ObjectiveTo explore the suitable method for isolation and maintenance of primary cultures of human gallbladder epithelial cells (GECs) for establishing the basis of research works in physiological function of gallbladder and its related diseases.MethodsGECs were isolated with collagenase type Ⅳ and blunt separation.The dishes were coated with fibronectin, laminin and polyDlysine respectively.Additional 10 ng/ml epidermal growth factor was added to DMEM medium containing 20% fetal calf serum.The cells were studied under light and electron microscope to determine their shape and distribution.ResultsEach gallbladder yielded approximately (1-5)×107columnar epithelial cells,greater than 95% of which were viable by trypan blue exclusion.The cells grew vigorously within one week which was flat and multangular in shape. CK19 expressed positive.Electron microscope showed typical gallbladder epithelia with microvilli,tight junctions and mucus droplets.ConclusionCombination of collagenase type Ⅳ，mechanical blunt separation and twostep attachment is of great benefit for separating and harvesting GEC.Fibronectin coated culture dish and DMEM medium containing 20% calf serum and 10 ng/ml hEGF is of great benefit for culturing gallbladder epithelial cells.
Objective To investigate inter-observer reproducibility in the pathologic diagnosis of breast intraductal proliferative lesions (BDPL). Methods Forty three BDPL patients were diagnosed by criterion of Page. Every specimen from each case was sorted randomly. All slides were classified as mild usual hyperplasia, moderate-severe usual hyperplasia, mild atypical hyperplasia, moderate-severe atypical hyperplasia, ductal carcinoma in situ, or ductal carcinoma in situ with invasion. Inter-observer agreement of the two groups was statistically analyzed using Kappa test. Then we compared all the diagnoses of individual pathologist with the consensus opinion confirmed by two breast pathologists to analyze the diagnostic accuracy and undue diagnosis. Results Inter-observer reproducibility of the trial group was higher than that of the control group (The total K value of 6, 3, and 2 diagnoses in the two groups were 0.289 3, 0.337 1, 0.492 8, 0.100 3, 0.150 3 and 0.340 3, respectively). When the categories were simplified, inter-observer reproducibility increased. There were still undue diagnoses of different degrees among pathologists of the trial group. Conclusion Using the same criteria is an important method to increase the diagnostic reproducibility and accuracy. More practice is needed to familiarize with these criteria.
In order to enhance the anticoagulant properties of decellularized biological materials as scaffolds for tissue engineering research via heparinized process, the decellularized porcine liver scaffolds were respectively immobilized with heparin through layer-by-layer self-assembly technique (LBL), multi-point attachment (MPA) or end-point attachment (EPA). The effects of heparinization and anticoagulant ability were tested. The results showed that the three different scaffolds had different contents of heparin. All the three kinds of heparinized scaffolds gained better performance of anticoagulant than that of the control scaffold. The thrombin time (TT), prothrombin time (PT) and activated partial thromboplastin time (APTT) of EPA scaffold group were longest in all the groups, and all the three times exceeded the measurement limit of the instrument. In addition, EPA scaffolds group showed the shortest prepared time, the slowest speed for heparin release and the longest recalcification time among all the groups. The decellularized biological materials for tissue engineering acquire the best effect of anticoagulant ability in vitro via EPA heparinized technique.
The emergence of regular short repetitive palindromic sequence clusters (CRISPR) and CRISPR- associated proteins 9 (Cas9) gene editing technology has greatly promoted the wide application of genetically modified pigs. Efficient single guide RNA (sgRNA) is the key to the success of gene editing using CRISPR/Cas9 technology. For large animals with a long reproductive cycle, such as pigs, it is necessary to screen out efficient sgRNA in vitro to avoid wasting time and resource costs before animal experiments. In addition, how to efficiently obtain positive gene editing monoclonal cells is a difficult problem to be solved. In this study, a rapid sgRNA screening method targeting the pig genome was established and we rapidly obtained Fah gene edited cells, laying a foundation for the subsequent production of Fah knockout pigs as human hepatocyte bioreactor. At the same time, the method of obtaining monoclonal cells using pattern microarray culture technology was explored.