RESUMO
The aim of this study was to investigate different gas-permeable membranes for culturing human islets. Dynamic insulin release was used to assess islet functional quality. Islets isolated from cadaveric pancreata (n = 8) using standard isolation methods were stained with dithizone, counted, and cultured on five different commercially available medical-grade membranes reported to have high permeability to O2, CO2, and other gases. Fraction 1 (20,000 islet equivalents [IEQ] purity >70%; viability >85%) was cultured using serum-free medium in nonadherence tissue culture flasks (group I) and custom-made chambers with membranes (group II). Each vessel contained 5000 IEQ at a density of 30 IEQ/cm2 and 69 IEQ/cm2 for groups I and II, respectively. Islets were cultured for 48 to 90 hours at 37 degrees C in 5% CO2. In vitro dynamic insulin response to low glucose (3 mmol/L), high glucose (16.7 mmol/L), and 25 mmol/L KCI was measured. Stimulation indices were calculated by dividing average of initial response over basal insulin release; basal insulin release defined as average of the first seven values. Islets cultured on MG7 (n = 3) showed a higher stimulation index (3.49 +/- 0.37) compared with flasks (2.44 +/- 0.22), indicating better specific functional quality. Islets cultured on other membranes proved to show similar or worse functional quality than those cultured in flasks. In fact, islets cultured on MG6 (n = 2) were not tested owing to complete disintegration. Islet functional quality was improved when cultured on selected biocompatible gas-permeable membranes; however, finding the best membrane requires further investigation before clinical application.
Assuntos
Técnicas de Cultura de Células/métodos , Gases/análise , Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/fisiologia , Membranas Artificiais , Cadáver , Dióxido de Carbono/análise , Contagem de Células , Técnicas de Cultura de Células/instrumentação , Meios de Cultura Livres de Soro , Humanos , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Transplante das Ilhotas Pancreáticas , Oxigênio/análise , Permeabilidade , Doadores de TecidosRESUMO
Pefabloc and Trasylol are serine protease inhibitors that have been used during islet isolation to inhibit endogenous proteases (trypsin, chymotrypsin, and elastase) and improve islet yields. Using in vitro studies, we evaluated the effects of Pefabloc and Trasylol on the activities of these proteases and the effect of Pefabloc on islet function. In addition, it has been reported that Protector Solution (PS) enhances the efficiency of Pefabloc. Thus, we evaluated the efficacy of Pefabloc in the presence or absence of PS. EnzCheck protease assay was used to measure the activities of trypsin, chymotrypsin, elastase, liberase, and thermolysin in the presence or absence of 0.4 mmol/L Pefabloc (with or without PS) or 0.43 micromol/L Trasylol. We also tested switch samples containing the highest concentration of enzymes. Pefabloc significantly inhibited trypsin, chymotrypsin, elastase, and switch, but not liberase or thermolysin. Trasylol significantly inhibited all enzymes except for elastase and switch sample. Unexpectedly, the potency of Pefabloc was abrogated when diluted first in PS. Insulin release was diminished when islets were incubated or perifused with Pefabloc. In conclusion, Pefabloc when added appropriately significantly blocked in vitro protease activity. Unfortunately, Pefabloc also decreased islet insulin secretion, making it unsuitable for islet isolation. Trasylol cannot be used with collagenase because it impaired both liberase and thermolysin.
Assuntos
Pâncreas/enzimologia , Peptídeo Hidrolases/metabolismo , Aprotinina/farmacologia , Quimotripsina/metabolismo , Humanos , Cinética , Pâncreas/citologia , Fosfopiruvato Hidratase/metabolismo , Inibidores de Proteases/farmacologia , Inibidores de Serina Proteinase/farmacologia , Sulfonas/farmacologia , Inibidores da Tripsina/farmacologiaRESUMO
Assessing islet cellular composition and beta cell viability using Flow Cytometry (FC) and Laser Scanning Cytometry (LSC) may aid in determining the transplant quality of islets. Human islets (2500 IEQ, n = 44, purity >or=80%) dissociated into a single cell suspension were stained with ductal marker CA19, with Newport Green (NG) and FluoZin3 (FL3) for beta-cell identification, with TMRE to assess mitochondrial membrane potential, with DAPI to identify live vs. dead cells, and with Annexin-V/DAPI to differentiate apoptotic and necrotic cells. For LSC, cell preparations (n = 9) were stained for insulin (beta-cells), glucagon (alpha-cells), somatostatin (delta cells), and pancreatic polypeptide (ppp cells). Fluorescence microscopy (EtBr/FDA) and insulin response were also measured. DAPI- staining was 73.78% +/- 1.37, while EtBr/FDA was 96% +/- 0.48. 52.5% +/- 3.73 of all cells were NG+, of which 58.08% +/- 2.61 were NG+/TMRE+. Annexin-V/DAPI staining (n = 26) showed 13.8% +/- 0.89 apoptotic, 27.2% +/- 2.0 necrotic, and 51.9% +/- 2.22 live cells. 26.0% +/- 5.19 of cells were CA19 positive (n = 17), of which 45.5% +/- 4.37 were also TMRE+, and 5.2% +/- 1.2 of the TMRE+ were also NG+/CA19+. NG and FL3 showed similar staining (n = 8). Comparison of short-term (Assuntos
Citometria de Fluxo/métodos
, Ilhotas Pancreáticas/citologia
, Citometria de Varredura a Laser/métodos
, Técnicas de Cultura de Células
, Separação Celular/métodos
, Sobrevivência Celular
, Corantes
, Humanos
, Células Secretoras de Insulina/citologia
RESUMO
UNLABELLED: Pancreas preservation using an oxygenated two-layer method (TLM) has been reported to improve islet yields, as has supplementation of Liberase with Pefabloc. We hypothesized that using both TLM and Pefabloc could enhance islet yield as compared with preservation in University of Wisconsin (UW) or Histidine-Tryptophan Ketoglutarate (HTK) solution. METHODS: Ninety-eight pancreata with no significant differences of age, body mass index, or cold ischemia time preserved randomly with UW (n = 40), TLM (n = 48), or HTK (n = 10) were processed with (n = 36) or without (n = 66) Pefabloc. RESULTS: The total islet equivalent (IEQ) from TLM-preserved pancreata processed with Pefabloc (n = 12) showed lower yields versus those processed without Pefabloc (n = 36): 216,120 +/- 27,906 vs. 301,427 +/- 21,447 IEQ (P < .05). Islets from 1 of 12 (8.33%) pancreata processed with Pefabloc in TLM were transplanted, in contrast with 15/36 TLM (41.67%) pancreata processed without it. Islet yields were not significantly different among pancreata preserved in UW and processed with Pefabloc (n = 17) versus without Pefabloc (n = 23): 342,693 +/- 45,588 versus 266,609 +/- 29,006 IEQ (P = .149). The number of transplants from UW-preserved pancreata was 3/17 (17.65%) when processed with Pefabloc and 4/23 (17.39%) without. Among the HTK group, there was no significant difference in islet yields between pancreata processed with (n = 7) versus without Pefabloc (n = 3): 248,227 +/- 65,294 versus 483,555 +/- 144,070 IEQ (P = .118). CONCLUSIONS: Pefabloc showed no benefit to improve islet yields. Pancreata preserved in TLM provided better transplant quality islets when processed in the absence of Pefabloc.