ABSTRACT
Objective@#To study the effects of different purification method on the ratio of each component in the suspension of fat granules and the cell viability.@*Methods@#Fat granule suspension was harvested under conventional swelling anesthesia, and divided into staticseparation group, centrifugation group, washing and filtration group.①staticseparation group: based on different gravity separation time (30 s, 1 min, 5 min, 10 min, 30 min, 1 h, 4 h, 12 h, 24 h), the group was subdivided into 9 groups.②Centrifugation group: based on different centrifugation times (3, 5, 10 min) combined withdifferent centrifugation rates (1 000, 2 000, 3 000, 4 000 r/min), this group was subdivided into 12 groups.③Washing and filtration group: According to the layers of gauze (1 layer, 2 layers, 5 layers), this group was subdivided into three groups. Adipocyte activity was measured by Calcein-AM / Hoechst 33342 dual fluorescence staining, and the viable cell rate was calculated. T test was introducedto compare between two samples. One-way AVOVA analysis was used to compare between two groups of samples.@*Results@#Staticseparation group: After gravity separation, the fat granules were separated intooil layer, fat granules layer and liquid layer, and stabilized at 5 min after staticseparation. ②Centrifugation group: The viable cell ratewas highest at 3 min in 2 000 r/min group.③Washing and filtration group: More volume of fat particles were collected by filtration with 2-layer gauze, and the viable cell rate was not significantly reduced.Compared with static 5 min method (86.75±6.29)%, 2 000 r/min 3 min (89.05±7.16)% and double gauze filtration rinse, the cell viability rate was (87.85±5.92)%, and there was no significant difference between the three groups (P> 0.05).@*Conclusions@#5 min static separation, 2-layer gauze filter and 2 000 r/min 3 min fat cell survival rate is the highest among each group, and no significant difference in viable cell rate between these three method . Clinically, 2 000 r/min 3 min is suitable for a small amount of fat filling, washing and filtration is suitable for massive fat filling.
ABSTRACT
Objective To investigate the methods and feasibility of islets isolation and purification,and to get more islets with high purity and good function for clinical transplantation.Methods After being weighted,human pancreatic islets were isolated with type Ⅴ collagenase,and purified by Ficoll's discontinuous density gradient centrifugation.The yield and purity of islets were evaluated by dithizone(DTZ) staining under microscope,and the viability was assessed by insulin release assay in vitro.Results The average number of islets was about 3 600 ± 447 per gram pancreas after isolation and it was about 2140 ±207 after purification with more than 70％ purity.2,4,6 days after islet cell culture,the basal insulin concentration of the culture medium was measured,and it was 3.302 ± 1.63,3.504 ±1.10,and 2.921 ±1.13 (mIU/L/100 islets) respectively.Conclusion Collagenase digest and Ficoll's discontinuous density gradient centrifugation are effective methods for isolation and purification of human pancreatic islets.