In this article we will discuss about equipment and materials used in animal cell culture.
Basic equipment used in cell culture lab are dependable, Unless unlimited funds are available, it will be necessary to prioritize the specific needs of a tissue culture laboratory: (1) you cannot perform tissue culture reliably without this basic equipment used in cell culture lab; (2) culture would be done better, more efficiently, quicker, or with less labor; and (3) items that would improve working conditions, reduce fatigue, enable more sophisticated analyses to be made, or generally make your working environment more attractive.
Sterile Work Area
For processing the animal tissues for culture purpose a sterile or aseptic area is basic equipment used in cell culture lab (tissue culture laboratory). This working place must be free from any kind of contamination. Two types of sterile work areas are generally recommended.
a. Laminar flow cabinet
It is a specially designed chamber inside which animal tissue for culture purpose is being handled in an aseptic condition. It is completely open in front to allow the researcher to work comfortably and handle the basic equipment used in cell culture lab present inside the laminar flow cabinet. A motor blows air into the laminar flow cabinet through a coarse filter, where large dust particles are separated. This air then passes through a 0.3 μm HF.PA (High Efficiency Particulate Air). This keeps all contaminants away from the work surface. Such arrangement does not give protection to researcher against pathogenic organisms. Hence, laminar flow cabinet cannot be used in any cell or tissue culture which may contain a human pathogen (disease causing organism).
b. Bio-safety cabinet
Bio-safety cabinet provides a sterile environment for tissue culture in addition to making provision for the safety of researcher against human pathogen.
Figure 1: Equipment and materials used in animal cell culture
A cell culture incubator is designed to maintain a constant temperature and high humidity for the growth of tissue culture cells under a CO2 atmosphere. Typical temperature settings range from 4⁰C to 50⁰C, and CO2 concentrations run from 0.3 to 19.9%. Dry incubators are relatively cost-effective, but the cell cultures are needed to be incubated in sealed flasks to avoid evaporation. In a dry incubator, if the water dish is placed, it can supply some humidity however, they do not provide appropriate control of atmospheric conditions in the incubator.
Both refrigerators and freezer are very essential for storage of liquid media at 2–8°C and for enzymes (e.g. trypsin) and some media components (e.g., glutamine and serum) at –5°C to –20°C. There is high possibility for genetic instability in cell lines of continuous culture as their passage number increases, hence, it is necessary to prepare working stocks of the cells and preserve in cryogenic storage.
It is to be noted that the cells should not be stored in 20⁰C or -80⁰C freezers as their viability reduces when they are not stored at these temperatures. Liquid nitrogen freezers permit storage in the vapour phase just above the liquid at temperature between -140⁰C and -180⁰C, or submerged in the liquid at a temperature below -196⁰C.
A simple inverted microscope is basic equipment used in cell culture lab. It cannot be overemphasized that it is vital to look at cultures regularly to detect morphological changes and the possibility of microbiological contamination.
Make certain that the stage is large enough to accommodate large roller bottles, if required, between it and the condenser. It is worth getting a microscope with a phototube for digital recording or viewing linked to a monitor, but it need not be a large and expensive research microscope. Long working-distance phase-contrast optics is required to compensate for the thickness of plastic flasks. The increasing use of fluorescent tags (e.g., green fluorescent protein, GFP) for viewing live cells means that fluorescence optics may be considered as well.
A ring marker (Nikon) is a useful accessory to the inverted microscope. This device is inserted in the nosepiece in place of an objective and can be used to mark the underside of a dish to locate a colony or patch of cells.
Cell culture Vessels
Almost every type of cell culture vessel, together with support consumables such as tubes and pipettes, are commercially available as single use, sterile packs. The use of such plastic ware is more cost effective than recycling glassware, enables a higher level of quality assurance and removes the need for validation of cleaning and sterilization procedures. Plastic tissue culture flasks are usually either treated to provide a hydrophilic surface to facilitate attachment of anchorage dependent cells or untreated to provide a hydrophobic surface for the culture of cells in suspension.
Even if all tissue culture plastic ware should support cell growth maximally, it is necessary to make sure that the new supplier facilitates the growth of cultures. Cells can be kept in petri dishes or flasks (25 cm2 or 75 cm2) , that have added the benefit that the flasks can be gassed and then sealed so that a CO2 incubator should not be used.
Glassware such as pipettes should be immersed in a suitable detergent, then passed through a strict washing procedure with thorough soaking in distilled water prior to drying and sterilizing. Pipettes are often stuffed with non-absorbent cotton wool before being placed in sterilization containers. Glassware such as pipettes, conical flasks, beakers (covered with foil of aluminium) is sterilized for one hour in a hot air oven at 160°C. All other basic equipment used in cell culture lab, like automatic pipette tips and bottles (lids loosely attached) are sterilized by autoclaving at 121°C for 20 min.
For preparation of media, and rinsing glassware, a double distilled or reverse osmosis water supply is required. The pH of the double distilled water should be checked regularly, as this can vary in some instances. Variations in the quality of water used may account for variations in outcomes, so it is necessary to use water from one source. Water is sterilized for 20 minutes at 121 °C by autoclaving.
Media which cannot be autoclaved must be sterilized through a membrane filter of 0.22 μm pore size. These can be obtained in different designs to filter a wide range of volumes. They can be bought as sterile disposable filters, or they can be sterilized in appropriate filter holders by autoclaving.
Periodically, to increase the concentration of cells or to wash off a reagent, cell suspensions require centrifugation. For most purposes, a small bench-top centrifuge, preferably with proportionally controlled braking, is enough for tissue culture laboratory. Refrigeration is not necessary, although, set at room temperature, it can be used to prevent overheating of cell samples. At 80 to 100 g, cells sediment satisfactorily; higher g may cause damage and encourage pellet agglutination.