Characterization of isolated liver sinusoidal endothelial cells for liver bioengineering
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The liver sinusoidal capillaries play a pivotal role in liver regeneration, suggesting they may be beneficial in liver bioengineering. This study isolated mouse liver sinusoidal endothelial cells (LSECs) and determined their ability to form capillary networks in vitro and in vivo for liver tissue engineering purposes.
Methods and results
In vitro LSECs were isolated from adult C57BL/6 mouse livers. Immunofluorescence labelling indicated they were LYVE-1+/CD32b+/FactorVIII+/CD31−. Scanning electron microscopy of LSECs revealed the presence of characteristic sieve plates at 2 days. LSECs formed tubes and sprouts in the tubulogenesis assay, similar to human microvascular endothelial cells (HMEC); and formed capillaries with lumens when implanted in a porous collagen scaffold in vitro. LSECs were able to form spheroids, and in the spheroid gel sandwich assay produced significantly increased numbers (p = 0.0011) of capillary-like sprouts at 24 h compared to HMEC spheroids. Supernatant from LSEC spheroids demonstrated significantly greater levels of vascular endothelial growth factor-A and C (VEGF-A, VEGF-C) and hepatocyte growth factor (HGF) compared to LSEC monolayers (p = 0.0167; p = 0.0017; and p < 0.0001, respectively), at 2 days, which was maintained to 4 days for HGF (p = 0.0017) and VEGF-A (p = 0.0051). In vivo isolated mouse LSECs were prepared as single cell suspensions of 500,000 cells, or as spheroids of 5000 cells (100 spheroids) and implanted in SCID mouse bilateral vascularized tissue engineering chambers for 2 weeks. Immunohistochemistry identified implanted LSECs forming LYVE-1+/CD31− vessels. In LSEC implanted constructs, overall lymphatic vessel growth was increased (not significantly), whilst host-derived CD31+ blood vessel growth increased significantly (p = 0.0127) compared to non-implanted controls. LSEC labelled with the fluorescent tag DiI prior to implantation formed capillaries in vivo and maintained LYVE-1 and CD32b markers to 2 weeks.
Isolated mouse LSECs express a panel of vascular-related cell markers and demonstrate substantial vascular capillary-forming ability in vitro and in vivo. Their production of liver growth factors VEGF-A, VEGF-C and HGF enable these cells to exert a growth stimulus post-transplantation on the in vivo host-derived capillary bed, reinforcing their pro-regenerative capabilities for liver tissue engineering studies.
KeywordsLiver sinusoidal endothelial cells Cell marker characteristics In vitro angiogenesis assays Spheroid formation Growth factor production In vivo implantation into a vascularized tissue engineering chamber
The authors acknowledge the assistance of the Experimental Medical and Surgical Unit (Sue Mc Kay, Anna Deftereos, Liliana Pepe, and Amanda Rixon) at St Vincent’s Hospital Melbourne. Assoc/Prof Alice Pébay (Centre for Eye Research Australia) who provided the cytospin equipment; and Dr Guei-Sheung Liu (Centre for Eye Research Australia), Prof Shyh-Ming Kuo (Department of Biomedical Engineering, I-Shou University, Kaohsiung, Taiwan) and Dr Shiang Lim (O’Brien Institute Department of St Vincent’s Institute, Melbourne) who supplied the porous collagen scaffolds. The authors also gratefully acknowledge the facilities of the Centre for Microscopy, Characterization and Analysis at The University of Western Australia, and the scientific and technical assistance of Ms. Lyn Kirilak and Associate Professor Peta Clode for the scanning electron microscope image used in this publication. We also thank Mr. Phil Francis and Dr. Chaitali Dekiwadia for preliminary work at the RMIT Microscopy and Microanalysis Facility, and Mr Jonathan Clarke, Centre for Eye Research Australia for assistance with the fluorescence microscopy.
This research was funded by National Health and Medical Research Council of Australia Project Grants (1023187 and 1125233); St Vincent’s Hospital Melbourne, Research Endowment Fund; the Australian Catholic University; the Stafford Fox Foundation, Australia; and the Victorian State Government’s Department of Innovation, Industry and Regional Development’s Operational Infrastructure Support Program. AMD was supported by an Australian Post Graduate Award. KKY is supported by scholarships from the National Health and Medical Research Council of Australia; Australia and New Zealand Hepatic, Pancreatic, and Biliary Association; and St Vincent’s Institute Foundation.
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