Why Are Lipids Challenging to Provide to Cells?
Lipids have many important functions in the cell. They are an energy source, are involved in cell
signaling, and are building blocks of cell membranes. In bioproduction, their primary function is
as a building block of the outer cell membrane and organelle membranes, including those of the
endoplasmic reticulum and Golgi apparatus. Although cells can synthesize cholesterol and some
fatty acids on their own, providing lipids from an outside source can improve cell growth and
metabolism in culture. Studies show that cells supplied with lipids in culture media demonstrate
more efficient metabolism, which results in higher levels of recombinant protein expression.
Thus, many bioproduction scientists aim to supply lipids to cells. However, they often observe
minimal impact to protein productivity, or worse, reduced cell viability, because of the challenges
associated with providing lipids in cell culture media.
The Challenge
There are several challenges associated with adding lipids to cell culture media. First, free fatty
acids can become oxidized in cell culture media, making them toxic to cells. Second, and the
topic that will be discussed here, is that lipids are hydrophobic, which makes solubility in media
an issue. There are two different formats to provide solubilized cholesterol currently on the
market: solubilization in ethanol or binding to cyclodextrin. Most free fatty acids are solubilized in
ethanol, but cholesterol can be found either solubilized in ethanol or bound to cyclodextrin. .
Importantly, both ethanol and cyclodextrin have a downside in bioproduction.
Ethanol is an adequate solvent for fatty acids, but once the supplement is added to the media,
the ethanol is diluted. Once the solvent is diluted, fatty acids become unstable and degrade
and/or stick to the bioproduction vessel. Adding more lipid to the media to compensate for the
short half-life of the lipids can be toxic to the cell. Cyclodextrin-bound cholesterol has also been
seen to bind to vessel walls. Binding of lipids, whether dissolved in ethanol or bound to
cyclodextrin, is an issue in both standard vessels made of glass and steel, and even more so in
single-use plastic systems. In studies performed by Merck, 90% of cholesterol was bound to
single-use film in 72 hours, while 75% of cholesterol was bound to glass and steel in 72 hours (1,
2). For the cyclodextrin-bound cholesterol, this is particularly problematic, as cyclodextrin has
the ability to bind cholesterol and then becomes a cholesterol sink, removing cholesterol from
the cell.
Conclusions and Solutions
Fatty acids and cholesterol are critical to cell function. Therefore, providing fatty acids and
cholesterol in cell culture has the potential to provide many benefits to bioproduction, for
example increased protein productivity and improved protein efficacy. However, it is a challenge
to provide stable, soluble fatty acids and cholesterol to cells. Bioproduction scientists have
attempted to overcome these challenges through labor-intensive titrations and dilutions and
careful monitoring of cell health and productivity.
Cell-Ess® media supplement utilizes a novel a proprietary method to deliver fatty acids and
cholesterol to cells. The novel delivery method limits fatty acids and cholesterol from oxidation,
degrading, and sticking to bioproduction vessels while allowing ready uptake of the lipids by the
cells. Recent data generated using Cell-Ess show a greater than 25% increase in protein
production in various cell lines and media schemes. This increase was observed in shake flasks
and single-use bioreactors.
References
1Altaras
GM, Eklund C, Ranucci C, Maheshwari G. Quantitation of interaction of lipids with
polymer surfaces in cell culture. Biotechnol Bioeng. 2007. 96(5): 999-107.
2Okonkonwski J,
Balasubramanian U, Seamans C, Fries S, etc. Cholesterol Delivery to NS0 Cells:
Challenges and Solutions in Disposable Linear Low-Density Polyethylene-Based Bioreactors.
Biosci and Bioeng. 2007. 103(1): 50-59.
Contact us and learn more about Cell-Ess and the potential benefits it provides to bioprocessing
engineers by email [email protected] or phone 609-323-7838.