Background: It has been suggested that mesenchymal stem cells (MSCs) have immunomodulatory effects through interaction with immune system cells. We aimed to define a suitable condition for co-culture of equine adipose-derived MSCs and neutrophils. Materials and Methods: Frozen equine adipose-derived MSCs (AMSCs) were thawed and their purity and characteristics were confirmed at passage 5. Equine Polymorphonuclear neutrophils (PMN) were isolated by density and hypotonic lysis. Cells were resuspended and neutrophil suspensions were perfused over adherent AMSC (at AMSC to neutrophil ratios of 1:50) in direct contact for 24 h in 12-well plates. After that, probability of pure isolation of AMSCs and neutrophils, and cell viability were checked. Results: AMSCs showed typical characteristics of MSC such as marker expression (CD29, CD90 and CD44) as well as trilineage differentiation potentials. Neutrophils were not affected by isolation procedure and their normal morphology was confirmed under light microscope. AMSCs strictly adhere to the plates whereas neutrophils were in suspended status during 24 h of co-culture. At the end, supernatants on plate wells containing neutrophils were removed and purified neutrophils were isolated. In next step, pure and alive adherent AMSC was collected using trypsin. Conclusion: We conclude that MSC can co-culture easily with neutrophils and this culture system can be used for studying interactions between MSc and Neutrophils in vitro.

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