Tips for practising safe cells - Part 2

Part 1 of this article looks at ways to improve aseptic techniques and to preserve the integrity of cell cultures and sterile liquid transfers. Here in Part 2 we look at the lab environment and how it can impact on safety.

Working in hoods

Biological safety cabinets and laminar flow hoods provide containment and protection for the personnel, environment and cell cultures or products from biohazards and cross-contamination during routine procedures. Many different types and classification of safety cabinets and hoods exist to meet the specific needs of any cell culture laboratory.

Product selection will depend on the nature of the cell culture work and the biosafety level of the materials being used and processed. Horizontal laminar flow hoods should not be used for cell culture procedures.

• Always have the biosafety cabinets certified at the time of installation and re-certify if moved or repaired. It is also recommended to routinely test the quality of the airflow and filter integrity every 6 to 12 months.
• Biosafety cabinets may be equipped with germicidal UV lights for decontaminating work surfaces. However, the efficacy of UV lamps has been challenged. The UV light rays must directly strike a microorganism in order to destroy it. Over time, the UV output and germicidal capacity from the tube diminishes. Finally, there are safety concerns related to the exposure to UV light. UV exposure is damaging to the eyes and skin; therefore, the UV light should never be on while the cabinet is in use.
• Biosafety cabinets and hoods should be turned on 15 minutes prior to use each day. Alternatively, keep hoods running 24 hours a day during the work week. Work surfaces should be wiped down with 70% ethanol, or other suitable disinfectant, before and after each use and between cell lines.
• Wipe down bottles and flasks with 70% ethanol or other suitable disinfectant before being placed in the cabinet.
• Wear a clean lab coat when working in a hood. This coat should be for hood use only and not be worn anywhere else in the laboratory.
• Limit people access to area around the hood while working. This reduces levels of airborne contaminants, unnecessary distractions and talking.
• Avoid unnecessary talking while working in the hood. Talking generates microbial-laden aerosols that can then enter into the hood. Consider wearing a mask if talking is necessary or if you have a cold.
• Avoid moving materials in or out of the hood while work is in progress.
• Keep the hood work area clean and uncluttered. Do not use hoods as storage cabinets. Clutter makes it very difficult to clean the work surface properly and can disrupt the laminar flow around the work area.
• Do not use open flames, especially Bunsen burners, in laminar flow hoods. The heat plume from the flame will disrupt the moving curtain of filtered air provided by the hood and increase the risk of contamination. It is also a major safety issue. Serious hood explosions, fires and injuries have resulted from gas leaking from Bunsen burners or an open flame igniting alcohol used as a disinfectant.
• Doors in the culture area should be kept closed while the hood is in use. Opening a door can create a back draft and disrupts laminar flow in hoods. Consider replacing traditional doors with sliding doors to eliminate this problem, especially in heavy traffic areas.

Transporting cultures

• Minimise transport distances and avoid using common hallways to reduce contact with airborne contaminants. Ideally, incubators should be placed in close proximity to the culture area to restrict the movement of cells within the facility. Contamination resulting from transport is far more likely in unsealed vessels, such as dishes and microplates, than in flasks.
• Use flasks with vented caps whenever possible, especially for long-term cultures.
• Transport and incubate unsealed vessels, such as dishes and microplates, in plastic boxes or trays. 240 mm square culture dishes can be used as trays for 96-well plates and smaller dishes; 150 mm dishes can accommodate 35 mm dishes.

Culture area

• Ideally, cell and tissue culture should be conducted in a specifically designated room which has minimal traffic.
• Since microorganisms attach to dust particles, reducing the amount of dust and dirt in the culture area is always a priority. Do not open windows or use window fans that allow in outside air. If possible, cover incoming air vents with HEPA filters. Wash floors and benchtops frequently with an appropriate disinfectant. Sticky mats placed on the floor outside the entry to a culture room can also reduce the amount of dirt carried inside.
• Frequently clean water baths used for warming media or solutions. Better yet, avoid water baths entirely by using an incubator room or a bath filled with metal beads for warming media and solutions.
• Periodically empty and carefully clean incubators. Use cleaning agents that do not leave residues or corrode the metal interior or shelves. Never use metal cleaners that contain waxes.
• Use a pest management program to keep rodents and insects, such as flies, ants or cockroaches, under control. Insects can enter opened packages of sterile products. There have been reported instances of insects found inside incubators. Also, plants should always be kept out of the culture area. They attract insects and their soil is a rich source of contamination.
• When possible, avoid placing refrigerators in the culture area, especially near safety cabinets. Their external condenser cooling coils collect dust particles which then get carried into the room during the cooling cycle. Vacuum cleaning the coils several times a year will reduce the problem and allow them to run more efficiently.

All personnel should receive training and demonstrate proficiency in the laboratory practices for conducting and supporting sterile procedures and facility safeguards. Education and technical training programs need to be ongoing to ensure the quality of work in the long term.

Finally, aseptic technique must be supported by good laboratory design, selection of proper equipment and facility safeguards to protect laboratory workers and to reliably achieve successful sterile manipulation and processing of cell cultures.