Amino Acids for Cell Culture

Amino acids are compounds with amino groups (-NH₂) and carboxyl groups (-COOH) in their molecules. They have a common basic structure and contain complex functional groups. They are the basic units that make up proteins. Amino acids can be divided into α-, β-, γ-, ω-...amino acids according to the different positions of the amino group on the carbon chain, but the amino acids obtained after protein hydrolysis are all α-amino acids or imino acids. According to their different chemical structures, they can be divided into aliphatic, aromatic and heterocyclic amino acids.

Amino acids are essential for the culture of mammalian cells. In large-scale cell culture, amino acids play an important role in the state of cells, the maintenance of high density, and protein yield and quality. During the cell culture process, amino acids will change and be consumed depending on the proteins expressed by the cells. It is usually necessary to add amino acids of different components and proportions for supplementation at different stages of the cell culture process.

Cell Culture

Cell culture refers to a method of simulating the in vivo environment (sterility, suitable temperature, pH and certain nutritional conditions, etc.) in vitro so that cells can survive, grow, reproduce and maintain their main structure and function. The industrial preparation of antibody drugs, vaccines and proteins is inseparable from the cell culture process. Cell culture generally uses basal culture medium and feed medium. The basal culture medium can artificially simulate the in vivo growth environment of animal cells, provide suitable pH and osmotic pressure for cell survival and proliferation, and various nutrients that the cells themselves cannot synthesize to promote cell growth. Feed medium is used to supplement the consumption of nutrients during cell culture. Generally, feed medium is added about 2 to 4 days after cell inoculation. Cell culture in production usually adopts appropriate feeding strategies to increase cell growth rate and vitality, thereby increasing product yield. The formula of cell culture medium generally contains 70-100 different chemical components, including sugars, amino acids, vitamins, inorganic salts, trace elements, growth-promoting factors, etc. It is necessary to determine the formula components suitable for cell growth by analyzing cell characteristics and process tests, which often requires repeated and extensive experimental demonstration and scientific analysis.

Cell Culture Medium

Culture medium is not only the basic substance that provides cell nutrition and promotes cell proliferation, but also the environment for cell growth and proliferation. Cell culture medium is based on balanced salt solution (BSS), with added carbohydrates, amino acids, lipids, inorganic salts, vitamins, trace elements and cell growth factors. According to the development history of cell culture medium, it can be roughly divided into several categories, such as balanced salt solution, natural cell culture medium, synthetic cell culture medium, serum-free cell culture medium, and cell culture medium with defined chemical composition.

• Balanced Salt Solution (BSS)

BSS is mainly composed of inorganic salts and glucose. Its function is to maintain the osmotic pressure balance of cells, keep pH stable and provide simple nutrition. It is mainly used for cell rinsing, preparation of other reagents, etc. D-Hank's and Hank's are commonly used balanced salt mixtures. A major difference between D-Hank's and Hank's is that the former does not contain Ca²⁺ and Mg²⁺, so D-Hank's is often used to prepare pancreatic enzyme solutions. Because Ca²⁺ and Mg²⁺ are important components of cell membranes and participate in functions such as cell adhesion, using BSS without Ca²⁺ and Mg²⁺ can avoid cell agglomeration. In addition, Hanks solution and Earle solution are commonly used BSS basic solutions. The former has a weaker buffering capacity and is suitable for closed culture; the latter has a stronger buffering capacity and is suitable for 5% CO₂ culture conditions.

• Natural Culture Medium

Natural culture medium refers to a type of culture medium that comes from animal body fluids or is extracted by tissue separation, such as plasma, serum, lymph fluid, chicken embryo extract, etc. Its advantages are rich nutrients and good culture effects, but its disadvantages are complex ingredients, limited sources, complex production processes, and large batch differences. The most widely used natural culture medium is serum. In addition, various tissue extracts and collagen substances that promote cell adhesion are also essential for culturing certain special cells. At present, the serum used for cell culture is mainly bovine serum, and human serum and horse serum are also used to culture certain special cells. Serum contains various plasma proteins, peptides, fats, carbohydrates, growth factors, hormones, inorganic substances, etc. These substances achieve physiological balance in promoting cell growth or inhibiting growth activity. In addition, serum contains some substances that are toxic to cells, such as polyamine oxidase, which can react with polyamines from highly proliferative cells (such as spermine and spermidine) to form polyspermine with cytotoxic effects. Complement, antibodies, bacterial toxins, etc. can affect cell growth and even cause cell death. At present, serum is mostly used as an added ingredient mixed with synthetic culture medium, and the usage concentration is generally 5 to 20%, with 10% being the most commonly used.

• Synthetic Medium

Synthetic medium, also known as combined medium, is prepared by sequentially adding accurately weighed high-purity chemical reagents and distilled water. The ingredients (including trace elements) and their amounts are known exactly. Synthetic medium is generally used in laboratory research with high quantitative requirements such as nutrition, metabolism, genetics, identification and bioassay. Synthetic medium is a medium that is synthesized, artificially designed and prepared using chemical substances based on the composition of natural culture medium. The earliest developed basal medium (minimal essential medium, MEM) is essentially a pH-buffered isotonic mixture containing salts, amino acids, vitamins and other essential nutrients. On this basis, various synthetic cell culture media such as DMEM, IMDM, HAM F12, PRMI1640, etc. have been continuously developed.

• Serum Free Medium (SFM)

After natural culture medium and synthetic culture medium, serum-free culture medium and serum-free culture have become a major trend in the field of cell culture today. The use of serum-free culture can reduce production costs, simplify separation and purification steps, and avoid the harm caused by virus contamination. Serum-free culture medium is generally based on synthetic culture medium, with completely or partially defined serum replacement components added to achieve the purpose of meeting the requirements of animal cell culture and effectively overcoming the problems caused by the use of serum. Some additional components are usually added to serum-free culture medium to help cells grow by adhering to the wall, including the following major categories of substances:

• Adhesion-promoting substances: Generally extracellular matrix, such as fibronectin, laminin, etc. They are also important mitogens and differentiation factors that maintain normal cell function, and play an important role in the reproduction and differentiation of many cells.
• Growth factors and hormones: Different growth factors are added for different cells. Hormones are also important substances that stimulate cell growth and maintain cell function. Some hormones are essential for many cells, such as insulin.
• Enzyme inhibitors: Cells that grow by adhering to the wall need to be digested and subcultured with trypsin. Enzyme inhibitors are required in serum-free culture media to terminate the digestion of enzymes and protect cells. The most commonly used is soybean trypsin inhibitor.
• Binding proteins and transport proteins: Common ones include transferrin and bovine serum albumin. The addition of bovine serum albumin is relatively large, which can increase the viscosity of the culture medium and protect cells from mechanical damage. Many serum-free culture media for rotary culture contain bovine serum albumin.
• Trace elements: Selenium is the most common.

Amino Acids for Cell Culture

Amino acids are the basic building blocks of proteins and logically constitute all protein materials of cells, including cytoskeleton, protein components of enzymes, receptors and signaling molecules. In addition, amino acids are used for cell growth and maintenance. Therefore, amino acids are key components of cell culture media, especially in chemically defined media. Studies have shown that slight changes in the amino acid composition in cell culture media can change growth curves and product titers, and can also significantly affect the glycosylation pattern of products. In the production of fusion proteins, the optimization of amino acid concentration in the culture medium using metabolic flux analysis can increase cell peak density and titer by more than 50% and 25%, respectively. The role of amino acids in cells is mainly reflected in the following aspects:

• As the basic building block of protein, amino acids participate in the synthesis of all proteins in cells, including cytoskeleton, enzymes, receptors and signaling molecules.
• Amino acids can be used to synthesize certain nitrogen-containing compounds with important physiological effects, such as nucleic acids, nicotinamide, etc.
• Some amino acids also have unique physiological effects, such as glycine participating in biotransformation, alanine and glutamate participating in the transport of intracellular ammonia, etc.
• Amino acids can also be converted into sugars and fats, participate in oxidation pathways and provide energy for cell metabolism.

Which Amino Acid is Used in Cell Culture?

Amino acids are divided into left-handed and right-handed, which are defined as L-amino acids (left-handed amino acids) and D-amino acids (right-handed amino acids). Amino acids synthesized and used in nature are generally L-type amino acids. D-type amino acids can only be used by certain bacteria and fungi. Some D-type amino acids are toxic and are rarely used in culture media. Therefore, L-type amino acids have biological activity and can be absorbed and used by animals and plants and used in the preparation of culture media. Different cells have different requirements for amino acids, but based on whether the cells can synthesize them themselves, the 20 common amino acids can be divided into essential amino acids and nonessential amino acids.

• Essential Amino Acids

Essential amino acids cannot be synthesized by themselves and must be provided by the external environment. They can be synthesized by mammalian cells in culture. In the addition of cell culture supplements, the concentration of essential amino acids is usually high. The optimization of amino acid concentration is the most important link in the development of culture medium. Generally, 20 kinds of amino acids are added to the culture medium, and a slight change in their concentration can significantly affect cell growth and protein expression.

• Non-essential Amino Acids

During the development of culture medium, due to the high expression level of the target product, once the rate of the cell's own synthesis of non-essential amino acids cannot keep up with the synthesis rate of the target protein, mismatching is likely to occur, affecting the expression of the product. Adding appropriate concentrations of non-essential amino acids can reduce the cell's own synthesis burden and improve the utilization rate of other nutrients. In recombinant cells, most amino acids are used to synthesize proteins, and some amino acids are used to synthesize other substances, such as nucleic acids and lipids.

BOC Sciences offers a wide range of amino acids, including essential, non-essential, and conditional amino acids, as well as custom formulations to meet the specific needs of our customers. We can tailor amino acid formulations to meet specific customer requirements. These formulations can include specific ratios of amino acids, additional nutrients and growth factors, and other additives to optimize cell culture conditions for specific cell types or experimental protocols. Our team of experienced scientists and technicians can work with you to develop a custom formulation that meets your specific needs and ensures the success of your cell culture experiments. Contact us today to learn more about our amino acid products and how we can help you succeed in your cell culture experiments.

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