Trehalose: a cryoprotectant that enhances recovery and preserves function of human pancreatic islets after long-term storage.
The scarcity of available tissue for transplantation in diabetes and the need for multiple donors make it mandatory to use an optimal cryopreservation method that allows maximal recovery and preservation of beta-cell function. We have developed a method to cryopreserve islets with excellent survival of endocrine cells. Current methods use DMSO as cryoprotectant. Our method involves introducing both DMSO and the disaccharide trehalose into the cells during cooling. Uptake and release of trehalose occurred during the thermotropic lipid-phase transition measured in pancreatic endocrine cells between 5 degrees and 9 degrees C, using [14C]trehalose. Recovery of adult islets after cryopreservation with 300 mmol/l trehalose was 92 vs. 58% using DMSO alone. In vitro function, in terms of insulin content and release in response to secretagogues, was indistinguishable from fresh islets. Grafts from islets cryopreserved with trehalose contained 14-fold more insulin than grafts from islets cryopreserved without trehalose. Results with human fetal islet-like cell clusters (ICCs) were more pronounced: recovery from cryopreservation was 94%, compared with 42% without trehalose. Complete functionality of fetal cells was also restored; tritiated thymidine incorporation and insulin content and release were similar to fresh tissue. After transplantation in nude mice, there was a 15-fold increase in insulin content of grafts from ICCs cryopreserved with trehalose compared with ICCs cryopreserved without trehalose. Thus, the addition of trehalose to cryopreservation protocols leads to previously unobtainable survival rates of human pancreatic endocrine tissue.
Beattie
G M
GM
Whittier Institute, Department of Pediatrics, University of California at San Diego School of Medicine, La Jolla 92037, USA.
Crowe
J H
JH
Lopez
A D
AD
Cirulli
V
V
Ricordi
C
C
Hayek
A
A
D000328:Adult; D000704:Analysis of Variance; D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D004121:Dimethyl Sulfoxide; D016332:Fetal Tissue Transplantation / Q000502:physiology*; D005333:Fetus; D005947:Glucose; D006801:Humans; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology / Q000502:physiology; D016381:Islets of Langerhans Transplantation / Q000502:physiology*; D007668:Kidney; D051379:Mice; D008819:Mice, Nude; D014183:Transplantation, Heterologous; D016042:Transplantation, Heterotopic; D014199:Trehalose / Q000494:pharmacology
D003451:Cryoprotective Agents; D007328:Insulin; D014199:Trehalose; D005947:Glucose; D004121:Dimethyl Sulfoxide