BME 2 RGF Organoid Matrix Enables Long-Term Culture of Liver Organoids
Background:
AMSBIO has been working with the variability of the cellular microenvironment and how it affects the physiological relevance of cell culture. Factors contributing to this variability include: organ specific stromal cells,
growth factors, proteoglycan and protein composition, and stiffness or tensile strength of the basement membrane extract or extracellular matrix. Matrices from AMSBIO not only support cells and cell layers, but also
play an essential role in tissue organization that affects cell adhesion, migration, proliferation, and differentiation.
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These aspects can be studied with kits from AMSBIO incorporating their wealth of experience in this area. Cultrex® Basement Membrane Extract (BME) is a soluble form of basement membrane purified from
Engelbreth-Holm-Swarm (EHS) tumour. The extract gels at 37°C to form a reconstituted basement membrane. Major components of BME include
laminin,
collagen IV, entactin, and heparan sulfate proteoglycan.
These extracted proteins can be used in multiple applications, under a variety of cell culture conditions, for maintaining growth or promoting differentiation of primary endothelial, epithelial, smooth muscle and
stem cells. BME can also be utilized in cell attachment, neurite outgrowth, angiogenesis,
in vitro cell invasion and in vivo tumorigenicity assays. The new BME 2 is a proprietary formulation that has a
higher tensile strength than our original BME 1.
"We have obtained culture conditions that allow us to long-term expand genetically stable human donor liver cells in organoid culture. One of the clues to this success is the use of ECM that allows the cells to grow in 3D.
BME 2 has been our ECM of choice for these experiments."
Dr. Meritxell Huch (Gurdon Institute, University of Cambridge, UK)
Background:
In their recent publication in Cell, Dr Meritxell Huch, Prof Hans Clevers et al. have
used Cultrex® BME 2 reduced growth factor (organoid growth matrix) to enable long-term (>1 year) organoid cultures of genome-stable bipotent stem cells from
adult human liver. These results open up new experimental avenues towards the use of human liver material expanded in vitro for use in disease modeling, toxicology studies, drug testing, regenerative medicine and gene therapy.
Liver diseases (ranging from genetic inherited disorders to viral hepatitis, liver cancer, and obesity-related fatty liver disease) are the twelfth-leading cause of death in the United States. Failure in the management
of liver diseases can be attributed to the shortage of donor livers as well as to our poor understanding of the mechanisms behind liver pathology. The value of any cultured cell as a disease model or for cell therapy transplantation depends on the fidelity and robustness of its expansion potential as well as its ability to maintain a normal genetic and epigenetic status.
This research by Huch, Clevers et al. shows that:
- Primary human bile duct cells can readily be expanded in vitro into 3D liver organoids using AMSBIO’s reduced growth factor Cultrex®
BME 2 as extracellular matrix (ECM)
- Adult liver stem cells maintain their ability of self-renewal; differentiating into functional hepatocyte cells in vitro and generating bona fide hepatocytes upon transplantation.
- Expanded cells preserve their genetic integrity over months in culture (agreeing with the authors’ previous observations in a mouse model).
- Organoids derived from patients with genetic disorders can be used to model liver disease in vitro.
Liver Organoids Grown on BME 2 (RGF):
Figure 1: Long-term culture of Human Liver Organoids on BME 2 RGF. (Clonal cultures obtained by seeding sorted cells at one cell per well)
(Image courtesy of Meritxell Huch, Gurdon Institute, University of Cambridge, UK)
Figure 2: Marker Expression in Human Liver Organoids on BME 2 (RGF). Confocal image stained for ECAD and the hepatocyte marker HNF4. Nuclei
counterstained with Hoechst. (Image courtesy of Meritxell Huch, Gurdon Institute, University of Cambridge, UK)
Figure 3: Differentiation of Organoids into Hepatocytes (liver cells) on BME 2 (RGF). Expression of hepatocyte genes in human
liver organoid after 11 days on differentiation medium (DM). Immunofluorescence for albumin (ALB, red) and ZO-1 (green); nuclei counterstained with Hoechst (Blue) (Image courtesy of Meritxell Huch,
Gurdon Institute, University of Cambridge, UK)
Dr Meritxell Huch is based at the Gurdon Institute, University of Cambridge, UK; and Professor Hans Clevers is Professor and Director of the Hubrecht Institute and President of the Royal Netherlands
Academy of Arts and Sciences. Professor Clevers co-authored the seminal 2009 paper by Sato et al. on mouse organoid culture;
and Dr Huch won the 2013 NC3Rs 3Rs Prize for the advance with the most potential to reduce the use of animals in science for her earlier work growing “mini-liver” organoids from adult mouse stem cells. Using the new method,
cells from one mouse could be used to test 1000 drug compounds to treat liver disease, and reduce animal use by up to 50,000. (See video below of Dr. Huch talking about this research, courtesy of the National Centre for the 3Rs):
References:
Huch, M., Gehart, H., van Boxtel, R., Hamer, K., Blokzijl, F., Verstegen, M. M., ... & Clevers, H. (2014). Long-Term Culture of Genome-Stable Bipotent Stem Cells from Adult Human Liver. Cell , 160, 299 - 312
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