Reproduction, Fertility and Development | VOL. 28
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227 MESENCHYMAL STEM CELL ISOLATION AND CULTURE FROM ADIPOSE TISSUE OF A DEAD DOG

Publication Date Jan 1, 2016

Abstract

Isolation of cells or stem cells from clinically dead animals may serve applications such as revival of the animal through somatic cell nuclear transfer (SCNT) or cryopreservation of their cells for a long period so that cells can be used in the future. Thus, combining isolation of cells from clinically dead animals and SCNT of germplasm of elite animals could benefit research into endangered or extinct species. In the present study, we tried to isolate and culture adipose-derived mesenchymal stem cells (ADSC) from a clinically dead dog. Adipose tissues were collected surgically from the abdomen of a dead dog after 3 h and processed tissues within 10 h of death. The isolated tissues were washed in 70% ethanol for 30 s and washed 5 times in Dulbecco’s PBS supplemented with 50 µg mL–1 gentamicin. These fat tissues were minced to very small pieces and washed in DMEM by centrifugation at 800 rpm for 3 min. The tissue pellet was subjected to enzymatic digestion (collagenase 1 mg mL–1 of Dulbecco’s PBS) at 37°C in CO2 incubator for 1 h, with intermittent shaking after every 10 min. The digestive enzyme was inactivated by equal volume of DMEM/F-12 supplemented with fetal bovine serum (20%) and centrifuged at 1000 rpm for 10 min. The pellet was resuspended in DMEM/F-12 with 10% fetal bovine serum and cultured at 1 × 106 cells mL–1 in 25-cm2 tissue culture flasks. The medium was changed after every 48 h. Mesenchymal stem cells (MSC) were observed under an inverted microscope after 6 days. These cells were subcultured and a confluen...

Concepts

Somatic Cell Nuclear Transfer Mesenchymal Stem Cells Adipose-derived Mesenchymal Stem Cells Cryogenic Vial Fetal Bovine Serum Characterisation Of Mesenchymal Stem Cells Intermittent Shaking Tissue Pellet Dead Dog Automatic Cell Counter

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