獣医科学と技術

In vitro Effects of Canine Wharton’s Jelly Mesenchymal Stromal Cells and Micellar Nanoparticles on Canine Osteosarcoma D17 Cell Viability

Abstract

Mary Lynn Higginbotham, Dudley L.McCaw,Raja Rachakatla,Gwi Moon Seo,Marla Pyle, Dery,L.Troyer,Carla L.Goad and Kimberly B.Reeds

Objectives: To isolate and maintain canine Wharton’s jelly mesenchymal stromal cells (WJMSC) in culture, determine the effects of micellar nanoparticles containing doxorubicin (DOX) on WJMSC and canine osteosarcoma (OSA) D17 cells, and determine the effects of WJMSC loaded with micellar nanoparticles containing DOX on OSA D17 cell viability.

Procedures: Stromal cells were isolated from canine umbilical cords. Micellar nanoparticles containing DOX were prepared and added to individual culture plates containing canine WJMSC and OSA D17 cells to determine DOX in micelles (DOX-M) effects on cell growth and viability. Conditioned media (CM) from culture plates containing canine WJMSC incubated with various DOX-M concentrations was added to OSA D17 cells. An MTT assay was performed to assess osteosarcoma D17 cell viability. A trypan blue stain was utilized to perform cell counts to determine the effect of the DOX-M on WJMSC growth.

Results: WJMSC were successfully isolated and maintained in culture. Micellar nanoparticles containing DOX decreased viability of OSA D17 cells. Osteosarcoma D17 cell viability decreased following incubation with CM obtained from WJMSC loaded with DOX-M. Significant decreases in OSA D17 cell viability were observed after incubation with the CM of canine WJMSC loaded with 10 μM DOX-M at 96 hours (p=0.0037). Significant decreases were also observed with the CM from WJMSC loaded with 1 μM DOX-M at 96 hours (p=0.0222). WJMSC numbers decreased in a dose dependent manner following incubation with DOX-M. The decrease in WJMSC number was not secondary to cytotoxicity as all variables produced similar percentages of dead WJMSC.

Conclusions: Canine WJMSC can be isolated and maintained in culture. DOX-M produces OSA D17 cytotoxicity and slows proliferation of canine WJMSC. WJMSC containing DOX-M cause OSA D17 cell cytotoxicity. These data support in vivo experiments utilizing canine WJMSC and micellar nanoparticles.