The in vitro cell culture involving primary cells and immortalized cell lines, has had a tremendous impact on scientific pursuit. Cell lines have been beneficial and easy to use due to their unlimited life span. Primary cells are directly isolated and cultured directly from native tissue of humans or animals. Primary cell culture has been the key player in research owing to the retention of tissue-based characteristics by primary cells. The primary cell culture facilitated the understanding of the basic cell biology, delineation of signaling pathways, editing of the genome, and screening of drugs.
It has been particularly useful in the drug development process for evaluating the efficacy, metabolism, and toxicity of drug candidates. The maintenance of primary cells in culture is challenging and requires an appropriate culture medium. The medium supports survival, growth, and proliferation of cells to avail them for research purposes. The optimization of medium composition avoided its interference with the cellular processes and also with the studies on cells.
History of Cell Culture Medium
In the initial stages of research, scientists used to experiment directly on animal tissues and organs. To maintain these tissue fragments, they began employing bodily fluids like blood, lymph, tissue fluid, and serum. Afterwards, they even proceeded with the development of balanced salt solutions. These solutions comprised inorganic salts and buffers that ensured adequate pH and osmotic pressure of the medium. Additionally, researchers also added glucose as the energy source. Sydney Ringer’s solution was the first balanced salt solution formulated in the late 19th century. The isolation of cells from the tissue necessitated the formulation of proper cell culture medium. To decode the components of cell culture media, scientists began identifying the constituents of plasma and serum. In addition, they also lowered the concentration of natural medium substituting it with synthetic or chemically defined components.
Components of Cell Culture Medium
As the scientists experimented with different elements of the media, they established the following components for cell culture medium:
Glucose
It is a carbohydrate (sugar) and serves as a source of energy.
Amino Acids
Scientists identified amino acids as the first component in natural mediums like plasma and serum, that were essential for cell growth. The addition of essential amino acids is particularly crucial since cells could not synthesize them. Also, non-essential amino acids in the medium lessen the burden of biosynthesis of those amino acids on cells. L-Glutamine is an unstable amino acid that dissolves into a form that cannot be utilized by the cells. Therefore, it is added just before the use, or it is added in the form of Glutamax, a dipeptide of alanine and glutamine. It has higher stability than glutamine and also minimizes ammonia that accumulates due to glutamine degradation.
Buffer
Buffer is vital to maintain the pH. Although CO2 has buffering capacity, additional buffers like HEPES or sodium bicarbonate. Sodium bicarbonate is not available in the powdered media as it dissolves into gas during the process. Therefore, for powdered media, sodium bicarbonate is added exogenously.
Phenol Red
It is a pH indicator that can be identified visually. It changes the media color to yellow at low pH (acidic) and bright pink or fuschia at high pH (alkaline). But it can affect colorimetric assays, and therefore, cells should be suspended in buffer for such studies. Moreover, it also mimics estrogen, which can affect the growth of cells with estrogen receptors.
Vitamins
Vitamins are precursors to several cofactors, and few vitamins have antioxidant properties. Thus, they are crucial for cell growth and proliferation.
Inorganic Salts
They are essential for maintaining osmotic pressure and membrane potential of the cell by providing ions like sodium, potassium, and calcium.
Proteins
Several proteins have been found in serum and play diverse roles. For example, blood albumin binds to biomolecules to facilitate their transport. Transferrin is essential for iron transport. Fibronectin supports the attachment of cells to substrates.
Trace Elements
These include minerals like zinc, iron, copper, etc. They act as active centers of various enzymes and also participate in electron transfer reactions.
Lipids
Lipids are components of cell membrane and are responsible for the transport of biomolecules and signaling pathways. Cells don’t possess all the enzymes for lipid synthesis and therefore require exogenous lipids.
Serum
Serum contains growth factors, carbohydrates, proteins, vitamins, hormones, and trace elements. The serum constituents promote cell growth and proliferation while also supporting substrate attachment and pH maintenance. Some media contain serum while chemically-defined media are supplemented with chemical components mimicking serum.
Types of Cell Culture Media
Media components depend on the cell type and the research applications.
Serum Containing Media
Sources of serum are fetal bovine serum (FBS) and calf serum (FCS). FBS contains fewer cell growth inhibitors as opposed to FCS. Therefore, FCS is employed in contact inhibition growth studies and FBS for highly proliferating cells. Serum is added to the basal medium, which requires additional components for cell proliferation.
Eagle Media
Harry Eagle formulated the Basal Medium Eagle (BME) in 1955. It contained minimal amino acids and vitamins to support proliferation. Afterwards, he modified the BME to Minimal Essential Medium (MEM) by supplementing BME with an increased number and concentration of amino acids. Dulbecco’s Modified Eagle Medium (DMEM) is an altered version of MEM that consists of a higher concentration of amino acids, vitamins, and glucose. It is available in low and high glucose versions. Stanners also amended MEM to form α-MEM by adding lipoic acid and pyruvate along with more vitamins and amino acids. Iscove’s Modified Dulbecco’s Medium (IMDM) consists of additional components than DMEM, like biotin, transferrin, and lipids with increased concentrations of amino acids and vitamins. It also contains HEPES and is useful for cells with increased proliferation capacity.
RPMI 1640
Moore et al. formulated RPMI 1640 with low calcium and magnesium, but a high amount of phosphate. This media was specifically prepared for suspension cells like lymphocytes and hybridomas.
Serum-Free Media
Although serum has several essential components that support cell proliferation, it’s undefined, and batch-to-batch variation affects the uniformity of media and also causes reproducibility issues. Therefore, researchers use chemically defined media without serum addition.
Ham’s F12
It contains substitutes like linoleic acid and putrescine for serum elements. It is the first chemically defined medium created to support the colony formation of Chinese hamster ovary (CHO) cells.
M199
It comprises amino acids, vitamins, and precursors of nucleic acid. CMRL1066 is the modified form of M199 containing reducing substances like glutathione, coenzymes, and nucleic acid precursors. It removes the fat-soluble vitamins and proteins of M199 for protein-free culture.
Types of Primary Culture and Their Media
Several options for media are available for primary cell culture. But the unique characteristics and applications of primary cells prompted the addition of extra components to support normal cell functions. Different types of primary cells require customized media for their optimal culture.
Epithelial Cells
Epithelial cells are present in the outer layer of skin. They maintain the barrier and homeostasis in the skin. Therefore, in culture, they need additional components such as insulin, hydrocortisone, triiodothyronine, epidermal growth factor (EGF), and transferrin for their proper growth and functioning.
Endothelial Cells
Endothelial cells compose blood vessels and remain quiescent in vivo. For in vitro culture, they require several growth factors like EGF, vascular endothelial growth factor (VEGF), insulin growth factor 1 (IGF1), and fibroblast growth factor (FGF), along with ascorbic acid, hydrocortisone, and heparin.
Fibroblasts
Fibroblasts secrete the extracellular matrix (ECM) components. They have applications in wound healing, inflammation, tumor response, and cell signaling research. Their culture medium is a mixture of DMEM and F-12 media supplemented with non-essential amino acids and fibroblast growth factor 2 (FGF2).
Neuronal Cells
Research in neurological diseases has grown in the past decades, mandating the optimization of neuronal cell culture. The culture media should support the cell survival, growth, and proliferation as well as their electrophysiological activity. Thus, the culture media has a B27 supplement comprising glutathione, progesterone, albumin, putrescine, triiodothyronine, and insulin-transferrin-selenite (ITS).
Stem Cells
Medium for stem cell culture should promote their self-renewal capacity. Therefore, their medium has reduced serum concentrations to avoid the effects of growth factors and hormones. Their medium includes ascorbate, ITS, and basic fibroblast growth factor (bFGF). The differentiation media for stem cells contain growth factors that drive cell transformation towards specific cell lineages.
Immune Cells
RPMI 1640 media have been synthesized specifically for immune cells to facilitate their proliferation and cytokine secretion.
Liver Cells
Liver cells or hepatocytes perform functions like glucose consumption, urea depletion, and anaerobic glycolysis. To support hepatocyte functions, media is supplemented with hepatocyte growth factor (HGF), fibroblast growth factor 4 (FGF4), and oncostatin M.
Conclusion
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FAQs
Q: What are natural and synthetic media?
Natural media is extracted from the natural sources and is devoid of synthetic constituents. Plasma, serum, and lymph are some examples of natural media. Synthetic media has exogenously added chemical components. It may or may not contain a certain quantity of natural media.
Q: What is the role of serum in the media?
Serum is an undefined component of media. It consists of vitamins, growth factors, hormones, carbohydrates, polyamines, minerals, and fatty acids. It supports cell proliferation, cell attachment to the substrate, and cell functioning.
Q: What are the disadvantages of adding serum in media?
Serum varies from batch to batch and lacks specified composition. This affects the uniformity of the media and might cause repeatability problems. Additionally, it can be a source of contamination and may contain growth inhibitors that negatively impact cell growth.
Q: Why is phenol red added in the media?
The pH of the culture media can be visually determined by the addition of phenol red. It turns yellow at low pH, indicative of media depletion or contamination. But it interferes with fluorescence-based or colorimetric assays requiring the cells to be suspended in buffer for such assays.
