Cell Culture Environment

Introduction

One of the major advantages of cell culture is the ability to manipulate the physicochemical (i.e., temperature, pH, osmotic pressure, O2 and CO2 tension) and the physiological environment (i.e., hormone and nutrient concentrations) in which the cells propagate. With the exception of temperature, the culture environment is controlled by the growth media.

While the physiological environment of the culture is not as well defined as its physicochemical environment, a better understanding of the components of serum, the identification of the growth factors necessary for proliferation, and a better appreciation of the microenvironment of cells in culture (i.e., cell-cell interactions, diffusion of gases, interactions with the matrix) now allow the culture of certain cell lines in serum-free media.

Adherent vs Suspension Culture:

A. Adherent Culture:

Appropriate for most cell types, including primary cultures.

Requires periodic passaging, but allows easy visual inspection under inverted microscope.

Cells are dissociated enzymatically (e.g., TrypLE™ Express, trypsin) or mechanically .

Growth is limited by surface area, which may limit product yields.

Requires tissue-culture treated vessel.

Used for cytology, harvesting products continuously, and many research applications.

B. Suspension Culture:

Appropriate for cells adapted to suspension culture and a few other cell lines that are nonadhesive (e.g., hematopoietic).

Easier to passage, but requires daily cell counts and viability determination to follow growth patterns; culture can be diluted to stimulate growth.

Does not require enzymatic or mechanical dissociation.

Growth is limited by concentration of cells in the medium, which allows easy scale-up.

Can be maintained in culture vessels that are not tissue-culture treated, but requires agitation (i.e., shaking or stirring) for adequate gas exchange.

Used for bulk protein production, batch harvesting, and many research applications.

Media:

The culture medium is the most important component of the culture environment, because it provides the necessary nutrients, growth factors, and hormones for cell growth, as well as regulating the pH and the osmotic pressure of the culture. Although initial cell culture experiments were performed using natural media obtained from tissue extracts and body fluids, the need for standardization, media quality, and increased demand led to the development of defined media. The three basic classes of media are:-

1. Basal media,

2. Reduced-serum media, and

3. Serum-free media.

which differ in their requirement for supplementation with serum.

Serum is vitally important as a source of growth and adhesion factors, hormones, lipids and minerals for the culture of cells in basal media. In addition, serum also regulates cell membrane permeability and serves as a carrier for lipids, enzymes, micronutrients, and trace elements into the cell.

However, using serum in media has a number of disadvantages including high cost, problems with standardization, specificity, variability, and unwanted effects such as stimulation or inhibition of growth and/or cellular function on certain cell cultures.

Basal Media:

The majority of cell lines grow well in basal media, which contain amino acids, vitamins, inorganic salts, and a carbon source such as glucose, but these basal media formulations must be further supplemented with serum.

Reduced-Serum Media:

Another strategy to reduce the undesired effects of serum in cell culture experiments is to use reduced-serum media. Reduced-serum media are basal media formulations enriched with nutrients and animal-derived factors, which reduce the amount of serum that is needed.

Serum-Free Media:

Serum-free media (SFM) circumvents issues with using animal sera by replacing the serum with appropriate nutritional and hormonal formulations. Serum-free media formulations exist for many primary cultures and cell lines, including recombinant protein producing lines of Chinese Hamster Ovary (CHO), various hybridoma cell lines, the insect lines Sf9 and Sf21 (Spodoptera frugiperda), and for cell lines that act as hosts for viral production (e.g., 293, VERO, MDCK, MDBK), and others. One of the major advantages of using serum-free media is the ability to make the medium selective for specific cell types by choosing the appropriate combination of growth factors.

Advantages:

Increased definition.

More consistent performance.

Easier purification and downstream processing.

Precise evaluation of cellular functions.

Increased productivity.

Better control over physiological response.

Enhanced detection of cellular mediators.

Disadvantages:

Requirement for cell type-specific media formulations.

Need for higher degree of reagent purity.

Slower growth.

pH:

Most normal mammalian cell lines grow well at pH 7.4, and there is very little variability among different cell strains. However, some transformed cell lines have been shown to grow better at slightly more acidic environments (pH 7.0–7.4), and some normal fibroblast cell lines prefer slightly more basic environments (pH 7.4–7.7). Insect cell lines such as Sf9 and Sf21 grow optimally at pH 6.2.

CO2:

The growth medium controls the pH of the culture and buffers the cells in culture against changes in the pH. Usually, this buffering is achieved by including an organic (e.g., HEPES) or CO2-bicarbonate based buffer. Because the pH of the medium is dependent on the delicate balance of dissolved carbon dioxide (CO2) and bicarbonate (HCO3 – ), changes in the atmospheric CO2 can alter the pH of the medium. Therefore, it is necessary to use exogenous CO2 when using media buffered with a CO2-bicarbonate based buffer, especially if the cells are cultured in open dishes or transformed cell lines are cultured at high concentrations. While most researchers usually use 5–7% CO2 in air, 4–10% CO2 is common for most cell culture experiments. However, each medium has a recommended CO2 tension and bicarbonate concentration to achieve the correct pH and osmolality; refer to the media manufacturer’s instructions for more information.

Temperature:

The optimal temperature for cell culture largely depends on the body temperature of the host from which the cells were isolated, and to a lesser degree on the anatomical variation in temperature (e.g., temperature of the skin may be lower than the temperature of skeletal muscle). Overheating is a more serious problem than under heating for cell cultures; therefore, often the temperature in the incubator is set slightly lower than the optimal temperature.

Most human and mammalian cell lines are maintained at 36°C to 37°C for optimal growth.

Insect cells are cultured at 27°C for optimal growth; they grow more slowly at lower temperatures and at temperatures between 27°C and 30°C. Above 30°C, the viability of insect cells decreases, and the cells do not recover even after they are returned to 27°C.

Avian cell lines require 38.5°C for maximum growth. Although these cells can also be maintained at 37°C, they will grow more slowly.

Cell lines derived from cold-blooded animals (e.g., amphibians, cold-water fish) tolerate a wide temperature range between 15°C and 26°C.

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