Improved cryopreservation of human hepatocytes using a new xeno free cryoprotectant solution

AIM:To optimize a xeno-free cryopreservation protocol for primary human hepatocytes.METHODS:The demand for cryopreserved hepatocytes is increasing for both clinical and research purposes.Despite several hepatocyte cryopreservation protocols being available,improvements are urgently needed.We first compared controlled rate freezing to polystyrene box freezing and did not find any significant change between the groups.Using the polystyrene box freezing,we compared two xeno-free freezing solutions for freezing of primary human hepatocytes:a new medium(STEM-CELLBANKER,CB),which contains dimethylsulphoxide(DMSO) and anhydrous dextrose,both permeating and non-permeating cryoprotectants,and the frequently used DMSO-University of Wisconsin(DMSOUW) medium.The viability of the hepatocytes was assessed by the trypan blue exclusion method as well as a calcein-esterase based live-dead assay before and after cryopreservation.The function of the hepatocytes was evaluated before and after cryopreservation by assessing enzymatic activity of 6 major cytochrome P450 isoforms(CYPs):CYP1A2,CYP2C9,CYP2C19,CYP2D6,CYP3A4 and CYP3A7.RESULTS:The new cryoprotectant combination preserved hepatocyte viability significantly better than the standard DMSO-UW protocol(P < 0.01).There was no significant difference in viability estimation between both the trypan blue(TB) and the Live-Dead Assay methods.There was a correlation between viability of fresh hepatocytes and the difference in cell viability between CB and DMSO protocols(r 2 = 0.69) using the TB method.However,due to high within-group variability in the activities of the major CYPs,any statistical between-group differences were precluded.Cryopreservation of human hepatocytes using the cryoprotectant combination was a simple and xeno-free procedure yielding better hepatocyte viability.Thus,it may be a better alternative to the standard DMSO-UW protocol.Estimating CYP activities did not seem to be a relevant way to compare hepatocyte function between different groups due to high normal variability between different liver samples.CONCLUSION:The cryoprotectant combination may be a better alternative to the standard DMSO-UW protocol in primary human hepatocyte cryopreservation.

过表达IDO骨髓间充质干细胞分泌外泌体通过不同分子轴调节DC及T细胞

目的探讨过表达吲哚胺2,3双加氧酶(IDO)间充质干细胞分泌外泌体(BMSC^(IDO)-exosome)抑制T及DC细胞激活机制。方法将BMSC^(IDO)-exosome与DC、T细胞共培养设立为实验组。检测共培养DC及T细胞内miR-540-3p、CD74、CD28、CCL4、p-ERK1/2、p-NF-κB、p-AKT表达。验证miRNA-540-3p与CD74基因的靶向结合。结果实验组中DC细胞内miR-540-3p表达增高,CD74、p-p65表达降低。T细胞内miR-540-3p表达增高,CD74、p-AKT表达降低。miRNA-540-3p与CD74可以靶向结合。结论BMSC^(IDO)-exosome通过miRNA-540-3p(+)/CD74(-)/p-NF-κB分子轴抑制DC细胞的激活。通过miRNA-540-3p(+)/CD74(-)/p-AKT(-)分子轴抑制T细胞的激活。

河南省部分地区溴氰菊酯抗性中华按蚊代谢酶活性的调查研究

目的了解河南省部分地区溴氰菊酯抗性中华按蚊的代谢酶活性,探讨中华按蚊对溴氰菊酯的代谢抗性机制。方法 2010年8月在河南省桐柏、淮滨县和永城市采集中华按蚊样本,子1代成蚊用接触筒内成蚊药膜滤纸接触法,按照WHO标准,区分溴氰菊酯抗性与敏感样本,并用微量滴定板法测定抗性样本的非特异性酯酶、谷胱甘肽S转移酶(GST)和P450单加氧酶活性。以实验室敏感品系蚊为对照。结果淮滨、永城和桐柏三地中华按蚊抗性样本的A、B型非特异性酯酶活性均高于对照组(t=2.41,3.30,10.31,7.67,7.90,11.17,均P<0.05);永城和淮滨地区中华按蚊抗性样本GST活性高于对照组的敏感品系(t=3.687,2.484,均P<0.05);淮滨、永城及桐柏三地的中华按蚊抗性样本和敏感品系的P450单加氧酶活性的差异均无统计学意义(t=-1.489,0.397,-0.413,均P>0.05)。结论永城、淮滨两个地区中华按蚊的溴氰菊酯抗性与酯酶和GST活性增高有关;桐柏地区中华按蚊的溴氰菊酯抗性与酯酶活性增高有关。