Cryopreservation of freshly isolated porcine islet cells.
BACKGROUND
The use of xenogenic islet cells may be a possibility to overcome the shortage of human donor organs to treat diabetes. Microencapsulation seems to be a promising method for immunoprotection. Since isolation, purification, encapsulation, and transplantation of islet cells are labor intensive, cryopreservation has emerged as an attractive system of islet banking. The aim of this study was to determine the influence of three different freezing media (FM) on viability of freshly isolated porcine islet cells (FIPIC).


METHODS
FIPIC were isolated using a modified Ricordi method and purification performed using a Lymphoprep density gradient. Viability of FIPIC prior to freezing and after thawing was determined using the MTT-based Cell Growth Determination Kit. Insulin production was detected using enzyme-linked immunosorbent assay. Three different FM containing dimethylsulfoxide (DMSO) or glycerol and sucrose were used for cryoprotection of FIPIC.


RESULTS
Isolation and purification of FIPIC resulted in 95% +/- 1.3% viability and 97% +/- 1.4% purity. Cryopreservation with FM I (containing DMEM, FCS, DMSO) yielded 98.4% and FM III (containing DMEM, FCS, glycerol) 93.1% viability, whereas only 85.6% were alive when cryoprotection is performed with FM II (containing DMSO, BM). Glucose stimulation revealed a loss of 2.8% and 1.9% of insulin secretion per microgram DNA when working with FM I and FM III, but a decrease in glucose-dependent insulin secretion of 7.8% (P < .05) when FIPIC were stored in FM II.


CONCLUSIONS
Low concentrations of DMSO or the use of glycerol and sucrose seem to be equivalent to cryopreserve FIPIC.
Stiegler
P
P
Department for Transplantation Surgery, University Clinic for Surgery, Auenbruggerplatz 29, 8036 Graz, Austria. philipp.stiegler@klinikum-graz.at
Stadlbauer
V
V
Schaffellner
S
S
Florian
H
H
Lackner
C
C
Iberer
F
F
Tscheliessnigg
K
K
D000818:Animals; D002214:Capsules; D015925:Cryopreservation / Q000379:methods; D003451:Cryoprotective Agents / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005990:Glycerol / Q000494:pharmacology; D006801:Humans; D007515:Islets of Langerhans / Q000166:cytology* / Q000187:drug effects; D016381:Islets of Langerhans Transplantation; D013395:Sucrose / Q000494:pharmacology; D013552:Swine; D014183:Transplantation, Heterologous
D002214:Capsules; D003451:Cryoprotective Agents; D013395:Sucrose; D005990:Glycerol; D004121:Dimethyl Sulfoxide