Islet neoformation in tissue culture

We have devised a tissue culture system that permits de novo formation of islets. Neonatal rat pancreata are enzymatically dissociated into single cells. The cell suspension is filtered through polyester cloth with 20 microns pores to exclude cell aggregates as well as preformed islets and a single cell suspension is then plated into tissue culture dishes at a density precluding reaggregation. Pancreatic cells proliferate forming numerous colonies of epithelioid cells. After a confluent monolayer, cells proliferate into a third dimension, the space occupied by the culture medium. Third dimensional proliferation occurs from basal monolayers of epithelioid colonies. At about 9 days in culture, numerous hillocks are visible that are spaced at about 1 mm from one another. Islets are observed to bud from the hillock surfaces. In 1 micron-thick sections, secretion granules are detected with the light microscope in some islet cells. With the electron microscope three basic cell types are seen. One peripherally located cell type is sparsely granulated and appears to be a precursor cell. The other peripherally located cell type shows a homogeneous population of secretion granules characteristic of A-cells. The third cell type is found in the interior of islets containing granules characteristic of B-cells. Islet cells, but not hillock cells, react immunocytochemically for insulin and glucagon. The cultures secrete 2 to 10-fold the amounts of glucagon present in fresh medium. It is concluded that differentiation of A- and B-cells occurs in neoformed islets.