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Glucose in Cell Culture

Importance and uses of glucose in serum-free eukaryotic, including hybridoma and Chinese Hamster Ovary (CHO) cell, cultures

Glucose, a Serum-Free Medium Supplement, Useful In Biomanufacturing; Tissue Engineering and Specialty Media:

Glucose is a soluble hexose sugar added to all cell culture media including Ames' Medium; Basal Medium Eagle (BME); BGJb Medium Fitton-Jackson Modification; Click's Medium; CMRL-1066 Medium; Dulbecco's Modified Eagle's Medium (DMEM); DMEM/Ham's Nutrient Mixture F-12 (50:50); F-12 Coon's Modification; Fischer's Medium; H-Y Medium (Hybri-Max®); Iscove's Modified Dulbecco's Medium (IMDM); McCoy's 5A Modified Medium; MCDB Media; Medium 199; Minimum Essential Medium Eagle (EMEM); NCTC Medium; Nutrient Mixture, Ham's F-10; Nutrient Mixture, Ham's F-12; Nutrient Mixture Ham's F-12 Kaighn's Modification (F12K); RPMI-1640; Serum-Free/Protein Free Hybridoma Medium; Waymouth Medium MB; Williams Medium E and various proprietary media. L-15 Medium contains galactose in place of glucose.

The amount of glucose in cell culture formulations ranges from 1 g/L (5.5 mM) to as high as 10 g/L (55 mM). Many classical media are supplemented with approximately 5.5 mM D-glucose which approximates normal blood sugar levels in vivo. These media include: Ames' Medium; Basal Medium Eagle (BME); Click's Medium; CMRL-1066 Medium; Fischer's Medium; Medium 199; Minimum Essential Medium Eagle (EMEM); NCTC Medium; and Swim's S-77 Medium. The MCDB media series contain glucose in the range from 3.9 to 10 mM. F-12 Coon's Modification contains 10 mM and RPMI-1640 and Williams Medium E contain 11 mM glucose.

Concentrations of glucose approaching 10 mM are pre-diabetic levels. Concentrations of glucose above 10 mM are analogous to a diabetic condition within the cell culture system. This is important because the same processes that can affect cells and molecules in vivo can occur in vitro. The consequence to growing cells under conditions that are essentially diabetic is that cells and cell products are modified by the processes of glycation and glyoxidation. These processes cause post-translational secondary modifications of therapeutic proteins produced in cell cultures.

Several important media used as the base media for the design of proprietary media used in bio-manufacturing and tissue engineering contain diabetic levels of glucose supplementation. DMEM/Ham's Nutrient Mixture F-12 (50:50) contains 17.5 mM of D-glucose; DMEM (Hi), GMEM and IMDM all contain 25 mM levels of D-glucose; and H-Y Medium (Hybri-Max®) and Serum-Free/Protein Free Hybridoma Medium contain 22.6 and 28.9 mM D-glucose, respectively. These levels of glucose require special formulation strategies to protect the cells and cell products from glucose mediated oxidative and carbonyl stress. For a more complete discussion of D-glucose as a cell culture additive, visit our Media Expert®.

Primary Functions of Glucose in Cell Culture Systems:

Animal cells, heterotrophs, derive their energy from coupled oxidation-reduction reactions. Glucose is a primary fuel for heterotrophs. Energy derived from glucose is stored in the form of high-energy phosphate bonds in ATP, or other nucleotide triphosphates, and as energy-rich hydrogen atoms associated with the co-enzymes NADP and NAD. The metabolic pathways involved with the production and utilization of these high-energy intermediates are the

  • cytoplasmic glycolytic pathway
  • cytoplasmic pentose phosphate shunt, PPP
  • cytoplamic:mitochondrial aspartate:malate shuttle
  • cytoplamic:mitochondrial glycerol:phosphate shuttle
  • mitochondrial tricarboxylic acid cycle, TCA
  • mitochondrial electron transport chain

Efficient energy metabolism and maintenance of a reduced cellular environment depend upon the fine balance of these pathways in response to environmental factors. It is important to understand how and when these pathways operate within the compartments of the cell as it responds to changing environmental conditions.

Cell Entry:

Glucose is unable to diffuse across the cell membrane without the assistance of transporter proteins. At least 13 hexose transporter proteins with different functions have been identified. Some hexose transporters allow glucose to flow passively from high to low concentration without requiring the expenditure of cell energy. Those that move glucose against its concentration gradient consume energy, generally in the form of ATP. In vitro, the external glucose concentration exceeds the intracellular concentration. Under these conditions, passive hexose transporters are important. The transporters Glut-2, Glut-3 and Glut-5 are found primarily in specialized tissue. Glut-1 and Glut-4 are found on a wide range of cell types. Many cell types have more than one type of hexose transporter.

Cell Defense:

Once inside the cell, glucose is phosphorylated to glucose-6-phosphate (G6P) primarily by hexokinase. G6P can be converted to glucose-1-phosphate (G1P), or enter the glycolytic or pentose phosphate pathway. The amounts of G6P that are metabolized by the glycolytic and pentose phosphate pathways (PPP) are controlled by the relative activities of phosphofructokinase (PFK) and glucose-6-phosphate dehydrogenase (G6PD). The balance of PFK and G6PD activities is regulated at many levels.

Glucose provides the reducing power needed to neutralize oxidative species (oxidative stress) that form in vivo and in vitro. Cytoplasmic NADP induces G6PD to convert G6P to 6-phospho-gamma-lactone and initiates the PPP. Concurrently, G6PD reduces NADP to NADPH. NADPH is the primary reductant for glutathione, and thioredoxin, two very important molecules for the management of oxidative stress. If a cell is unable to maintain a reduced intracellular environment, it will enter apoptosis.

Metabolism of glucose via the oxidative branch of the pentose phosphate pathway provides the reducing power needed to maintain the pool of NADPH. It should be noted that G6PD metabolizes G6P before any glucose metabolites that can engage in energy metabolism are produced. Cells in vitro are subjected to oxidative stress and their ability to survive and grow is likely to be significantly affected by their capacity to generate NADPH through the PPP.

Cell Energy:

When glucose levels are sufficient, its metabolites move through the PPP and glycolytic pathways to form glyceraldehyde-3-phosphate (G3P). Energy from glycolysis that is ultimately stored as ATP is derived from reactions that occur at the level of glyceraldehyde-3-phosphate and below. Pyruvate and the vitamin NAD have distinct roles in the transfer of reducing equivalents into the mitochondrial systems. NAD delivers reducing equivalents to the electron transport system by shuttles that bypass the TCA cycle and pyruvate delivers reducing equivalents through the TCA cycle.


The glycolysis of G3P to pyruvate is initiated by glyceraldehye-3-phosphate dehydrogenase (G3PD). This multi-step process generates cytoplasmic ATP and NADH. The G3PD reaction depends upon the availability of oxidized cytoplasmic NAD. NAD does not move between the mitochondria and the cytoplasm and the amount of cytoplasmic NAD is limited. Under conditions of high glucose, glyceraldehyde-3-phosphate will build up in the cell unless cytoplasmic NADH is continuously re-oxidized. Cells oxidize cytoplasmic NADH by a combination of three pathways, the aspartate:malate shuttle, the glycerol:phosphate shuttle and during the conversion of pyruvate to lactate.

Aspartate:Malate Shuttle:

The aspartate:malate shuttle transports reducing equivalents from cytoplasmic NADH, produced during the oxidation of G3P, into the mitochondria matrix as malate. While doing this, it regenerates NAD that can be used by G3PD to keep glycolysis going. This system is driven, in part, by glutamine.

Glycerol-Phosphate Shuttle:

Cells may regenerate cytoplasmic NAD via the glycerol:phosphate shuttle. This shuttle depends upon the activities of cytoplasmic ( and mitochondrial glycerol phosphate dehydrogenases (EC These enzymes interconvert dihydroxyacetone phosphate and glycerol-3-phoshate inside and outside the mitochondria and effectively transfer reducing equivalents from cytoplasmic NADH to a dehydrogenase complex located between the inner and outer mitochondrial membranes. Many transformed cells lack cytoplasmic glycerol phosphate dehydrogenase. This system is not driven by amino acids.

Cell Lactic Acid Dehydrogenase

Cell lactic acid dehydrogenase (LDH) can re-oxidize cytoplasmic NADH by converting pyruvate to lactic acid. This is a wasteful process that leads to a metabolic dead-end. NADH that has not been oxidized by the aspartate:malate or gylcerol:phosphate shuttle is metabolized by LDH. A buildup of lactic acid in cell culture systems is evidence that the shuttles are unable to re-oxidize all of the NAD required to support G3P catabolism. At high levels, lactic acid becomes toxic to cells.


Glucose is a potential source of energy for cells when its metabolite, pyruvate, enters the mitochondria and is decarboxylated to acetyl-CoA. Mitochondrial acetyl-CoA is converted into citrate which either feeds the tricarboxylic acid cycle (TCA) or exits the mitochondria into the cytoplasm. If citrate enters the TCA cycle, it can be further metabolized to generate ATP and mitochodrial NADH. Mitochondrial NADH is the delivery molecule of reducing equivalents into the respiratory chain. Most, but not all, ATP is formed in the cell mitochondria during the process of oxidative phosphorylation. Citrate can exit the mitochondria via the tricarboxylic acid transport system and donate its acetyl groups for the synthesis of fatty acids or isoprenoids. Pyruvate may not enter the mitochondria. It may be reduced to lactic acid by lactic acid dehydrogenase. This reaction is driven when the cell’s need to oxidize NADH to NAD for use as a substrate to keep glycolysis working. Pyruvate reacts with hydrogen peroxide and forms water, carbon dioxide and acetic acid. This non-enzymatic reaction helps the cell defend itself from oxidative intermediates.

Chemical Attributes of D-Glucose that make it a Useful Serum-Free Medium Supplement:


Molecular formula: C6H12O6

Molecular weight: 180.16

Glucose is a six-carbon aldehyde sugar that exists in various stereoisomeric forms and as a linear or cyclized molecule. D-Glucose is the natural form used by animal cells. The D refers to the asymmetry around the C5 carbon atom. When glucose is dissolved in water it exists almost exclusively in its cyclic form. The designations α and β refer to the asymmetry around the C1 carbon when glucose is in its cyclized form. The α- and β- forms can interconvert. The process is called mutarotation.

Glucose is very soluble and chemically stable in normal physiological solutions and media. However, glucose is a reducing sugar that participates in Schiff base formation and Amadori rearrangements that cause protein glycation and carbonyl stress in cell culture. The ability of glucose to promote glycation and carbonyl stress is an important contributor to the secondary modifications of bio-manufactured molecules, especially proteins.

Glucose Products that Enhance the Growth of Hybridoma, Chinese Hamster Ovary (CHO) and other Mammalian Eukaryotic Cells in Serum-free Cultures.

Our Cell Culture Media Expert® provides in depth discussion of this and other serum-free and protein-free media supplements. The Media Expert® contains additional sections on raw materials, component use recommendations, formulation strategies and references. Whenever you have a questions about or problems with your eukaryotic mammalian cell culturing system visit the Media Expert® for helpful guidance.