Objective To build the models of viral transfection and hypoxia-reoxygenation induced cellular injury in AMCMs which are isolated by a non-Langendorff method. Methods AMCMs were isolated, extracted, sedimented and wall-plated by a non-Langendorff method. The morphology and survival rate of isolated cells was evaluated at 2 h, 24 h, 48 h and 72 h after wall-plating. The isolated AMCMs were infected with adenoviruses carrying RFP-expressing vector. Fluorescence photographs were taken at 36 h and 48 h post infection and used for calculation of transfection efficiency. The cells were cultured under hypoxic for 45 min and reoxygenated for 24 h. The cells were then stained with propidium iodide (PI) to verify the establishment of hypoxia-reoxygenation injury model. Results the survival rate of AMCMs at 2 h, 24 h and 48 h after plating were comparable, whereas this number at 72 h was significantly reduced. More than 80% of cells were transfected with adenovirus at 48 h. The hypoxia-reoxygenation treatment induced 42% of cells stained by PI, suggesting successful establishment of AMCMs injury model. Conclusions In this study, we developed a non-Langendorff method for quickly and easily isolating AMCMs with high cell viability. The isolated cells can be efficiently infected with adenovirus and are response to hypoxia-reoxygenation injury. Our findings provide a systematic method for isolating AMCMs as well as gene modification and hypoxia-reoxygenation injury in these cells.