Production of islet cell sheets using cryopreserved islet cells.

ABSTRACT

BACKGROUND: To establish novel islet-based therapies, our group has recently developed technologies to create a contiguous, monolayered sheet made from freshly dispersed islet cells. Islet cell sheets generated from freshly isolated cells are easily transplantable for engraftment into subcutaneous sites in rodents. The use of a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm), grafted culture dishes with laminin-5 coating is an important feature of this process. To expand the utility of this protocol, the present study was performed to assess whether sheets generated using cryopreserved islet cells maintained viability and normal cellular phenotypes. METHODS: Dispersed islet cells obtained from Lewis rats were, cryopreserved using University of Wisconsin solution and 10% dimethyl sulfoxide. Specially coated plastic dishes were prepared by covalently immobilizing PIPAAm onto the culture plastic, followed by a coating of rat laminin-5. After 1 month of cryopreservation, the thawed cells were plated onto the PIPAAm-coated dishes. RESULTS: Viability of the thawed islet cells as assessed by trypan blue exclusion test was 86% ± 5%. Thawed dispersed islet cells favorably attached to PIPAAm dishes could be harvested as a contiguous cell sheet using a simple change in culture temperature conditions. Electron microscopy showed the harvested islet cell sheet to retain cell-cell connections and numerous secretion granules. CONCLUSIONS: The present data indicated that dispersed islet cells, which were appropriately frozen and thawed, represent another viable cells source to create functional islet sheets for tissue engineering and potential clinical applications.