Module 3: Culture Medium

Culture medium from: https://bioscience.lonza.com/lonza_bs/US/en/cell-culture-media

This is the third module in a four part series titled “Zero to One on the basic science behind cultivated meat”. You can click on these links to access: Course Overview, Module 1 (Cell Culture Basics), Module 2 (Cell Sources), Module 4 (Process monitoring).

By the end of this module, you should understand:

  • Basal culture medium composition and sources;
  • Serum-free medium;
  • The use of growth factors in culture medium.

*Grammar/syntax note: The words for culture ‘media’ vs. ‘medium’ are often used interchangeably. Media is simply the plural form of medium. Don’t worry too much about it.

The culture medium is probably the most important cell culture component, as it provides the liquid environment and nutrients that cells need to stay alive. All cell culture medium starts with a ‘basal medium’ that is composed of the essential ingredients that most types of cells will need to support their growth and metabolism, while providing a liquid environment that mimics the chemistry found in the blood and the fluids that surround cells inside the body’s tissues. Read this web page on cell culture media (Sigma-Aldrich/Merck), which provides a good overview of different types of culture medium and the essential components.

Of these culture medium types, DMEM (Thermo Fisher Scientific) is the most commonly used basal medium. This can be purchased as pre-mixed bottles of liquid, or as a powder that you can dissolve yourself and then filter to sterilize. This website also has a Cell Culture Media Formulation Tool, where you can choose different options that you need and it will tell you which type of DMEM is right for you. For an overview of what different components are included in DMEM (in this case, high glucose DMEM), you can see a detailed description of the formulation.

Using commercially-supplied culture medium for large-scale cell manufacture is prohibitively expensive, therefore various cultivated meat companies and organizations are investigating different approaches to make more cost-effective formulations from scratch. The Shojinmeat project has a recipe for DIY culture medium on its website, and Canadian company Future Fields has developed a serum free growth medium for cultivated meat. Many of the current cultivated meat companies are either using commercially available culture medium, developing their own formulations in-house, or developing formulations with commercial collaborators. The latter is Mosa Meat’s approach, where they are working with an animal feed supplier called Nutreco to source raw ingredients. This is mentioned in this article on Upscaling in the race to cultured meat (European Biotechnology).

Supplements to consider adding to your basal medium are antibiotics and antimycotics. The most commonly used supplements for mammalian culture are penicillin-streptomycin (Pen-Strep) and antibiotic-antimycotic (A/A) (both are from Thermo Fisher Scientific). A/A is especially versatile because it contains penicillin, streptomycin, and an anti-fungal component. Although it is generally considered undesirable to use antibiotics for producing food products, realistically you will probably need to use them while developing your initial prototype in a standard cell culture lab. In the pharmaceutical industry they are able to avoid using antibiotics and antimycotics by operating within extremely clean and well controlled facilities, where most of their processes happen inside closed systems. When you are working in a typical biological laboratory, your cultures are much more exposed to the external environment, even when using the most careful sterility precautions. This is especially true if you are working with primary cells extracted from a tissue biopsy, which are more prone to contamination in the initial processing steps.

Study questions:

1. Why does culture medium need to be buffered?

2. What is the main energy source for cells?

3. Which component of the culture medium is used by the cells to make proteins?

For the majority of cell culture applications, using a basal medium such as DMEM is not sufficient to keep cultured cells alive and proliferating. Therefore most culture medium formulations will include serum, typically at 10–20% by volume, indicating that serum is an extremely important component of cell culture medium.

Refer back to this web page on cell culture media (Sigma-Aldrich/Merck), and revisit the section on serum. The fact that serum is listed first in this article on cell culture medium gives an indication of how important it has traditionally been for maintaining mammalian cell culture. The most commonly used type of serum is FBS (fetal bovine serum), which is extracted from fetal calf blood. Because serum is derived from animal sources, this is a component that must be replaced for cultivated meat production in order to create a truly animal-free product. Serum can be difficult to replace because it is ‘undefined’ — this means that its components are not fully known or understood, and there is batch-to-batch variation. Therefore culture media with serum replacements are often referred to as ‘defined’ media, where every component is known.

There are commercially-available serum replacements, but these are often very expensive because they include either purified or recombinant proteins to replace the most essential contents of serum. They are able to justify the high costs because the typical applications for their products are biomedical research and pharmaceutical development. Thermo Fisher Scientific offers products including KnockOut serum replacement which is used for culturing embryonic stem cells and induced pluripotent stem cells (there is a xeno-free option, meaning it’s animal-free), and a variety of complete serum-free media, where the stem cell media may be most relevant for cultivated meat. However, as you’ll see from the price per bottle, in the long-term you’re going to need to find a cheaper alternative. Other established companies such as Peprotech are also making animal-free serum replacements, and a few newer companies such as Agulos Biotech and Defined Bioscience are developing serum replacements for cell culture applications, although their products are not specifically geared towards cultivated meat.

This article on the need for the industry to ditch serum (Wired) is a few years old, but it provides a good overview of the issue and some of the efforts towards finding serum replacements.

What’s also exciting is that Merck has become interested in cultivated meat, and is actively working on developing serum-free medium for cultivated meat applications. Several other companies working on serum replacements or serum-free media specifically for cultivated meat include Future Fields (mentioned in the previous section), Multus Media, Heuros, and Biftek.

Multus Media: Animal-free serum substitute

Due to the lack of cost-effective serum replacements on the market, many of the early cultivated meat companies have had no choice but to use the expensive commercially available products, or develop their own culture medium formulations in-house or via exclusive partnerships. Unfortunately, this means that a lot of R&D and innovation in this area has happened behind closed doors, and likely won’t be shared with the public unless these companies decide to patent their formulations.

Study questions:

4. What are the main components of serum?

5. Are there any serum alternatives for cultivated meat that are currently available for purchase?

6. Will it be possible to create a ‘universal’ serum replacement that works for all cell types? Explain why or why not.

One of the most important components of serum is growth factors, which is why it is extremely important to supplement culture medium with growth factors in the absence of serum. Also, certain cell types will require additional specific growth factors to be added to the culture medium to support their growth and differentiation. Typical amounts of growth factors present in culture medium are around several nanograms/mL, which doesn’t sound like a lot, but growth factors are typically sold as a few micrograms for several hundred dollars. It really adds up!

This short article on growth factors (Britannica) provides a good overview of what growth factors are and why they are important. This web page on growth factors and cytokines (Sigma) further explains how growth factors act upon cells, and why you may need to add growth factors to your cell culture medium.

From Sigma Aldrich: General Growth Factor and Cytokine Signaling Mechanism

Growth factors are traditionally harvested from animal sources, therefore in order to use non-animal growth factors, they must be produced by recombinant methods (News Medical Life Sciences).

This article from 2018 on animal-free cultured meat (C2W International) discusses some of the issues and cost considerations around growth factors, while describing Mosa Meat’s approach to incorporating growth factors in their culture medium (note: because this article is from 2018, it might not be fully relevant now). According to a recent Mosa Meat blog post, their scientists have been able to reduce their overall culture medium costs by 88 times, although of course they won’t tell us exactly how they did this!

Similar to the previous section, it’s possible to purchase growth factors from standard cell culture supply companies like Sigma-Aldrich/Merck, Thermo Fisher Scientific, and Peprotech, some of which will be animal-derived and some recombinant. However, these products will all be prohibitively expensive and available only at very small quantities. A variety of new companies have emerged in order to address the issues of cost and scale for producing recombinant growth factors, which are not specifically targeted towards cultivated meat but to biomedical technology more broadly (Tiamat Sciences, NUProtein, Richcore Lifesciences). One new company, Integriculture, is developing a unique platform where certain growth factor-secreting cell types are grown and their ‘conditioned’ medium containing these growth factors is then used to grow your cell type of interest.

Optional extra reading: Liz Specht’s white paper on culture medium (GFI) provides an excellent and detailed resource on the issues behind producing all of the components of culture medium for cultivated meat, where growth factors are noted as the main contributor to overall cost.

Study questions:

7. What are the main effects of growth factors on cells?

8. List a few different ways to produce recombinant proteins.

9. According to Mark Post, which industries (compared to laboratory/pharmaceutical) can produce recombinant proteins much more cheaply?

10. (Optional) — which two growth factors are highlighted in Liz Specht’s white paper?

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