Culture Medium Development

Definition and Types of Culture Medium

Culture medium is the key raw material of cell culture, which is essentially a liquid or gel that supports cell growth and provides cells with an appropriate amount of growth factors, vitamins, minerals, glucose, amino acids, and other nutrients. It can be simply divided into two categories: medium with serum and medium without serum.

• Serum culture medium

Serum medium is one of the most commonly used mediums in which fetal bovine serum (FBS) is the only or major additive component of the base medium. In general, the small animal serum is superior to human adult serum because it contains less gamma-globulin and has fewer antibody interactions. As a result, it has fewer negative effects on cell growth. The serum contains protein, vitamins, minerals, growth factors, hormones, and other large numbers of biologically active ingredients, which can provide essential nutrients for cells, promote cell proliferation, and improve cell function. It also contains a variety of vectors such as albumin and transferrin, which can transport vitamins, hormones, and lipophilic molecules into the cell. In addition, the serum improves cell adherence through fibronectin, in which protease inhibitors and some metals (such as calcium, iron, zinc, and magnesium) protect cells from proteolytic hydrolysis. Meanwhile, the serum acts as a viscosity enhancer and buffer, protecting cells from shear stress and extreme acid/base environmental damage. However, there are some drawbacks to the serum:

1. Large differences between batches and poor reproducibility. This difference is due to the unclear types and amounts of bioactive ingredients in serum, which may contain various growth factors, trace elements, hormones, and proliferative transcription factors. These components vary widely from batch to batch of serum and can cause inconsistent cell growth and alter the cell's response to chemical substrates. As a result, results from different study groups or laboratories are poorly comparable.

2. Serum is an important source of microbial pollution, including mycoplasma, virus, prion, fungus and yeast, endotoxin, etc.

3. Some serum components can be metabolically combined into cells and expressed as molecules such as antibodies and receptors, thus adversely affecting the interaction and reaction between cells and the surrounding environment.

4. In terms of ethics and morality, the method of collecting serum from calves, fetal cows, and other animals through heart puncture will cause pain to the animals and is not humane.

5. The economic cost is relatively high. Although some manufacturers are switching to the serum-free culture medium, the demand for serum-containing culture medium continues to rise. Moreover, the cost of serum has remained high due to the need to invest in infrastructure to meet GMP standards.

6. Some components in the serum can interact with other medium additives to form highly toxic compounds. For example, polyamine oxidase can interact with polyamines in the medium (e.g. spermine and spermidine) to form highly toxic polyspermine.

• Serum-free culture medium

The second category is serum-free medium, which can be further subdivided into serum-free medium, animal-free medium, protein-free medium, and chemically defined medium.

• Serum-free medium: using other additives to replace serums, such as discrete large protein molecules derived from plant or animal tissues. However, it still contains some substances of unknown composition like animal or plant hydrolysates.

• Animal-free medium: no animal or human components, but has catalysts, non-animal peptones, enzymes, plant hydrolysates, and some cell culture-derived recombinant proteins such as hormones, growth factors, and cytokines.

• Protein-free medium: only hydrolyzed or digested proteins, and some proteins with low molecular weight like insulin. These mediums not only support cell growth and product expression but also reduce the burden of downstream separation and purification. However, the hydrolysate and lipid components could not be identified one by one, so the chemical composition of the medium was not clear.

• Chemically defined medium: contains only organic or inorganic components that are chemically and structurally defined. They may also contain recombinant proteins produced by specific cell lines, bacteria, or yeast, such as hormones, growth factors, and cytokines. Its main advantages are good reproducibility, clear composition, and no ethical problems. As a result, many biopharmaceutical companies are shifting from existing animal-derived products to the chemical-specific culture medium. However, it also has some disadvantages: higher cost, cell type specificity, and a longer period of cellular adaptation.

New methods for culture medium development

• "Omics" technology

At present, several new methods have been introduced in the field of culture medium optimization, including a variety of "omics" techniques such as proteomics, genomics, epigenomics, and metabolomics. In addition, transcriptomics and glycomics contribute to a better understanding of cell metabolism and its molecular mechanisms and accordingly develop strategies to improve cell production mechanisms. For example, gene expression in cells producing proteins under different medium formulations can be compared to identify key components that influence gene expression and product yield. Then, key components are optimized based on transcriptomic data to improve the expression levels of genes responsible for differences, thereby improving cell growth and increasing production.

Proteomics is used to optimize the composition of the medium to minimize the accumulation of toxic byproducts and growth inhibitors. For example, high-performance liquid chromatography (HPLC) and mass spectrometry (MS) are combined to detect medium components and intracellular and extracellular metabolites, thereby characterizing metabolic pathways and metabolites that are adverse to cell growth and product expression. In the process with well-controlled levels of lactic acid, NH⁴⁺, and osmotic pressure, nuclear magnetic resonance (NMR) and MS are used to qualitatively and quantitatively analyze the harmful metabolites produced by the metabolism of nutrients such as amino acids, to optimize the nutrient addition strategy in the medium and meet the needs of cell metabolism without producing harmful metabolites. According to the identification, the accumulation of some toxic metabolites and the consumption of nutrients will activate autophagy, apoptosis, and other signaling pathways, so autophagy inhibitors or apoptosis inhibitors can be added accordingly to improve the cell state and prolong the culture cycle. However, these inhibitors are quite expensive. Therefore, simple substances such as insulin, IGF-1, and LongR3 can be added to inhibit autophagy and apoptosis, promote cell growth, and increase productivity.

• Chemical additive

Some chemicals can be added to the culture medium as well. For example, adding histone deacetylase inhibitors such as sodium valproate and sodium butyrate, on the one hand, can block cells in the G1 phase to increase the number of cells in the production phase; on the other hand, the cell state can be more conducive to gene transcription, to improve cell production rate. However, the concentration and time of these chemical additions need to be carefully optimized to avoid adverse effects on cell growth and productivity. Besides, adding some nucleotides can also increase the yield. For instance, the addition of deoxycytidine, deoxyuridine, thymidine, and other pyrimidine nucleosides can also significantly increase the production of proteins.

• Optimize culture medium and fed-batch strategies

Optimization of medium and fed-batch strategies based on the rate of nutrient consumption by cells can effectively reduce inhibitory metabolites and wastes, minimize osmotic pressure, and thus promote cell growth and increase production. For example, a strategy of high-end pH-controlled delivery of glucose significantly improved cell growth rate and total yield. Specific consumption rates can be calculated based on the results of online cell density or glucose concentration measurements, from which culture medium optimization and dynamic feeding strategies can be developed to significantly increase yield. Optimizing the culture medium for pH changes and using lactic acid and pyruvate to control pH reduced CO₂ injection and NH⁴⁺ accumulation, thereby increasing cell density and protein production.