (pBM-MSCs) Cryopreserved with Dimethyl Sulfoxide (DMSO)

옥선아, 노규진

국립축산과학원 바이오공학과, 국립경상대학교 수의과대학 수의산과학연구실

Abstract

Adult stem cell transplantation has been increased every year, because of the lack of organ donors for regenerative medicine. Therefore, development of reliable and safety cryopreservation and bio-baking method for stem cell therapy is urgently needed. The present study investigated safety of dimethyl sulfoxide (DMSO) such as common cryoprotectant on porcine bone marrow derived mesenchymal stem cells (pBM-MSCs) by evaluating the activation of Caspase-3 and -7, apoptosis related important signal pathway. pBM-MSCs used for the present study were isolated density gradient method by Ficoll-Paque Plus and cultured in A-DMEM supplemented 10% FBS at $38.5^{circ}C$ in 5% $CO_2$ incubator. pBM-MSCs were cryopreserved in A-DMEM supplemented either with 5%, 10% or 20% DMSO by cooling rate at $-1^{circ}C$/min in a Kryo 360 (planner 300, Middlesex, UK) and kept into $LN_2$. Survival rate of cells after thawing did not differ between 5% and 10% DMSO but was lowest in 20% DMSO by 0.4% trypan blue exclusion. Activation of Caspase-3 and -7 by Vybrant FAM Caspase-3 and -7 Assay Assay Kit (Molecular probes, Inc.OR, USA) was analyzed with a flow cytometer. Both of cryopreserved and control groups (fresh pBM-MSCs) were observed after the activation of Caspase-3 and -7. The activation did not differ between 5% and 10% DMSO, but was observed highest in 20% DMSO. Therefore 5% DMSO can be possibly used for cell cryopreservation instead of 10% DMSO.

Keywords: porcine, mesenchymal stem cells, apoptosis, cryopreservation, DMSO, caspase-3 and -7