227 MESENCHYMAL STEM CELL ISOLATION AND CULTURE FROM ADIPOSE TISSUE OF A DEAD DOG

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 confluent monolayer was obtained. We have already standardized the protocol of MSC culture and characterisation as we are treating wounded and paralysed dogs using these MSC in a pet clinic. Characterisation of MSC was performed with specific surface marker genes of CD44, CD29, and CD166 in PCR and by immunocytochemistry of MSC-specific marker of CD44. Differentiation of these MSC into osteogenesis and chondrogenesis were observed after 3 weeks. Chondrogenic differentiation was confirmed by positive expression of chondrocyte-specific marker genes Aggrecan F-TTGGACTTTGGCAGAATACC and R-CTTCCACCAATGTCGTATCC and Collagen II F-AACCCTGGAACTGACGGAAT and R-CTCACCCGTTTGACCTTTCG primer in PCR. The MSC were cryopreserved after 80% confluency was reached. The monolayer cells were scraped out from the culture flask and pelleted down. The pellet was resuspended in DMEM containing 10% DMSO and 20% fetal bovine serum. The number of cells was determined by trypan blue staining using an automatic cell counter and 10⁵ cells mL^–1 were added to a 2-mL cryogenic vial. The cryogenic vials were kept in a cryobox at –80°C for slow cooling. Then these vials were transferred to liquid nitrogen tanks after 12 h for long-term storage. We conclude that ADSC were successfully cultured from adipose tissue of a dog within 10 h of death and further subcultured under in vitro conditions. The cells could be used for SCNT to revive the dead animal and cryopreserve these cells for use in the future.