CELL CULTURE BASICS

Cell culture is the laboratory technique of growing and maintaining the cells of multicellular organisms (plants and animal cells inclusive) in a favourable artificial environment conducive for growth. It is the maintenance of animal cells (inclusive of plant and human cell lines) in vitro. Such an artificial environment or growth medium mimics the internal environment of the organism from which the cell was obtained from; and they support the propagation of the cells to be cultured in vitro. Cell culture is also the removal of cells from an organism (i.e. from their parent tissue) and their subsequent growth in vitro under special controlled growth conditions. This process of removing cells or tissues from their normal in vivo environment (i.e. from their parental host organism) and maintaining their growth in vitro in an artificial growth environment is generally known as explantation. Such cells or tissues are said to be explanted since they now survive outside their host organism. An explant is simply defined as a fragment of tissue that is transplanted from its original (parental) host organism and maintained in an artificial growth medium in vitro. The survival or viability of cells or tissues outside of their host or parental organism after explantation is critical because once a cell or tissue is explanted from its normal physiological in vivo environment, maintaining its optimal growth in vitro is fundamental to the success of the experimentation.

Cell culture techniques are also used for the study and cultivation of viruses, protozoa and other obligate intracellular parasites such as Chlamydia and Rickettsia. To ensure viability of the explanted cells or tissues, the in vitro artificial growth environment must be inundated and fashioned with all necessary requirements that mimic the normal in vivo environment from which the cells/tissues was obtained from. Culture is generally the microbiological laboratory technique in which the growth of microorganisms in a growth medium (solid, liquid or broth) is enhanced for visibility and easy study. Such a medium (which is usually placed in a Petri dish) contains nutritive substances/materials that support the growth of the organisms under certain conditions. For example, bacteria grow best at 37oC for 18-24 hrs while fungi grow best at 28oC for 18-24 hrs. It is noteworthy that the medium for the growth of a bacterium and fungus varies, and so does the temperature at which they grow. Though cells that can be grown through cell culture can be of prokaryotic or eukaryotic origin, in research the term “cell culture” refers mainly to the culturing of cells that are either of human, microbial, animal or plant origin.

The culturing of fungal or bacterial cells must not be mistaken for cell culture. But for the purpose of this topic, the term cell culture and tissue culture shall be used interchangeably.  Cells derived from either animals, humans or plants will continue to grow if supplied with the correct nutrient and environmental conditions necessary for growth to take place. The cultured cells are capable of dividing and increasing in size until their growth is limited by some environmental conditions such as the depletion of growth nutrients. Cell culture encompasses organ culture and other in vitro culture techniques in which cells derived from their parent tissues (as dispersed cells) or from particular cell lines/strains and from primary cell cultures are cultivated in vitro in specialized growth medium that mimic the natural environment from which the cells were initially derived from. Cell culture techniques have several applications. It is applied in tissue engineering, toxicological research, immunological research, proteomics, pharmacology (especially in drug design), and in other advanced molecular biology manipulations.

PHYSIOCHEMICAL ENVIRONMENTAL FACTORS THAT AFFECT CELL CULTURE

Cell or tissue culture experimentations should not be carried out in the regular laboratory space where other laboratory investigations are undertaken. This is critical to avoid contamination of cells in the cell culture plates and also to ensure that all the physiochemical environmental factors that encourage optimal growth of the cells are provided. Thus, cell culture experimentations should be carried out in a specialized laboratory or an area in the regular laboratory that is secluded from the usual laboratory area in order to achieve optimal result.  Some key environmental conditions (i.e. the physiochemical or physico-chemical environmental growth factors) must be met in order to achieve optimum cell/tissue culture technique. Some of these environmental conditions that must be met for cell culture to take place smoothly are highlighted in this section.

The microenvironment in which cell culture technique is basically carried out is unique and quite different from the traditional microbial cultures that also occur in vitro in the sense that tissue culture support the growth of “living cells or cultures” derived from their parent cells. Such cells are manipulated and grown in such a way that they mimic the actual natural environment of the host organism from which these cells or tissues where extracted from. Therefore, it is critical that certain physiochemical and environmental factors including temperature, oxygen, pressure and CO2 of the growth medium (i.e. the cell culture growth medium) is performing at optimal levels that allow these cells to be manipulated in vitro. Controlling these physiochemical environmental factors (even though they may not always be defined in most cell culture techniques) at an equilibrium state is vital to the unperturbed growth of the cells. The growth nutrients for the cells in the culture also provide some supplements such as serum that contribute to the optimal growth of the cells or tissue in the culture.

EFFECT OF TEMPERATURE ON CELL CULTURE

The temperature condition of tissue culture varies with the site of extraction of the cells i.e. the temperature of the actual body site the cells or tissues was extracted or obtained from. It is vital to always ensure that the extracted cells are cultivated in vitro while maintaining the original body temperature of the animal or body site of the animal from which the cells was originally extracted from. This is critical in cell culture techniques because the body temperature of an animal varies from one site of the body to another. For example, the temperature of the scrotal area (i.e. the scrotum that harbours the testes) which is covered is quite different from the temperature of the skin which is rationally exposed to the atmosphere.

However, most cell culture techniques especially those that has to do with the in vitro cultivation of cells obtained directly from animals are mainly carried out at 37oC since the normal body temperature of animals inclusive of humans is around this range. The temperature range for cell culture should be optimally maintained. The CO2 humidified incubator used for incubating cell culture plates should not be under-heated or overheated during incubation as this may affect the growth of the cultured cells and other physiochemical factors. The spatial distribution of cell culture flasks in the CO2 humidified incubator in such a way that they are not overcrowded is critical because this practice ensures that the air within the incubator is evenly distributed and that temperature is maintained at ambient or optimal levels for the growth of the cells.

EFFECT OF HUMIDITY ON CELL CULTURE

Humidity is moistness or wetness of the air. It gives an estimate of the amount of water in the atmosphere. Humidity is a critical physiochemical factor considered for cell culture techniques. The incubator used for cell culture techniques is humidified in nature. This implies that the incubator maintains temperature levels that ensures optimum wetness or moisture level required for the growth of the cultured cells or tissues. Cells or tissues in cell culture plates or flasks require high humidity for growth but the humid level of the incubator should be controlled to avoid the growth of fungi within the CO2 humidified incubator. To avoid fungal contamination of the incubator, such humidified incubators should be routinely cleaned using disinfectants especially those that are antifungal in action.

EFFECT OF GAS ON CELL CULTURE

Gases inclusive of oxygen (O2) and carbondioxide (CO2) are critical for the optimal growth of cells in cell culture plates or flasks. The oxygen requirement for cell culture techniques varies across the different types of cells or tissues to be cultured even though most of the cells require O2 in vivo for their respiratory activities. Optimal O2-tension levels are critical for growth. Due to the difficulty experienced in the diffusion of O2 through the cell culture medium it is important that sustainable measures (e.g. the use of O2 packs or O2 carriers) be employed to ensure that the optimal amount of O2 required for the growth of the cells or tissues in culture is provided. CO2 tension is another critical physiochemical factor of the gas-phase of tissue culture that must be considered when contemplating cell culture experimentations.

The CO2 is mainly generated from the cell culture medium especially from its sodium bicarbonate (NaHCO3) component and even from atmospheric CO2. The CO2 tension level should be maintained at optimal levels. The caps of some cell culture flasks or bottles are vented and have holes which allow excess CO2 generated within the vessel to escape while others are not vented and have no holes on their caps so that excess CO2 generated within the CO2 humidified incubator does not infiltrate into the cell culture flask or bottle. The regulation of CO2 in this manner helps to regulate the pH of the cell culture medium for the optimal growth of the cells or tissues of interest.

EFFECT OF PRESSURE ON CELL CULTURE

The osmotic pressure of cell culture flasks is usually measured using a special piece of equipment or instrument known as the osmometer. Cells or tissues cultured in vitro via cell culture techniques should be cultivated at optimal osmotic pressure tension levels in order to ensure that the osmotic pressure in the growth environment mimics that of the site or organism from which the cells were originally extracted from. The addition of extra growth nutrients (e.g. serum) and other substances into the growth or cell culture flasks should be optimally and cautiously done because these factors may affect and destabilize the normal osmotic pressure of the cell culture process. And this is not too good for optimal performance of the cells or tissues in the cell culture flasks.

EFFECT OF NUTRIENTS ON CELL CULTURE

Dulbecco’s Modified Eagle’s Medium (DMEM) is a commonly used cell culture medium for a variety of cell/tissue culture techniques (Figure 1). This medium contains vital nutrients and/or growth factors that support the optimal growth of cells or tissues in cell cultures. DMEM is a basal or general purpose medium used for most cell/tissue culture techniques. It can also be supplemented with other nutritional components such as amino acids, vitamins, serum, glucose, hormones and proteins in order to improve its quality. Antibiotics, lipids, minerals, organic and inorganic substances are other components that can be included to a basal cell culture medium to increase its nutritional base. The addition of these extra growth nutrients makes the media a complex cell culture medium.

Figure 1: Dulbecco’s Modified Eagle Medium (DMEM) for cell culture experimentations.

Effect of pH on cell culture

The hydrogen ion concentration (pH) of a cell culture medium varies according to the type of cells or tissues to be cultured in vitro. However, some cells have optimal pH level in the range of 5.5 – 7.4 and even up to 7.8 in some cases. To maintain an optimal physiological pH level required for the successful in vitro cultivation of particular cells or tissues using cell culture techniques, it is critical to ensure the appropriate buffering of the culture media through the removal, adjustment and/or addition of materials and other factors within the growth media that will help to ensure a balanced pH level for growth.

CELLS THAT CAN BE CULTURED IN VITRO USING CELL CULTURE TECHNIQUE

The living cells that are cultured may include:

  • Cells – (e.g. blood and microbial cells)
  • Tissues – (e.g. skin)
  • Organs – (e.g. heart)

When a whole organ or intact organ fragments of an organism (plant or animal) are removed from an organism and cultured in vitro for the purpose of studying their function and development, the process is called organ culture. The culturing of cells, tissues or organs occurs in vitro i.e. in glass or cell culture dishes or flasks (Figure 2); and this is opposed to in vivo processes (i.e. natural processes that occur inside the whole organism from which the cells or tissues where obtained from). The artificial environment that cell culture provides, and in which cells are engineered to grow in, usually consists of a suitable plastic or glass culture vessel containing liquid or semi-solid medium that supplies all essential growth nutrients for the survival of the cells, tissues or organs being cultured in vitro.

Several sizes of these flasks (usually in the range of 10 – 180 cm2) exist for cell (tissue) culture techniques. While the cap of some of these flasks is vented i.e. some flasks have openings on their caps others are not vented and have no openings on their caps. Some tissue (cell) culture flaks are multilayered (e.g. some have up to 5 layers) while others are single layered. Vented cell/tissue culture flasks provide or allow excess CO2 generated within the culture flask to exit the flask or to allow CO2 to enter the flask when incubated inside a CO2 humidified incubator. Other types of vessels used for cell (tissue) culture apart from the vented and non-vented flasks shown here include the stirrer flasks, multiwall plates, screw-capped vials/phials, Petri dishes and screw-capped conical flasks.

Figure 2: Illustration of a typical cell/tissue culture flask containing cell culture medium.

REASONS FOR DOING CELL CULTURE

Why is cell, tissue or organ culture embarked upon despite the ethical issues surrounding its development, acceptance and usage? Cell culture has application in a variety of biomedical sciences and even in the industry. It has become one of the major tools used in the life sciences world over. Its application has greatly revolutionized the field of medicine, biotechnology and even agriculture owing to the many useful products derived from it. Cells are cultured for so many reasons. Some of these reasons are highlighted in this section.

  • Cell culture is an important source of pharmaceuticals such as vaccines, proteins, antibodies, hormones and even the anti-diabetic hormone called insulin.
  • It helps to study the metabolism of living cells including cells of microbes, plants and animals.
  • Cell culture helps to provide evidence on the function and development of specific cells.
  • It is used for the isolation and growth of cancerous cells for further studies.
  • Cell culture gives a clue as to how cancerous cells develop and spread.
  • It can be used for the screening of putative drugs in the pharmaceutical industry during novel drug development.
  • It is a tool for investigating the numerous processes relating to human health and diseases.
  • Cell culture is used to test new products including drugs, cosmetics, vaccines and other pharmaceuticals for toxicity.

Cytotoxicity

Cytotoxicity is the cellular damage of metabolic pathways, structures and intracellular processes of a living organism which ultimately result in the loss of function or impaired metabolic function. In some cases, cytotoxicity may lead to the loss of viability of the lining cells or tissues. Most in vitro experimentations (particularly cell culture techniques) are mainly aimed at determining the probable toxicity or cytotoxicity of substances (e.g. drugs, cosmetics and vaccines) that is being tested using cell culture techniques. And because these tested materials or substances are used in vivo by living organisms (inclusive of humans) for treatment and other beneficial purposes, it is critical that they are certified safe to the host’s body and thus portend no danger or toxicity when used. Cytotoxicity testing is used to determine the level of toxicity of a substance at the cellular level either in vivo or in vitro. It can be carried out in vivo (i.e. in laboratory animals) or in vitro (as is applicable with cell culture). Cytotoxicity testing is crucial for the testing of products such as drugs and vaccines as aforementioned before they are released into the market for public consumption.

CLASSIFICATION/TYPES OF CELL CULTURE

In the cell culture laboratory, there are many cell culture techniques that are routinely engaged or carried out. These various types of cell culture techniques are highlighted in this section.

  1. Primary cell culture: These are cells obtained directly from an organism and are directly plated in a cell culture dish or flask. They comprise cells of a tissue or organ obtained from an organism and immediately transferred to a suitable cell culture environment conducive for growth. Such cells will attach to the medium, divide and grow exponentially. They are generally termed primary cell cultures. Primary cell cultures have a limited life span. They will only last for a short period of time (usually days to weeks). Their only advantage is that they may exhibit some physiological behaviour similar to that obtainable in vivo because they are freshly isolated cells. Primary cell cultures are usually unstable and require some time to adapt to the in vitro environment they are introduced to.

In addition, some cells in primary cell culture may sustain injury during their isolation and preparation, and thus eventually die in the process. In primary cell cultures, a series of enzymatic and mechanical disruptions of the tissues or organs and selection steps are usually employed to isolate the cells of interest from a heterogeneous population of cells. Some examples of primary cell cultures or cell lines includes: macrophages, natural killer (NK) cells, B and T cells, dendritic cells and cells of the spleen (splenocytes).  These cells are all cells of the immune system. They are used in primary cell cultures to decode the effects of some certain substances (e.g. drugs) on the functions and proliferation of cells of the immune system.

  1. Secondary cell culture: These are cells taken from a primary cell culture and are passaged (or subcultured) into a new and fresh cell culture flask/disk containing new growth medium. Passaging which can also be referred to as sub-culturing is the transplantation of cells from one cell culture vessel to another. Passaging gives cells the chance to expand and increase in population. A higher cell growth is usually achieved due to the addition of fresh growth medium and the introduction of other environmental conditions. Normally, the number of cells obtained from a primary cell culture are may not be enough to create sufficient cells required for a graft, and this warrant the need for Passaging of cells obtainable in secondary cell culture.

Secondary cell cultures are transformed and immortalized cell lines with infinite growth and proliferation capacity. They are usually derived from human carcinomas/tumours. Such cells have been transformed in the sense that they have lost sensitivity to factors associated with growth control and thus can grow unlimited. Secondary cell cultures are more easily cultured than the primary cell cultures. Some sources of secondary cell cultures include: embryos and tumours or transformed cells such as HeLa cells and Chinese hamster ovary (CHO). Secondary cell culture has applications in a range of areas such as in vaccine production and drug screening.

  1. Suspension cell cultures: These are cells that grow freely and unattached to any surface. Such cells are cultured in suspensions of growth medium. They are maintained in a cell culture flask without any adherence to any surface. Examples of cells cultured in suspension include the cells of the blood such as hematopoietic cells. Such cells are engineered to grow in suspensions. They grow in a very much higher proportion.
  2. Adherent cell cultures: These are cells that attach or adhere to the surfaces of the cell culture flask used for their culturing. They are referred to as anchorage-dependent cells. These cells are cultivated in suitable growth medium that is specially suited and treated to allow adhesion and the spreading of the cells. The cell culture flask used for adherent cells are usually coated with materials that increase their adherence features and provide signals needed for their growth and proliferation in the cell culture medium.

BASIS FOR A SUCCESSFUL CELL CULTURE

Having a cell culture without any form of contamination is paramount in the cell culture laboratory. Such success is usually achieved when the laid down principles and aseptic techniques for undertaking a cell culture procedure are conscientiously followed. A successful cell culture technique depends on a number of factors. These factors are highlighted in this section.

  • The quality of cell lines you are working with.
  • The quality of reagents and cell culture media used.
  • The aseptic technique used.
  • The quality of the laboratory equipment used and their operation.
  • The experience of the researcher.

When these factors are met and made available prior to undertaking cell culture experimentation, it is expected that the cell culture technique will be successful.

OVERVIEW OF CELL LINES

A cell line is a cell that has undergone mutation and series of genetic manipulations, and will not undergo apoptosis after a limited number of passages (sub-culturing). Apoptosis is defined as programmed cell death. It is a cell death that occurs by a biologically-controlled intracellular process that involves the fragmentation and cleavage of the host cells nucleic acid (particularly the DNA). When cells from the first culture (usually taken from the organism) are used to make subsequent cultures, a cell line is established. Immortal cell lines can replicate indefinitely due to manipulations of their genetic material and the maintenance and sustenance of optimum nutrient and environmental conditions. Cell lines are cells or cell cultures obtained after the first subculture of a primary cell culture.

This primary cell culture becomes known as a cell line or sub-clone after the first subculture has taken place. Cell lines derived from primary cultures usually have a limited life span. But as these cells are passaged, those cells with the highest growth capacity predominate. This will result in a degree of genotypic and phenotypic uniformity of the population of cells that will be produced. Such cells produced in this way are generally referred to as cell lines. Cell lines are obtained or sourced for research purposes in any of the following ways:

  • Through primary culture
  • Through Passaging or sub-culturing
  • Through buying and borrowing either from already established cell collection centers such as the American Type Culture Collection (ATCC). Cell lines can also be sourced from research laboratories and institute in possession of cell lines. Cell lines obtained by borrowing are usually not too good because they can be contaminated by bacteria or mycoplasmas

Some established cell lines include: Chinese hamster ovary (CHO), HeLa cell lines, 3T3 cell lines and BHK21 cell lines. These established cell lines can be commercially obtained and used for cell culture experimentations.

PRECAUTIONS TAKEN TO AVOID CONTAMINATION IN CELL CULTURE TECHNIQUES

Contamination is a great enemy in the cell culture laboratory. Thus all aseptic techniques must be dutifully followed in order to knock out all sources of contamination in the cell culture. Since contamination by microbes is a major factor in most cell culture techniques it is critical to ensure sterility and/or aseptic techniques at every stage of the experiment in order to get optimum results. Aseptic techniques in the cell culture laboratory ensure that all cell culture protocols are performed to a standard that will prevent contamination from microorganisms (bacteria, fungi and mycoplasmas inclusive) and cross-contamination with other cell lines. Aseptic techniques are all the precautionary measures taken during an experiment to avoid contamination of the work and the researcher. It ensures amongst other things the maintenance of strict sterility in the course of the research. Even though an absolute sterility could not be observed in most cell/tissue culture techniques, it is critical that the researcher imbibe and carry out all the necessary aseptic measures required for that particular experiment in order to avoid the introduction of exogenous or environmental organisms into the cell culture flasks.

Normally, all cell culture works are undertaken inside a tissue culture hood (laminar flow cabinet) that provides an aseptic environment for work. The face area of the hood is covered with a removable glass panel which acts as a physical barrier that helps to maintain a particulate free environment and laminar flow of air within the hood. The hood is fitted with an ultraviolet (UV) light which is switched on after work (i.e. at night) and off before work. The UV light is switched off prior to usage and allowed for about 2-3 minutes for air flow patterns to filter and establish within the hood. The hood is sprayed with 70% ethanol and wiped dry, and every reagent bottles and equipment taken into the hood must be decontaminated by spraying with 70% ethanol as well. All work in the cell culture laboratory is normally performed within the hood, and after work all disposables should be disposed. The hood should be sprayed with 70% ethanol after work, and the UV light turned on at night. Some of the safety and aseptic techniques employed in the cell culture laboratory are highlighted in this section.

  • It is critical to start the cell culture experiment on an entirely clear and clean surface. Work area should be properly disinfected prior to and after the experiment using 70 % ethanol and recommended disinfectants.
  • More than one cell line should not be handled at a time to avoid cross contamination of cells.
  • Always use sterile equipment for cell culture experimentations.
  • The cell culture laboratory should be as quiet as possible and unnecessary walking around should also be avoided during experimentation.
  • All equipment to be used for the cell culture experiment should be close by.
  • Reagents and media should not be shared with other people or among different cell lines.
  • All cell culture tubes should be opened inside the hood.
  • Researchers should ensure good personal hygiene’s. They should always were laboratory gowns, eye goggles and other personal protective wears during the experiment. Researchers should always work with gloved hands.
  • Do not breathe or talk into the hood (i.e. the biosafety laminar flow cabinet).
  • Growth or culture media should be stored according to manufacturer’s instructions.
  • Splashing of media and other reagents within the hood should be minimized as much as possible.
  • Cell culture media and cells should be transported carefully. Cell cultures should be routinely tested for Mycoplasma, a common contaminant of cell culture experiments.
  • Cell culture flasks should be kept apart from each other to avoid falling over. This will also help to avoid overcrowding which will prevent improper flow of air within the incubator.
  • Disposable plastic pipettes or glass pipettes (generally known as plugged pipettes) should be used for cell culture experiments, and used pipettes should not be reused. Mouth pipetting should not be allowed in the cell culture laboratory.
  • Pipettes should be opened within the hood. Mouth pipetting is not advisable because it may introduce contaminants into the cell culture flasks. Mouth pipetting also exposes the researcher to hazardous substances in the laboratory.
  • Pouring or transferring of cell culture media and reagents should be done based on laid down laboratory standard operating procedures (SOPs). Sterile media should not be poured from one sterile container to another unless such containers are to be used once. Disposable pipettes should be used for media and reagent transfer during cell culture experimentations.
  • The hood should be effectively decontaminated with 70 % ethanol prior to and after use.

SOME IMPORTANT EQUIPMENT USED IN CELL CULTURE TECHNIQUES

  1. Laminar flow cabinet (hood): The laminar flow biological safety cabinet which can also be called hood or cell culture hood is one of the most important equipment in the cell (tissue) culture laboratory. This piece of equipment helps to provide sterile environment to work with cells without any fear of contamination (Figure 3). It is a filtration system which protects a technician/scientist working in the cell culture laboratory from microorganisms or contamination by cells being handled within the hood. The cell culture hood helps to prevent aerosols from coming in contact with the researcher. The constant and steady air filtration system of the hood which is distributed throughout the entire working surface of the laminar flow biological safety cabinet ensures that the whole working surface of the hood is adequately protected from extraneous contaminants and dust particles that might compromise the experimentation.

Working in a clean environment (which the hood provides) is important to avoid personnel contamination and to keep everyone around safe. The air filtration system in the hood is usually of two types: the vertical airflow and the horizontal airflow. This air filtration system is also used in classifying the hood as either a vertical airflow hood or a horizontal airflow hood. In the vertical airflow system, the air blows down from the top of the biosafety laminar flow cabinet to the entire work surface within the hood and re-circulated. But in the horizontal airflow system, the air is drawn from the sides of the hood facing the researcher.

This type of air filtration is not re-circulated as is obtainable in the vertical airflow system. While hoods with vertical airflow system gives more protection to the researcher, those with horizontal airflow systems gives lesser protection to the worker and more sterile protection to the reagents and culture media in the hood. The horizontal airflow hood gives the most stable airflow since the air it produces is not vented or re-circulated as is the case with the vertical airflow hood.

Figure 3: Biological Safety Cabinet or Hood. The hood is used to maintain a sterile environment for working aseptically in the laboratory.

  1. Inverted phase microscope: The inverted phase microscope unlike the normal light microscope does not require any staining of cells prior to its usage (Figure 4). Trypan blue dye is used for diluting or mixing cells to be viewed under the inverted microscope. It is inverted because the objective lenses are located below the stage; and condenser is located above the stage too. Cells or tissues in a cell culture flask or dish are viewed directly without being removed for staining (as is normally the case in light microscopy). Inverted microscopy allows scientists to view cells in a cell culture flask or bottle directly (Figure 5). Staining is not usually allowed in the cell culture lab because it can kill the cells being stained. The microscope is used to check for cell growth or viability, contamination and the health of the cells. It also helps the researcher to ensure that he or she is working with the correct cell line. Another type of microscope used in the cell culture laboratory is the contrast microscope. The inverted microscope is a basic piece of equipment required in the setting up of a cell (tissue) culture laboratory. This type of microscope allows scientists to routinely check cell culture bottles for possible signs of deterioration especially in the face of microbiological infection.

Figure 4: Illustration of an inverted microscope.

The inverted microscope is usually fitted with digital camera and phase-contrast optics which improves the magnification of the object. Unlike in the normal light microscopy, the condenser of the inverted microscope is above the stage while the objective lenses are below the stage as shown in Figure 4. Inverted microscopes can also be fitted with CCD (closed-circuit digital) cameras as aforementioned, and they are usually connected to computer monitors which enables digital viewing of cells in the cell culture bottle as well as allow the scientists to print an image of the cells.

Figure 5: Cell culture flask being viewed with an inverted phase contrast microscope (arrow).

  1. Incubator: The incubator used in the cell culture laboratory is a CO2 humidified incubator. The CO2 humidified incubator provides and maintains CO2 level (at 5-10%), humidity and temperature (at 37oC) to stimulate in vivo conditions necessary for the optimum growth of cells in the cell culture medium. Cell culture flasks should be evenly spaced when placed inside the CO2 incubator to avoid overcrowding and ensure even distribution of air or gases. Overcrowding of cell culture flasks within the CO2 incubator should be avoided so as to ensure proper gas exchange within the incubator. Very tall stacks of culture flasks should be avoided within the incubator in order to avoid them falling over. The CO2 incubator for cell culture experiments should be routinely cleaned to remove fungal growths, traces of detergents, dust particles and dampness or wetness common with humidified incubators used for cell culture experimentations (Figure 6).

Figure 6: Illustration of CO2 incubator. Cell/tissue culture flasks is shown and properly stacked in the CO2 humidified incubator.

  1. Centrifuge: The centrifuge is important in the cell culture laboratory because it is used to sediment particles or cells suspended in fluid (Figure 7). Centrifugation is a routine in the cell/tissue culture laboratory because suspensions of cells are often centrifuged to either wash-off a reagent or increase the concentration of cells required for a particular analysis.

Figure 7: Illustration of a centrifuge.

  1. Waste container/bin: The waste container is used for collecting wastes including used pipettes, used cotton wools, broken dishes, used hand gloves and other wastes that arise during cell culture procedures (Figure 8). Old culture flasks/dishes and other wastes should be autoclaved first before their disposal.
  2. Pipette: Pipette is used for collecting and transferring cells and media. Mouth pipetting is not allowed in the cell culture laboratory; and thus pipette pumps are used for the aspiration and passaging of cells during cell culture experiments (Figure 9). Micropipettes or pipette is used for the collection and transferring of reagents or cell cultures in the cell culture laboratory; and this is made possible with the pipette pump – which is used to hold the pipette meant for the aspiration of the cells to be passaged or transferred. Plugged pipettes or disposable plastic pipettes are the pipettes routinely used for cell culture experimentations. Pipette pumps are used with plugged pipettes and this helps the scientist to draw certain concentration or amount of solution without using mouth pipetting method.

Figure 8: Waste collection bin.

Figure 9: Illustration of plugged pipettes and pipette pumps.  

  1. Cell counting machine (haemocytometer): The haemocytometer is used for counting cells from cell cultures (Figure 10). It counts cells manually under the microscope. There are other automatic cells counting machines such as the countess machine which can be used to count cells in the cell culture lab. Cell growth can also be measured by using spectrophotometer. Spectrophotometer is a device that measures the optical density of cells in suspension. The results of a spectrophotometer are extrapolated in order to determine the growth of the cells. A haemocytometer has an indention down the center where cells can be suspended in a liquid, and then the numbers of cells in a particular sized square are counted to calculate the overall cell count.

Figure 10: Illustration of haemocytometer with the squared area for counting particular cell types.

  1. Refrigerator: The refrigerator is used for the storage of cell culture reagents and media (Figure 11). Reagents and media should always be stored according to the manufacturer’s instructions. All reagents for cell culture experimentation should always be stored at the appropriate temperature to avoid spoilage.

Figure 11: Laboratory refrigerator.

  1. Water bath: Water bath is used to incubate bottles of cell culture media or other reagents and liquids in flasks in order to bring them to a particular or optimum temperature prior to their usage (Figure 12). Flasks should always be kept upright in the water bath, and the neck of the flasks should always be above the water in the water bath. Reagents in water baths should not be left more than is necessary because excessive warming could lead to the loss of activity in the media or reagents being warmed.

Figure 12: Water bath.

USE OF ANTIBIOTICS IN CELL CULTURE LABORATORY

Bacterial contamination of cells was one of the major threats encountered in the culturing of animal cells in the cell culture laboratory. Microorganisms are naturally ubiquitous. The ever-present nature of microbial organisms should be considered and eliminated as much as possible whenever any cell culture protocol is being contemplated. Cells can be easily contaminated in the cell culture laboratory especially during collection and passaging. Microbial contamination in the cell culture laboratory should therefore be taken seriously and avoided as much as possible because it can affect the healthiness of the resulting cell lines. The microorganisms that usually affect cell cultures and freshly isolated cells, tissues or organs are: mycoplasmas, bacteria, and fungi. Antibiotics is therefore used and applied during the collection, transportation and the dissection of organs or tissues prior to establishing and getting the primary cell culture. The antibiotics help to eliminate any form of microbial contamination of cells, tissues or biopsies. Decontamination of contaminated cells or their total discarding should be adopted whenever cells are contaminated so as to avoid spreading to other uninfected cells. Antibiotics should be eliminated once the primary cells have been established because long contact of antibiotics with cells can affect some vital eukaryotic cells.

Careful physical and microscopic examination of cells can help to detect infections by bacteria and fungi. But this is not the case for mycoplasmas which require more specific screening test for mycoplasmas (which are one of the most serious contaminations in the cell culture lab). Because of the several disadvantages which they portend in cell (tissue) culture experimentations, antibiotics are rarely used for cell culture techniques. The availability and versatility of the biosafety laminar flow cabinet (i.e. the hood) which provides a sterile environment to perform tissue culture experiments have relegated the use of antibiotics in cell culture techniques. Antibiotics may encourage poor aseptic technique and they may also cover mycoplasma infections in cell culture flasks or bottles. Some antibiotics may cross-react with mammalian cells, and thus they may impede the result of the tissue culture experiments when used to prevent contamination of the cells. These reasons and some others have restricted the usage of antibiotics in most cell (tissue) culture experiments. However, the usage of antibiotics as a preventive measure especially to contain bacterial and/or microbial contamination in cell (tissue) culture flasks is not entirely abandoned. The hood should be thoroughly checked and cleaned with 70 % alcohol to reduce contamination within it.

ADVANTAGES OF CELL CULTURE TECHNIQUES

  1. Cell culture is economical since it is carried out in vitro (i.e. in culture flasks or dishes). It requires small portions of reagents and media unlike in vivo techniques that require a whole organism.
  2. Homogenous cells are produced since the growth environment can be controlled in vitro.
  3. The physiology and biochemistry of cells can be studied and manipulated in vitro.
  4. In cell culture techniques there are no ethical, moral or legal issues as is the case in experimentation that involves the use of a whole animal.
  5. Cells in a culture flask/dish can be exposed to chemicals or drugs directly.

DISADVANTAGES OF CELL CULTURE TECHNIQUES

  1. Cell culture techniques are usually capital intensive. They should be undertaken only when necessary.
  2. Cells in cell culture flasks are denied of some in vivo materials such as hormones and other supporting structures that the isolated cell interact with in vivo.
  3. Success in cell culture techniques requires expertise to know the behaviour of cells in culture. The aseptic techniques involved usually takes some time to learn.
  4. The artificial condition in vitro may cause the cells to produce different substances (e.g. proteins) from the ones they produce in vivo.
  5. It is almost impossible to reproduce an in vivo process in an in vitro technique like cell culture.

SAFETY IN THE CELL CULTURE LABORATORY

Safety is paramount in any cell culture laboratory. It protects the researcher from possible contamination and it also helps to ensure that healthy cell lines are produced in the process. Failure to dutifully follow aseptic techniques and other safety measures in the cell culture laboratory can result in the contamination of cells. Some safety measures to be observed in the cell culture laboratory include:

  • All new cells that arrives the cell culture lab should be first quarantined, and treated as potentially infected.
  • Carcinogenic substances and materials should be handled with care and caution.
  • Existing cell culture stocks should be regularly screened.
  • New cells should also be screened to lookout for infection.
  • Do not eat, smoke or drink in the cell culture working area.
  • Used gloves should not be worn outside the cell culture laboratory.
  • Do not bring in friends in to the cell culture laboratory for chatting or making phone calls.
  • Protective clothing or lab coat should always be worn when in the cell culture laboratory.
  • All equipment, reagents and media should be handled with care and according to the manufactures instructions.
  • Hands should always be washed with detergents/antiseptics or soap before and after work and even before leaving the cell culture laboratory.
  • Treat all liquid waste with bleach before disposing.
  • Care should be taken when handling infectious cells (e.g. prions and viruses).
  • The cell culture hood must always be used in cell culture experimentations to avoid cross contamination.

APPLICATIONS OF CELL CULTURE TECHNIQUE

Cell culture technique is defined as the process by which prokaryotic, eukaryotic or plant cells are grown or cultured in vitro under controlled laboratory conditions. Cell culture techniques have applications in the following fields:

  • Disease diagnosis: Cell culture techniques are applied in clinical medicine for the diagnosis of infectious diseases especially diseases caused by pathogenic viruses. Cell culture techniques aid in rapid viral detection from clinical samples. It also aids in the early treatment of viral infections once the causative viral agent have been detected. Over the years, viral disease diagnosis has traditionally relied on the isolation of viral pathogens in cell cultures which some perceive as being slow and requires special technical expertise. However, advances in cell culture-based viral diagnostic products and techniques including but not limited to cryopreserved cell cultures, centrifugation-enhanced inoculation, precytopathogenic effect detection, co-cultivated cell cultures, and transgenic cell lines have made cell culture to be useful for the diagnosis of viral diseases.
  • Biomedical research: In biomedical research, cell culture techniques are most preferable than the use of animals for research. Since the use of animals such as monkeys and chimpanzees for research could lead to the extinction of these animals, cell culture techniques is a good alternative and replacement to prevent the extinction of some wildlife. Cell culture techniques can be applied in biomedical research especially in the area of studying some molecular disease processes, and finding out ways via which these diseases of non-microbial origin could be better treated. With the application of cell culture techniques in biomedical research, improved and prompt ways of detecting disease causative agents could be developed. Cell culture techniques could also be used as model system to study basic cell biology, metabolism and the physiology of living systems.
  • Virology: In the field of virology, animal cell culture techniques can be used to replicate the viruses used for vaccine production instead of using animals for this purpose. Cell culture techniques can also be used to detect and isolate pathogenic viruses from clinical samples. It can also be used to study the growth and development cycle of viruses. Cell culture techniques can also be used in virology to study the mode of infection of viral disease agents.
  • Genetic engineering: In genetic engineering, cultured animal cells can be used to introduce new genetic material like DNA or RNA into another cell. Such exchange of genetic information amongst cells or organisms can be used to study the expression of new genes and its effect on the health of the recipient host cell. The recipient host cell starts expressing novel proteins that could be of immense industrial and medical importance. Animal cell cultures are used to produce commercially important genetically engineered proteins or immunobiologicals such as monoclonal antibodies, polyclonal antibodies, insulin, anticancer agents and hormones.
  • Model systems: Cell culture techniques are used in model systems to study the effect of drugs in human or animal host. It can also be used to study the process of aging in humans. In model systems, cell culture techniques are used to study the major triggers for ageing in man. It can also be used to study how host cell and disease causing agents like bacteria, fungi and viruses interact in vivo.
  • Cancer research: Cell culture techniques is used in cancer research to study the basic difference between normal cells and cancer cells since both cells can be cultured in vitro in the laboratory. Normal cells can be converted into cancer cells by using radiation, chemicals and viruses. This allows the mechanism and cause of cancer to be studied in vitro using cell culture techniques. Cell culture techniques can also be used to determine the effective chemotherapeutic drugs that can selectively destroy only cancer cells without harming the host cells since most cancer drugs have several untoward effects on the host.
  • Toxicity testing of novel drugs: Cell culture techniques can be used to study the effects of novel drugs, cosmetics and other chemical agents in order to determine not just their efficacy but also the level of their toxicity (i.e. cytotoxicity). The toxicity of the newly developed drugs to vital organs of the body such as the liver and kidney (that are involved in drug metabolism) is also evaluated using cell culture techniques. Drug dosages for novel drugs can also be determined using cell culture techniques.
  • Gene therapy: Gene therapy is an experimental technique that uses genes to treat or prevent disease especially molecular or non-infectious diseases such as cancer. It allows clinicians to treat a genetic disorder by inserting a functional gene (to replace a dysfunctional gene) into a patient’s cells instead of using the conventional treatment methods such as the use of drugs, chemotherapy or surgery. In gene therapy techniques, a dysfunctional gene is replaced with a functional gene. Through cell culture techniques, cultured animal cells are genetically altered and made functional so that they can be used in gene therapy techniques. Briefly, cells are removed from the patient lacking a functional gene or missing a functional gene; and the extracted cells are cultured in vitro through cell culture techniques. These dysfunctional genes are replaced by functional genes. Gene therapy uses a vector, typically a virus, to deliver a gene to the cells where it is needed. Once inside the host cell, the host cell’s gene-reading machinery uses the information in the introduced functional gene to build ribonucleic acid (RNA) and protein molecules which will now replace the lost activities of the replaced dysfunctional gene.
  • Vaccine development: Cell culture techniques can be used in vaccine development since they help to culture animal cells in vitro. Cultured animal cells are in turn used in the production or propagation of viruses that are used to produce vaccines. These vaccines are used clinically for the prevention of communicable diseases caused by pathogenic viruses including measles, polio, rabies, hepatitis and chicken pox and other preventable viral diseases.

ISSUES SURROUNDING THE USE OF ANIMALS IN SCIENTIFIC RESEARCH

Animals such as mice, rats, rabbits, monkeys and primates are used in biomedical research to test the efficacy and toxicity of novel drugs and vaccines prior to their general usage in humans and animals for therapeutic purposes. The use of animals to do research whose results will in turn be extrapolated to humans is hinged on many factors. For example, human beings and animals share similar physiology and biochemistry in terms of the makeup of their body systems. In addition, the metabolic activities of animals are to some extent akin to those of humans who also possess eukaryotic cells like the animals. In the long run, these animals used for biomedical and/or medical research are usually scarified or killed during the course of the research or at the end of the research. These factors are some of the issues that surround the use of animals for research, and the global call for the discontinuance of the use of animals in research.

However, animal-based research cannot be extrapolated to humans because of species differences and is therefore misleading and counterproductive. Testing a drug or chemical on an animal provides no evidence that it is safe for humans because animals do not react in the same way to drugs as animals do. There is also a wide variety of differences in the metabolic activities (e.g. absorption and distribution of substances such as drugs) that occur in animals when compared to humans. The mechanisms of disease development and spread in animal population can also not be fully extrapolated to that in humans. Nevertheless, pharmaceutical companies and other drug research institution still claim that novel drugs must be tested on animals in order to ensure their efficacy and safety before they are given to human populations for which they were actually meant for. The scientific community believes that the use of animals in biomedical research leads to their extinction, and as such, an alternative scientific technique should be developed for techniques that normally require animals.

The use of laboratory animals in biomedical/scientific research (vivisection) has been a subject of controversy owing to much opposition to its usage. Medical discoveries and innovations no doubt usually begin with clinical observations that eventually make use of laboratory animals which mimic and explain internal conditions of humans. Yet, the use of laboratory animals for biomedical/scientific research has been greatly criticized even till date. It should also be noted that there are some human diseases that do not naturally occur in animals, and thus such pathologies are difficult to study using animal models. So much has been learnt in the medical profession through the use of animals to test new drugs and other chemicals. Also, new knowledge on how the physiology and biochemistry of the human body works has also been learnt through the usage of animals for medical research. The physiology and biochemistry of the human body relates very much to that of animals. For these reasons, animals have helped in the preclinical testing of most drugs and other biomedical innovations meant for animal and human usage. Many believe that the use of animals for scientific research leads to their extinction. And there is some element of truth to this since the animals used for research are usually sacrificed or killed at the end of the research as aforesaid.

To many, animals should not be used for biomedical research because of their religious and cultural values. Others believe that the animals go through pains in their usage and that their consent is not always sought (as is the case in humans) before being used for scientific research. Antivivisectionists also believe that most animals used for scientific research (e.g. cats, rabbits and other animals) are kept as pets and as such no experiments should be performed on them because it also leads to their death. Vivisection is the use of animals for biomedical or medical research. Antivivisectionists those opposed to the use of animals for biomedical research. They believe that the use of animals for scientific research should be undertaken with caution while ensuring less pain on the animals. It is also believed by the medical research community that ethical guideline should be strengthened in medical/academic institutions and always sought before using animals for scientific research. Animals meant for scientific research should be given optimum veterinary care pre- and post-experimentation. They should be handled with care while ensuring that their sufferings and pain are minimized as much as possible in the course of the research.

Vivisectionists believe that the care of animals is everyone’s business. However, since consent can never be given when it comes to animals unlike humans, animals should be given all humane and ethical treatment before their usage. Their alternatives for biomedical research should be developed. Nevertheless, the use of animals for scientific research and the extrapolation of results from these animals to humans are still being used. Until more reliable hi-tech in vitro techniques (e.g. genetically modified mice and transgenic animals) are developed, animals will still be used for biomedical research. In addition, the use of animals has helped in the advancement of biomedical/scientific research, and this has impacted positively on the health benefits of both animals and humans.

THE GUIDING PRINCIPLES IN THE USE OF ANIMALS FOR BIOMEDICAL/SCIENTIFIC RESEARCH

The use of laboratory animals including mice, rats, rabbits and primates for scientific/biomedical research is guided by some principles and ethical guidelines that ensure the optimum care, protection and security of animals used in scientific investigations. Animal research is a very important procedure in biomedical research through which novel drugs are tested and scientific knowledge acquired and improved upon. It is important that researchers imbibe certain internationally accepted ethical guidelines in their use of animals for scientific investigations.

Russell and Birch in 1953 came up with some guiding principles known as the “3Rs”, and which should guide any clinical, scientific or biomedical research involving animals. According to Russell and Birch, the use of laboratory animals for research should be focused and result-oriented while bearing in mind some important factors to the benefit of the animals, humans and the environment. These factors include: ensuring best practices in the use of animals for scientific research and putting the animal’s wellbeing (health and feeding) into consideration before any other gain that accrue from the research.

The “3Rs” proposed by Russell and Birch in 1953 for the use of animals for scientific research is an internationally accepted laboratory guideline for animal research industry across the world, and it stands for: REDUCTION, REFINEMENT and REPLACEMENT.

  • Reduction: Reduction involves the use of fewer animals for the undertaking of any laboratory research. It advocates for a reduction in the number of animals used for scientific research, and that more information (results) should be actualized by the use of lesser animals than being contemplated in any anticipated biomedical research that involves animals. The aim is to eliminate unwanted killings of animals for research, thus reducing the number of animals required to carry out a particular laboratory experiment. This principle encourages the development of novel laboratory methods or procedures such as cell culture and other in vitro techniques that require fewer animals for scientific/biomedical research.
  • Refinement: Refinement involves the modification of already existing scientific research involving animals in order to reduce pain, stress and other discomforts experienced by the animal in the course of the laboratory procedure. It involves the use of techniques that reduce the invasiveness of animal’s bodies and other such methods that involves their timely killing for result taking. Refinement helps to ensure that animals meant for scientific research are adequately taken care of, fed, and treated humanely.
  • Replacement: Replacement is the optimum guideline for scientific research because it encourages the development and use of procedures that does not involve animals. It involves the use of techniques such as cell culture, Limulus Amoebocyte Lysate (LAL), computer modeling, mathematical modeling, and other alternative high-tech immunoassay testing techniques that rules out the usage of laboratory animals. Replacement advocates a partial or a total exclusion of animals from laboratory research that will eventual cause pain, stress and death of laboratory animals.

RATIONALE FOR THE CONTINUED USE OF ANIMALS FOR RESEARCH

Animals including (primates, dogs, cats, rabbits, mice) are still being used to conduct scientific/biomedical research because of some of the following reasons:

  • There is still no complete alternative to the use of animals in testing drugs and other chemicals meant for human and animal consumption.
  • Scientific experimentation using animals draw grant, and is thus a lucrative business for researchers.
  • It helps researchers to evade all potential legal battery that might accrue from the usage of animals for research especially when there is death or disability of the animal.
  • Scientific/biomedical experimentations involving the use of animals are more easily published than non-animal experimentation.
  • Experimentation involving the use of laboratory animals appears to be more scientific and result-oriented than non-animal experimentation since animal and humans share some similarities in physiology and biochemistry.
  • Researcher’s professional status in terms of finance and research grants is usually tied to animal experimentation in some quarters.
  • Results from animal research can easily be extrapolated to humans than in vitro research carried out in non-animal hosts.

REFERENCES

Verma P.S and Agarwal V.K (2011). Cytology: Cell Biology and Molecular Biology. Fourth edition. S. Chand and Company Ltd, Ram Nagar, New Delhi, India.

Weaver R.F (2005). Molecular Biology. Third edition. McGraw-Hill Publishers, USA.

World Health Organization (1993). Laboratory Biosafety Manual, 2nd edn. Geneva: WHO.

Lederberg J (editor): Encyclopedia of Microbiology, 4 vols. Academic Press, 1992.

Lindee, S.M.  2007.  The Culture of Cell Culture.  Science; 316(5831): 1568-1569

Lodish H, Berk A, Matsudaira P, Kaiser C.A, Kreiger M, Scott M.P, Zipursky S.L and Darnell J (2004). Molecular Cell Biology. Fifth edition. Scientific American Books, Freeman, New York, USA.

Marcovic O and Marcovic N (1998). Cell cross-contamination in cell cultures: the silent and neglected danger. In Vitro Cell Dev Biol. 34:108.

Mather J and Barnes D (1998). Animal cell culture methods, Methods in cell biology. 2rd eds, Academic press, San Diego.

Fleming, D. O., Richardson, J. H., Tulis, J. I. and Vesley, D. (eds) (1995). Laboratory Safety: Principles and practice. Washington DC: ASM press.

Freshney R.I (2005). Culture of Animal Cells, a Manual of Basic Technique, 5th Ed. Hoboken NJ, John Wiley and Sons Publishers.

Health Services Advisory Committee (HSAC) (2003). Safe Working and the Prevention of Infection in Clinical Laboratories. HSE Books: Sudbury

Caputo J.L (1996). Safety Procedures. In: Freshney, R.I., Freshney, M.G., eds., Culture of Immortalized Cells. New York, Wiley-Liss, Pp. 25-51.

Cooper G.M and Hausman R.E (2004). The cell: A Molecular Approach. Third edition. ASM Press.

Alberts B, Bray D, Johnson A, Lewis J, Raff M, Roberts K and Walter P (1998). Essential Cell Biology: An Introduction to the Molecular Biology of the Cell. Third edition. Garland Publishing Inc., New York.

Alberts B, Bray D, Lewis J, Raff M, Roberts K and Watson J.D (2002). The molecular Biology of the Cell. Fourth edition. New York, Garland, USA.

Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K., eds (2002). Short Protocols in Molecular Biology, 5th edn. John Wiley & Sons, New York.

 

Leave a Reply

Your email address will not be published. Required fields are marked *