Continuous Gradient Method for Porcine Islet Purification

To begin, set a pump to a flow rate of 200 milliliters per minute and load 125 milliliters of high-density gradient solution into the continuous gradient former, and then into the cell processor. Start the cell processor centrifuge function at 1, 000 RPM. Release air from the system and rep-rime it with a high-density gradient solution.

Then add 125 milliliters of high-density gradient solution and 130 milliliters of low-density solution to the gradient former. Load 100 milliliters of digested tissue suspension. Increase the cell processor speed to 2, 000 RPM for three minutes.

To prepare 16 50 milliliter conical tubes for collecting purified islet fraction, add 25 milliliters of RPMI medium to each tube and arrange them in ascending order. Collect the purified islet fractions in the prepared conical tubes. To stain the islets, collect 100 to 200 microliter aliquots from each purified islet fraction, and assess islet content using DTZ staining in a 24-well tissue culture plate.

Inspect each fraction using light microscopy at 4X where the islets will appear dark red to black. Using this protocol, pancreatic islet isolation was performed on three pigs. Brightfield images showed purified pancreatic islets, which were characterized for viability using live/dead staining.

Representative islet size distribution after the islet isolation showed size distribution around 50 to 100 microns. Further, yield and purity were evaluated using an islet cell counter, indicating efficient porcine pancreatic isolations.