Cryopreservation of specific pathogen-free (SPF) pig islet cells: Effect of culture time before cryopreservation and after thawing

The aim of this study was to determine the optimal conditions (effect of culture time before and after cryopreservation) for cryopreservation of specific pathogen-free pig islet cells. Methods. (1)Glucose-induced insulin secretion by fresh islet cells cultured for 10 days was compared to that by islet cells cryopreserved 7 days after isolation and cultured 3 days after thawing. (2) Islet cells were cryopreserved 1, 7, or 14 days after isolation and cultured 3, 7, 14, or 21 days after thawing. Islet cell number, insulin content, and insulin response under perifusion tests were investigated. Results. (1) Insulin response by cryopreserved islet cells was identical to that by fresh islet cells (basal/ stimulation index: 2.13 +/- 0.19 vs 2.17 +/- 0.16, n = 4, NS), although the amount of secreted insulin was reduced by 40% (area under the curve: 2136 +/- 198 pM/10(4) cells/180 min vs 3564 +/- 636 pM/10(4) cells/180 min, P = 0.104). (2) Cell number 6 days after thawing was reduced by 54, 40, and 63% when cryopreservations were carried out at D1, D7, and D14. (3) Insulin content in cultured or cryopreserved islet cells increased between 7 and 14 days of culture. (4) Whatever the culture time before and after cryopreservation, insulin secretion in response to glucose was maintained. The insulin release was the highest for islet cells cryopreserved 14 days after isolation and cultured 14 days after thawing (stimulation index: 6.19 +/- 2.68). Conclusions. SPF pig islet cells remained functional after cryopreservation in polyethylene glycol and it may be important to culture islet cells over 14 days before and after cryopreservation, (C) 1999 Academic Press.