Applications

Large Cells/Cell Clusters: Pancreatic Islet Cells

COPAS: a breakthrough approach to islet isolation and quality control
The COPASTM PLUS instrument is able to analyze and sort large objects (30 – 700 microns) on the basis of size, density, and fluorescence signals. The gentle pneumatic sorting mechanism used by the COPAS PLUS provides a method for analyzing and handling sensitive tissues such as islets with a high level of recovery and a low level of lethality. Early experiments have shown that the COPAS instrument may be used in a broad range of islet research applications in the area of Diabetes research.

Background
In insulin-dependent diabetes (Type 1 Diabetes), a diabetic's body fails to produce insulin, an essential hormone for the metabolism of glucose. Insulin is normally produced and secreted by the b cells of the pancreas in response to blood glucose. These cells are organized in cell clusters, called the Islets of Langerhans (or simply, islets), and are located within the pancreas. Type 1 Diabetes occurs when the islets are unfortunately destroyed, resulting in the absence of insulin-producing b cells.

One of the most successful approaches to treating insulin-dependent Diabetes has been the transplantation of islets. It has been shown that transplantation of pure islets has advantages over transplantation of the whole pancreas. The promising results of these early transplants have led to increased research aimed at discovering new methods for improved islet isolation procedures and better transplantation protocols for reducing rejection. Researchers are working with human islets as well as islets from many different animals including mice, dogs, and pigs.

Limitations of current islet research

  1. Quantity: There is a limited supply of human pancreas donors.
    About one million islet cells are required for a successful transplant in an average-sized person, which typically requires two donor pancreases, using current islet cell isolation procedures. Due to the large requirement for islets, researchers continue to search for new sources of donor islet cells, including from animal donors such as pigs.
  2. Quality: Current methods of quality control during islet isolation are time-consuming and subjective.
    In order to break apart the pancreatic tissue to release the islets, the pancreas is injected with digestive enzymes and then incubated and physically agitated,. If this isolation procedure is too vigorous, islets may fall apart resulting in limited yield and varying quality. Therefore, quality of the isolated islets has to be constantly monitored through out the purification procedure and before transplantation. The current method employs manual, under-the-microscope examination of the purified islets, which is both time consuming and subjective to human errors
  3. Purity: Pancreatic islet isolation is a laborious and time-consuming process.
    The pancreas is a mix of endocrine and exocrine glands that control many homeostatic functions. Endocrine cells form clusters called islets (Islet of Langerhans), which are comprised of four cell types: a, b, g and polypeptide cells. The b cells form the core of the islets and are responsible for producing insulin. The islets comprise about 2% of the total pancreas mass, thereby making pancreatic islet identification and extraction a laborious and time-consuming process. The current purification method is based on Ficoll density gradient separation, which is both time-consuming and slightly toxic. About 40-50% of the islets are lost in the process and the purity is only around 85%.

Early Experiments with COPAS
An alternative to manual sorting (under a microscope), COPAS systems sort and dispense objects based on size and fluorescence parameters. Automating this process offers increased speed, sensitivity, quantification, and repeatability of experiments. We anticipate that the COPAS instrument will have application in both the areas of quality control and the purification process. Early experiments for quality control have shown that COPAS is able to measure the size and multiple fluorescence signals of an individual islet at the same time. Therefore, by using a combination of different dyes identified in research literature, the various parameters for indicating the quality of the isolated islets can be measured simultaneously with a speed as high as 20 islets per second.

An Overview of COPAS™ Large Particle Flow Cytometry for the Analysis and Sorting (QTN-014)