First isolated from raisins in 1747 by German chemist Andreas Marggraf, glucose and metabolism have been widely studied ever since. Whether it’s mammalian cells, bacteria, or yeast, most living things on earth utilize glucose as their primary sugar source for metabolism. And since it’s often the limiting factor in cell culture, optimal glucose concentrations are crucial in healthy cell growth and efficient product yield. As the primary limiting factor in most cell cultures, it is very easy to overcompensate but the costs are not just financial if it’s done incorrectly.
Understanding what it is and how it works can help raise your bioprocessing game to the next level, but glucose utilization is a deceptively complex topic. If there is not enough in culture media then cells starve, but simply adding more isn’t always the answer.
What is Glucose
One of the most vital molecules in biochemistry, glucose is a simple six-carbon sugar. It is the most abundant monosaccharide on earth and similar to heavier amino acids in terms of its size. Able to diffuse freely through the plasma membrane, glucose is the primary fuel source in cell culture.
The energy contained in its chemical bonds is used to synthesize adenosine triphosphate (ATP) both in interconnected and independent ways. ATP is important because it carries energy within the cells and is often referred to as the energy currency of the cell. All known living things use ATP, and in addition to serving as the energy source it is also important in signal transduction pathways for cell communication.
In glycolysis, a single glucose molecule generates two pyruvate molecules, two nicotinamide adenine dinucleotide (NADH) molecules, and two ATP molecules.
C6H12O6 → 2 C3H4O3 + 2 ATP + 2 NADH
The pyruvate generated by the entry of glucose into cells can then take different paths depending on if the cell is prokaryotic or eukaryotic. In prokaryotic (or eukaryotic under specific conditions) cells, pyruvate enters the fermentation pathway.
If the cell is eukaryotic and has normal energy requirements, pyruvate is moved to the inside of the mitochondria where it takes part in the Krebs cycle. Two ATP result from glycolysis, the Krebs cycle adds 2 ATP, and the electron transport chain adds 34 ATP for a total of 38 ATP per molecule of glucose completely consumed in the three metabolic processes.
Each step along the way is important and it all starts with glucose. If there is not enough glucose in the media then the entire cycle stops, but it is also well known that high glucose levels have detrimental effects on many cell types.
Why Glucose Concentrations Matter
In cell culture there can be too much or too little glucose in the media and the results are greater than just slow cellular metabolism or overpopulation.
If the glucose level in cell culture media is too low the cells will undoubtedly starve but there are other issues at play.
In instances where certain bacteria are being cultured, a lack of glucose is exactly the type of hostile environment that can cause endospore formation. These spores are resistant to heat, chemicals, and desiccation and can be formed in as few as 6 hours after the cells are exposed to external stress. This can be reversed by returning the cells to favorable conditions but the experiment may be compromised by that point.
The osmotic pressure due to the difference in glucose concentration across the plasma membrane is also something that has to be considered. Molecules will move across the membrane when there is osmotic change, and water molecules in low glucose media can move into the cell at volumes too high for the cell to manage causing lysis.
If the glucose concentration in the media is too high, there are not the same issues with lack of nutrients but there are still problems. This is especially true in mammalian cell culture. High glucose concentrations are known to have detrimental effects on many cell types.
Studies have shown that high glucose in cell media can decrease proliferation rates and increase apoptosis in rat mesenchymal stem cells. There is also strong evidence that high glucose may suppress growth factor production by rat multipotent adult progenitor cells and it is further suspected to diminish production of Human Growth Factor, Vascular Endothelial Growth Factor, and Fibroblast Growth Factor 2 in response to TNF-α, LPS, and hypoxia.
Simply adding glucose to a deficient cell culture may work in some circumstances like stirred tank bioreactors where extra media equals extra volume for the cells, but the same technique will likely not work in hollow-fiber systems where the cells are attached to the inside of perfusion cartridges. In hollow-fiber cell culture the cells are densely packed in small spaces and glucose can drop quickly. If the feed rate is turned too high to compensate for the high glucose uptake rate, it can literally wash the cells out of the cartridge.
In hollow-fiber cell culture the process of increasing the feed rate, and thus the glucose concentration, must be done gradually over several days or even weeks. To do this properly, glucose concentration must be closely monitored.
The monitoring of metabolic compounds in cell cultures can provide real-time information of cell line status, and the subtle differences between too much and too little glucose are incredibly important in industrial cell culture where any misstep is amplified downstream.
What this means is that monitoring glucose in cell culture is more important and more complicated than many people think. It is vital for cell health that the correct levels of glucose are present at the right stages, and monitoring cell culture conditions is an especially important element in bioprocesses such as fermentation or mammalian cell culture where the results are not the cells themselves but the proteins they secrete.