To prepare the sample for perfusion culture using the 3D-printed bioreactor. In a laminar flow cabinet, place the isolated porcine carotid artery samples in cold transport media. Using a scalpel blade, remove excess connective tissue and trim the ends of the tissue.
Then wash the tissue twice in cold transport media. Next, place the tissue in transport media on an orbital shaker for a minimum of 30 minutes for a thorough final washing. After that, connect a non-branching segment of the washed artery to the fabricated bioreactor system using two barbed lure connections.
And secure it using a vessel bond made of vascular silicone ties. Using a small syringe attached to the first lure connection, gently flow media through the artery to ensure its patency. After securing the artery to the fabricated insert, fill the reaction space with perfusion media.
Next, carefully fill the luminal circulation loop with media, removing any remaining air from the system. Lastly, connect the reaction space to the assembled perfusion system, completing the circulation. To perform the perfusion culture, place the perfusion system in an incubator.
After connecting it to a peristaltic pump, connect any additional acquisition systems, such as pressure sensors. Allow the system to equilibrate overnight with a low media flow rate of approximately 10 to 15 milliliters per minute. The following day, incrementally increase the flow until a final flow rate of 35 milliliters per minute is achieved.
Every three days, replace 50%of the media by connecting one syringe with fresh media to the media exchange port closer to the pump and an empty syringe to the port closer to the reservoir to collect the spent medium. After the experiment, using sterile surgical scissors, harvest the tissue from the reaction chamber by trimming the tissue ends connected to the bioreactor. Confocal imaging using endothelial and smooth muscle-specific markers revealed that the normal structure of an artery was well preserved and maintained throughout, starting from the time of harvest, to cleaning and processing, and even after perfusion culture.
However, incorrect handling during processing resulted in the loss of the endothelium ahead of the culture, and the application of non-physiological culture conditions like abrupt initiation of high flow showed luminal damage. Histological evaluation of the tissue using hematoxylin and eosin staining and immunofluorescence staining at the time of harvest and after seven days of perfusion culture revealed that the morphology and overall distribution of the cells in the vessel wall were maintained throughout.
