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Characterization of isolated liver sinusoidal endothelial cells for liver bioengineering
Aaron M. Dingle Kiryu K. Yap Yi-Wen Gerrand Carolyn Taylor Efthimia Keramidaris Zerina Lokmic A.M. Kong H.L. Peters Wayne Morrison Geraldine Mitchell
Aaron M. Dingle
Kiryu K. Yap
Yi-Wen Gerrand
Carolyn Taylor
Efthimia Keramidaris
Zerina Lokmic
A.M. Kong
H.L. Peters
Wayne Morrison
Geraldine Mitchell
Abstract
Background
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.
Conclusion
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.
Keywords
liver sinusoidal endothelial cells, cell marker characteristics, in vitro angiogenesis assays, spheroid formation, growth factor production, in vivo implantation into a vascularized tissue engineering chamber
Date
2018
Type
Journal article
Journal
Angiogenesis
Book
Volume
21
Issue
3
Page Range
581-597
Article Number
ACU Department
School of Behavioural and Health Sciences
Faculty of Health Sciences
School of Nursing, Midwifery and Paramedicine
Faculty of Health Sciences
School of Nursing, Midwifery and Paramedicine
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Source URL
Event URL
Open Access Status
License
File Access
Controlled