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Browsing by Author "Chen, Angela M."
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Item Comparison of porcine corneal decellularization methods and importance of preserving corneal limbus through decellularization(PLOS, 2021-03-05) Isidan, Abdulkadir; Liu, Shaohui; Chen, Angela M.; Zhang, Wenjun; Li, Ping; Smith, Lester J.; Hara, Hidetaka; Cooper, David K. C.; Ekser, Burcin; Surgery, School of MedicineBackground: The aim of this study is to compare the three previously applied, conventional porcine corneal decellularization methods and to demonstrate the importance of preserving the corneal limbus through decellularization. Methods: Fresh, wild-type (with or without) limbus porcine corneas were decellularized using three different methods, including (i) sodium dodecyl sulfate (SDS), (ii) hypertonic saline (HS), and (iii) N2 gas (NG). Post-treatment evaluation was carried out using histological, residual nuclear material, and ultrastructural analyses. Glycerol was used to help reduce the adverse effects of decellularization. The corneas were preserved for two weeks in cornea storage medium. Results: All three decellularization methods reduced the number of keratocytes at different rates in the stromal tissue. However, all methods, except SDS, resulted in the retention of large numbers of cells and cell fragments. The SDS method (0.1% SDS, 48h) resulted in almost 100% decellularization in corneas without limbus. Low decellularization capacity of the NG method (<50%) could make it unfavorable. Although HS method had a more balanced damage-decellularization ratio, its decellularization capacity was lower than SDS method. Preservation of the corneoscleral limbus could partially prevent structural damage and edema, but it would reduce the decellularization capacity. Conclusion: Our results suggest that SDS is a very powerful decellularization method, but it damages the cornea irreversibly. Preserving the corneoscleral limbus reduces the efficiency of decellularization, but also reduces the damage.Item DELAYED KIDNEY TRANSPLANTATION AFTER 83 HOURS OF COLD ISCHEMIA TIME IN COMBINED LIVER-KIDNEY TRANSPLANT(Wolters Kluwer, 2019-02) Ekser, Burcin; Chen, Angela M.; Kubal, Chandrashekhar A.; Fridell, Jonathan A.; Mihaylov, Plamen; Goggins, William C.; Powelson, John A.; Surgery, School of MedicineItem Differences in platelet aggregometers to study platelet function and coagulation dysregulation in xenotransplantation(Wiley, 2021-01) Isidan, Abdulkadir; Chen, Angela M.; Saglam, Kutay; Yilmaz, Sezai; Zhang, Wenjun; Li, Ping; Ekser, Burcin; Surgery, School of MedicineXenotransplantation (ie, cross-species transplantation) using genetically engineered pig organs could be a limitless source to solve the shortage of organs and tissues worldwide. However, despite prolonged survival in preclinical pig-to-nonhuman primate xenotransplantation trials, interspecies coagulation dysregulation remains to be overcome in order to achieve continuous long-term success. Different platelet aggregometry methods have been previously used to study the coagulation dysregulation with wild-type and genetically engineered pig cells, including the impact of possible treatment options. Among these methods, while thromboelastography and rotational thromboelastometry measure the change in viscoelasticity, optical aggregometry measures the change in opacity. Recently, impedance aggregometry has been used to measure changes in platelet aggregation in electrical conductance, providing more information to our understanding of coagulation dysregulation in xenotransplantation compared to previous methods. The present study reviews the merits and differences of the above-mentioned platelet aggregometers in xenotransplantation research.Item Genetic engineering of porcine endothelial cell lines for evaluation of human-to-pig xenoreactive immune responses(Springer Nature, 2021-06-23) Li, Ping; Walsh, Julia R.; Lopez, Kevin; Isidan, Abdulkadir; Zhang, Wenjun; Chen, Angela M.; Goggins, William C.; Higgins, Nancy G.; Liu, Jianyun; Brutkiewicz, Randy R.; Smith, Lester J.; Hara, Hidetaka; Cooper, David K.C.; Ekser, Burcin; Surgery, School of MedicineXenotransplantation (cross-species transplantation) using genetically-engineered pig organs offers a potential solution to address persistent organ shortage. Current evaluation of porcine genetic modifications is to monitor the nonhuman primate immune response and survival after pig organ xenotransplantation. This measure is an essential step before clinical xenotransplantation trials, but it is time-consuming, costly, and inefficient with many variables. We developed an efficient approach to quickly examine human-to-pig xeno-immune responses in vitro. A porcine endothelial cell was characterized and immortalized for genetic modification. Five genes including GGTA1, CMAH, β4galNT2, SLA-I α chain, and β2-microglobulin that are responsible for the production of major xenoantigens (αGal, Neu5Gc, Sda, and SLA-I) were sequentially disrupted in immortalized porcine endothelial cells using CRISPR/Cas9 technology. The elimination of αGal, Neu5Gc, Sda, and SLA-I dramatically reduced the antigenicity of the porcine cells, though the cells still retained their ability to provoke human natural killer cell activation. In summary, evaluation of human immune responses to genetically modified porcine cells in vitro provides an efficient method to identify ideal combinations of genetic modifications for improving pig-to-human compatibility, which should accelerate the application of xenotransplantation to humans.Item Organoids and Spheroids as Models for Studying Cholestatic Liver Injury and Cholangiocarcinoma(Wolters Kluwer, 2021) Sato, Keisaku; Zhang, Wenjun; Safarikia, Samira; Isidan, Abdulkadir; Chen, Angela M.; Li, Ping; Francis, Heather; Kennedy, Lindsey; Baiocchi, Leonardo; Alvaro, Domenico; Glaser, Shannon; Ekser, Burcin; Alpini, Gianfranco; Medicine, School of MedicineCholangiopathies, such as primary sclerosing cholangitis, biliary atresia, and cholangiocarcinoma, have limited experimental models. Not only cholangiocytes but also other hepatic cells including hepatic stellate cells and macrophages are involved in the pathophysiology of cholangiopathies, and these hepatic cells orchestrate the coordinated response against diseased conditions. Classic two-dimensional monolayer cell cultures do not resemble intercellular cell-to-cell interaction and communication; however, three-dimensional cell culture systems, such as organoids and spheroids, can mimic cellular interaction and architecture between hepatic cells. Previous studies have demonstrated the generation of hepatic or biliary organoids/spheroids using various cell sources including pluripotent stem cells, hepatic progenitor cells, primary cells from liver biopsies, and immortalized cell lines. Gene manipulation, such as transfection and transduction can be performed in organoids, and established organoids have functional characteristics which can be suitable for drug screening. This review summarizes current methodologies for organoid/spheroid formation and a potential for three-dimensional hepatic cell cultures as in vitro models of cholangiopathies.Item Oxygenation Profiles of Human Blood, Cell Culture Medium, and Water for Perfusion of 3D-Bioprinted Tissues using the FABRICA Bioreactor Platform(Nature Research, 2020-04-29) Chen, Angela M.; Lashmet, Matthew; Isidan, Abdulkadir; Sterner, Jane L.; Walsh, Julia; Koehler, Cutter; Li, Ping; Ekser, Burcin; Smith, Lester; Surgery, School of MedicinePersistent and saturated oxygen distribution from perfusion media (i.e., blood, or cell culture media) to cells within cell-dense, metabolically-active biofabricated tissues is required to keep them viable. Improper or poor oxygen supply to cells within the tissue bulk severely limits the tissue culturing potential of many bioreactors. We added an oxygenator module to our modular FABRICA bioreactor in order to provide stable oxygenation to biofabricated tissues during culture. In this proof of concept study of an oxygenated and perfused bioreactor, we characterized the oxygenation of water, cell culture medium, and human blood in the FABRICA as functions of augmenting vacuum (air inlet) pressure, perfusion (volumetric flow) rate, and tubing/oxygenator components. The mean oxygen levels for water and cell culture media were 27.7 ± 2.1% and 27.6 ± 4.1%, respectively. The mean oxygen level for human blood was 197.0 ± 90.0 mmHg, with near-physiologic levels achieved with low-permeability PharMed tubing alone (128.0 ± 14.0 mmHg). Hematologic values pre- and post-oxygenation, respectively were (median ± IQR): Red blood cell: 6.0 ± 0.5 (106/μL) and 6.5 ± 0.4 (106/μL); Hemoglobin: 17.5 ± 1.2 g/dL and 19.2 ± 3.0 g/dL; and Hematocrit: 56.7 ± 2.4% and 61.4 ± 7.5%. The relative stability of the hematologic parameters indicates that blood function and thus blood cell integrity were maintained throughout oxygenation. Already a versatile research tool, the now oxygenated FABRICA provides easy-to-implement, in vivo-like perfusion and stable oxygenation culture conditions in vitro semi-independently of one another, which means the bioreactor has the potential to serve as a platform for investigating the behavior of 3D tissue models (regardless of biofabrication method), performing drug toxicity-testing, and testing pharmaceutical efficacy/safety.Item Oxygenation Profiles of Human Blood, Cell Culture Medium, and Water for Perfusion of 3D-Bioprinted Tissues using the FABRICA Bioreactor Platform(Nature, 2020-04-29) Chen, Angela M.; Lashmet, Matthew; Isidan, Abdulkadir; Sterner, Jane L.; Walsh, Julia; Koehler, Cutter; Li, Ping; Ekser, Burcin; Smith, Lester; Surgery, School of MedicinePersistent and saturated oxygen distribution from perfusion media (i.e., blood, or cell culture media) to cells within cell-dense, metabolically-active biofabricated tissues is required to keep them viable. Improper or poor oxygen supply to cells within the tissue bulk severely limits the tissue culturing potential of many bioreactors. We added an oxygenator module to our modular FABRICA bioreactor in order to provide stable oxygenation to biofabricated tissues during culture. In this proof of concept study of an oxygenated and perfused bioreactor, we characterized the oxygenation of water, cell culture medium, and human blood in the FABRICA as functions of augmenting vacuum (air inlet) pressure, perfusion (volumetric flow) rate, and tubing/oxygenator components. The mean oxygen levels for water and cell culture media were 27.7 ± 2.1% and 27.6 ± 4.1%, respectively. The mean oxygen level for human blood was 197.0 ± 90.0 mmHg, with near-physiologic levels achieved with low-permeability PharMed tubing alone (128.0 ± 14.0 mmHg). Hematologic values pre- and post-oxygenation, respectively were (median ± IQR): Red blood cell: 6.0 ± 0.5 (106/μL) and 6.5 ± 0.4 (106/μL); Hemoglobin: 17.5 ± 1.2 g/dL and 19.2 ± 3.0 g/dL; and Hematocrit: 56.7 ± 2.4% and 61.4 ± 7.5%. The relative stability of the hematologic parameters indicates that blood function and thus blood cell integrity were maintained throughout oxygenation. Already a versatile research tool, the now oxygenated FABRICA provides easy-to-implement, in vivo-like perfusion and stable oxygenation culture conditions in vitro semi-independently of one another, which means the bioreactor has the potential to serve as a platform for investigating the behavior of 3D tissue models (regardless of biofabrication method), performing drug toxicity-testing, and testing pharmaceutical efficacy/safety.Item PUBLISHER CORRECTION: Genetic engineering of porcine endothelial cell lines for evaluation of human-to-pig xenoreactive immune responses(Springer Nature, 2021-08-16) Li, Ping; Walsh, Julia R.; Lopez, Kevin; Isidan, Abdulkadir; Zhang, Wenjun; Chen, Angela M.; Goggins, William C.; Higgins, Nancy G.; Liu, Jianyun; Brutkiewicz, Randy R.; Smith, Lester J.; Hara, Hidetaka; Cooper, David K.C.; Ekser, Burcin; Surgery, School of MedicineItem Use of PET-CT Imaging to Identify Femoral–Popliteal Stent Infection(Sage, 2019-03) Chen, Angela M.; Serafin, Emily M.; Motaganahalli, Raghu L.; Surgery, School of Medicine