Islet cells were isolated using a modification of the automated Ricordi method17
and purified by continuous gradient with Biocoll (Biochrom, Berlin, Germany) and a cooled COBE 2991 cell processor (Terumo BCT, Lakewood, CO, USA). After isolation and purification, the cell preparations were cultured in cell culture flasks T175 (Sarstedt, Nümbrecht, Germany) at 37°C in a humidified incubator (5% CO2) in a Ham’s F10 based medium (Lonza, Bazel, Switzerland).
Preparations were characterized immediately after purification and after a 1- to 5-day culture period by their beta cell number, insulin content, and insulin purity, as described previously17
. Beta cell number was calculated from the total nuclear count (NucleoCounter YC-100; ChemoMetec, Allerod, Denmark) and the percentage of insulin positive cells [immunocytochemistry with guinea pig anti-insulin (1/2,000, in-house produced) on 1.5 µm araldite sections; >2 × 103 cells counted; pictures were captured using Nikon Eclipse Ti microscope and analyzed with NIS-Elements AR v5.21 software (Nikon Europe, Amsterdam, The Netherlands;Fig. 1 )]. Yield immediately post purification was also expressed as islet equivalent (IEQ), calculated using a volume based method after dithizone staining19
.
and purified by continuous gradient with Biocoll (Biochrom, Berlin, Germany) and a cooled COBE 2991 cell processor (Terumo BCT, Lakewood, CO, USA). After isolation and purification, the cell preparations were cultured in cell culture flasks T175 (Sarstedt, Nümbrecht, Germany) at 37°C in a humidified incubator (5% CO2) in a Ham’s F10 based medium (Lonza, Bazel, Switzerland).
Preparations were characterized immediately after purification and after a 1- to 5-day culture period by their beta cell number, insulin content, and insulin purity, as described previously17
. Beta cell number was calculated from the total nuclear count (NucleoCounter YC-100; ChemoMetec, Allerod, Denmark) and the percentage of insulin positive cells [immunocytochemistry with guinea pig anti-insulin (1/2,000, in-house produced) on 1.5 µm araldite sections; >2 × 103 cells counted; pictures were captured using Nikon Eclipse Ti microscope and analyzed with NIS-Elements AR v5.21 software (Nikon Europe, Amsterdam, The Netherlands;
.