3 research outputs found
Scalable Spheroid Model of Human Hepatocytes for Hepatitis C Infection and Replication
Developing effective new drugs against
hepatitis C (HCV) virus
has been challenging due to the lack of appropriate small animal and <i>in vitro</i> models recapitulating the entire life cycle of
the virus. Current <i>in vitro</i> models fail to recapitulate
the complexity of human liver physiology. Here we present a method
to study HCV infection and replication on spheroid cultures of Huh
7.5 cells and primary human hepatocytes. Spheroid cultures are constructed
using a galactosylated cellulosic sponge with homogeneous macroporosity,
enabling the formation and maintenance of uniformly sized spheroids.
This facilitates easy handling of the tissue-engineered constructs
and overcomes limitations inherent of traditional spheroid cultures.
Spheroids formed in the galactosylated cellulosic sponge show enhanced
hepatic functions in Huh 7.5 cells and maintain liver-specific functions
of primary human hepatocytes for 2 weeks in culture. Establishment
of apical and basolateral polarity along with the expression and localization
of all HCV specific entry proteins allow for a 9-fold increase in
viral entry in spheroid cultures over conventional monolayer cultures.
Huh 7.5 cells cultured in the galactosylated cellulosic sponge also
support replication of the HCV clone, JFH (Japanese fulminant hepatitis)-1
at higher levels than in monolayer cultures. The advantages of our
system in maintaining liver-specific functions and allowing HCV infection
together with its ease of handling make it suitable for the study
of HCV biology in basic research and pharmaceutical R&D
Scalable Spheroid Model of Human Hepatocytes for Hepatitis C Infection and Replication
Developing effective new drugs against
hepatitis C (HCV) virus
has been challenging due to the lack of appropriate small animal and <i>in vitro</i> models recapitulating the entire life cycle of
the virus. Current <i>in vitro</i> models fail to recapitulate
the complexity of human liver physiology. Here we present a method
to study HCV infection and replication on spheroid cultures of Huh
7.5 cells and primary human hepatocytes. Spheroid cultures are constructed
using a galactosylated cellulosic sponge with homogeneous macroporosity,
enabling the formation and maintenance of uniformly sized spheroids.
This facilitates easy handling of the tissue-engineered constructs
and overcomes limitations inherent of traditional spheroid cultures.
Spheroids formed in the galactosylated cellulosic sponge show enhanced
hepatic functions in Huh 7.5 cells and maintain liver-specific functions
of primary human hepatocytes for 2 weeks in culture. Establishment
of apical and basolateral polarity along with the expression and localization
of all HCV specific entry proteins allow for a 9-fold increase in
viral entry in spheroid cultures over conventional monolayer cultures.
Huh 7.5 cells cultured in the galactosylated cellulosic sponge also
support replication of the HCV clone, JFH (Japanese fulminant hepatitis)-1
at higher levels than in monolayer cultures. The advantages of our
system in maintaining liver-specific functions and allowing HCV infection
together with its ease of handling make it suitable for the study
of HCV biology in basic research and pharmaceutical R&D
Functionally Enhanced Human Stem Cell Derived Hepatocytes in Galactosylated Cellulosic Sponges for Hepatotoxicity Testing
Pluripotent
stem cell derived hepatocyte-like cells (hPSC-HLCs)
are an attractive alternative to primary human hepatocytes (PHHs)
used in applications ranging from therapeutics to drug safety testing
studies. It would be critical to improve and maintain mature hepatocyte
functions of the hPSC-HLCs, especially for long-term studies. If 3D
culture systems were to be used for such purposes, it would be important
that the system can support formation and maintenance of optimal-sized
spheroids for long periods of time, and can also be directly deployed
in liver drug testing assays. We report the use of 3-dimensional (3D)
cellulosic scaffold system for the culture of hPSC-HLCs. The scaffold
has a macroporous network which helps to control the formation and
maintenance of the spheroids for weeks. Our results show that culturing
hPSC-HLCs in 3D cellulosic scaffolds increases functionality, as demonstrated
by improved urea production and hepatic marker expression. In addition,
hPSC-HLCs in the scaffolds exhibit a more mature phenotype, as shown
by enhanced cytochrome P450 activity and induction. This enables the
system to show a higher sensitivity to hepatotoxicants and a higher
degree of similarity to PHHs when compared to conventional 2D systems.
These results suggest that 3D cellulosic scaffolds are ideal for the
long-term cultures needed to mature hPSC-HLCs. The mature hPSC-HLCs
with improved cellular function can be continually maintained in the
scaffolds and directly used for hepatotoxicity assays, making this
system highly attractive for drug testing applications