Polyvinyl pyrrolidone: a novel cryoprotectant in islet cell cryopreservation.
The present study was performed on the basis of the hypothesis that the low molecular weight (MW) compounds, DMSO and glycerol, permeate the cell and interact hydrophobically with intracellular proteins, thereby perturbing the cytoskeletal architecture of frozen cells and diminishing islet cell integrity and function. Isolated rat islets were cultured overnight (18-24 h) at 37 degrees C in RPMI medium supplemented with 10% fetal calf serum and 1% mixture of penicillin/streptomycin. Using a programmable temperature controller, samples of precounted islets were then frozen under liquid nitrogen, in the presence of either 2 M DMSO (MW = 0.078 kDa), 3 M glycerol (MW = 0.092 kDa), 5% polyethylene glycol (PEG, MW = 20 kDa), or 10% polyvinylpyrrolidone (PVP, MW = 40 kDa), and stored at -80 degrees C for 1 week. Following thawing and overnight (18-24 h) culture, intact islet recovery was determined by islet counting after dithizone staining. Islet function was assessed by determination of glucose-stimulated insulin secretion in perifusion experiments with Krebs-Ringer bicarbonate buffer, pH 7.4, containing either basal (3.3 mM) or high (16.7 mM) glucose concentrations. The assessment of islet recovery and function of all cryopreserved samples was performed only after thawing and overnight culture (18-24 h) of islets. The mean +/- SEM percent intact islet recovery was higher with PVP compared with DMSO (82 +/- 4.6 vs. 62.7 +/- 3.1%, respectively, p < 0.005, n = 9). Furthermore, the glucose stimulation index of insulin secretion by islets taken from samples frozen with PEG and PVP, after thawing and overnight culture, was comparable to that of freshly isolated islets, in contrast to DMSO and glycerol. There was no significant difference in intact islet recovery and function between samples frozen with PVP and those frozen with PEG. Samples frozen with DMSO and glycerol had similar results in islet recovery and function. These data show that PVP is a new and potent cryoprotectant for islet cell freezing.
El-Shewy
Hesham M
HM
Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA.
Kendall
William F
WF
Darrabie
Marcus
M
Collins
Bradley H
BH
Opara
Emmanuel C
EC
D000818:Animals; D017690:Cell Transplantation / Q000379:methods*; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D003599:Cytoskeleton / Q000187:drug effects; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D005947:Glucose / Q000378:metabolism; D005990:Glycerol / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology* / Q000187:drug effects; D008297:Male; D009584:Nitrogen / Q000494:pharmacology; D010592:Pharmaceutic Aids / Q000494:pharmacology; D011205:Povidone / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013696:Temperature; D013997:Time Factors
D003451:Cryoprotective Agents; D007328:Insulin; D010592:Pharmaceutic Aids; D011205:Povidone; D005947:Glucose; D009584:Nitrogen; D005990:Glycerol; D004121:Dimethyl Sulfoxide