Insight

To absorb nutrients, synthesize self-constituent substances and complete proliferation, cells require fundamental materials, including inorganic matters such as water and inorganic salts, as well as organic matters like proteins, nucleic acids, lipids and carbohydrates. These substances form the structural foundation of cells, covering cell membrane, cytoplasm, organelles, nucleus and other structures. Any cultured cell with growth and proliferation capacity must be supplied with these substances at basic concentrations. As engineered cell factories for production, CHO cells evidently need additional nutritional ingredients to support the synthesis of target products.

The composition of cell culture medium is highly complex, mainly including amino acids, vitamins, inorganic salts, trace elements, carbohydrates, lipids, hormones, growth factors and other categories of ingredients.

Carbohydrates mainly serve as energy substances, with glucose being the primary representative added in medium.

Amino acids are responsible for supporting cell growth and protein expression, and most or all types are supplemented in conventional medium.

Vitamins act as coenzymes and cofactors, with most water-soluble vitamins and part of fat-soluble vitamins routinely added.

Inorganic salts function to maintain cell membrane potential and osmotic pressure, mainly including sodium, potassium, magnesium, calcium, chlorides, phosphates, carbonates, sulfates, nitrates and other types.

Trace elements participate in metabolic regulation, and common types involve iron, copper, zinc, manganese, molybdenum, selenium, vanadium and so on.

Lipids constitute the main components of cell membranes and are added selectively according to culture requirements.

Growth factors act as cellular signaling molecules and are also supplemented selectively.

Other non-nutritive components are used to stabilize the physical and chemical environment of the medium, including buffers, surfactants and antifoams, all of which are added selectively.

All life activities of cells are inseparable from energy supply, and cell growth, proliferation and protein expression consume a large amount of energy. Studies have shown that the average dry weight of a single cell is about 400 pg, of which more than 50% is protein. Protein synthesis relies on the connection of amino acids through peptide bonds, and the formation of each peptide bond consumes 3 ATP molecules, while the complete oxidation of one glucose molecule produces only about 30 ATP molecules. It is evident that tremendous energy is consumed for cells to synthesize their own proteins. For high-expression cell lines, the specific productivity Qp can reach more than 50 pg, meaning each cell can express 50 pg of protein per day, accounting for nearly a quarter of its total endogenous protein.

As the primary energy substance in medium, glucose is absorbed and utilized by cells. It first enters the cell through transporter proteins on the plasma membrane, and is decomposed into pyruvate via multiple steps in the cytoplasm, a process defined as glycolysis. Part of pyruvate enters the mitochondria and is completely oxidized into water and carbon dioxide to generate energy, which is the TCA cycle — the main way for cells to produce energy. During the rapid growth phase of cells, about 90% of pyruvate is converted into lactate under the action of lactate dehydrogenase and secreted out of the cell, resulting in lactate accumulation at this stage and reduced glucose utilization efficiency. In addition, glucose can generate ribose phosphate through the pentose phosphate pathway, and ribose phosphate is an important raw material for nucleotide synthesis.

Glutamine is the second largest energy substance after glucose. Apart from being used to synthesize cellular self-components and target proteins, most glutamine is converted into glutamate, which is further transformed into α-ketoglutarate to participate in the TCA cycle for energy production. It is worth noting that GS-system CHO cells have been transfected with exogenous glutamine synthetase genes, so they do not need to obtain glutamine from the external environment. The expressed glutamine synthetase can synthesize glutamine from intracellular glutamate.

Amino acids are the molecular basis for cells to synthesize proteins, including structural functional proteins and target recombinant proteins. Amino acids are divided into essential amino acids and non-essential amino acids. Non-essential amino acids can be synthesized by mammalian cells themselves, while essential amino acids cannot be synthesized intracellularly and must be provided by the external environment. In the development of culture medium, due to the high expression level of target products, once the rate of cellular self-synthesis of non-essential amino acids cannot keep up with the synthesis rate of target proteins, amino acid mismatches are prone to occur and affect product expression. Therefore, nearly all 20 types of amino acids are generally added to the medium.

Optimization of amino acid concentration is the most critical link in medium development; tiny changes in amino acid concentration can significantly affect cell growth and protein expression. For recombinant cells, most amino acids are used for protein synthesis, and part of them are diverted to synthesize other substances such as nucleic acids and lipids. Amino acids share common metabolic characteristics due to similar structural properties, while their metabolic pathways differ owing to structural variations.

The main metabolic pathways of amino acids include multiple categories. The first is deamination, which produces α-keto acids and ammonium ions, with the typical reversible reaction between α-amino acid and α-ketoglutarate generating glutamate and α-keto acid, and glutamate further decomposing into α-ketoglutarate and ammonium ions. The second is decarboxylation, which generates amines or polyamines, such as histidine converting to histamine and ornithine converting to putrescine. The third is transmethylation, participating in the synthesis of creatine and choline, with methionine as the main involved amino acid.

Some amino acids undergo metabolic transformation to form taurine and cystine, for instance cysteine can be converted into cystine and taurine. Some can be converted into one-carbon units for the synthesis of purines and pyrimidines, mainly including serine, glycine, histidine and tryptophan. Phenylalanine and tyrosine can be metabolically transformed into neurotransmitters such as DOPA and dopamine as well as melanin. Tryptophan metabolism can produce pyruvate. Valine, leucine and isoleucine can be converted into succinyl-CoA and acetyl-CoA through metabolism.

It can be seen that most amino acids produce corresponding α-keto acids and ammonium ions via deamination, so the accumulation of ammonium ions is a non-negligible factor in cell culture. In addition, some amino acids can generate other amino acids through transamination, which all belong to non-essential amino acids. Due to the poor solubility of tyrosine and cysteine, they are conventionally supplemented in fed-batch medium in the form of alkaline solution. At present, some easily soluble alternatives have been launched on the market, such as disodium tyrosine phosphate and L-cysteine sulfate developed by Merck.

All life activities of cells rely on enzymes, the biological catalysts. Vitamins serve as binding cofactors of enzymes, combining with enzymes to catalyze cellular physiological processes such as metabolism and energy transfer. Cultured cells cannot synthesize vitamins by themselves, so vitamins are essential ingredients to be added in medium, especially B vitamins, including thiamine, riboflavin, pantothenic acid, pyridoxine, biotin, folic acid, cyanocobalamin, inositol and vitamin C.

Vitamin B1, namely thiamine, acts as the coenzyme for α-keto acid oxidative decarboxylase. Vitamin B2, or riboflavin, participates in biological oxidation processes. Vitamin PP, namely niacinamide, takes part in biological oxidation and serves as the coenzyme of dehydrogenase. Vitamin B6, pyridoxine, is the coenzyme for amino acid decarboxylase, transaminase, alanine synthetase and enzymes involved in the catabolism of homocysteine. Pantothenic acid is a component of coenzyme A, participating in acyl group transfer and fatty acid synthesis. Biotin acts as the prosthetic group of carboxylase and participates in cell signal transduction and gene expression regulation. Folic acid is mainly involved in the transfer of one-carbon units. Vitamin B12, cyanocobalamin, promotes methyl transfer, facilitates DNA synthesis and participates in the generation of succinyl-CoA. Vitamin C, ascorbic acid, takes part in hydroxylation reactions and antioxidant processes, and can also promote iron absorption.

Some medium is supplemented with an appropriate amount of fat-soluble vitamins such as tocopherol and retinol, or precursors of vitamin D like cholesterol. Although these fat-soluble vitamins have certain impacts on cell growth, they are generally not prioritized for component optimization considering the complexity of addition and poor solubility.

Cytoplasmic matrix fills the gaps between tangible cellular structures, and its chemical composition mainly includes water, inorganic salts, lipids, carbohydrates, amino acids, nucleotides and proteins. It provides a suitable ionic environment and material substrates for various organelles to maintain normal structure and function. Therefore, appropriate concentration of inorganic salts is essential for cells to sustain osmotic pressure and membrane potential.

Common inorganic salts in medium include sodium, potassium, magnesium, calcium, chloride, phosphate, sulfate and bicarbonate. Among them, calcium ions play an important role in intracellular signal transduction. Phosphate not only acts as a buffer salt, but also serves as one of the constituent components of nucleotides, nucleic acids and phospholipids. As the counter ion of ATP, magnesium ion is added to the medium at a relatively high concentration, usually at the millimolar level. Relevant literature has reported that a proper concentration ratio of sodium to potassium is the basis for cells to maintain normal physiological activities.

Trace elements also act as coenzyme factors to regulate cellular physiological activities. In the early stage of medium development, the water used for medium preparation had low purity and naturally contained a certain amount of trace elements. Nowadays, the purity of preparation water has been greatly improved, making the development and precise addition of trace elements increasingly important. Common trace elements used in medium include zinc, copper, iron, manganese, selenium, molybdenum and others.

Iron is an important constituent of iron-sulfur proteins, peroxidase and catalase, playing a vital role in gas transport, biological oxidation and enzymatic reactions. Iron ions are utilized by cells mainly in the form of complexes; free or improperly chelated iron may produce toxicity to cells. In addition, superoxide radicals or reducing agents such as vitamin C in the medium can reduce ferric iron to ferrous iron. Considering the source limitation and cost of transferrin, ferric ammonium citrate is now widely used as the iron source in medium.

Zinc is a component of zinc metalloenzymes and is related to protein folding. It can effectively bind to disulfide bonds of proteins and affect protein stability and activity. It also plays an indispensable role in RNA and DNA synthesis, mRNA stabilization and cellular anti-apoptosis processes. Meanwhile, zinc ions participate in the activation of glutathione and antioxidant enzymes such as superoxide dismutase and catalase, protecting cells from the attack of reactive oxygen species. Relevant literature has shown that adding zinc ions at a concentration of 25 mg/L can improve protein expression of CHO cells. It should be noted that when the medium is under oxidative stress, zinc ions may form precipitates with oxides, peroxides and sulfides in the medium and thus be lost. Other components in the medium such as EDTA may also reduce the utilization rate of zinc through chelation.

Copper ions exist in the medium in a balanced state of monovalent reduced copper and divalent oxidized copper. It can oxidize other components in the medium such as cysteine and vitamin C, and form chelate precipitates with cystine, resulting in the loss of cysteine and cystine in the medium. The deficiency of cysteine in the medium will suspend the synthesis of proteins and glutathione. In addition, the metabolic shift from lactate production to lactate consumption during culture of certain cell lines requires the presence of copper ions at a specific concentration.

Lipids are one of the main constituent parts of cell membranes. Cell membranes maintain the relative stability of the intracellular environment, regulate and selectively control the entry and exit of substances, act as a barrier for free substance exchange, and also play an important role in material transport and protein secretion. The composition of cell membranes is extremely complex, mainly including glycerophospholipids, sphingolipids, glycolipids, cholesterol, proteins and carbohydrates. Among glycerophospholipids, phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine occupy the highest content. Their synthesis takes glycerol, fatty acids, phosphate, choline, serine and inositol as basic raw materials. The main synthetic raw materials of sphingolipids include acyl-CoA and serine, and the synthesis also requires the participation of coenzymes such as pyridoxal. Therefore, raw materials such as choline, ethanolamine and inositol need to be added to the medium.

Fatty acids and glycerol are mainly converted from glucose metabolism. Mammalian cells cannot synthesize unsaturated bonds with carbon chains longer than 9 carbons. Appropriate concentrations of linoleic acid, linolenic acid or arachidonic acid can be added to the medium as required. However, considering the source limitation and complex addition process of these substances, their medium development and application need to be treated with caution. As one of the basic structural components of cell membranes, cholesterol is an important substance determining cell membrane properties, and can also be converted into precursors of fat-soluble vitamin D3.

Oxidative stress occurs in all cells during culture, generating oxidative intermediate products, and this phenomenon is more obvious in serum-free medium systems due to the lack of inherent antioxidant substances. With the increase of reactive oxygen species concentration, superoxide radicals and hydrogen peroxide will be produced. These substances can damage lipids, proteins and DNA, causing harm to cells.

Cells have two major antioxidant systems. One is the enzymatic antioxidant system, including superoxide dismutase, catalase and glutathione peroxidase. The other is the non-enzymatic antioxidant system, covering vitamin C, vitamin E, glutathione, lipoic acid, carotenoids, as well as trace elements copper, zinc and selenium.

In the design of culture medium, on the one hand, antioxidant substances are supplemented to endow cells with the ability to resist oxidative environment; on the other hand, it can reduce the oxidation probability of unstable components in the medium. Glutathione, composed of glutamate, cysteine and glycine, is a commonly added antioxidant in medium. In the process of recombinant protein expression, the higher the expression level of target protein, the stronger the oxidative stress that cells need to cope with, and the more oxidized glutathione will be produced. Relevant studies have shown that the accumulation of oxidized glutathione is an early apoptotic signal of cell death, which is another key factor inducing cell apoptosis besides ammonium ions and lactate.

The synthesis of purine and pyrimidine nucleotides by cells requires amino acids as raw materials. Purine synthesis needs glycine, asparagine, glutamine and one-carbon units, while pyrimidine synthesis relies on asparagine, glutamine and one-carbon units. This is one pathway for cells to synthesize nucleotides, known as the de novo synthesis pathway. The other is the salvage pathway, which synthesizes nucleotides based on exogenous nucleosides and bases.

DHFR can reduce dihydrofolate to tetrahydrofolate, and tetrahydrofolate is a key substrate in the de novo nucleotide synthesis pathway. DHFR-deficient cell lines therefore lose the ability of de novo nucleotide synthesis and can only survive when exogenous nucleosides such as hypoxanthine and thymidine are added to the medium. CHO-DHFR cell lines are designed based on this principle. By exogenously inserting DHFR and target genes, cells can grow in medium without hypoxanthine and thymidine. Meanwhile, the addition of DHFR inhibitors can increase the copy number of DHFR and target genes. Nucleosides generally account for less than 5% of cell dry weight, and can be synthesized by cells themselves, so the addition amount in medium is very low or no addition is required.

Growth factors can promote cell growth and proliferation. Typical representatives such as insulin can regulate cellular metabolic processes, boost the uptake and utilization of glucose by cells, and simultaneously promote the synthesis of fatty acids and proteins. Insulin is conventionally added to medium, but considering the cost of medium development, alternative compounds are being widely explored nowadays.

In addition to maintaining proper osmotic pressure, the physicochemical environment of cell culture also needs appropriate viscosity. F68 commonly used in medium is a polyoxyethylene-polyoxypropylene ether block copolymer, belonging to non-ionic surfactants, with washing and defoaming functions. The higher the proportion of polyoxyethylene, the stronger the hydrophilicity, better cell buffering effect and higher washing activity; the higher the proportion of polyoxypropylene, the stronger the defoaming capacity, accompanied by increased toxicity. The addition of poloxamer at a concentration of 1–2 g/L in medium can effectively enhance the shear resistance of cells, while the disadvantage is that it is easy to generate foam.

Buffers are used to maintain a stable pH range for medium, and common types include carbonate buffer system, phosphate buffer system and HEPES. Dextran sulfate is also a common additive in medium as an anti-aggregation agent.