<abstract xmlns="http://www.w3.org/1999/xhtml">This study was initiated in order to test a mini-invasive method of mesenchymal stem/progenitor cells (MS/PCs) isolation from a rat bone marrow (BM), and subsequently their expansion, differentiation, and evaluation of their immunophenotypic characteristics; and later their preservation as donor cells in an optimal condition for potential autotransplantation. The study group comprised of 6 adult male Sprague-Dawley (S-D) rats, weighing 480—690 g. The rats were anaesthetised by isoflurane with room air in a Plexiglas box and maintained by inhalation of a mixture of isoflurane and O2. Their femurs were surgically exposed and their diaphyses double-trephined. Then BM cells were flushed out by saline with heparin and aspirated into a syringe with a solution of DMEM (Dulbecco’s modified eagle’s medium) and heparin. The mononuclear cells from the BM were isolated by centrifugation and expanded in a standard culture medium supplemented with ES-FBS (es-cell-qualified foetal bovine serum), L-glutamine and rh LIF (recombinant human leukemia inhibitory factor). Following 14 days of passaging cultures, the cells were split into 2 equal parts. The first culture continued with the original medium. The second culture received additional supplementation with a human FGFβ (fibroblast growth factor beta) and EGF (epidermal growth factor). The populations of these cells were analysed by light-microscopy, then the mean fluorescence intensities (MFIs) of CD90 and Nestin were evaluated by a tricolour flow cytometry using monoclonal antibodies. The type of general anaesthesia used proved to be appropriate for the surgical phase of the experiments. All rats survived the harvesting of the BM without complications. The total number of mononuclear cells was 1.5—4.0 × 106 per sample and the proportion of CD90/Nestin expressing cells was &lt; 1 %. Following 14 days of expansion, the cells became larger, adherent, with fibrillary morphology; the proportion of cells expressing CD90/Nestin increased to almost 25 %, i. e. they earned basic phenotypic characteristics of MSCs. Throughout the further cultivation a gradual decrease of the CD90/Nestin expression occurred. This suggested that the suitability of rat bone marrow derived MS/PCs for replacement therapy would probably be the highest between days 12—15 of cultivation and then would diminish.</abstract>

This study was initiated in order to test a mini-invasive method of mesenchymal stem/progenitor cells (MS/PCs) isolation from a rat bone marrow (BM), and subsequently their expansion, differentiation, and evaluation of their immunophenotypic characteristics; and later their preservation as donor cells in an optimal condition for potential autotransplantation. The study group comprised of 6 adult male Sprague-Dawley (S-D) rats, weighing 480—690 g. The rats were anaesthetised by isoflurane with room air in a Plexiglas box and maintained by inhalation of a mixture of isoflurane and O2. Their femurs were surgically exposed and their diaphyses double-trephined. Then BM cells were flushed out by saline with heparin and aspirated into a syringe with a solution of DMEM (Dulbecco’s modified eagle’s medium) and heparin. The mononuclear cells from the BM were isolated by centrifugation and expanded in a standard culture medium supplemented with ES-FBS (es-cell-qualified foetal bovine serum), L-glutamine and rh LIF (recombinant human leukemia inhibitory factor). Following 14 days of passaging cultures, the cells were split into 2 equal parts. The first culture continued with the original medium. The second culture received additional supplementation with a human FGFβ (fibroblast growth factor beta) and EGF (epidermal growth factor). The populations of these cells were analysed by light-microscopy, then the mean fluorescence intensities (MFIs) of CD90 and Nestin were evaluated by a tricolour flow cytometry using monoclonal antibodies. The type of general anaesthesia used proved to be appropriate for the surgical phase of the experiments. All rats survived the harvesting of the BM without complications. The total number of mononuclear cells was 1.5—4.0 × 106 per sample and the proportion of CD90/Nestin expressing cells was < 1 %. Following 14 days of expansion, the cells became larger, adherent, with fibrillary morphology; the proportion of cells expressing CD90/Nestin increased to almost 25 %, i. e. they earned basic phenotypic characteristics of MSCs. Throughout the further cultivation a gradual decrease of the CD90/Nestin expression occurred. This suggested that the suitability of rat bone marrow derived MS/PCs for replacement therapy would probably be the highest between days 12—15 of cultivation and then would diminish.

Acquisition and Expansion of Adult Rat Bone Marrow Multipotent Mesenchymal Stromal Cells

Department of Anatomy, Histology and Physiology, University of Veterinary Medicine and Pharmacy, Komenského 73, 041 81 Košice,

Department of Neurosurgery, P. J. Šafárik University, Faculty of Medicine, Tr. SNP 1, 040 11 Košice,

Department of Pharmacology, P. J. Šafárik University, Faculty of Medicine, Tr. SNP 1, 040 11 Košice,

Folia Veterinaria

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

This study was initiated in order to test a mini-invasive method of mesenchymal stem/progenitor cells (MS/PCs) isolation from a rat bone marrow (BM), and...

Acquisition and Expansion of Adult Rat Bone Marrow Multipotent Mesenchymal Stromal Cells

Published Online: Apr 04, 2017

Page range: 15 - 22

Received: Oct 26, 2016

Accepted: Dec 13, 2016

DOI: https://doi.org/10.1515/fv-2017-0003

Keywords
adult rat, bone marrow, expansion, isolation, multipotent cells

© 2017 Šulla, I. et al., published by De Gruyter Open

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Acquisition and Expansion of Adult Rat Bone Marrow Multipotent Mesenchymal Stromal Cells

Published Online: Apr 04, 2017

Page range: 15 - 22

Received: Oct 26, 2016

Accepted: Dec 13, 2016

DOI: https://doi.org/10.1515/fv-2017-0003

Keywordsadult rat, bone marrow, expansion, isolation, multipotent cells

© 2017 Šulla, I. et al., published by De Gruyter Open

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Keywords
adult rat, bone marrow, expansion, isolation, multipotent cells