Purification method using iodixanol (OptiPrep)-based density gradient significantly reduces cytokine chemokine production from human islet preparations, leading to prolonged beta-cell survival during pretransplantation culture.
Purification is one of the most important steps in human islet isolation. Although Ficoll-based density gradients are widely used, OptiPrep-based density gradients are used in few centers. Cytokine/chemokine production from human islet preparations varies widely. Some cytokines/chemokines have been reported to have adverse effects on human islet preparations. Control of cytokine/chemokine production may be a key to improve islet quality and quantity, leading to better transplantation outcomes. The aim of the present study was to investigate the effects on islet preparations of purification methods using various density gradients on viability, cellular composition, and proinflammatory cytokine/chemokine production. After the digestion phase, the extracts were divided into 2 groups for purification using a semiautomated cell processor with Ficoll-based or OptiPrep-based density gradients. Islet preparations cultured for 2 days were assessed regarding islet cell viability (fluorescein diacetate/propidium iodide [FDA/PI]), fractional beta-cell viability by FACS, and beta-cell content using iCys. Cytokine/chemokine production from islet preparations was also measured by Bio-plex. After purification, the purity, islet equivalents (IEQ), and islet recovery rates were comparable between the 2 groups. Although FDA/PI and fractional beta-cell viability showed no significant difference, survival of beta cells during culture was significantly higher in the OptiPrep compared with the Ficoll-based density gradient group. There were significantly lower tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, interferon (IFN)-gamma, IL-6, and MIP-1beta productions from the OptiPrep-based density gradient group. OptiPrep-based density gradients reduced cytokine/chemokine production by islet preparations. In addition, OptiPrep-based density gradient purification significantly reduced the loss of beta-cell mass during pretransplantation culture.
Mita
A
A
Cell Transplant Center, Diabetes Research Institute, University of Miami Leonard M. Miller School of Medicine, Miami, Florida 33136, USA.
Ricordi
C
C
Miki
A
A
Barker
S
S
Khan
A
A
Alvarez
A
A
Hashikura
Y
Y
Miyagawa
S
S
Ichii
H
H
D000241:Adenosine; D000493:Allopurinol; D001331:Automation; D018929:Cell Culture Techniques / Q000379:methods; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology*; D018925:Chemokines / Q000096:biosynthesis; D003287:Contrast Media / Q000494:pharmacology*; D016207:Cytokines / Q000096:biosynthesis; D005978:Glutathione; D006801:Humans; D007328:Insulin; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000187:drug effects / Q000637:transplantation*; D007515:Islets of Langerhans / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D019188:Organ Preservation Solutions; D011887:Raffinose; D014283:Triiodobenzoic Acids / Q000494:pharmacology*
D018925:Chemokines; D003287:Contrast Media; D016207:Cytokines; D007328:Insulin; D019188:Organ Preservation Solutions; D014283:Triiodobenzoic Acids; C055206:University of Wisconsin-lactobionate solution; D000493:Allopurinol; D005978:Glutathione; C044834:iodixanol; D000241:Adenosine; D011887:Raffinose