iv. Harvesting Cells

Harvesting is the process where tissue containing cells of interest is isolated and then processed in some way to create a solution of viable cells. The cells that we would like to isolate are smooth muscle cells, which are present in blood vessels and some organs. It is necessary to get smooth muscle cells in an isolated and sterile form. To do this, we must get tissue that contains them, disaggregate the tissue (i.e. turn it into a collection of individual cells), and then grow a sufficient quantity of the cells for our purpose. Here, we will harvest our smooth muscle cells from a swine aorta.

The aorta is made up of 3 cell layers:

• adventitia (outer layer)
• media (middle layer of smooth muscle cells
• intima (inner layer of endothelial cells)

We are going to need the following items: a culture dish, scalpel, large (50 ml) centrifuge tubes, motorized pipette bottles for media and other reagents, and a pan or dish to hold the aorta.

IMPORTANT: Harvesting should be done under a laminar flow hood to maintain sterility.

The steps in harvesting the cells from the aorta are:

1. Remove section of aorta from swine heart and place in culture dish
2. Remove adventitia (outer layer)
3. Cut down the length of the aorta to expose the intima (inner layer)
4. Scrape off the endothelial cells (not visible to naked eye) which make up the intima
5. Chop remaining tissue (called media) into fine pieces and transfer to centrifuge tube
6. Add collagenase and incubate for 24 hours at 37° to break down the tissue into individual cells
7. Centrifuge the now disaggregated cells and remove supernatant
8. Add media to prepare the cells for seeding