Organoids are key 3-dimensional tissue structures and can be developed in many different types, they are used widely in drug discovery and various medical treatments due to their flexibility and how they can reflect cellular characteristics. When culturing organoids two growth factors are key to development, Wnt3a and FGF.
Afamin/Wnt3a
Why the Wnt signalling pathway is important
Wnt signalling is known to be involved in the early development, maintenance and regeneration of stem cells, and in cancer formation. Wnt signalling has also been found to play an important role in the growth and maintenance of these processes. In particular, Wnt3a has been revealed to be an essential niche component for maintaining the proliferation of Lgr5-positive stem cells in intestinal epithelial cells. It is used for the production of various digestive organoids such as the small intestine, large intestine, stomach, pancreas and liver. Although Wnt3a has been conventionally used for the culture of gut organoids, it is a fat-soluble protein, so it forms aggregates in serum-free medium and can not exert its activity sufficiently. In 2016, Mihara et al. found that high Wnt3a activity can be maintained by forming a complex with Wnt3a by Afamin, which is one of the components of serum. In addition, by using Afamin and Wnt3a complex for organoid culture, long-term culture of organoids becomes possible.
Mechanism of Wnt3a stabilisation by Afamin proteins
Due to Wnt3a proteins exhibiting strong hydrophobic properties, they are difficult to store and purify. This is the main issue for commercial Wnt3a products. MBL offers the solution to these issues with their stable and highly reactive Afamin/Wnt3a cell culture medium. When conjugated with Afamin, not only is the high activity maintained, but the ligand also does not aggregate in the aqueous solution.
Afamin/Wnt3a CM increased Lgr5 Positive Stem Cells Expressed td Tomato
Images on the top panels show a bright field of human colour organoids. Images on the bottom panels show fluorescent LGR5 positive stem cells that express td tomato regulated by Lgr5 promoter. Afamin/Wnt3a CM maintained LGR5 positive stem cell growth is seen at greater levels compared with cell growth in commercial recombinant Wnt3a (300ng/mL).
Recombinant Afamin/Wnt3a increased LGR5 Positive Stem Cells
An organoid strain expressing tdTomato regulated by Lgr5 promoter was cultured in the presence of Recombinant Afamin/Wnt3a and Recombinant Wnt3a of competitor A. It was confirmed that Recombinant Afamin/Wnt3a maintains LGR5-positive cells even at low concentrations.
MBL’s Afamin/Wnt3a Conditioned Medium vs Existing Wnt3a products
| MBL’s Afamin/Wnt3a Conditioned Medium | Recombinant Wnt3a | Wnt3a Conditioned Medium made in-house from Recombinant Wnt3a | |
| Wnt3a activity | High activity stabilised by Afamin protein | Insufficient activity for long-term organoid culture | High activity stabilised by serum |
| Quality/stability | Completed evaluation of Wnt signal activity sufficient for organoid culture | Wnt signal activity evaluation completed | Lot-to-lot variation causes large differences in activity evaluation |
| Presence or absence of serum? | Serum-free | Serum-free | Contains serum |
Afamin/Wnt3a is different from the wild-type proteins and using advanced techniques from Japan, has been designed to improve organoid culture. MBL has the exclusive commercial use license of Afamin/Wnt3a and is developing many new products to further simplify organoid culturing.
References
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