Cell culture is a foundational tool in biological research, providing a controlled environment to study cellular behaviour, test drug responses, and understand disease mechanisms. While fetal bovine serum (FBS) is the most commonly used supplement in cell culture, other animal sera like horse serum offer unique benefits that are often overlooked. This blog will explore the role of horse serum in cell culture, its applications, advantages, and how it compares to more commonly used sera.

What is Horse Serum?

Horse serum is a biologically rich component derived from the clotted whole blood of horses, typically collected via vein puncture. This serum contains a diverse array of bioactive molecules, including growth factors, hormones, proteins, and lipids, which are crucial for supporting cellular functions in vitro.

The collection process involves drawing blood from healthy adult horses, followed by allowing the blood to clot naturally. The resulting serum is then separated from the clot, purified, and sterilised to ensure it is free from contaminants and suitable for use in cell culture. Due to the controlled collection process, horse serum provides a stable and consistent source of nutrients necessary for cell growth, differentiation, and maintenance.

Horse serum is particularly valued in research and industrial applications for its ability to support a wide range of cell types. It plays a critical role in cell culture by providing the necessary components that mimic the natural extracellular environment found in living organisms. The rich composition of horse serum makes it especially effective in maintaining primary neuronal cultures, supporting the growth of hematopoietic progenitor cells, and facilitating the differentiation of muscle cells, among other specialised uses.

Advantages of Horse Serum Over FBS

Horse serum can be an effective alternative to FBS in specific cell culture applications,
though its composition differs significantly. For instance, horse serum typically contains lower
levels of growth factors and higher levels of immunoglobulins than FBS. It also has about
twice the total protein content of FBS but lower concentrations of certain trace metals
necessary for optimal cell growth. These differences mean that while horse serum can
substitute for FBS, its use may require adjustments to culture conditions based on the
specific needs of the cells being cultured. Below are the key advantages of horse serum
over FBS:

1. Affordability and Cost-Effectiveness: Horse serum is generally more affordable than FBS, making it a cost-effective option for laboratories looking to manage expenses without compromising quality. This cost advantage is particularly beneficial in large-scale experiments or routine cell culture applications where the high price of FBS may be prohibitive.
2. Ethical Considerations and Animal Welfare: The production of horse serum does not involve the sacrifice of fetal animals, addressing a significant ethical concern associated with FBS. Instead, horse serum is collected from living, healthy adult horses, which can reduce ethical objections related to animal welfare. This makes horse serum a more ethically favourable choice for researchers and institutions that prioritise humane treatment of animals in their research practices.
3. Lower Risk of Bovine Virus Contamination: Horse serum has a reduced risk of contamination by bovine viruses, a concern inherent to FBS. This lower contamination risk makes horse serum a safer alternative in applications where the presence of bovine pathogens could compromise experimental outcomes or pose biosafety risks. This is particularly valuable for researchers working in fields like virology, vaccine development, or cell therapy.
4. Consistency and Limited Batch Variability: Horse serum generally exhibits lower batch-to-batch variability compared to FBS, leading to greater consistency in experimental results. This consistency is crucial in research and industrial applications where reproducibility is essential. By minimising variability in serum composition, horse serum helps ensure more reliable and accurate data across different experiments and over time.
5. Suitability for Specialised Applications: Horse serum is particularly effective in supporting the growth of certain cell types, especially those derived from equine species, making it an excellent choice for veterinary research and studies involving horse-derived cells. Additionally, its unique composition may offer advantages in specific niche applications, such as cultivating neuronal and glial cells or inducing myogenesis.

Applications of Horse Serum in Cell Culture

Horse serum is a versatile supplement with a range of applications across various cell culture systems. Its unique composition supports specific cell types and experimental protocols, making it a valuable tool in both basic research and applied biotechnology.

1. Primary Neuronal Cell Cultures: Horse serum is particularly effective in the culture of primary neuronal, where it plays a crucial role in supporting cell differentiation and long- term maintenance. The rich array of growth factors and hormones present in horse serum promotes the maturation and survival of these cells, making it a preferred supplement in neurobiological studies. Researchers use horse serum to culture primary neurons, astrocytes, oligodendrocytes, and microglia, providing a supportive environment that mimics the natural extracellular matrix found in the central nervous system. Typically, a concentration of 10% horse serum is included in the culture medium to ensure the optimal growth and maintenance of primary neuronal cultures.
2. Supplement in Bacteriological Media: Horse serum is frequently used as a supplement in bacteriological media to support the growth of fastidious organisms—those that are difficult to culture under standard conditions. Its rich supply of nutrients and growth factors creates an optimal environment for bacteria that require specific conditions to thrive. For instance, horse serum is commonly added to media used to culture Helicobacter pylori, a bacterium linked to gastric ulcers and cancer, and Mycoplasma species, which are implicated in respiratory and urogenital infections. The use of horse serum in these contexts enhances the growth and viability of these challenging organisms, facilitating their study in the laboratory. For example, H. pylori strains are often grown on brain heart infusion (BHI) agar supplemented with 7% horse serum, which provides the essential nutrients needed for robust bacterial growth. Similarly, Mycoplasma pneumoniae is cultured in Hayflick media, which contains 16% horse serum, supporting the organism’s specific nutritional requirements. By incorporating horse serum, researchers can more effectively study these pathogens and develop targeted treatments.
3. Culturing Pheochromocytoma (PC12) Cells: Pheochromocytoma (PC12) cells, derived from a rat adrenal medulla tumour, are a model system for studying neurobiology, particularly neuronal differentiation, neurotransmitter release, and neurotoxicity. Horse serum is commonly used in the culture of PC12 cells, where it aids in their proliferation and differentiation, as it provides essential support for maintaining the viability and functionality of these cells over extended periods. In a study that investigates the effects of varying concentrations of horse serum on the morphology and attachment of PC12 cells, it was found that the higher percentages of horse serum (10-15%) lead to more loosely attached, rounded cells, whereas lower percentages result in more attached, polygonal cells. Although these morphological changes do not significantly impact the cells’ ability to undergo nerve growth factor (NGF)-mediated differentiation, the findings suggest that the choice of horse serum concentration may influence cell culture conditions and warrants further investigation into the specific components of horse serum responsible for these effects.
4. Maintenance of Hematopoietic Progenitor Cells: Horse serum plays a crucial role in the maintenance and culture of hematopoietic progenitor cells, which are precursors to various blood cell types such as erythrocytes, leukocytes, and platelets. These progenitor cells require a precisely balanced environment to stay viable and retain their capacity to differentiate into specific lineages. Horse serum contributes to creating the optimal conditions for maintaining these cells in culture, helping to keep them in a less differentiated, progenitor state. In one study, the ability of horse serum to support the self-renewal of multipotent murine hematopoietic progenitor FDCP-Mix cells was found to be correlated with the concentration of specific fatty acid products of phospholipase A2, highlighting the importance of horse serum in preserving the undifferentiated state of these cells for further research and therapeutic applications.
5. Induction of Myogenesis: Myogenesis, the process by which muscle cells (myocytes) are formed, can be effectively induced in vitro using horse serum. During myogenesis, horse serum supports the differentiation of precursor cells, such as myoblasts, facilitating their fusion into myotubes—a critical step in the development of skeletal muscle. This application is particularly important in muscle biology research, where understanding the mechanisms of muscle formation, regeneration, and disease can lead to novel therapeutic approaches for muscle-wasting conditions. One widely used model for studying skeletal muscle differentiation is the C2C12 cell line, which consists of mouse skeletal myoblasts. C2C12 cells are typically cultured in a serum-rich medium containing 10% FBS to promote their proliferation as myoblasts. However, to induce differentiation, the medium is switched to a serum-reduced formulation containing 2% horse serum. This change triggers the myoblasts to exit the cell cycle and begin differentiating, rapidly leading to the formation of myotubes. The ability of horse serum to induce this critical step highlights its utility in research focused on muscle development and the study of muscle-related diseases.

Figure 1: Schematic representation of myogenic differentiation in C2C12 myoblast cells.
Credit: Katayama T., Chigi Y and Okamura D, doi: 10.3389/fcell.2023.1193634
Reproduced under the Creative Commons license

Choosing Between Horse Serum and Fetal Bovine Serum (FBS)

Selecting the appropriate serum for cell culture is crucial to the success of any biological research or application. Both horse serum and FBS are popular choices, each offering distinct benefits depending on the specific requirements of the experiment. Horse serum is known for its affordability, ethical advantages, and suitability for specialised applications, while FBS is renowned for its rich content of growth factors and nutrients, supporting a broad range of cell types. Understanding the key differences between these two options can help researchers make an informed decision on which to use.

Comparison of Horse Serum Vs Fetal Bovine Serum

Donor Horse Serum vs Normal Horse Serum

Donor horse serum refers to the serum that is collected from horses that are specifically chosen and maintained as donors for serum production. The term “donor” indicates that the horses are typically kept under controlled conditions to ensure the consistency, quality, and safety of the serum produced.

Here are some key points about donor horse serum:

1. Controlled Environment: The donor horses are usually housed in regulated environments where their diet, health, and overall well-being are monitored to minimise variability in the serum composition.
2. Regular Screening: Donor horses undergo regular health screenings to ensure they are free from diseases that could affect the quality of the serum or introduce contaminants into cell cultures.
3. Batch Consistency: Using a consistent group of donor horses helps to produce serum batches with more uniform characteristics, reducing variability in cell culture experiments.
4. Ethical Considerations: The management of donor horses also involves ethical considerations, ensuring that the animals are treated humanely and that their health and well-being are prioritized throughout the serum collection process.

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Normal horse serum typically refers to horse serum collected from horses that are not specifically maintained as donor animals under controlled conditions. Here’s what it generally entails:

1. General Population Collection: Normal horse serum is often obtained from a broader population of horses, not specifically designated as serum donors. These horses may come from various sources, such as farms, and might not be under the same level of control as donor horses.
2. Less Stringent Controls: Unlike donor horse serum, the conditions under which normal horse serum is collected may be less tightly regulated. This could mean more variability in the diet, health, and environment of the horses, potentially leading to greater variability in the serum’s composition.
3. Routine Serum: The term “normal” indicates that the serum is not treated or selected for any particular enhancement or purpose, such as the heightened consistency required for specialized applications. It’s considered a standard or general-use serum.
4. Common Use: Normal horse serum might be used in less sensitive applications where the highest levels of consistency and quality control are not as critical. It could also be used as a blocking agent in immunohistochemistry or ELISA assays.
5. Ethical and Quality Considerations: Since the horses are not specifically managed as donors, normal horse serum might raise additional concerns about variability and ethical considerations regarding the horses’ health and treatment.

Applications: Donor horse serum is collected from horses specifically maintained under controlled conditions, ensuring consistent quality and minimising variability for sensitive cell culture applications. In contrast, normal horse serum is sourced from a broader population of horses, with less stringent controls, leading to potential variability in its composition. While donor horse serum is ideal for precise and demanding experiments such as a supplement in cell culture, normal horse serum is more suited for general or less sensitive laboratory uses, such as for blocking or saturating generalised binding interactions for immunodetection methods, such as immunohistochemistry (IHC), immunoblotting, immunofluorescence, or ELISA.

Horse serum, with its unique composition and versatile applications, plays a critical role in
cell culture, offering an alternative to more commonly used supplements like FBS. Its
affordability, ethical advantages, reduced risk of contamination, and consistency make it a
valuable resource for various research and industrial applications. Whether used in
specialised cell cultures, such as neuronal or hematopoietic progenitor cells, or in broader
applications like bacteriological media and myogenesis, horse serum proves to be a reliable
and effective supplement. By understanding the distinctions between donor and normal
horse serum, researchers can make informed choices to optimise their experiments,
ensuring both the quality and ethical integrity of their work.

References

• Ditz T, Schnapka-Hille L, Noack N, et al. Phospholipase A2 products predict the hematopoietic support capacity of horse serum. Differentiation. 2019;105:27-32. doi:10.1016/j.diff.2018.12.002

• Goshi N, Kim H, Girardi G, Gardner A, Seker E. Electrophysiological Activity of Primary Cortical Neuron-Glia Mixed Cultures. Cells. 2023;12(5):821. Published 2023 Mar 6. doi:10.3390/cells12050821

• HAYFLICK L, CHANOCK RM. MYCOPLASMA SPECIES OF MAN. Bacteriol Rev. 1965;29(2):185-221. doi:10.1128/br.29.2.185-221.1965

• Jiang X, Doyle MP. Growth supplements for Helicobacter pylori. J Clin Microbiol. 2000;38(5):1984-1987. doi:10.1128/JCM.38.5.1984-1987.2000

• Katayama T, Chigi Y, Okamura D. The ensured proliferative capacity of myoblast in serum-reduced conditions with Methyl-β-cyclodextrin. Front Cell Dev Biol. 2023;11:1193634. Published 2023 May 12. doi:10.3389/fcell.2023.1193634

• Meth JL, Schoenfeld AR. Higher percentage of horse serum in culture media blocks attachment of PC12 cells. Biotechniques. 2019 Dec;67(6):256-258. doi: 10.2144/btn-2019-0073. Epub 2019 Oct 17.
• Subbiahanadar Chelladurai K, Selvan Christyraj JD, Rajagopalan K, Yesudhason BV, Venkatachalam S, Mohan M, Chellathurai Vasantha N, Selvan Christyraj JRS. Alternative to FBS in animal cell culture – An overview and future perspective. Heliyon. 2021 Jul 28;7(8):e07686. doi: 10.1016/j.heliyon.2021.e07686.