6.3: Cell Culture Equipment

The Biosafety Cabinet

The cell culture facility requires a place to aseptically manipulate cells. This is achieved using a biosafety cabinet (BSC; i.e. a cell culture hood). The BSC is an enclosed, ventilated workspace for safely working with biological materials that require a defined biosafety level. There are three main types of BSCs, with each providing different levels of protection to the user, the environment, and the biological product. Table \(\PageIndex{1}\) compares the three types of BSCs.

It is important not to confuse a BSC with a laminar flow hood. A laminar flow hood draws air in from the environment, passes it through a HEPA filter and then moves the cleaned air across the work surface from back to front, protecting the materials in the hood from contamination. However, this curtain of air is blown out the hood at the worker placed in front of the hood. Therefore, the laminar flow hood provides no protection to the worker using the hood. In contrast, the air flow inside of a BSC is designed to protect the materials inside the cabinet, in addition to the worker seated in front of it.

Class II Biosafety Cabinets

The most commonly used type of BSC in the cell culture laboratory is the Class II cabinet (Figure \(\PageIndex{1}\)). The interior of this class of BSC, including the work surface, is made of stainless steel and can be disassembled for sterilization through autoclaving. The front of the work surface is a large grill that collects air from the environment. A grill located at the back of the work surface collects air from the interior of the cabinet. At the front of the class II BSC is a glass sash that can be raised and lowered. When working in the cabinet, the sash is partially raised to allow for manipulation of the materials inside the hood but limit the entrance of room air with contaminants. A HEPA filter, exhaust manifold and a blower motor are inside the top of the cabinet. Inside the cabinet are visible and ultraviolet (UV) light bulbs. The UV light can be turned on for short periods of time to help sterilize the work surface when it is not in use. A control panel at the front of the cabinet turns on the lights and the blower motor for airflow. It also provides key information about the status of the cabinet.

The Class II BSC creates a unique pattern of airflow within the cabinet that protects both the material inside of the cabinet and the worker using it (Figure \(\PageIndex{2}\)). Contaminated room air is drawn in through the open sash into the front grill. The air flows beneath the work surface and up the back of the cabinet. It enters a HEPA filter at the top of the cabinet where the air is filtered. A portion of the filtered air is exhausted out to the environment and the remainder is blown down over the work surface from the front of the cabinet to the back. This air then flows through the back grill to mix with the room air flowing up to the HEPA filter. The air pattern over the work surface creates a “curtain” to protect the worker and efficiently moves potentially contaminated air out of the work area.

There four types of Class II biosafety cabinets are classified as A1, A2, B1 and B2, with each type having unique specifications (Table \(\PageIndex{2}\)). Most cell culture facilities use a either a type A1 or A2 biosafety cabinet because they are cheaper to operate since they do not require a hard connection to the exhaust system of the room’s building.

The Culture Incubator

Cultured cells are maintained and grown within an incubator that provides the cells with a sterile, humidified, and warm environment (Figure \(\PageIndex{3}\)). The incubator is like a biosafety cabinet in that it relies upon a flow of HEPA-filtered air to prevent contamination. Air inside the incubator is filtered through a HEPA filter, warmed by the incubator and then moved through the interior of the incubator before being pulled back through the HEPA filter for filtration and recirculation. The majority of the air that moves through the incubator is taken from the environment and is a mixture of oxygen (~20%), carbon dioxide/CO2 (~0.04%), and nitrogen (~78%). The CO2 levels found in this air are inadequate for cell culture. Therefore, additional CO2 is injected into the incubator from a cylinder of compressed CO2 gas. CO2 levels for most cell cultures will range between 5% and 10%, depending on the needs of the cultured cell. Sensors inside of the incubator constantly measure the CO2 concentration and adjust the levels by injecting additional compressed CO2. Humidity levels inside of the incubator can be monitored and adjusted by the incubator. However, most incubators provide humidity to the air using a pan of sterilized water placed at the bottom of the incubator.

The air of the incubator is typically warmed to 37°C as most mammalian cells grow well at this temperature. To keep the temperature inside of an incubator stable, the incubator is “jacketed”, meaning that there is an inner chamber (where the cells are incubated) surrounded by an outer chamber (the "jacket"). The jacket prevents significant drops in temperature when the door is opened and closed. There are two kinds of jackets: water and air. The inner chamber of the air-jacketed incubator (also known as a direct heat incubator) contains air, warmed to 37°C. This type of incubator is light and cost-effective in comparison to the water-jacketed incubator. In a water-jacketed incubator, the jacket is filled with sterile water and heated to 37°C. This jacket keeps the temperature of the incubator more stable versus an air-jacketed incubator, as the water jacket will return the inner chamber's temperature to its set point much faster (i.e. within 10 minutes for water-jacketed vs. 1 hour for air-jacketed). However, the water jacket is an excellent source of contamination by micro-organisms like fungus. In addition, a water-jacketed incubator is extremely heavy and must be drained in order for the incubator to be moved.

Cell counting systems

Cell counting systems are used to determine the number of cells within a culture. The most common and inexpensive type of counting system is the hemacytometer. Originally designed and used for counting red blood cells, the hemocytometer is used for the manual counting of numerous types of cells. Refer to Lab Technique: Cell Culture for a protocol on the use of a hemocytometer. The hemocytometer consists of a thick glass microscope slide with a rectangular indentation that creates a counting chamber of 10^-4 mL (Figure \(\PageIndex{4}\)). This chamber is engraved with a laser-etched grid of perpendicular lines. Cells are added to the counting chamber and visualized under the microscope. Those falling over certain parts of the counting grid are counted. With this cell count and the volume of the counting grid (i.e. 10^-4 mL), the number of cells per mL can be determined. This concentration can be used to determine the total number of cells within the culture.

Automated cell counters use advanced technology to accurately and efficiently count and analyze cells isolated from a culture. Most counting systems are image-based, using cameras and microscopes to capture high-resolution images of the sample. Software processes the images to identify and count cells based on their size, shape, or staining. Other systems use measures changes in electrical resistance when the cells pass through a small aperture or channel cells through a micro-fluidic device where they are imaged, electrically measured, or optically analyzed. Automated counting systems eliminate the manual labor and potential for human error associated with traditional methods like hemacytometry. They are significantly more accurate and faster and easier to use. However, they are markedly more expensive that hemacytometers.

Inverted microscope

An inverted microscope is a type of light microscope where the optical arrangement is reversed compared to a conventional (upright) microscope. The light source, with its condenser, is located above the stage, while the objectives are positioned below the stage (Figure \(\PageIndex{5}\)). This design is ideal for observing living cells in their culture dishes, culture flasks, or multi-well plates.

Cryopreservation Systems

Cell culture facilities will often remove cells from culture in order to store them over the long-term. Storing cultures allows the facility to remove cells from culture when they are not needed and re-introduce them when they are. This saves time, materials, and money. Long-term storage of cell cultures is done at extremely low temperatures and is referred to a cryopreservation. The simplest system for cryopreservation is a liquid nitrogen tank or Dewar. The liquid nitrogen inside of the tank (approximately -320°F or -196°C) provides the optimal temperature for long-term storage of cells. Modern tanks can hold hundreds of cryopreservation samples. The samples are placed into boxes and the boxes are stacked onto racks that are lowered into the tank. The bottom of the tank holds several inches of liquid nitrogen that produces a vapor layer that is cold enough to keep the samples frozen. The liquid nitrogen inside of the tank is replenished on a regular basis.

Additional Culture Equipment

1. Water bath: necessary to warm reagents used in cell culture
2. Centrifuge: for the collection of culture cells
3. Refrigerator (4°C) and freezer (–20°C): for storage of culture reagents
4. Sterilizing equipment (i.e., autoclave)
5. Aspiration pump (peristaltic or vacuum)