Tissue Purification
Islet and non-islet tissue have specific and different densities. Using this density difference has allowed islet separation for decades and remains today the standard for purification of islets.
Tissue purification SOPs: Preparing for pump-made density gradient islet separation, Density gradient-based islet separation using computer-controlled pumps
Pump-made density gradients, tissue loading & Fraction collection
As opposed to most islet isolation labs, our lab uses technology providing automated density gradient production using a bag system. Computer-controlled, pump-made UW/ficoll density gradients (modified from Transplantation 2007;84: 1200-1203) are then created to separate the islets from otherwise unwanted tissue. The tissue is then loaded onto the density gradient and allowed to separate based on its specific density, so-called isopycnic separation.
After centrifugation in the COBE the separated tissue volume is collected into separate fractions to maximize the capture of pure islets. Islets are typically lighter and, when evacuated from the system, are in the earilier fractions collected. The more an islet is embedded by exocrine (unwanted) tissue, the more difficult it becomes to separate from impure fractions. The flasks used for collection are filled with cold washing media to dilute the density solution and provide a more tolerable environment for captured tissue.
Schematic for automated
gradient making system. Bags containing different
density solution are pumped onto a spinning COBE 2991 cell
separator by two independant computer-controlled pumps. No openings
or magnets are necessary. Reproduced with permission (Cell
Transplant. 2008;17(12):1305-13).
Video - Programming pumps for making densitygradient
Computer-controlled pumps need instructions for dosing high and low density solutions at specific time points. This precise dosing ensures consistent gradient production.
This method has been adopted and adapted by other islet isolation centers as a standard practice for density gradient production.