Cell culture is one of the major techniques in the life sciences. It is the general term used for the removal of cells, tissues or organs from an animal or plant and their subsequent placement into an artificial environment conducive to their survival and/or proliferation. Basic environmental requirements for cells to grow optimally are: controlled temperature, a substrate for cell attachment, and appropriate growth medium and incubator that maintains correct pH and osmolality. The most important and crucial step in cell culture is selecting appropriate growth medium for the in vitro cultivation. A growth medium or culture medium is a liquid or gel designed to support the growth of microorganisms, cells, or small plants. Cell culture media generally comprise an appropriate source of energy and compounds which regulate the cell cycle. A typical culture medium is composed of a complement of amino acids, vitamins, inorganic salts, glucose, and serum as a source of growth factors, hormones, and attachment factors. In addition to nutrients, the medium also helps maintain pH and osmolality.

Animal cells can be cultured either using a completely natural medium or an artificial/synthetic medium along with some natural products.

Natural media consist solely of naturally occurring biological fluids. Natural media are very useful and convenient for a wide range of animal cell culture. The major disadvantage of natural media is its poor reproducibility due to lack of knowledge of the exact composition of these natural media.

Artificial or synthetic media are prepared by adding nutrients (both organic and inorganic), vitamins, salts, O₂ and CO₂ gas phases, serum proteins, carbohydrates, cofactors [1]. Different artificial media have been devised to serve one or more of the following purposes: 1) immediate survival (a balanced salt solution, with specific pH and osmotic pressure); 2) prolonged survival (a balanced salt solution supplemented with various formulation of organic compounds and/or serum); 3) indefinite growth; 4) specialized functions.

Artificial media are grouped into four categories:

Fetal bovine serum is the most common supplement in animal cell culture media. It is used as a low-cost supplement to provide an optimal culture medium. Serum provides carriers or chelators for labile or water-insoluble nutrients, hormones and growth factors, protease inhibitors, and binds and neutralizes toxic moieties.

Presence of serum in the media has many drawbacks and can lead to serious misinterpretations in immunological studies [2, 3]. A number of serum-free media have been developed [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 [6], for stem cells [7], and incorporate defined quantities of purified growth factors, lipoproteins, and other proteins, which are otherwise usually provided by the serum [8]. These media are also referred to as ‘defined culture media’ since the components in these media are known.

These media contain contamination-free ultra pure inorganic and organic ingredients, and may also contain pure protein additives, like growth factors [9]. Their constituents are produced in bacteria or yeast by genetic engineering with the addition of vitamins, cholesterol, specific amino acids, and fatty acids [10].

Protein-free media do not contain any protein and only contain non-protein constituents. Compared to serum-supplemented media, use of protein-free media promotes superior cell growth and protein expression and facilitates downstream purification of any expressed product [11-13]. Formulations like MEM, RPMI-1640 are protein-free and protein supplement is provided when required.

Culture media contain a mixture of amino acids, glucose, salts, vitamins, and other nutrients, and available either as a powder or as a liquid form from commercial suppliers [14]. The requirements for these components vary among cell lines, and these differences are partly responsible for the extensive number of medium formulations [15]. Each component performs a specific function, as described below:

Regulating pH is critical for optimum culture conditions and is generally achieved by one of the two buffering systems:

In a natural buffering system, gaseous CO₂ balances with the CO₃/HCO₃ content of the culture medium. Cultures with a natural buffering system need to be maintained in an air atmosphere with 5-10% CO₂, usually maintained by a CO₂ incubator. A natural buffering system is low-cost and non-toxic [16].

Chemical buffering using a zwitterion, HEPES, has a superior buffering capacity in the pH range 7.2-7.4 and does not require a controlled gaseous atmosphere [17]. HEPES is relatively expensive and toxic at a higher concentration for some cell types. HEPES has also been shown to greatly increase the sensitivity of media to phototoxic effects induced by exposure to fluorescent light [18].

Most of the commercially available culture media include phenol red as a pH indicator, which allows constant monitoring of pH [19]. During the cell growth, the medium changes color as pH is changed due to the metabolites released by the cells. At low pH levels, phenol red turns the medium yellow, while at higher pH levels it turns the medium purple. Medium is bright red for pH 7.4, the optimum pH value for cell culture. However, there are certain disadvantages of using phenol red as described below: 1) Phenol red mimics the action of some steroid hormones, particularly estrogen [20]. Thus it is advisable to use media without phenol red for studies using estrogen-sensitive cells like mammary tissue. 2) Presence of phenol red in some serum-free formulations interferes with the sodium-potassium homeostasis. This effect can be neutralized by the inclusion of serum or bovine pituitary hormone in the medium [21]. 3) Phenol red interferes with detection in flow cytometric studies.

Inorganic salt in the media helps to retain the osmotic balance and help in regulating membrane potential by providing sodium, potassium, and calcium ions [22].

Amino acids are the building blocks of proteins, and thus are obligatory ingredients of all known cell culture media. Essential amino acids must be included in the culture media as cells can not synthesize these by themselves. They are required for the proliferation of cells and their concentration determines the maximum achievable cell density. L-glutamine, an essential amino acid, is particularly important [23]. L-glutamine provides nitrogen for NAD, NADPH and nucleotides and serves as a secondary energy source for metabolism. L-glutamine is an unstable amino acid, that, with time, converts to a form that can not be used by cells, and should thus be added to media just before use [24]. Caution should be used when adding more L-glutamine than is called for in the original medium formulation since its degradation results in the build-up of ammonia, and ammonia can have a deleterious effect on some cell lines. L-glutamine concentrations for mammalian cell culture media can vary from 0.68 mM in Medium 199 to 4mM in Dulbecco’s Modified Eagles’s Medium. Invertebrate cell culture media can contain as much as 12.3 mM L-glutamine. Supplements like glutamax are more stable and can replace glutamine for long term culturing of slow cells. Typical suppliers of L-glutamine for cell culture is MilliporeSigma (G7513) [25].

Nonessential amino acids may also be added to the medium to replace those that have been depleted during growth. Supplementation of media with non-essential amino acids stimulates growth and prolongs the viability of the cells.

Carbohydrates in the form of sugars are the major source of energy. Most of the media contain glucose and galactose, however, some contain maltose and fructose.

The most commonly used proteins and peptides are albumin, transferrin, and fibronectin. They are particularly important in serum-free media. Serum is a rich source of proteins and includes albumin, transferrin, aprotinin, fetuin, and fibronectin. Albumin is the main protein in blood acting to bind water, salts, free fatty acids, hormones, and vitamins, and transport them between tissues and cells. The binding capacity of albumin makes it a suitable remover of toxic substances from the cell culture media.

Aprotinin is a protective agent in cell culture systems, stable at neutral and acidic pH and resistant to high temperatures and degradation by proteolytic enzymes. It has the ability to inhibit several serine proteases such as trypsin. Fetuin is a glycoprotein found in fetal and newborn serum at larger concentrations than in adult serum. It is also an inhibitor of serine proteases. Fibronectin is a key player in cell attachment. Transferrin is an iron transport protein that acts to supply iron to the cell membrane.

They are particularly important in serum-free media as they are generally present in serum.

Many vitamins are essential for growth and proliferation of cells. Vitamins cannot be synthesized in sufficient quantities by cells and are therefore important supplements required in tissue culture. Again serum is the major source of vitamins in cell culture, however, media are also enriched with different vitamins making them suitable for a particular cell line. The B group vitamins are most commonly added for growth stimulation.

Trace elements are often supplemented to serum-free media to replace those normally found in serum. Trace elements like copper, zinc, selenium and tricarboxylic acid intermediates are chemical elements that are needed in minute amounts for proper cell growth [26]. These micronutrients are essential for many biological processes, e.g., the maintenance of the functionality of enzymes.

The complete growth media recommended for certain cell lines requires additional components which are not present in the basal media and serum. These components, supplements, help sustain proliferation and maintain normal cell metabolism [27, 28]. Although supplements like hormones, growth factors and signaling substances are required for normal growth of some cell lines, it is always best to take the following precautions: since the addition of supplement can change the osmolality of the complete growth media which can negatively affect the growth of cells, it is always best to recheck the osmolality after supplements are added. For most of the cell lines, optimal osmolality should be between 260 mOSM/kg and 320 mOSM/kg.

The shelf life of the growth media changes after the addition of supplements. Complete media containing protein supplement tend to degrade faster than basal media alone.

Although not required for cell growth, antibiotics are often used to control the growth of bacterial and fungal contaminants [29]. Routine use of antibiotics for cell culture is not recommended since antibiotics can mask contamination by mycoplasma and resistant bacteria [30, 31]. Moreover, antibiotics can also interfere with the metabolism of sensitive cells. Penicillin-streptomycin preparations from Life Technologies (15140163) and MilliporeSigma (P0781) are typical choices [25]. Plasmocin can eliminate mycoplasma contamination [32], and has been used in culturing glioma cell lines TS603, TS516, and BT260 [25].

In addition, primary cell culture can also be contaminated by fungi, protozoa, and plant cells (for animal cell culture), due to the host or other circumstances. Roundup/glyphosate isopropylamine/(2-Oxo-2-hydroxyethyl)aminomethylphosphonic acid・isopropylamine can be applied for plant cells; amphotericin B (fungizone) and/or fluconazole for fungi; and pentamidine and/or atovaquone for protozoa.

Serum is a complex mix of albumins, growth factors and growth inhibitors [33]. Serum is one of the most important components of cell culture media and serves as a source for amino acids, proteins, vitamins (particularly 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 are commonly used to support the growth of cells in culture [34]. Fetal serum is a rich source of growth factors and is appropriate for cell cloning and for the growth of fastidious cells [35]. Calf serum is used in contact-inhibition studies because of its lower growth-promoting properties. Normal growth media often contain 2-10% of serum. Supplementation of media with serum serves the following functions [36] :

• Serum provides the basic nutrients (both in the solution as well as bound to the proteins) for cells.
• Serum provides several growth factors and hormones involved in growth promotion and specialized cell function.
• It provides several binding proteins like albumin, transferrin, which can carry other molecules into the cell. For example: albumin carries lipids, vitamins, hormones, etc. into cells.
• It also supplies proteins, like fibronectin, which promote the attachment of cells to the substrate. It also provides spreading factors that help the cells to spread out before they begin to divide.
• It provides protease inhibitors which protect cells from proteolysis.
• It also provides minerals, like Na+, K+, Zn2+, Fe2+, etc.
• It increases the viscosity of the medium and thus, protects cells from mechanical damages during agitation of suspension cultures.
• It also acts a buffer.

Due to the presence of both growth factors and inhibitors, the role of serum in cell culture is very complex. Unfortunately, in addition to serving various functions, the use of serum in tissue culture applications has several drawbacks [12, 37, 38]. Table 2 shows the advantages and disadvantages of using serum in the media.

For suspension culture, Pluronic F-68 at 0.1% can be added to reduce the water shear force and reduce foaming [39].

Culture medium is available in three forms from commercial suppliers:

1. Powdered form: it needs to be prepared and sterilized by the investigator.
2. Concentrated form: to be diluted by the investigator.
3. Working solution: to be used directly without further manipulation.

Powdered medium is the least expensive but needs to be sterilized [40]. It is advisable to filter-sterilize it prior to the addition of serum as the foaming that occurs in the presence of serum denatures the protein. Fetal bovine or horse sera can be added after filtration. Media should always be tested for sterility by placing it in a 37^oC CO₂ incubator for 72 hours prior to utilization to ensure that the lot is contamination-free. Medium should be stored at 4^oC. Since several components of the medium are light-sensitive, it should be stored in the dark.

The choice of cell culture media is extremely important, and significantly affects the success of cell culture experiments [41]. The selection of the media depends on the type of cells to be cultured and also the purpose of the culture and resources available in the laboratory [42, 43]. Different cell types have highly specific growth requirements, therefore, the most suitable media for each cell type must be determined experimentally [44]. In general, it’s always good to start with MEM for adherent cells and RPMI-1640 for suspension cells. Table 3 describes commonly studied cell lines and recommended growth media.

Primary cell culture provides unique and valuable research data, but most of the time cell number is the limitation. For such critical samples, especially from diseased human biopsies, a quality medium is required. Most of the life sciences companies are providing complete and ready to use, fully supplemented conditioned medium. This reduces the risk of contamination as well as save time, labor and money by eliminating the preparation steps and supplementation required. Moreover, all of these media are subjected to comprehensive quality control tests and each lot is routinely tested for growth promotion, the absence of cytotoxicity, and physical parameters such as osmolality and pH level. Table 4 describes the recommended media provided by different companies for commonly used primary cells.

Most commonly used culture media include the following.

DMEM has almost twice the concentration of amino acids and four times the amount of vitamins as EMEM, as well as ferric nitrate, sodium pyruvate, and some supplementary amino acids. The original formulation contained 1,000 mg/L of glucose and was first reported for culturing embryonic mouse cells. A further variation with 4500 mg/L of glucose has been proved to be optimal for the culture of various types of cells. DMEM is a basal medium and contains no proteins or growth promoting agents. Therefore, it requires supplementation to be a “complete” medium. It is most commonly supplemented with 5-10% Fetal Bovine Serum (FBS). DMEM utilizes a sodium bicarbonate buffer system (3.7 g/L) and therefore requires artificial levels of CO₂ to maintain the required pH. Powdered media is formulated without sodium bicarbonate because it tends to gas off in the powdered state. Powdered media requires the addition of 3.7 g/L of sodium bicarbonate upon dissolving it in water. DMEM was used initially for the culture of mouse embryonic stem cells. It has been found to be widely applicable in primary mouse and chicken cells, viral plaque formation and contact inhibition studies. It can also be used to culture hybridomas [46].

RPMI-1640 is a general purpose media with a broad range of applications for mammalian cells, especially hematopoietic cells. RPMI-1640 was developed at Roswell Park Memorial Institute (RPMI) in Buffalo, New York. RPMI-1640 is a modification of McCoy’s 5A and was developed for the long-term culture of peripheral blood lymphocytes. RPMI-1640 uses a bicarbonate buffering system and differs from the most mammalian cell culture media in its typical pH 8 formulation. RPMI-1640 supports the growth of a wide variety of cells in suspension and grown as monolayers. If properly supplemented with serum or an adequate serum replacement, RPMI-1640 has a wide range of applications for mammalian cells, including the culture of fresh human lymphocytes, fusion protocols and growth of hybrid cells like mouse hybridoma cells for antibody preparations. It is also often used to preserve/suspend peripheral blood mononuclear cells [47].

EMEM was among the first widely used media and was formulated by Harry Eagle from a simpler basal medium (BME). EMEM contains balanced salt solution, nonessential amino acids, and sodium pyruvate. It is formulated with a reduced sodium bicarbonate concentration (1500 mg/l) for use with 5% CO₂. Since EMEM is a non-complex medium, it is generally fortified with additional supplements or higher levels of serum making it suitable for a wide range of mammalian cells. Huang H et al, for example, maintained HepG2 (HB-8065)in EMEM medium from ATCC [48]. EMEM was used to maintain HeLa cells [49].

These were originally developed to support the clonal outgrowth of Chinese hamster ovary (CHO) cells. There have been numerous modifications to the original formulation including Hams’s F-12 medium, a more complex formulation than the original F-10 suitable for serum-free propagation. Mixtures were formulated for use with or without serum supplementation, depending on the type of cells being cultured.

Ham’s F-10: It has been shown to support the growth of human diploid cells, for example, human fibroblast cells [50], and white blood cells for chromosomal analysis.

Ham’s F-12: It has been shown to support the growth of primary rat hepatocytes and rat prostate epithelial cells. Ham’s F-12 supplemented with 25 mM HEPES provides more optimum buffering.

Coon’s modification of Ham’s F-12: It consists of almost two times the amount of amino acids and pyruvate as compared to F-12 and also includes ascorbic acid. It was developed for culturing hybrid cells produced by viral fusion.

DMEM/F12: It is a mixture of DMEM and Ham’s F-12 and is an extremely rich and complex medium. It supports the growth of a broad range of cell types in both serum and serum-free formulations. HEPES buffer is included in the formulation at a final concentration of 15 mM to compensate for the loss of buffering capacity incurred by eliminating serum.

IMDM is a highly enriched synthetic media well suited for rapidly proliferating, high-density cell cultures. IMDM is a modification of DMEM containing selenium, and has additional amino acids, vitamins and inorganic salts as compared to DMEM. It has potassium nitrate instead of ferric nitrate and also contains HEPES and sodium pyruvate. It was formulated for the growth of lymphocytes, for example the differentiation of monocytes into macrophages [51], and hybridomas. Studies have demonstrated that IMDM can support murine B lymphocytes, hemopoietic tissue from bone marrow, B cells stimulated with lipopolysaccharide, T lymphocytes, hybrid cells, and others [52]. HAP1 cells from Horizon Discovery, a cell line derived from near-haploid chronic myeloid leukaemia KBM7 cells, and knockout cell lines derived form HAP1 are usually maintained in IMDM media [53-55].

Table 5 describes different cells/cell lines which can be cultured using above mentioned media:

The complexity of the composition of cell culture media provides many challenges to optimize individual components of media. Most of the classical culture media were devised for small-scale low-density cultures and often require serum as a key nutrient. However, in the biotechnology industry where there is a need to sustain high cell densities and increase cellular productivity, development and optimization of culture media is very critical [56]. Typically, media for the biotechnology industry are serum-free and have a much higher concentration of nutrients than classical media [57, 58]. Optimization of media requires the following parameters to be considered:

The type of product needed will determine the medium optimization strategy.

For the rapid generation of cell numbers, cell growth rate and viability are critical. So, cell culture media should support maximal cell growth and sustain cell viability at increasing cell densities.

For the production of virus, not just high cell densities are required but there must be abundant nutrients in the media to sustain virus replication after infection, for example, DMEM High Glucose H-21 for Lenti-X 293T cells [59].

For the production of recombinant protein, high cell density is required. However, nutrients required for the cell growth can compete with those required for the production of proteins. It is, therefore, very important to carefully determine the maximum cell densities a given medium can sustain for a required level of productivity. In addition, it is very important to consider that changes to the medium during optimization must not affect product quality.

Different cell lines have different nutritional requirements because of the difference in metabolism which dictates media optimization methods. The most common cell lines used in the biotechnology industries are CHO cells, BHK-21, hybridoma cells, myeloma cells, and normal diploid fibroblasts. Certain cell lines have specific nutritional requirements, such as cholesterol for NS0 myeloma cells. Normal diploid fibroblasts require attachment factors to adhere and spread out on a surface for growth. They grow too much lower densities and therefore do not need nutrients in high quantities. Hybridoma cells lines are generally highly dependent on glutamine. They typically lack a stationary phase after reaching a peak cell density and then decline rapidly in viability. Optimization of medium, thus, would reduce the decline in viability and improve monoclonal antibody production.

Manufacturing process mode would not only affect the choice of cell culture medium but also approaches to optimization. Different manufacturing processes used are:

Batch Process: A single medium is used to sustain cell growth and productivity. Medium should therefore be rich in nutrients but remain in physiological limits of the cells.

Fed-batch: Several kinds of media are used over the course of the cell culture, depending on the stage of the process. A growth medium is designed in such a way that it has lower nutrient concentrations when cell densities are low during inoculation but maintain high rates of cell growth during culture scale-up and early production. A separate production medium which has increased nutrient concentrations can also be used when the culture reaches the production stage.

Labome conducts systematic surveys about reagents and instruments cited in formal publications. Labome has curated formal articles with citations of cell culture media. Table 6 lists the major types of the media and the main suppliers.

Invitrogen / Life Technologies is one of the major suppliers of DMEM medium. Its DMEM media were used to culture rat primary cortical astrocytes [45], A498 cells [48], A549 cells [76], C2C12 cells [25], COS-1 cells [64], COS-7 cells [77], HEK293T cells [48, 62, 75-77], Hela cells [48, 75], RCC4 cells [25], STHdh [75] and mouse embryonic fibroblasts [25].

MilliporeSigma DMEM media (including brands such as Merck, EMD, Sigma) were used for culturing 293T cells [25]. Other suppliers also provided the DMEM media, such as GE Healthcare [73], Biochrom [68, 69], Cellgro [78], Nissui Pharmaceutical Co [79], and Dundee Cell Products [80].

MEM (Minimum Essential Medium) can be used with a variety of suspension and adherent mammalian cells. Invitrogen was also the major supplier of MEM medium. Applications of their products range from ES cell culture to show proper organogenesis needs B-type lamins in mice [81] and other topics [82].

Other MEM medium suppliers were cited as well, including Lonza [83, 84], Irvine Scientific [85] and MilliporeSigma [86, 87].

Roswell Park Memorial Institute (RPMI)-1640 medium was originally developed to culture human leukemic cells. Invitrogen / ThermoFisher RPMI-1640 has been used for a variety of mammalian cells, including mouse hybridoma cells [88], K562 [49] and SK-N-BE(2) cells [25], T47D, RENCA and CT26.WT cells [63]. ATCC [89], Athena Enzyme Systems [90], Lonza [91], Mediatech [92], Nacalai Tesque [79] and US Biological [93] also provided RPMI-1640 media.

DMEM/F12 is a 1:1 mixture of DMEM and Ham's F-12. It is an extremely rich complex medium. Most of the DMEM/F12 media cited in this survey were provided by Invitrogen. They were used to support the growth of a wide range of cell types and study their biological characteristics, such as CHO cells [94] and A431, COLO-16, SCC25 and KJD cells [63]. Advanced DMEM/F-12 (ADMEM/F-12) allows the culture of mammalian cells with reduced FBS supplementation. Vaidyanathan S et al maintained human upper-airway basal stem cells in ADMEM/F-12 from Thermo Fisher, supplemented with B27 supplement [95].

Other suppliers also provided the DMEM/F12 medium, such as STEMCELL Technologies [96].

Ham's F-12 Nutrient Mixture (F-12) has been used for serum-free growth of CHO cultures as well as serum-supplemented growth of other mammalian cells. Invitrogen offered a variety of F-12 modifications for a range of cell culture applications [97, 98].

Besides, products from MilliporeSigma [99] and Wako [100] were cited as well.

Neurobasal medium is a basal medium that meets the special cell culture requirements of post-natal and adult neuronal cells. All the neuralbasal medium cited in the survey were provided by Invitrogen. It was used to grow neuronal cells from hippocampus [62, 64], cortex [75], and other regions of the brain [101], and iPS-derived neural progenitors [75]. More recently, in a comparison study with Neurobasal medium, Brainbits NBACTIV4 media was shown to be a better one in maintaining microglia in a relatively quiescent state in a co-culture glial-neuronal system.

McCoy's 5A medium is a general purpose medium that supports the propagation of many types of cells. Invitrogen provided most of the McCoy's 5A medium in the surveyed cohort for SKBR3 cells [63], U2OS cells [49] and others [79].

Commercial suppliers of cell lines often provide specially-formulated media. For example, D Wrapp et al maintained FreeStyle293F cells and HEK293-S cells in FreeStyle293 expression media from Thermo Fisher Scientific and ExpiCHO-S cells in ExpiCHO expression media from Thermo Fisher Scientific [102].

Other media include Dynamis medium from Thermo Fisher for human embryonic kidney cell line 293T [74], Essential 8 (E8) media from Thermo Fisher for expanding hESCs [66, 103] or iPSC lines [75, 104], StemFlex culture media for human H9 and H1 ES cells and iPS cells (IMR90-4) [105], Airway Epithelial Cell basal medium from Promocell for human primary airway epithelial cell culture [72], alpha-modified minimum essential medium (α-MEM) from Invitrogen for OP9M2 stroma and human haematopoietic stem cell co-culture [106] and from Wako Pure Chemical Industries for primary dental pulp stem cells [107], StemMacs iPS-Brew media from Miltenyi Biotech for (ESC line H1 and iPSC line EC11 [108], TeSR-E8, mTeSR1, mTeSR Plus medium from STEMCELL Technologies for human iPS cell expansion and culture [6, 109], GMEM (Glasgow Minimum Essential Medium) from MilliporeSigma for ES cell culture [110], Opti-MEM I from Thermo Fisher for HEK293 cells [94], SmGM-2 from Lonza for human bronchial smooth muscle cells [94], fibroblast growth media / FGM from Lonza for lung fibroblast cells [94], StemPro-34 serum free growth medium from Thermo Fisher for LAD2 cells [94], Eagle’s Minimum Essential Medium from MilliporeSigma (M2279) for Caco-2 cells [76], , mTESR1 medium from Stem Cell Technologies for feeder-free maintenance of human embryonic stem cells line H1 [111] and human iPS cell line [112], ECGM-2 media from Promocell for primary murine lung endothelial cells, EGM2-Bullet kit medium from Lonza for HUVEC cells [113], Williams’ Medium E from Gibco for primary human hepatocytes [114], medium M254 from Thermo Fisher Scientific for melanocytes [115], CnT07 media from CellnTEC for keratinocytes [115], TNM-FH media for Sf9 insect cells [25], mammary epithelial cell growth basal medium (MEBM) from Lonza for human mammary epithetelial cells [25], complete skeletal muscle media from ZenBio for primary human skeletal myoblasts [25], NeuroCult NS-A basal medium from StemCell Technologies for glioma cell lines TS603, TS516, and BT260 [25], Ham's F-10 [50] and Fibroblast Growth Kit-Low Serum from ATCC for primary dermal fibroblasts [116].