Major Discoveries In Animal Cell Culture Media

History of Animal Cell Culture Media

In 1882, Sydney Ringer developed Ringer’s solution, a balanced salt solution with composition close to that of bodily fluids, to keep beating frog hearts after dissection. This is considered as the first witnessing of in vitro animal tissue cultivation. In the wake of Ringer’s report of the animal Cell Culture Media solution, balanced salt solutions were developed including Locke’s solution, Tyrode’s solution, Krebs–Ringer bicarbonate solution, Earle’s solution, and Hanks’ solution. The composition of these balanced salt solutions generally included only inorganic salts, sometimes with glucose. The pH, osmolarity, and salt concentrations were calibrated and optimized to physiological solutions to keep cells and tissues in vitro successfully for a few days.

Gradually, the focus was shifted from making animal cell culture media to maintaining the cells for more time as the cells did not usually survive for more days and rarely showed healthy proliferation. In 1907, Ross G. Harrison did a successful monitoring of an apparent nerve fiber outgrowth for several weeks in freshly drawn lymph fluid from an adult frog. In the history of Animal Cell Culture media, this was considered as a milestone.

The first success of animal cell growth culture by Harrison inspired Montrose T. Burrows to work under him and during his work, he found that plasma is better suitable for animal cell culture from warm‐blooded animals instead of lymphatic fluid. Burrows successfully cultivated chicken embryonic cells by using plasma and later went ahead with mammalian cell culture. In 1913, Carell discovered that adding embryonic extract to plasma can increase proliferation and culture period of fibroblasts. In due course, scientific inquiry regarding the components of plasma, lymph, and embryonic extract was popular to find the factors affecting survival and growth of animal tissues and cells. 

Warren H. Lewis and Margaret H. Lewis, demonstrated that the Locke–Lewis solution (modified Locke’s solution with additional amino acids, glucose, and nutrients) is more effective for embryo cell culture than simple balanced salt solutions. They reported that glucose and partially hydrolyzed proteins effectively promote the animal cell culture growth. In 1940, Wilton R. Earle et al. used carcinogens to successfully surpass the Hayflick’s limit to create immortal mouse Fibroblasts and due to the emergence of established continuous cell lines, scientists started examining and quantifying the differences in the animal cell culture media effects. The need for advanced animal cell culture media thus crept up and scientists started looking intently into understanding and determining the specific components in animal cell culture media instead of naturally derived media components of unknown composition.

The first strategy to understand was using dialyzed serum and adding defined components for the animal cell culture support while the second strategy involved creating the formulation using exclusive definite media components. The first strategy was popularly used by Fischer and he found that low‐molecular‐weight fraction (amino acids being the key component) was essential for the enhanced survival of animal cells. On the basis of Fischer’s method, Harry Eagle studied the minimum necessary amounts of low‐molecular‐weight components used for animal cell growth culture, in 1995. Upon finding that glucose, 13 amino acids, and 8 vitamins are necessary, he developed the minimum essential medium (MEM). This composition was then modified by several scientists like Dulbecco, Vogt, Stanners, Iscove, and Melchers. Later, Thomas A. McCoy et al. suggested the use of pyruvate for specific cells in his 5A medium but the Roswell Park Memorial Institute (RPMI) modified it further in terms of its Ca and Mg concentrations to come up with RPMI 1640 used in lymphocyte culture.

The other strategy was popularly researched by Philip R. White, who developed a chemically defined medium composed of glucose, inorganic salts, amino acids, iron, vitamins, and glutathione, without protein. Some researchers suggested that this media still needed 10%‐20% serum for obtaining similar animal cell culture reports to White. Later, Connaught Medical Research Laboratories (CMRL), developed Medium 199 and further went ahead to compose a chemically defined medium, CMRL1066, consisting of 58 components after several modifications. Following that, other chemically defined mediums like NCTC109 and MB 752/1 were prepared to optimize animal cell culture. 

In 1963, G. Ham developed Ham’s F‐10 medium with two serum protein fractions (albumin and fetuin) instead of serum, and successfully cultured Chinese hamster ovary (CHO) cell lines. However, its proliferative capacity was less than that of CHO cell lines in serum‐containing media. To further elevate his research, Ham replaced albumin and fetuin with low‐molecular‐weight linoleic acid and putrescine to develop Ham’s F‐12, a synthetic defined animal cell culture medium. 

With time, growth factors like nerve growth factor, epidermal growth factor, insulin‐like growth factor, fibroblast growth factor (FGF), platelet‐derived growth factor, and transforming growth factor (TGF) were discovered to increase cellular proliferation but their effect on animal cell culture proliferation was always enhanced with some amount of serum in the culture media. But researchers were bent on discovering optimum serum-free medium and that led to some interesting discoveries. Ham discovered that selenite (trace element) is required for the serum‐free culture of human diploid cells whereas Guilbert and Iscove showed that transferrin and albumin combination, besides selenite, can be a good serum substitute. Prompted by these animal cell culture media discoveries, several attempts were made to optimize cell culture media as per researcher’s interest and application. 

Significant Fuel for Animal Cell Culture Research

• In 1982, clinical application of recombinant human insulin expressed in E. coli led researchers to find different expression systems as glycosylated proteins were not possible in prokaryote systems. Therefore, researchers started realizing the application of animal cell culture in genetic engineering and the production of recombinant proteins. Among several host cell lines used, CHO and NS0 cells gained popularity in the field of biopharmaceutical manufacturing due to a few reasons: (a) technological advances in scaled-up culture methods (b) knowledge regarding virology of the cell lines and (c) advances in high‐expression derived sublines. The production efficiency in these cases required that the animal cell culture medium contains none or a minimal amount of natural biological ingredients, like serum, as they hamper protein purification. This is one research direction that led scientists to come up with several culture media modification strategies based on medium component and byproduct concentration, genomics and proteomics-based approaches, and host-cell modifications. Due to the patent rights and commercial value of these media compositions, biopharma companies have not disclosed them over years. 
• The biomedical community is well-aware of the special place that embryonic stem cells and pluripotent stem cells hold due to their usefulness in basic and clinical regenerative medicine studies. Owing to the huge demand, the need for culturing these cells in a low‐cost and highly productive way led to modifications in animal cell culture media. The animal cell culture media involved a layer of feeder cells in a serum‐ or KSR‐supplemented medium but soon, research began in order to come up with feeder-free media like E8 medium. Further research is being conducted to develop more media compositions for the long‐term growth of stem cells using low-molecular weight compounds. 
• In 1978, the first successful in vitro fertilization application used Ham’s F-10 media with serum supplements for human zygote culture. Later scientists found that hypoxanthine and the trace elements in Ham’s F‐10 negatively affected the embryos as metabolism of a pre‐implantation embryo differs from that of somatic cells. With further research, the usually preferred composition of the embryo culture media was similar to G1/G2 medium or the KSOMAA medium for optimum cell culture practice.

Aside from the mentioned milestones, there were numerous other applications like viral culture technique, cancer research, and acellular matrix bioscaffold model, which led to major turning points in the course of developing optimum animal cell culture media to keep up with the ever-evolving pace of scientific and clinical studies.