Equipment and materials used in animal cell culture
What are the Basic equipments required for animal cell culture?
There are various equipments used in animal cell culture and the basic equipments required to carry out the animal cell culture are enlisted as follows:
Essential equipments
Beneficial equipments
Useful additional equipments
Incubator
Laminar flow hood
Low-temperature freezer
Microscope
Cell counter
Glassware washing machine
Sterilizer
Vacuum Pump
Colony counter
Washing up instrument
CO2 incubator
Closed-circuit machine
Sterilizing and drying oven
Preparation and quality control
Cell sizing
Centrifuge
Temperature recording
Time-lapse
Water purification
Bulk culture
Controlled-Rate cooler
Cell freezing
Pipette aids and automatic pipetting
Cinemicrography
Centrifugal elutriator
Fluorescence activated Cell sorter
List of Basic Equipments needed in animal cell culture lab:
Sterile Work Area/Cell culture hood (i.e., laminar-flow hood or biosafety cabinet)
Incubator (humid CO2 incubator recommended)
Water bath
Centrifuge
Refrigerator and freezer (–20°C)
Cell counter (e.g. Automated Cell Counter or hemocytometer)
Inverted microscope
Liquid nitrogen (N2) freezer or cryostorage container
Sterilizer (i.e., autoclave)
1. Sterile work area required for cell culture:
In order to maintain clean cell culture work, it is necessary to prepare a separate room or space if possible.
This room should be devoid of traffic, and if possible it should be equipped with an air flow cabinet that provides filtered air surrounding to the work surface.
A HEPA (High Efficiency Particle Air Filter) filtered air is appropriate but is not economical.
The laboratory must be especially designated for clean culture work and it should be strictly restricted to culture the primary animal tissue and micro-organisms in or near the cell culture laboratory.
The laboratory coats should be placed at the entry gate of the laboratory and should not be used outside the lab.
A laminar flow hood (i.e. biosafety cabinet) is supposed to the simplest and the most cost effective way to supply aseptic conditions.
While permitting the containment of infectious splashes or aerosols produced by many microbiological processes, the laminar flow hood provides an aseptic work area.
In order to meet the diversified research and clinical needs, Three kinds of laminar flow hoods, have been designated as Class I, II and III.
When used with proper microbiological techniques, Class I laminar flow hoods supplies essential levels of protection to laboratory workers and to the environment, but they do not protect cultures from contamination.
They are identical to chemical fume hoods in design and air flow characteristics.
For work that involves BSL-1, 2, and 3 materials, Class II laminar flow hoods are designed and they also allow an aseptic environment essential for cell culture experiments.
In order to handle exclusively harmful materials (e.g., primate-derived cultures, virally infected cultures, radioisotopes, carcinogenic or toxic reagents) a Class II biosafety cabinet should be employed.
Class III biosafety cabinets are gas-tight, and they supply the highest achievable level of protection to personnel and the environment.
A Class III biosafety cabinet is needed for work that involves known human pathogens and other BSL-4 materials.
Air-Flow Characteristics of Cell Culture Hoods:
The working environment is protected by the laminar flow hoods from dust and other airborne contaminants by retaining a constant, unidirectional flow of HEPA-filtered air over the work area.
The flow can be both horizontal, blowing parallel to the work surface, and it can be vertical, blowing onto the working surface from the top of the cabinet.
Clean Benches:
Vertical laminar flow or horizontal laminar flow “clean benches” are not biosafety cabinets.
These pieces of equipment discharge HEPA-filtered air from the back of the cabinet across the work surface toward the personnel, and the user might be exposed to potentially hazardous materials.
These devices only aids product protection.
2. Incubator:
An incubator will be needed in order to supply the suitable temperature environment for cell growth at 30-400 C.
Depending on the type of cells being cultured, the incubation temperature will vary.
An incubator that has been designated to permit CO2 to be supplied from a main supply or gas cylinder is needed in order to maintain an atmosphere of between 2-5% CO2 is maintained in the incubator.
In the medium, the concentration of CO2 is kept in the equilibrium with sodium bicarbonate.
In general, several cell lines can be retained in an atmosphere of 5% CO2: 95% air at 99% relative humidity.
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.
Humid CO2 incubators are relatively expensive, however it allows superior control of culture conditions.
They can be used to incubate cells that are cultured in petri-dishes or multiwell plates that needs a regulated atmosphere of high humidity and increased CO2 tension.
3. Refrigerators and freezer (-20 °C) for specimen storage:
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.
To store medium and buffers, a refrigerator or cold room is needed.
A freezer is required for keeping pre-aliquoted stocks of serum, nutrients and antibiotics.
Cryogenic Storage
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 20oC or -80oC freezers as their viability reduces when they are not stored at these temperatures.
Liquid nitrogen freezers permit storage in the vapor phase just above the liquid at temperature between -140oC and -180oC, or submerged in the liquid at a temperature below -196oC.
The possibility of leaky vials or ampules exploding during removal is highly reduced by use of vapor phase storage, however, the liquid phase systems generally have longer static holding times, and are thus, more cost-effective.
4. Microscopes:
In order to examine the cultures in flasks and dishes, a simple inverted microscope is needed.
The morphological changes in cultures should be recognized as they are the first indicators for the identification of deterioration of a culture.
Although, a microscope of very high quality will be needed for chromosome analysis or autoradiography work, a very simple light microscope with X100 magnification will suffice for routine cell counts in a hemocytometer.
5. Tissue culture ware:
A diverse tissue culture plasticware is found, the most general being specially treated polystyrene.
Even if all tissue culture plasticware 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.
This is especially useful in case if incubators fail.
6. Washing up and sterilizing facilities:
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 aluminum) is sterilized for one hour in a hot air oven at 160 ° C.
All other equipment, like automatic pipette tips and bottles (lids loosely attached) are sterilized by autoclaving at 121 °C for 20 min.
7. Water still or reverse osmosis apparatus:
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.
8. Filter sterilization
Media which can not 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.
9. Centrifuge
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.
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.
Other expanded equipments and additional supplies:
Other additional equipments and supplies needed in animal tissue culture lab are:
An aspirator is a form of ejector-jet pump, that creates vacuum by means of the Venturi effect.
Fluid (liquid or gaseous) passes through a tube in an aspirator that gets narrower and then expands in the cross-sectional area and thus volume.
The pressure of the fluid diminishes as the tube narrows.
The most popular type of aspirator is the cheap and simple water aspirator.
It is used in laboratories for chemistry and biology and consists of a tee fitting connected to a tap and has a hose barb on one side.
The water flow passes through the tee’s straight portion, which at the intersection where the hose barb is attached has a restriction.
To this barb, the vacuum hose should be attached.
Although historically popular for low-strength vacuums used in chemical bench work, they use a lot of water, and depending on what the vacuum is being used for, i.e. removal of solvents, by mixing these potentially dangerous solvents into the water stream, they can breach environmental protection laws such as RCRA, then rinsing them down a drain which often directly leads to the municipal sewer.
The intensity of the vacuum generated is restricted by the vapor pressure of the liquid (for water, 3.2 kPa or 0.46 psi or 32 mbar at 25o C or 77 F) if a liquid is being used as the working fluid.
This restriction does not exist if a gas is used.
The industrial steam ejector (also named the ‘steam jet ejector’, ‘steam aspirator’, or ‘steam jet aspirator’) uses steam as a working fluid.
2. pH meter:
PH meter is an electrical instrument for calculating the activity of hydrogen ions (acidity or alkalinity) in the solution.
A pH meter comprises necessarily of a voltmeter connected to a pH-responsive electrode and a reference (unvarying) electrode.
The pH-responsive electrode is normally glass, and a mercury-mercurous chloride (calomel) electrode is usually the reference, although sometimes a silver-silver chloride electrode is used.
The two electrodes act like a battery when they are submerged in a solution.
The electrical potential (charge) of the glass electrode is directly related to the hydrogen ion activity in the solution (59.2 millivolts per pH unit at 25o C [77 °F]) and the potential difference between the glass and reference electrodes is determined by the voltmeter.
3. Confocal microscope:
Confocal microscopy is a specialized type of standard fluorescence microscopy (also termed widefield fluorescence microscopy) that produces high-resolution images of material stained with fluorescent probes using specific optical components.
4. Flow cytometer:
Flow cytometry is a method for cell analysis that was first used in the 1950s to determine the volume of cells in a fluid stream that circulated quickly as they flowed in front of a viewing aperture.
Fluidics, optics and electronics are the three principal components of a flow cytometer.
The transport of the sample from the sample tube to the flow cell is the function of the fluidic system of the flow cytometer.
The sample is either sorted (in the case of cell sorters) or transported to waste after it has been through the flow cell (and past the laser).
The optical system components include excitation light source, lenses, and filters used to capture and move light around the instrument and the photocurrent-generating detection system.
The brains of the flow cytometer are the electronics.
Here the photocurrent from the detector is digitized and analyzed to be saved for subsequent analysis.
5. Cell culture vessels:
In order to shield cultures from the external environment while retaining the correct internal environment, culture vessels provide a contamination barrier.
The vessels have an effective and consistent cell attachment substrate for anchorage-dependent cells.
Simple access to cultures and optically transparent viewing surfaces are more features of vessels.
All cultural vessels were originally glass.
In comparison to plastic, glass drawbacks include heavy weight, cost, labor-intensive cleaning, and poor microscopic viewing.
Surface treatment techniques were developed for polystyrene in the 1960s, enabling plastic vessels to replace glass for most applications of cell culture.
a. Glass flasks:
In the 1920s, the first glass flasks were developed by Alexis Carrel.
The more conventional straight neck rectangular (also hexagonal) glass T-flasks were created by Harry Earle in the 1940s.
Today, with a number of growing areas, a variety of shapes, with several different neck designs, plastic flasks are available.
Description of glass flasks
Growth area(cm2)
Recommended working volume (ml)
Cell yield
T-25
25
5-10
2.5 X 106
T-75
75
15-25
7.5 X 106
T-150
150
30-50
15.0 X 106
T-175
175
35-60
17.5 X 106
T-225
225
45-75
22.5 X 106
b. Cell culture dishes:
The best economy and access to the surface of growth are provided by cell culture dishes.
Hence, they are regarded as the vessels of choice for cloning or other manipulations such as scraping that need the direct access to cell monolayer.
They should be used with incubators that regulate CO2 and humidity.
The majority of manufacturers provide dishes in four sizes: 35 mm, 60 mm, 100 mm and 150 mm.
These are nominal diameters and may not be the growth surface’s true diameter.
Cell culture dishes are accessible for growing anchorage-dependent cells with either specially treated surfaces, or untreated (native) surfaces for growing suspension cultures where attachment is not required.
Description of cell culture dishes
Growth well (cm2)
Working volume(ml)
Cell yield
96-well
0.32
0.1-0.2
0.32 X 105
48-well
1.00
0.3-0.6
0.8 X 105
24-well
1.88
0.5-1.2
1.9 X 105
12-well
3.83
1.0-2.4
3.8 X 105
6-well
9.40
2.0-3.0
9.5 X 105
c.Roller bottles:
To cultivate large numbers of anchorage-dependent cells, the roller bottle was created.
Today, they suppy a more cost-effective means of growing large volumes of cells using basically the same culture techniques as with flasks, but with substantially less labor.
In addition to the conventional smooth wall design, roller bottles are available with small ridges that roughly double the available surface area for developing cells without raising the bottle dimensions.
Description of roller bottles
Growth area (cm2)
Working volume(mL)
Cell yield
Small
490
100-150
4.9 X 105
Standard
850
170-250
8.5 X 105
Pharmaceutical
1750
340-500
17.5 X 105
d. Pipettes and pipettors:
The serological pipette is a relatively common laboratory instrument used for transferring milliliter volumes of liquid.
In order to calculate the volume of liquid being aspirated or dispensed, serological pipettes usually have gradations along their sides.
These instruments are most commonly used with a pipette dispenser, which enhances the liquid transfer through the development of a partial vacuum.
Depending on the amount of volume you want to pass, the same pipette dispenser can be used with a number of serological pipette sizes.
Serological pipettes are usually sterilizable and reusable, either plastic, sterile, and disposable or glass.
For the transfer of fluids, all serological pipettes involve the use of a pipette dispenser.
This primitive liquid transfer method is not recommended, as it may lead to liquid entering the oral cavity and some severe adverse side effects may be caused.
One type of dispenser, the pipette bulb supplies the least amount of accuracy and is usually used with glass serological pipettes in order to transfer non-specific volumes of liquid.
The pipette pump is often used for glass pipettes and enables the liquid volume to be controlled more precisely.
For dispensing repeated volumes of solution, pipette pumps are especially useful.
The most popular pipette dispenser type is the pipet-aid.
It consists of several primary components: the nose cone is where the pipette is attached and where the filter is also located, which protects the inside of the pipet-aid from fluid and retains sterility.
Two triggers can be found on the pipet-aid handle; the top trigger for aspirating liquids is depressed, the bottom for dispensing.
Pipet-aids are also fitted with settings that monitor the speed at which fluid is dispensed.
For example, the instrument can be fixed to dispense liquid using pressurized air, in a blow-out setting, and with no force, in a gravity setting.
Although there are cords in some pipet-aids, most are battery operated.
Some pipet-aids have a stand attached to the handle that allows the pipet-aid to rest on its side without removing the pipette.
