Preparation of Media for Animal Cell Culture

Introduction
The growing interest in products from animal cells has caused an extensive research effort for the development of media for cell cultivation. The basic components in the media used for cultivation of animal cells vary depending upon the character of the cells, and the cultivation method. Basic components consist of an energy source, nitrogen source, vitamins, fats, fatty acids and fatsoluble components, inorganic salts, nucleic acid, antibiotics, oxygen, pH buffering systems, hormones, growth factors serum, and extensive efforts are directed toward developing serum-free media, protein media, or (synthetic media) chemically defined media (e.g., MEM—minimum essential media).

Almost 50% of the biologicals produced today or planned to be produced in the future are of animal cell origin. Therefore, there is an increasing interest in developing technologies for cultivation of animal cells and production of a wide spectrum of biologicals. The worldwide activities and market of biologicals from mammalian cells were recently received.

Although the major achievements in the field of animal cell cultivation have been accomplished in the last 3 decades, it has a long history of about one hundred years. Apart from the development of various types and size of culturing vessels, research and development of optimal media for cell division are also carried out among most groups involved in the field of animal cell cultivation and production of biologicals. Media used for cell cultivation are considered to include 2 major parts.

1. Essential basal ingredients that fulfill all cellular requirements for nutrients, known as the basal growth medium.
2. A set of supplements that satisfies other types of cellular growth requirements and makes it possible for the medium to grow.

Water for Animal Cell Media
Out of the most important points for consideration when preparing media is the required high quality of the water. Water used for culture media should be pyrogen-free (especially if the product is for human or animal use it should have resistance of 1.5–2.0 m. Ohms, indicating a low salt content.)

It is highly recommended to use fresh ultrapure water, and not store water since in some storage tanks, organic materials, or irons from plastic or glass
may dissolve in the water.

Purity of Chemicals, Stability, and Shelf Life
Chemicals of the highest purity are required for preparation of media. Commercial chemicals, although pure, inevitably contain traces of contaminants. Some of the traces may be toxic (like Hg). With regard to stability of media ingredients, inorganic chemicals are indefinitely stable. Vitamins are the least stable. Hormones, several antibiotics, and growth factors are recommended to be stored frozen (–20°C) or refrigerated (0°C–4°C).

Several ingredients used in animal cell culture media are known for their instability, e.g., ascorbic acid and glutamine. Most factors affect the shelf life of media, among them are the following: Natural decay rates of unstable compounds, pH, moisture, storage temperature, access of oxygen, and an exposure to near-ultraviolet, day light, or inflorescence light. Most media should be stored at 41°C and in a dark place. Storage of media by freezing may cause loss of some purely soluble ingredients. Powdered media may be stored for several years.

Basic Components in Media

1. Energy sources—Glucose, fructose, amino acids.
2. Nitrogen sources—amino acids.
3. Vitamins: mainly water-soluble vitamins—B and C.
4. Fat and fat-soluble components: fatty acids, cholesterols.
5. Inorganic salts: Na⁺, K⁺, Ca₂⁺, Mg₂⁺
6. Nucleic acid precursors
7. Antibiotics.
8. pH and buffering systems.
9. Oxygen.
10. Hormones and growth factors.

Sera in Animal Cell Media
Sera is the most important and most problematic component in animal cell media. During more than 3 decays, sera has been an essential medium component with the following functions:

1. Provides nutrients.
2. Provides proteins that solubilize essential nutrients that do not dissolve readily.
3. Binds essential nutrients that are toxic when present in excessive amounts and releasing then slowly in a controlled manner.
4. Provides hormones and growth factors.
5. Modulates the physical and chemical properties of the medium (viscosity, rate of diffusion)—protect cells in agitated culture.
6. Has a pH-buffering function.

Despite these advantages, there are several problems associated with the use of serum for cell cultures.

1. Serum is the most expensive component.
2. Being highly viscous, sera slows down the sterilization by filtration of the media.
3. From time to time, there is a shortage in world supply of sera.
4. Possible availability of contaminants. For example, Mycoplamas, viruses.
5. Availability of serum in media increases the complicity of the downstream processing of the desired biological media.