Online citations, reference lists, and bibliographies.
Please confirm you are human
(Sign Up for free to never see this)
← Back to Search

3D Co-Culture With Vascular Cells Supports Long-Term Hepatocyte Phenotype And Function In Vitro

Asli Z. Unal, Sydney E. Jeffs, J. West
Published 2018 · Chemistry

Save to my Library
Download PDF
Analyze on Scholarcy
Share
Progress in liver tissue engineering depends on the ability to reliably culture hepatocytes in vitro. In this study, we designed an environment that can help support liver-specific functions of primary hepatocytes for long enough periods of time to use them in therapeutic devices and in vitro modeling. This was accomplished by encapsulating hepatocytes with endothelial cells (ECs) and pericytes in a cell-adhesive and enzymatically degradable poly(ethylene glycol) (PEG) hydrogel scaffold. We used these hydrogels to investigate the long-term effects of 3D co-culture of hepatocytes with non-parenchymal ECs and pericytes in the context of vascular networks. We found that 3D co-culture of hepatocytes with tubule-forming ECs and pericytes leads to the development of robust microvascular tubules with close approximation to hepatocytes, similar to structures found in vivo. Furthermore, hepatocytes help support vasculogenesis and long-term tubule stability. We show that 3D co-culture of hepatocytes with cells in these networks enhances and retains hepatocyte function and phenotype. Hepatocytes in our constructs were able to synthesize 6.3 ± 1.1 pg albumin/hepatocyte/day at day 7 and increased over 3-fold to 22.6 ± 1.5 pg albumin/hepatocyte/day by day 28. Other essential liver functions, like urea secretion and cytochrome P450 activity, were also retained in our constructs for at least 4 weeks. Finally, we found that some of the hepatocytes in these organoids expressed the proliferation marker, Ki-67. These results are promising for the development of new liver disease treatments, organ engineering, and drug-testing models.Lay SummaryHepatocytes are the parenchymal cells of the liver, and they are responsible for performing many essential liver functions. When they are isolated from the body and cultured without the correct biochemical cues, they tend to lose their liver-specific functions and dedifferentiate into fibroblast-like cells. Advancement in tissue engineering liver disease therapies and in vitro drug-testing models depends on the development of hepatocyte culture methods that can maintain hepatocyte phenotype and function. Here, we tailored a polymeric hydrogel in which we can 3D co-culture hepatocytes with stabilizing non-parenchymal cells and achieve organ-level albumin synthesis as well maintenance of urea secretion and cytochrome P450 enzymatic activity for at least a month.
This paper references
10.2144/000113317
Layered patterning of hepatocytes in co-culture systems using microfabricated stencils.
C. H. Cho (2010)
10.1002/BIT.20585
Laser-guided direct writing for three-dimensional tissue engineering.
Y. Nahmias (2005)
10.1006/EXCR.1993.1266
Effect of collagen gel configuration on the cytoskeleton in cultured rat hepatocytes.
R. Ezzell (1993)
10.1111/j.1749-6632.1980.tb29518.x
PHENOTYPIC STABILITY OF ADULT RAT HEPATOCYTES IN PRIMARY MONOLAYER CULTURE *
D. Bissell (1980)
Hepatocyte growth factor (HGF) stimulates the tyrosine kinase activity of the receptor encoded by the proto-oncogene c-MET.
L. Naldini (1991)
10.1039/c6tx00101g
Characterization of a functional C3A liver spheroid model† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6tx00101g
H. Gaskell (2016)
10.1021/bp980036j
Microfabrication of Hepatocyte/Fibroblast Co‐cultures: Role of Homotypic Cell Interactions
S. Bhatia (1998)
10.1111/J.1525-1594.1996.TB00725.X
Function of culturing monolayer hepatocytes by collagen gel coating and coculture with nonparenchymal cells.
M. Koike (1996)
10.1002/(SICI)1097-0215(20000215)85:4<563::AID-IJC19>3.0.CO;2-D
Nk4, a new HGF/SF variant, is an antagonist to the influence of HGF/SF on the motility and invasion of colon cancer cells
C. Parr (2000)
10.1002/adbi.201600021
Macrophages Influence Vessel Formation in 3D Bioactive Hydrogels
Erika M Moore (2017)
10.1021/bm401926k
Hepatocyte cocultures with endothelial cells and fibroblasts on micropatterned fibrous mats to promote liver-specific functions and capillary formation capabilities.
Y. Liu (2014)
10.1016/J.BIOMATERIALS.2006.08.043
Assessment of hepatocellular function within PEG hydrogels.
G. H. Underhill (2007)
10.1038/srep13079
Decellularized human liver as a natural 3D-scaffold for liver bioengineering and transplantation
G. Mazza (2015)
10.1021/acsbiomaterials.5b00064
Encoding Hydrogel Mechanics via Network Cross-Linking Structure
Ryan M. Schweller (2015)
10.1002/ADFM.201200976
Integration of Self-Assembled Microvascular Networks with Microfabricated PEG-Based Hydrogels.
Michael P. Cuchiara (2012)
10.1016/j.addr.2015.11.004
Studying the influence of angiogenesis in in vitro cancer model systems.
L. C. Roudsari (2016)
10.1016/0014-4827(75)90532-7
Primary culture of parenchymal liver cells on collagen membranes. Morphological and biochemical observations.
G. Michalopoulos (1975)
10.1089/TEN.1999.5.407
Polymer substrate topography actively regulates the multicellular organization and liver-specific functions of cultured hepatocytes.
C. Ranucci (1999)
10.1161/01.HYP.33.6.1379
Therapeutic angiogenesis induced by human recombinant hepatocyte growth factor in rabbit hind limb ischemia model as cytokine supplement therapy.
R. Morishita (1999)
10.1038/nrd1851
Predicting in vivo drug interactions from in vitro drug discovery data
L. Wienkers (2005)
10.1016/j.biomaterials.2010.01.104
Biomimetic hydrogels with pro-angiogenic properties.
J. Moon (2010)
10.1016/J.CBI.2007.01.001
Human hepatocytes: isolation, cryopreservation and applications in drug development.
A. Li (2007)
10.1016/J.BIOMATERIALS.2005.02.027
Fabrication of viable tissue-engineered constructs with 3D cell-assembly technique.
Yongnian Yan (2005)
10.1089/TEN.2006.12.1627
Endothelium-mediated hepatocyte recruitment in the establishment of liver-like tissue in vitro.
Y. Nahmias (2006)
10.1038/onc.2016.421
Blood vessel endothelium-directed tumor cell streaming in breast tumors requires the HGF/C-Met signaling pathway
E. Leung (2017)
10.1136/gut.2005.069153
Diagnosis of cirrhosis by transient elastography (FibroScan): a prospective study
J. Foucher (2006)
10.1096/fasebj.3.2.2914628
Hepatocyte function and extracellular matrix geometry: long‐term culture in a sandwich configuration
J. Dunn (1989)
10.1038/srep32726
A 3D Poly(ethylene glycol)-based Tumor Angiogenesis Model to Study the Influence of Vascular Cells on Lung Tumor Cell Behavior
L. Roudsari (2016)
10.1111/j.1440-1746.2012.07137.x
Therapeutic potential of mesenchymal stem cells overexpressing human forkhead box A2 gene in the regeneration of damaged liver tissues
Jong-Woo Cho (2012)
10.1001/ARCHSURG.135.11.1334
Dedifferentiation of human hepatocellular carcinoma up-regulates telomerase and Ki-67 expression.
T. Yeh (2000)
10.1152/AJPCELL.1994.266.6.C1764
Formation of extensive canalicular networks by rat hepatocytes cultured in collagen-sandwich configuration.
E. Lecluyse (1994)
10.1016/j.biomaterials.2010.01.068
Heparin-based hydrogel as a matrix for encapsulation and cultivation of primary hepatocytes.
Mihye Kim (2010)
10.2174/156802608783790983
Vascularization of engineered tissues: approaches to promote angio-genesis in biomaterials.
J. Moon (2008)
10.1002/JBM.10228
Novel approach for achieving double-layered cell sheets co-culture: overlaying endothelial cell sheets onto monolayer hepatocytes utilizing temperature-responsive culture dishes.
M. Harimoto (2002)
10.1073/PNAS.84.18.6379
Sequence of MET protooncogene cDNA has features characteristic of the tyrosine kinase family of growth-factor receptors.
M. Park (1987)
10.1038/srep25187
Characterization of primary human hepatocyte spheroids as a model system for drug-induced liver injury, liver function and disease
Catherine C. Bell (2016)
10.1006/EXCR.1996.0222
3-D coculture of hepatic sinusoidal cells with primary hepatocytes-design of an organotypical model.
A. Bader (1996)
10.1096/fasebj.9.15.8529831
Role of growth factors and cytokines in hepatic regeneration
N. Fausto (1995)
10.1242/dev.142794
Paracrine signals regulate human liver organoid maturation from induced pluripotent stem cells
Akihiro Asai (2017)
10.1152/ajpheart.00321.2009
Synergistic effects of autologous cell and hepatocyte growth factor gene therapy for neovascularization in a murine model of hindlimb ischemia.
Y. Yamamoto (2009)
10.1161/01.CIR.97.4.381
Potentiated angiogenic effect of scatter factor/hepatocyte growth factor via induction of vascular endothelial growth factor: the case for paracrine amplification of angiogenesis.
É. Van Belle (1998)
10.1387/IJDB.052072HB
Differentiation of human embryonic stem cells into hepatocytes in 2D and 3D culture systems in vitro.
H. Baharvand (2006)
10.1083/JCB.116.4.1043
Hepatocytes in collagen sandwich: evidence for transcriptional and translational regulation
J. Dunn (1992)
10.1016/J.BIOMATERIALS.2007.03.004
Poly(ethylene glycol) hydrogels conjugated with a collagenase-sensitive fluorogenic substrate to visualize collagenase activity during three-dimensional cell migration.
S. Lee (2007)
10.1371/journal.pone.0090571
Hepatocyte Produced Matrix Metalloproteinases Are Regulated by CD147 in Liver Fibrogenesis
S. Calabro (2014)
10.1038/nm.2170
Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix
Basak E Uygun (2010)
10.1007/s00204-013-1078-5
Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME
P. Godoy (2013)
10.1002/MABI.200600205
Patterned co-culture of primary hepatocytes and fibroblasts using polyelectrolyte multilayer templates.
Srivatsan Kidambi (2007)
10.1016/j.pharmthera.2012.12.007
Cytochrome P450 enzymes in drug metabolism: regulation of gene expression, enzyme activities, and impact of genetic variation.
U. Zanger (2013)
10.1507/ENDOCRJ.KR-113
Activin A: autocrine regulator of kidney development and repair.
A. Maeshima (2008)
10.1126/SCIENCE.1846706
Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product.
D. Bottaro (1991)
10.1038/nprot.2006.386
Micropatterning of living cells by laser-guided direct writing: application to fabrication of hepatic–endothelial sinusoid-like structures
Y. Nahmias (2006)
10.1089/10763270152044206
Liver tissue engineering: a role for co-culture systems in modifying hepatocyte function and viability.
R. Bhandari (2001)
10.1016/j.jmbbm.2015.10.016
Stiffness of hyaluronic acid gels containing liver extracellular matrix supports human hepatocyte function and alters cell morphology.
Daniel B. Deegan (2015)
10.1002/(SICI)1097-4636(199802)39:2<266::AID-JBM14>3.0.CO;2-B
Incorporation of adhesion peptides into nonadhesive hydrogels useful for tissue resurfacing.
D. Hern (1998)
10.1016/S0735-1097(03)00231-6
Obesity is associated with increased levels of circulating hepatocyte growth factor.
J. Rehman (2003)
10.1016/S0142-9612(02)00176-X
Photoencapsulation of osteoblasts in injectable RGD-modified PEG hydrogels for bone tissue engineering.
J. Burdick (2002)
10.1038/nm0603-661
Endothelial signaling during development
O. Cleaver (2003)
Hepatocyte growth factor increases expression of vascular endothelial growth factor and plasminogen activator inhibitor-1 in human keratinocytes and the vascular endothelial growth factor receptor flk-1 in human endothelial cells.
J. Wojta (1999)
10.1089/tea.2007.0143
Three-dimensional primary hepatocyte culture in synthetic self-assembling peptide hydrogel.
Sihong Wang (2008)
10.1096/fasebj.13.14.1883
Effect of cell–cell interactions in preservation of cellular phenotype: cocultivation of hepatocytes and nonparenchymal cells
S. Bhatia (1999)
10.1083/JCB.119.3.629
Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth
F. Bussolino (1992)
10.1093/OXFORDJOURNALS.JBCHEM.A124814
Hepatocyte growth factor stimulates liver regeneration and elevates blood protein level in normal and partially hepatectomized rats.
T. Ishii (1995)
10.1088/1758-5090/aa70c7
3D liver membrane system by co-culturing human hepatocytes, sinusoidal endothelial and stellate cells.
H. Ahmed (2017)
10.1089/ten.TEB.2009.0085
Vascularization strategies for tissue engineering.
Michael L. Lovett (2009)
10.1002/(SICI)1097-4636(199702)34:2<189::AID-JBM8>3.0.CO;2-M
Controlling cell interactions by micropatterning in co-cultures: hepatocytes and 3T3 fibroblasts.
S. Bhatia (1997)
10.1007/978-1-62703-125-7_17
Long-term culture and coculture of primary rat and human hepatocytes.
Maria Shulman (2013)
10.1111/cas.12581
Multitargeting strategy using lenvatinib and golvatinib: Maximizing anti-angiogenesis activity in a preclinical cancer model
Y. Nakazawa (2015)
10.1126/scitranslmed.3005975
Cell and tissue engineering for liver disease
S. Bhatia (2014)
10.2450/2008.0080-08
Clinical use of albumin in hepatology.
M. Garcovich (2009)
10.1016/S0002-9440(10)64058-8
Hepatocyte growth factor enhances vascular endothelial growth factor-induced angiogenesis in vitro and in vivo.
X. Xin (2001)
10.1126/SCIENCE.1063889
Liver Organogenesis Promoted by Endothelial Cells Prior to Vascular Function
K. Matsumoto (2001)
10.1021/BP00009A007
Long‐Term in Vitro Function of Adult Hepatocytes in a Collagen Sandwich Configuration
J. Dunn (1991)
10.1089/ten.TEC.2014.0152
Long-term coculture strategies for primary hepatocytes and liver sinusoidal endothelial cells.
S. Bale (2015)
10.1016/0968-0004(91)90096-E
Arginyl-glycyl-aspartic acid (RGD): a cell adhesion motif.
S. D'Souza (1991)
10.1016/0076-6879(82)82086-7
Collagen as a substrate for cell growth and differentiation.
S. Strom (1982)
10.1148/radiol.14131251
Determination of normal hepatic elasticity by using real-time shear-wave elastography.
C. Suh (2014)
10.3748/WJG.V10.I5.699
Primary hepatocyte culture in collagen gel mixture and collagen sandwich.
Y. Wang (2004)
10.1002/bit.21911
A new technique for primary hepatocyte expansion in vitro
C. H. Cho (2008)
10.1016/0270-9139(91)90068-7
Hepatocyte growth factor receptor and the c-met oncogene Bottaro DP, Rubin JS, Faletto DL, Chan AM-L, Kmiecik TE, Vande Woude GF, Aaronson SA. Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product. Science 1991;251:802?804
N. Fausto (1991)
10.1182/BLOOD-2002-06-1731
HGF receptor up-regulation contributes to the angiogenic phenotype of human endothelial cells and promotes angiogenesis in vitro.
S. Ding (2003)
10.1089/biores.2013.0021
Immobilization of Cell-Adhesive Laminin Peptides in Degradable PEGDA Hydrogels Influences Endothelial Cell Tubulogenesis
S. Ali (2013)
10.1089/ten.TEA.2012.0681
Characterizing the effects of heparin gel stiffness on function of primary hepatocytes.
J. You (2013)



This paper is referenced by
Semantic Scholar Logo Some data provided by SemanticScholar