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Development of culture conditions

5th December 2016

Posted By Paul Boughton


Amsbio reports on the recent publication in Nature Protocols1 by Dr Meritxell Huch**  and co-workers which describes development of culture conditions that allow the long-term expansion of adult primary tissues from the liver and pancreas into self-assembling 3D organoid cultures.

Traditionally adult somatic tissues have proven difficult to expand in vitro, largely because of the complexity of recreating appropriate environmental signals in culture.

Huch** and co-workers have overcome this problem and describe recently established protocols for the long-term (> 1 year) expansion and genetic manipulation of adult liver and pancreas cells in 3D organoid cultures from mouse and human models.

The authors provide a detailed protocol that describes how to grow adult mouse and human liver and pancreas organoids, from cell isolation and long-term expansion to genetic manipulation in vitro.

Human liver and pancreas cells were grown using Amsbio’s reduced growth factor basement membrane extract Cultrex BME2 as extracellular matrix (ECM) and a defined medium. Using Cultrex BME 2 the cells can self-organise into organoids that self-renew in vitro while retaining their tissue-of-origin commitment, genetic stability and potential to differentiate into functional cells in vitro (hepatocytes) and in vivo(hepatocytes and endocrine cells).

Genetic modification of these organoids opens up avenues for the manipulation of adult stem cells in vitro, which could facilitate the study of human biology and allow gene correction for regenerative medicine purposes.

The complete protocol, which can be conducted by personnel with basic scientific training, takes 1-4 weeks to generate self-renewing 3D organoids and to perform genetic manipulation experiments.

Commenting on this research, Dr Huch said: “This paper describes protocols for expanding, differentiating, characterizing and genetically manipulating human and mouse liver and pancreas organoids derived from Adult-Stem cells. These cultures represent the first organoid model for human and mouse liver and pancreas cultures derived from adult tissue; and the BME2 extracellular matrix supports very well the growth of these cultures”.

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 organisation that affects cell adhesion, migration, proliferation, and differentiation. 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 utilised 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 similar products such as Matrigel.

** Dr Meritxell Huch is a group leader at the Gurdon Institute, University of Cambridge, UK and winner of NC3Rs Award (2013) and Wellcome-Beit Prize Fellow (2014), Hamdan Award for Medical Research (2016)

Reference: 1. Nature Protocols, 11(9), 1724-1743, 25 August 2016  (this paper can be viewed online.





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