A method for mass harvesting islets (Brockmann bodies) from teleost fish.

Abstract

In certain species of fish, the insulin-producing tissue is uniquely located in separate structures called Brockmann bodies (BBs). Tilapia BBs have been shown to be a simple and inexpensive source of islet cells for xenotransplantation research. Each donor tilapia contains roughly 12-15 BBs, measuring from 0.3 to 5.0 mm in maximum dimension, in a triangular region of adipose tissue bounded by the liver, stomach, and spleen/gallbladder. At present, the larger BBs (usually 2-4) are harvested by microdissecting these "BB regions" using jeweler's forceps and microvascular scissors while being visualized with the aid of a dissecting microscope. It is a simple but time-consuming task that would not be applicable for harvesting massive amounts of BB tissue for large animal studies. Therefore, we have developed an easier and more efficient method of harvesting BBs based on a standard enzymatic method for isolating human adipocytes. BB regions are harvested from donor fish and pooled into a 50 mL plastic tube containing collagenase Type II (3 mg/mL) in Hank's balanced salt solution (HBSS); the tube is then placed in a 37 degrees C waterbath/shaker for roughly 15 min. The exact length of the digestion interval is determined by visual inspection of the tube to determine whether the BBs have been liberated. The digestion is then stopped by adding excess cold HBSS. The adipocytes float while the BBs and residual connective tissue (i.e., a few blood vessels, nerves, and bile ducts) form a pellet. The pellet is washed several times in HBSS and then placed in a culture dish. The BBs are easily handpicked with a siliconized pipette. Based on functional data and DNA content, this new method roughly doubles or triples our yield of BB tissue per donor fish. To determine whether BBs harvested in this manner functioned in a manner similar to those harvested by microdissection, we performed a series of transplants using mass-harvested BBs. Long-term normoglycemia was achieved in streptozotocin-diabetic nude mice and mean graft survival time was not altered in streptozotocin-diabetic euthymic balb/c mice. However, the total weight of donor fish required per recipient was decreased by 50% in both strains.