Biology subjects

Ekser, B.

Publications and source records attributed to Ekser, B..

2 recordsLinked to original sources

bioRxiv2022

Development of Scaffold-free 3D Cholangiocyte Organoids to Study the Progression of Primary Sclerosing Cholangitis

Organoids are novel in vitro models to study intercellular crosstalk between the different types of cells in the pathophysiology of disease. To better understand the underlying mechanisms driving the progression of primary sclerosing cholangitis (PSC), we developed scaffold-free multi-cellular 3D cholangiocyte organoids (3D-CHO) using primary liver cell lines derived from normal and PSC patients. Human liver samples from healthy donors and late-stage PSC patients were used to isolate primary cholangiocytes (EPCAM+/CK-19+), liver endothelial cells (LECs, CD31+), and hepatic stellate cells (HSCs, CD31-/CD68-/Desmin+/Vitamin A+). 3D-CHOs were formed using cholangiocytes:HSCs:LECs and kept viable for up to 1 month. Isolated primary cell lines and 3D-CHOs were further characterized by immunofluorescence (IF), qRT-PCR, and transmission electron microscopy. Gene expressions for cholangiocytes (SOX9, CFTR, EpCAM, AE, SCT, SCTR), fibrosis (ACTA2, COL1A1, DESMIN, TGF{beta}1), angiogenesis (PECAM, VEGF, CDH5, vWF), and inflammation (IL-6, TNF-) confirmed PSC phenotypes of 3D-CHOs. Since cholangiocytes develop a neuroendocrine phenotype and express neuromodulators, confocal-IF demonstrated that neurokinin-1 receptor (NK-1R, expressed by cholangiocytes and upregulated in PSC), was localized within CK-19+ cholangiocytes. Moreover, 3D-CHOs from PSC patients confirmed PSC phenotypes with upregulated NK-1R, tachykinin precursor 1, and downregulated membrane metalloendopeptidase. Our viable scaffold-free multiple-cell 3D-CHOs showed superiority as an in vitro model in mimicking PSC in vivo phenotypes compared to 2D cell culture, which can be used in PSC disease-related research.

bioengineering↗

bioRxiv2020

Comparison of porcine corneal decellularization methods and importance of preserving corneal limbus through decellularization

BackgroundThe 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. MethodsFresh, 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. ResultsAll 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. ConclusionOur 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.

bioengineering↗