Animal Cell Culture: Fundamentals, Facilities, Advantages and Everything Else

Read this article to learn about the fundamentals, facilities, aseptic conditions, advantages, disadvantages of animal cell culture and also about the risks & safety regulations in a tissue culture laboratory.

Introduction to Animal Cell Culture :

Animal cell culture basically involves the in vitro (in the laboratory) maintenance and propagation of animal cells in a suitable nutrient media. Thus, culturing is a process of growing cells artificially. Cell culture has become an indispensible technology in various branches of life sciences.

Historical Background:

It was in 1907, Ross Harrison first developed a frog tissue culture technique. He probably chose frog for two reasons—being a cold-blooded animal, no incubation is required and tissue regeneration is fast in frog. In 1940’s chick embryo tissue became a favorite for culture techniques.

Interest in culturing human tissues started in 1950’s after it was demonstrated (by HeLa; Gey) that human tumor cells could give rise to continuous cell lines. Among the various animal cell cultures, mouse cell cultures are the most commonly used in the laboratory.

Terminology in Cell Culture:

The term tissue culture is commonly used to include both organ culture and cell culture.

Organ culture:

The culture of native tissue (i.e. un-disaggregated tissue) that retains most of the in vivo histological features is regarded as organ culture.

Cell culture:

This refers to the culture of dispersed (or disaggregated) cells obtained from the original tissue, or from a cell line.

Histotypic culture:

The culturing of the cells for their re-aggregation to form a tissue—like structure represents histotypic culture.

Organotypic culture:

This culture technique involves the recombination of different cell types to form a more defined tissue or an organ.

Primary culture:

The culture produced by the freshly isolated cells or tissues taken from an organism is the primary culture. These cell are heterogenous and slow growing, and represent the tissue of their origin with regard to their properties.

Cell line:

The sub-culturing of the primary culture gives rise to cell lines. The term continuous cell lines implies the indefinite growth of the cells in the subsequent sub-culturing. On the other hand, finite cell lines represent the death of cells after several subcultures.

Facilities for Animal Cell Culture:

While designing the laboratory for animal cell culture technology, utmost care should be taken with regard to the maintenance of aseptic conditions. The facilities required with regard to infrastructure and equipment are listed below :

Minimal Requirements for Cell Culture:

i. Clean and quite sterile area

ii. Preparation facilities

iii. Animal house

iv. Microbiology laboratory

v. Storage facilities (for glassware, chemicals, liquids, small equipment).

Equipment:

Laminar-flow, sterilizer, incubator, refrigerator and freezer (-20°C), balance, C0₂ cylinder, centrifuge, inverted microscope, water purifier, hemocytometer, liquid nitrogen freezer, slow- cooling device (for freezing cells), pipette washer, deep washing sink.

Besides the basic and minimal requirements listed above, there are many more facilities that may be beneficial or useful for tissue cultures. These include air-conditioned rooms, containment room for biohazard work, phase-contrast microscope, fluorescence microscope, confocal microscope, osmometer, and high capacity centrifuge and time lapse video equipment.

Culture Vessels:

In the tissue culture technology, the cells attach to the surface of a vessel which serves as the substrate, and grow. Hence there is a lot of importance attached to the nature of the materials used and the quality of the culture vessels. The term anchorage dependent cells is used when the cells require an attachment for their growth. On the other hand, some cells undergo transformation, and become anchorage independent.

Materials used for culture vessels:

Glass:

Although glass was the original substrate used for culturing, its use is almost discontinued now. This is mainly because of the availability of more suitable and alternate substrates.

Disposable plastics:

Synthetic plastic materials with good consistency and optical properties are now in use to provide uniform and reproducible cultures. The most commonly used plastics are polystyrene, polyvinyl chloride (PVC), poly­carbonate, metinex and thermonex (TPX).

Types of culture vessels:

The following are the common types of culture vessels.

i. Multiwall plates

ii. Petridishes

iii. Flasks

iv. Stirrer bottles.

The actual choice of selecting a culture vessel depends on several factors:

1. The way cells grow in culture—monolayer or suspension.

2. The quantity of the cells required.

3. The frequency of sampling for the desired work.

4. The purpose for which the cells are grown.

5. The cost factor.

In general, for monolayer cultures, the cell yield is almost proportional to the surface area of the culture vessel. The flasks are usually employed for this purpose. Any type of culture vessel can be used to grow suspension cultures. It is necessary to slowly and continuously agitate the suspended cells in the vessel.

Treatment of culture vessel surfaces:

For improving the attachment of cells to the surfaces, and for efficient growth, some devices have been developed. It is a common observation that the growth of the culture cells is better on the surfaces for second seeding. This is attributed to matrix coating of the surfaces due to the accumulation of certain compounds like collagen and fibronectin released by the cells of the previous culture. There are now commercially available matrices (e.g. matrigel, pronectin, and cell-tak).

Feeder layers:

Some of the tissue cultures require the support of metabolic products from living cells e.g. mouse embryo fibroblasts. In this case, the growing fibroblasts release certain products which when fed to new cells enhance their growth.

Alternate substrates as substitutes of culture vessels:

In recent years, certain alternatives for culture vessels have been developed. The important alternative artificial substrates are micro carriers and metallic substrates.

Micro-carriers:

They are in bead form and are made up of collagen, gelatin, polyacrylamide and polystyrene. Micro-carriers are mostly used for the propagation of anchorage-dependent cells in suspension.

Metallic substrates:

Certain types of cells could be successfully grown on some metallic surfaces or even on the stainless steel discs. For instance, fibroblasts were grown on palledium.

Use of Non-Adhesive Substrates in Tissue Culture:

The growth of anchorage independent cells can be carried out by plating cells on non-adhesive substrate like agar, agarose and methyl cellulose. In this situation, as the cell growth occurs, the parent and daughter cells get immobilized and form a colony, although they are non-adhesive.

Contamination, Aseptic Conditions, and Sterilization:

There are several routes of contamination in the tissue culture laboratory (Table 33.1). These include the various materials (glassware, pipettes), equipment (incubators, refrigerators, and laminar-flow hoods), reagents (media, solutions), contaminated cell lines and poor techniques.

The routes of contamination are mostly associated with the laboratory environment, and operating techniques.

Types of microbial contamination:

Several species of bacteria, yeasts, fungi, molds and mycoplasmas, besides viruses are responsible for contamination. Major problems of conta­mination are linked to the repeated recurrence of a single species. Despite utmost care taken, no laboratory can claim to be totally free from contamination. It is necessary to continuously monitor for contamination and eliminate the same at the earliest.

Aseptic Conditions:

Maintenance of proper aseptic conditions is necessary to eliminate various contaminants (due to different microorganisms and viruses). The following measures are suggested for minimizing contamination, and maintenance of aseptic conditions.

i. Strict adherence to standard sterile techniques and code of practices.

ii. Checking of reagents and media for sterility before use.

iii. Checking of cultures by eyes, and microscopes (phase contrast) every time they are used.

iv. Use of media and separate bottles for each cell line is advised.

v. Maintenance of clean and tidy conditions at work places.

vi. Personal hygiene of the staff is very important.

Sterilization:

The sterilization procedures are designed to kill the microorganisms, besides destroying the spores.

There are three major devices for sterilization:

1. Dry heat

2. Moist heat (autoclave)

3. Filters.

In the Table 33.2, the sterilization of major equipment, apparatus and liquids is given.

Sterilization by dry heat:

This is carried out at a minimum temperature of 160°C for about one hour.

Sterilization by moist heat:

Certain fluids and perishable items can be sterilized in an autoclave at 121°C for 15-20 minutes. For effective moist heat sterilization, it is necessary that the steam penetrates to all the parts of the sterilizing materials.

Sterilization by filters:

The use of filters for sterilization of liquids often becomes necessary, since the constituents of these liquids may get destroyed at higher temperatures (dry heat or moist heat). Sterile filtration is a novel technique for heat- labile solutions. The size of micropores of the filters is 0.1-0.2 µm. Filters, made from several materials are in use. These materials include nylon, cellulose acetate, cellulose nitrate, polycarbonate, polyethersulfone (PES) and ceramics.

The filters are made in different designs-disc filters, cartridges and hollow fiber. In fact, many commercial companies (e.g. Millipore, Durapore) supply reusable and disposable filters, designed for different purposes of sterilization.

Advantages and Limitations of Tissue Culture:

Advantages of Tissue Culture:

Tissue culture technique has a wide range of applications.

The most important advantages of this technique are listed below:

1. Control of physicochemical environment- pH, temperature, dissolved gases (O₂ and CO₂), osmolarity.

2. Regulation of physiological conditions-nutrient concentration, cell to cell interactions, hormonal control.

3. The cultured cell lines become homogenous (i.e. cells are identical) after one or two subcultures. This is in contrast to the heterogenous cells of tissue samples. The homogenous cells are highly useful for a wide range of purposes.

4. It is easy to characterize cells for cytological and immunological studies.

5. Cultured cells can be stored in liquid nitrogen for several years.

6. Due to direct access and contact to the cells, biological studies can be carried out more conveniently. The main advantage is the low quantities of the reagents required in contrast to in vivo studies where most of the reagents (more than 90% in some cases) are lost by distribution to various tissues, and excretion.

7. Utility of tissue cultures will drastically reduce the use of animals for various experiments.

Limitations of Tissue Culture:

There are several limitations of tissue culture; some of them are given below.

1. Need of expertise and technical skill for the development, and regular use of tissue culture.

2. Cost factor is a major limitation. Establishment of infrastructure, equipment and other facilities are expensive.

3. It is estimated that the cost of production of cells is about 10 times higher than direct use of animal tissues.

4. Control of the environmental factors (pH, temperature, dissolved gases, disposal of biohazards) is not easy.

5. The native in vivo cells exist in a three- dimensional geometry while in in vitro tissue culture, the propagation of cells occurs on a two dimensional substrate. Due to this, the cell to cell interactive characters are lost.

6. The cell lines may represent one or two types of cells from the native tissue while others may go unrepresented.

7. Tissue culture techniques are associated with the differentiation i.e. loss of the characters of the tissue cells from which they were originally isolated.

8. This happens due to adaptation and selection processes while culturing.

9. Continuous cell lines may result in genetic instability of the cells. This may ultimately lead to heterogeneity of cells.

10. The components of homeostatic in vivo regulation (nervous system, endocrine system, metabolic integration) are lacking in vitro cultures. Addition of hormones and growth factors has been started recently.

Risks in a Tissue Culture Laboratory and Safety:

There are several risks associated with tissue culture technology. Most of the accidents that occur in culture laboratories are due to negligence and casual approach while dealing with biological and radiological samples, besides improper maintenance of the laboratory. A broad categorization of risks and the contributory factors is given in Table 33.5.

Safety regulations:

Some of the developed countries have formulated general safety regulations to minimize the risks associated with tissue culture laboratories.

Selected examples:

1. “Biosafety in microbiological and biomedical laboratories”, U. S. Department of Health and Human Sciences (1993).

2. “Safe working and the prevention of infection in clinical laboratories” U.K. Health Services Advisory Committee (1991).

Some of the general precautions for the safety of a tissue culture laboratory are listed here:

i. Strict adherence to recommendations of regulatory bodies.

ii. Periodical meetings and discussions of local safety committees.

iii. Regular monitoring of the laboratories.

iv. Periodical training of the personnel through seminars and workshops.

v. Print and make the standard operating procedures (SOPs) available to all staff.

vi. Good record keeping.

vii. Limited access to the laboratory (only for the trained personnel and selected visitors).

viii. Appropriate waste disposal system for biohazards, radioactive wastes, toxins and corrosives.

Biohazards:

The accidents or the risks associated with the biological materials are regarded as biohazards or biological hazards. There are two main systems that contribute to the occurrence of biohazards (Table 33.6).

1. The direct sources of the biological materials.

2. The processes or operations involved in their handling.

Control of biohazards:

Biohazards can be controlled to a large extent by strict adherence to the regulatory guidelines and maintenance programmes. Some important aspects are listed.

i. Microbiological safety cabinet or biohazard wood with pathogen trap filters have been developed.

ii. Vertical laminar-flow hood (instead of horizontal laminar-flow hood) is recently in use. This minimizes the direct exposure of the operator to the samples/processes.

iii. Pathogen containing samples are treated in separate rooms with separate facilities (centrifuge, incubator, cell counting etc.).

iv. Sterilization of all wastes, solid glassware etc. and their proper disposal.

v. Facilities for change of clothing while entering and leaving the rooms.

vi. Strict adherence to the access of designated personnel to the culture rooms.