Upgrading pretransplant human islet culture technology requires human serum combined with media renewal.
BACKGROUND.: The original Edmonton protocol used fresh islets, but for obvious logistic advantages most transplant centers have implemented pretransplant culture in human albumin. The aim of this study was to improve current pretransplant human islet culture techniques. METHODS.: Clinical-grade purified human islets from a total of 24 donors were directly resuspended after isolation in CMRL 1066-based media at 37 degrees C, and media additions and renewal were tested. At days 1 and 5 of culture, in vitro quality controls included islet viability, insulin content and function, apoptosis, and in vivo islet potency assay in nude mice. RESULTS.: Replacing human albumin with human AB serum improved 1- and 5-day preservation of islet function and viability which was further enhanced with antioxidant Stem Ease, leading to the iCulture medium (enriched CMRL: pyruvate, zinc sulfate, insulin, transferrin, selenium, 2.5% human AB serum and Stem Ease). Major damage occurs in the first day of culture and frequent media renewal (25% vol/hr) in this period further improved viability, apoptosis, islet recovery, and function in vitro and in vivo, compared with only changing medium after overnight culture. CONCLUSIONS.: The described human islet culture technique (iCulture medium+renewal) seems to be the best choice for clinical human islet culture when short (1 day) or long (5 days) periods are used. Media choice and dilution play a major role in the function and survival of human islets in culture.
Kerr-Conte
Julie
J
Université Lille Nord de France, F-59000 Lille, France. jkerr-conte@univ-lille2.fr
Vandewalle
Brigitte
B
Moerman
Ericka
E
Lukowiak
Bruno
B
Gmyr
Valery
V
Arnalsteen
Laurent
L
Caiazzo
Robert
R
Sterkers
Adrien
A
Hubert
Thomas
T
Vantyghem
Marie Christine
MC
Pattou
François
F
D000818:Animals; D017209:Apoptosis; D001769:Blood; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods / Q000592:standards; D002455:Cell Division; D002470:Cell Survival; D003470:Culture Media; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000032:analysis / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology* / Q000378:metabolism; D016381:Islets of Langerhans Transplantation / Q000379:methods*; D051379:Mice; D008819:Mice, Nude; D012709:Serum Albumin / Q000502:physiology
D003470:Culture Media; D007328:Insulin; D012709:Serum Albumin; D005947:Glucose