Cell culture media: An overview
In the field of life sciences, one of the most important methodologies is cell culture. The removal of cells, tissues, or organs from an animal or plant and the subsequent implantation of those cells, tissues, or organs in an artificial environment that is favorable to their survival and/or growth is what is meant by the phrase "cell culture." Controlled temperature, a substrate for cell attachment, an adequate growth medium, and an incubator that maintains the optimum pH and osmolality are the fundamental environmental needs for optimal cell development. Cells must have these conditions in order to grow to their full potential.
The selection of an adequate growth medium for in vitro cultivation is the stage in cell culture that is both the most critical and the most vital. A growth medium, also known as culture media, is a liquid or gel that has been formulated to encourage the development of organisms on a microscopic, cellular, or plant-like scale. The medium that is used for cultivating cells often contains an adequate supply of energy as well as substances that control the cell cycle. The main components of a culture media include amino acids, vitamins, inorganic salts, glucose, and serum. The serum is added to the medium because it acts as a source of growth factors, hormones, and attachment factors. In addition to providing nutrients, the medium also contributes to the upkeep of pH and osmolality levels.
Types of medium used in cell culture
Both human and animal cells have the potential to be grown in either an artificial or synthetic medium or an entirely natural media that is supplemented with natural elements. In the following, we will give you an overview of the different currently available media types.
Only biological fluids that exist in their natural state may be found in natural media. Natural media are very helpful and easy for the cultivation of a broad variety of animal cell types. The lack of understanding of the precise components that make up natural media is the primary factor contributing to the low repeatability of results obtained using natural media.
The preparation of artificial or synthetic media involves the addition of nutrients (both organic and inorganic), serum proteins, carbohydrates, cofactors, vitamins, and salts, as well as O2 and CO2 gas phases .
Various types of artificial media have been developed in order to fulfill one or more of the following functions: 1) Immediate survival (a balanced salt solution with a precise pH and osmotic pressure). 2) Prolonged survival (a balanced salt solution supplemented with different formulations of organic chemicals and/or serum). 3) Indefinite development. 4) Specialized functions.
There are four distinct classifications for artificial media:
Serum containing media
The most frequent kind of supplement found in medium used for growing animal cells is fetal bovine serum. It is added to the culture medium as a low-cost supplement in order to achieve the best possible growth conditions. In addition to acting as a transporter or chelator for nutrients that are unstable or water-insoluble, hormones and growth factors, protease inhibitors, and other substances, the serum also binds and neutralizes harmful molecules.
The presence of serum in the media has a number of disadvantages and has the potential to cause major errors in interpretation in immunological research [2, 3]. There have been a variety of different serum-free mediums created [4, 5]. These media are generally specifically formulated to support the culture of a single cell type, such as Knockout Serum Replacement and Knockout DMEM from Thermo Fisher Scientific, and mTESR medium from Stem Cell Technologies , for stem cells .
Additionally, these media incorporate defined quantities of purified growth factors, lipoproteins, and other proteins, which are otherwise typically provided by the serum . These media are often referred to as "defined cultural media" since the components that make up these media are well understood.
Chemically defined media
These media include ultra-pure inorganic and organic components that have not been contaminated by any kind of contamination. They may also include pure protein additions, such as growth factors.
The genetic modification of bacteria or yeast, together with the addition of particular fatty acids, vitamins, cholesterol, and amino acids, results in the production of their component parts .
Protein-free media are those that do not include any protein at all and instead only include non-protein elements. When compared to media with added serum, the use of media without added protein promotes greater cell proliferation and protein expression and makes it easier to purify any product generated in a downstream process [10-12]. Protein is not included in formulations such as MEM and RPMI-1640. However, a protein supplement might be administered if it is necessary.
Culture media and its basic components
Commercial culture medium may be purchased as a powder or a liquid and often include a variety of nutrients such as amino acids, glucose, salts, vitamins, and other dietary supplements.
The needs for these components are different for each cell line, and these variations are responsible for the wide number of different formulations of media. Each component is responsible for a certain function, which will be outlined in the following paragraphs:
To maintain optimal growing conditions, pH must be controlled, which is often done by one of two buffering systems:
Natural buffering system
The CO2/H2CO3 ratio in the atmosphere is equal to that of the medium, creating a natural buffering mechanism. In order to preserve their natural buffering mechanism, cultures must be kept in an air environment with 5-10% CO2, which is often achieved by using a CO2 incubator. One of the best things about using a natural buffer is how cheap and safe it is.
Chemical buffering using the zwitterion HEPES has a greater buffering capability in the pH range of 7.2-7.4 and does not need a regulated gaseous environment. For particular cell types, a larger dose of HEPES may be harmful. Media containing HEPES are likewise much more susceptible to the phototoxic effects of fluorescent light .
The pH indicator phenol red is often included in commercially available culture medium, allowing for continuous monitoring of pH. By expanding the cells, the metabolites produced by these cells cause a shift in pH and therefore a color change of the media. Phenol red has a dual effect on a medium's color, turning it yellow at acidic pH and purple at alkaline pH. pH 7.4, the optimal value for cell culture, causes the medium to appear fluorescent red.
But phenol red has a few drawbacks: First, phenol red is able to simulate the performance of a number of steroid hormones, primarily estrogen . Therefore, when studying estrogen-sensitive cells like breast tissue, a medium free of phenol red is recommended. Sodium-potassium balance is disrupted by the presence of phenol red in several serum-free formulations. Adding serum or bovine pituitary hormone to the media may counteract this effect . Thirdly, detection in flow cytometric experiments is hindered by the presence of phenol red.
Media containing inorganic salts, such as sodium, potassium, and calcium ions, assist maintain osmotic equilibrium and regulate membrane potential.
Since amino acids are the fundamental components of protein, they are an essential component of every single cell growth medium that has ever been conceived. Because cells are unable to produce certain amino acids on their own, it is important that the culture medium include essential amino acids. They are necessary for the proliferation of cells, and the concentration at which they are present determines the maximum cell density that may be attained. In particular, L-glutamine, an essential amino acid is especially crucial.
L-glutamine functions as a secondary source of energy for the metabolism and contributes nitrogen to the production of NAD, NADPH, and nucleotides. Due to the fact that L-glutamine is an unstable amino acid that, with time, changes into a form that cells are unable to utilize, it must be given to the medium.
In addition, non-essential amino acids may be supplied to the medium in order to refuel those that have been used up throughout the growth process. The growth of the cells is boosted and their viability is increased when the growth medium is supplemented with nonessential amino acids.
Carbohydrates in the form of sugars are the principal source of energy. Many of the media also include maltose and fructose in addition to the more common sugars of glucose and galactose.
Proteins and peptides
Albumin, transferrin, and fibronectin are the most commonly used proteins and peptides. They are especially significant in media that do not include serum. Albumin, transferrin, aprotinin, fetuin, and fibronectin are some of the proteins that may be found in serum, which is a rich supply of protein.
Albumin is the primary protein found in blood, and its function is to bind and transport various substances, including water, salts, free fatty acids, hormones, and vitamins, between different organs and cells. Albumin's ability to attach to chemicals makes it an effective candidate for removing harmful compounds from the medium in which cells are cultured.
Aprotinin is a protective agent in cell culture systems, since it is stable at neutral and acidic pH, as well as resistant to high temperatures and the destruction that may be caused by proteolytic enzymes. It is capable of inhibiting a number of serine proteases, including trypsin, amongst others.
Fetuin is a glycoprotein that may be detected in higher amounts in the serum of fetal and newborn animals compared to adult serum. In addition to that, it acts as a serine protease inhibitor. The protein fibronectin is an essential component in the process of cell adhesion. Transferrin is a protein that transports iron and is responsible for delivering iron to the membranes of cells.
Fatty acids and lipids
They play a crucial role in serum-free medium when serum is absent.
Numerous vitamins are necessary for cell development and proliferation. Vitamins cannot be produced in adequate amounts by cells and are thus essential in tissue culture as dietary supplements.
In cell culture, the serum is the primary source of vitamins; however, media are also treated with various vitamins to make them suited for a specific cell type. Most typically, the B group vitamins are used for growth stimulation.
Chemical elements such as copper, zinc, selenium, and tricarboxylic acid intermediates are known as trace elements. Trace elements are often added to media that does not include serum in order to replace those that are typically present in serum. These elements are important chemical components that are required for a healthy cell development. Many biochemical reactions depend on certain micronutrients, such as enzyme activity.
The full growth medium suggested for certain cell lines needs extra components that are absent from the baseline media and serum. These dietary supplements support cell growth and appropriate metabolic function.
Although hormones, growth factors, and signaling molecules are essential for the appropriate proliferation of particular cell lines, the following precautions should always be taken: Since the addition of supplements might alter the osmolality of the complete growth medium, which can inhibit cell development, it is always advisable to verify the osmolality after adding supplements. For the majority of cell lines, the optimum osmolality ranges between 260 and 320 mOSM/kg.
Antibiotics are often employed to inhibit the development of bacterial and fungal pollutants , although they are not essential for cell growth. Since antibiotics might conceal contamination by mycoplasma and resistant bacteria, their routine use is not suggested for cell culture [17, 18].
In addition, antibiotics may disrupt the metabolism of hypersensitive cells. The penicillin-streptomycin combinations made by MilliporeSigma and Life Technologies are often used. Plasmocin has been utilized in the culture of the glioma cell lines TS603, TS516, and BT260 , and it has been shown to be effective in removing mycoplasma contamination (20).
Albumins, growth factors, and growth inhibitors are all present in serum. Serum is one of the most significant components of cell culture medium because it provides amino acids, proteins, vitamins (especially fat-soluble vitamins such as A, D, E, and K), carbohydrates, lipids, hormones, growth factors, minerals, and trace elements.
Serum from fetal and calf bovine sources is often utilized to promote the development of cultured cells. Fetal serum is an abundant supply of growth factors and is suitable for cell cloning and the development of sensitive cells. Due to its diminished growth-promoting capabilities, calf serum is employed in contact-inhibition experiments. Normal growth mediums often include 2% to 10% serum. The addition of serum to culture medium serves the following purposes :
The serum delivers the essential nutrients for cells (both in solution and attached to proteins).
Several growth factors and hormones involved in growth promotion and specialized cell activity are included in serum.
It offers many binding proteins, like albumin and transferrin, that transport other chemicals into the cell. For example, albumin delivers fats, vitamins, hormones, etc. into cells.
It also provides proteins, such as fibronectin, which increase cell adhesion to the substrate. Additionally, it produces spreading elements that aid in cell expansion before division.
It delivers protease inhibitors that prevent proteolysis in cells.
It also contains minerals such as Na+, K+, Zn2+, and Fe2+.
It enhances the viscosity of the media, so protecting the cells from mechanical injury during suspension culture agitation.
It's also a buffer.
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