Cryopreservation of insulin-secreting INS832/13 cells using a wheat protein formulation.

Diabetes is a global epidemic that affects about 285million people worldwide. For severely-ill patients with type I diabetes, whole pancreas or islet transplantation is the only therapeutic option. Islet transplantation is hindered by the scarce supply of fresh functional islets and limitations in cryopreservation procedures. Thus, improved cryopreservation procedures are needed to increase the availability of functional islets for clinical applications. Towards this goal, this work developed a cryopreservation protocol for pancreatic cells using proteins that accumulate naturally in freezing-tolerant plants. A preincubation of cells with 1% lecithin-1% glycerol-1% N-methylpyrrolidone followed by cryopreservation with partially purified proteins from wheat improved the viability and insulin-secreting properties of INS832/13 cells, compared to cryopreservation with 10% dimethyl sulfoxide (Me2SO). The major factor that enhanced the cryoprotective effect of the wheat protein formulation was preincubation with the lipid lecithin. Expression profiles of genes involved in metabolic and signaling functions of pancreatic cells (Ins, Glut1/2/3, Pdx1, Reg1α) were similar between fresh cells and those cryopreserved with the plant protein formulation. This novel plant-based technology, which is non-toxic and contains no animal material, is a promising alternative to Me2SO for cryopreservation of insulin-secreting pancreatic cells.

Comparative expression of five Lea Genes during wheat seed development and in response to abiotic stresses by real-time quantitative RT-PCR.

Gene expression profiles of group 2 (dehydrins) and group 4 Late embryogenesis abundant (Lea) genes in developing seeds of Triticum durum and T. aestivum and in coleoptiles and coleorhizae of T. durum seedlings were monitored by real-time quantitative RT-PCR. The five genes exhibited clear differences in their accumulation pattern in wheat seed and in response to dehydration, low temperature, salinity and ABA. Td29b, Td16 and Td27e gene transcripts accumulate late in embryogenesis as expected for Lea genes, Td11 gene transcripts were present throughout seed development whereas no Td25a gene transcripts were detected in seeds. Drastic changes in the relative levels of Td29b, Td16, Td27e and Td11 transcripts occurred at the shift between the cell expansion and desiccation phases. All genes except the Td11 gene are more highly induced by dehydration in coleorhizae than in coleoptiles. In contrast, response to low temperature, salinity or ABA is higher in coleoptiles than in coleorhizae. Depending on both the gene and on the type of stress, a wide range of induction levels (8- to 100,000-fold) was observed.