Human Islet Isolation
Islets are specialised clusters of endocrine cells, which are located diffusely throughout the pancreas. Amongst other cell types, islets contain specialised cells that secrete insulin (the β cells). When islet cells are transplanted into the liver of patients with type 1 diabetes, they can produce insulin and successfully help to stabilise the recipients blood glucose levels. Islet cells only make up approximately 2% of the total pancreatic mass. Therefore, prior to transplantation, islets must be extracted (isolated) from the surrounding pancreatic tissue. The human islet isolation process is a multi-step procedure, which takes on average 5-6 hours to complete. The diagram below describes each stage of this sequential process.
Figure 1: Islet isolation is depicted from receipt of the donor pancreas to the infusion of islets into the recipient.
a) Pancreas procurement and dissection
With appropriate written consent, a pancreas is retrieved from the donor. From the donating hospital, the pancreas is shipped in a cold preservative solution (UW) to the DRWF Human Islet Isolation Facility at the Churchill Hospital in Oxford. Here, we must remove the spleen, duodenum (bowel) and any surface fat
b) Enzyme perfusion
The majority of the tissue that connects the islets to the rest of the pancreas is collagen derived. Therefore, in order to separate the islets from the rest of the pancreas, we use a commercially available enzyme that is able to digest collagen (‘collagenase’). This enzyme is infused (‘perfused’) into the pancreas, via the pancreatic ductal network
c) Pancreas digestion
The enzyme infused pancreas is loaded into a bioreactor device; the ‘Ricordi chamber’. Inside this chamber the pancreas is digested both enzymatically and mechanically, in a continuous sterile closed-loop circuit
d) Digest product
The digested pancreas consists of a product that has a liquid-like consistency. This product contains a mixture of islets (these can be seen in red, following staining with a special dye called dithizone) and non-islet tissue (pale yellow colour).
e) Density gradient purification
In order to separate the islets from the rest of the tissue, a specialised cell purification technique is used. This process exploits the fact that islets have different densities to the rest of the pancreatic cells. We load the pancreatic digest into a specialised centrifuge device called a COBE 2991 cell processor. A series of different liquid density interfaces are present within the COBE, and when the centrifuge spins it allows the islets to be purified away from the rest of the tissue
f) Islet culture and infusion into recipient
If for clinical use (i.e. transplantation) the purified islets are cultured for up to 48 hours, allowing sterility and quality tests to be performed, as well as allowing transplant logistics to be arranged. For more information regarding our clinical isolation programme please see ‘Clinical Islet Provision’. If the islets have been allocated for research use, we will package them up and ship them out to a number of our research recipient centres (please see 'Research Islet Provision' for more information on research islet distribution).
Images from DRWF Human Islet Isolation Facility, Oxford.
© Rebecca Spiers 2019