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A Deep Dive into Cell Culture Media

A Deep Dive into Cell Culture Media

A critical step in bioprocessing in determining the needs of your cells, starting with your growth medium. But with so many options, where does one start? The first thing is to understand what cell culture media is, the different classifications, and how we can take it to the next level.

What Is Cell Culture Media?

Culture medium is a nutrient-rich liquid or gel designed to support the growth of cells. It can also be formulated to help regulate environmental conditions, such as osmotic pressure and pH. 

In contrast to microbiological culture media, cell culture media are usually more complex due to the fact that cells derived from whole organisms often cannot grow without the addition of, for instance, hormones or growth factors which usually occur in vivo.

Cell Culture Media Components

Some of the components found in bioprocessing media include:

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    • Carbon source: This is an essential component of bioprocessing media as it provides the cells with the energy and carbon they need for growth and metabolic processes. Common carbon sources are glucose, fructose, sucrose, sorbitol, and mannitol.
    • Nitrogen source: Nitrogen is an essential component of amino acids and nucleic acids, and is therefore critical for growth and productivity. Common nitrogen sources include ammonium salts, nitrates, and urea.
    • Minerals: Cells require various minerals for growth, including iron, magnesium, calcium, and phosphate. These minerals can be provided through the use of salts such as ferric nitrate, magnesium sulfate, and calcium chloride.
    • Vitamins: Vitamins are essential components of bioprocessing media, as they play important roles in various metabolic processes. Common vitamins used in bioprocessing media include biotin, pantothenic acid, and thiamine.
    • Growth factors: Growth factors are compounds that support the growth and productivity of cells. Examples of growth factors include yeast extract, tryptone, and casamino acids.
    • Buffering System: We know that most cells can only grow well within a narrow pH range.  Most animal cells, mammalian included, are kept between a pH value of 7.2 to 7.4. However, these ideal values can vary depending on the cell type.  Regardless, cellular byproducts and environmental changes during handling can cause media to become more basic or acidic. To counteract this, some media contain chemical buffering systems. The most common buffer system for mammalian cells with minimal biological impact is sodium bicarbonate.
    • Phenol Red: Again related to maintaining the correct pH, some media contain indicators  that change color, providing a visual indictor that the pH has drifted outside the optimal range. The most common dye, phenol red (PR, also known as phenolsulfonphthalein) gradually transitions from yellow to red over a pH range of 6.2 to 8.2. The pH level is determined by matching the color of the solution to a color chart.
    • Antibiotics: Antibiotics are often used in cell culture as a means to prevent contamination, maintain aseptic conditions, or select for cells containing genetic modifications. PenStrep, a cocktail of both penicillin and streptomycin, is one of the most popular antibiotics used.


Different Classifications of Media

Media can be classified in a variety of ways.

1. According to Chemical Composition

Basal vs. Complex
Basal media typically consists of a minimal nutrient solution, providing only the essentials required for growth, such as carbon source, amino acids, and salts. Basal media are often used for the selective growth of specific cells, as they allow for the addition of specific nutrients or other factors required for growth.

In contrast, complex media provide a wider range of nutrients, including additional amino acids or vitamins. Complex media are often used when the nutritional requirements of the cells are unknown, or when the goal is to obtain a large quantity of cells. Complex media can also contain additional components, such as blood, serum, or tissue extracts, which can provide additional growth factors and support the growth of more fastidious cells.

Natural vs. Artificial
Natural media contains biological components derived directly through tissue extraction or bodily fluids. While it can be used for a wide variety of applications, the exact composition is not always well-defined and thus can be difficult to reproduce.  

Artificial media, also known as synthetic media, is made up of defined compounds. Often, this media is created to support a particular application, such as increased protein expression or cell longevity. 

Natural Media Coagulant or clots Plasma separated from heparinized blood, serum, and fibrinogen
Tissue extracts Extracts of chicken embryos, liver, and spleen and bone marrow extract
Biological fluids Plasma, serum, lymph, amniotic fluid, and pleural fluid
Synthetic Media Serum‐containing media Human, bovine, equine, or other serum is used as a supplement
Serum‐free media Crude protein fractions, such as bovine serum albumin or α‐ or β‐globulin, are used as supplements
Xeno‐free media Human‐source components, such as human serum albumin, are used as supplements but animal components are not allowed as supplements
Protein‐free media Undefined components, such as peptide fractions (protein hydrolysates) are used as supplements
Chemically defined media Undefined components, such as crude protein fractions, hydrolysates, and tissue extracts, are not appropriate as supplements, but highly purified components, such as recombinant proteins are appropriate supplements

Table 1. Categories of Animal‐cell Culture Media1


2. According to Physical State

Cell culture media can be purchased in liquid (broth), a solid (agar plates), or as a semi-solid (deeps). Solid and semi-solid media contain solidifying agents such as agar or gelatin. Biphasic culture medium is an option that contains both a solid part and a liquid part in a single bottle.

3. According to the Method of Preparation

Ready-to-use medium is a solid or liquid medium supplied in ready-to-use form or ready-to-use after reflow and supplementation.

Medium prepared from dehydrated formulations is purchased in dry form and requires rehydration and processing before use, resulting in either a complete medium or an incomplete medium to which supplements are added before use.

 

Media Optimization

While there are many options for cell culture media readily available on the market, media development and optimization continue to be important focuses for the those in bioprocessing. A properly optimized cell culture media can support high cell density with minimal changes in cellular phenotype, thus resulting in high bioactivity.

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Even with this in mind, a complete media optimization is often avoided due to the workload associated with the task. Bioprocess media optimization involves several key steps to enhance the growth and productivity of microorganisms or cells used in bioprocessing:

  1. Screening of base media: The first step in bioprocess media optimization is to screen a variety of base media to identify which ones support the growth and productivity of the cells or microorganisms. This can be done through simple empirical methods or through more complex statistical methods such as Design of Experiments (DOE).
  2. Identification of critical components: Once a suitable base media has been identified, it is important to determine which components are critical for growth and productivity. This can be achieved by systematically varying the concentration of individual components and observing the effect on cell growth and productivity.
  3. Optimization of critical components: Based on the results obtained in step 2, the concentrations of critical components can be optimized to achieve maximum growth and productivity. This can be done using a combination of empirical methods and mathematical models, such as response surface methodology.
  4. Validation of optimal conditions: Once the optimal conditions have been identified, it is important to validate them by repeating the experiments and confirming that the same results are obtained.
  5. Process scale-up: After the bioprocess media has been optimized, the process can be scaled up from laboratory to industrial scale, ensuring that the optimal conditions for growth and productivity are maintained throughout.
  6. Continuous monitoring and improvement: Bioprocess media optimization is an ongoing process, as changes in the bioprocessing conditions or the microorganisms or cells being used may require further adjustments to the media components. It is important to continuously monitor the performance of the bioprocess and make improvements as needed to ensure optimal growth and productivity.

Overall, bioprocess media optimization is a critical aspect of bioprocessing, as it has a direct impact on the cost, quality, and sustainability of the final product. The optimization process can be complex and time-consuming, but the results can be significant in terms of improved growth and productivity, reduced costs, and increased sustainability.

 


References
Yao T, Asayama Y. Animal‐cell culture media: History, characteristics, and current issues. Reprod Med Biol. 2017;16:99–117. https://doi.org/10.1002/rmb2.12024