Cell- and tissue culture

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The doors into the cell labs at IBM should always be closed!

Procedures for using the cell/tissue culture laboratories

Every cell/tissue culture laboratory has a room supervisor and a user supervisor. The room supervisor has overall responsibility for the use of the room as well as for HSE issues. The user supervisor has responsibility for training new users and approving cell cultures and other biological material that is used. The user supervisor is also responsible for following up HSE guidelines and ensuring that other guidelines drawn up by the room supervisor are followed. The user supervisor is also responsible for setting up a cleaning rota for each cell lab.

Only cell cultures which have been approved by the user supervisor may be cultivated, and under no circumstances must bacteria be brought into the cell lab. Work on approved viruses requires separate work procedures. Consult the user supervisor.

Changing clothes

The sluice room should be used for changing clothes.

Everyone must use lab coats which are designed for use in the cell lab. It is not permitted to use ordinary lab coats inside the cell lab. Used lab coats are delivered for cleaning every second week by the person appointed in the cleaning rota. You must also wear special shoes inside the cell lab. Should shoes be left lying about, the room will not be cleaned. Use the shoe rack.

LAF benches (laminar air flow)

Fans must be switched on for 10-15 minutes before benches can be used. Remember to close the UV light hatch.

The bench must be cleaned with 70% EtOH both before and after use. Keep as few objects as possible inside the bench when working.

Any spills of medium must first be removed using water and then cleaned using 70% EtOH.

All objects must be removed from the bench after use. Switch on the UV light.

See separate instructions for LAF benches.

Every six months (June + December)

Clean/sterilise the entire LAF bench.

• Take out the bench plates and clean them with mild soapy water. Rinse thoroughly with Milli-RO water before autoclaving them.
• Clean the inside of the bench with mild soapy water, then disinfect with 70% EtOH.
• Clean both sides of the front window with soapy water and 70% EtOH.
• Clean all exterior surfaces with soapy water and 70% EtOH.

If necessary, the whole LAF bench can be wrapped up and disinfected

Microwave oven

The microwave oven can be used to sterilise plastic/disposable equipment. Always add a little water (in a container) to avoid fires! Sterilise at full power for two minutes.

The microwave oven door must be left open after use in order to air it and thereby avoid rust.

Spills inside the microwave oven must be cleaned with soap and water. Then add 70% EtOH to a paper towel and wipe clean.

Never put metal or aluminium foil in the microwave oven!

Incubator cabinets

Spills must be cleaned up immediately! Wash first with water, then with alcohol. It is forbidden to defrost media etc. in incubators.

Everyone is responsible for refilling water in the containers (1 litre autoclaved Milli-RO water containing antibacterial agent). The water pans must be kept clean.

The incubator cabinets must be cleaned and sterilised every six months.

Every six months (June + December)

• Clean/sterilise the inside of the cabinets.
• Clean the whole interior with mild soapy water. Rinse thoroughly with Milli-RO water and wipe with 70% EtOH.
• Autoclave shelves, walls, ceiling panels and screws. If the cabinet has a self-sterilising function, this can be used instead of autoclaving.
• After sterilisation, the CO₂ level in the cabinet must be calibrated.

Every twelve months:

• Change the HEPA filter, the rubber tubing and the filter on the outlet for measuring gas concentrations.
• Change the water in the water jacket (~ 43,5 litres). Use chlorine-free distilled water.

Drawers and shelves

Cupboards and drawers are divided between the labs. The cupboard doors must be closed in order to avoid dust. Do not store objects on the floor or on top of the cupboards. Such objects gather dust and the floor will not be cleaned.

Microscopes

Remember to switch off the microscope after use. Replace the hood. In order to conserve bulb life, turn down the light intensity if the microscope is to be left on for a while. Training in the use of the microscope is available from the user supervisor. Eye make-up can easily dirty the ocular.

Glass equipment

All glass equipment which is to be used in the cell lab must be sterilised before use, either in the heating cabinet (2 hours, 180º C) or by dry autoclaving.

After use, the equipment must be rinsed in the sluice room and sent for washing.

Mycoplasma testing

All cell cultures that are thawed should be tested for mycoplasma. Cells which are cultivated over long periods of time must be tested every six months.

Mycoplasma are the smallest independent self-replicating prokaryote organisms (0.2-2 µm in diameter) and they infect many different cell lines. They lack cell walls, which means that antibiotics which target cell wall formation (e.g. penicillin) are ineffective against mycoplasma. A mycoplasma infection can be difficult to observe since it does not always cause turbidity in the medium or a change in pH, which is usual in the case of bacterial or fungal infections.

A mycoplasma infection can cause long-term effects such as:

• Reduced growth rate
• Morphological changes
• Chromosomal abnormalities
• Changes in gene expression
• Changes in amino acid and nucleic acid metabolism
• Changes in virus and antibody production

In order to prevent the spread of mycoplasma, all cell cultures should be checked regularly.

Waste

When working in the LAF bench, all leftover media/cells/liquids must be poured into a container which contains a little water. When you have finished your work, all the liquid must be poured into a glass bottle which is kept in the sluice room. Wash the container thoroughly and sterilise it (with a little water in the bottom) in the microwave oven before putting it away. The glass bottle containing cell remains must be autoclaved when full (at least every two weeks). After autoclaving, the contents can be poured out in the sink in the sluice room.

If you use a vacuum pump to remove used media, you must add a little liquid disinfectant (such as Klorin, a chlorinating disinfectant) to the bottle before use. If the liquid can be autoclaved without producing gas, do that as described above. If the liquid cant be autoclaved because it creates gas, it can be transferred into a 10 L can (the same as is used for hazardous waste), which contains disinfectant (virkon). When this is full it should be placed in a yellow box, and delivered as problematic waste.

Throw away all disposable equipment (such as pipette tips, plastic Pasteur pipettes, etc) in the box for special waste (located under the LAF bench). When the box is full, it must be closed up and placed in the sluice room. The person appointed by the cleaning rota takes it down to the waste room in the basement. Glass Pasteur pipettes can also be disposed of in this box.

Paper can be thrown in a regular bin.

Work with approved viruses requires separate work procedures. Consult the user supervisor.

Contaminated cell cultures must be poured into a new glass bottle and autoclaved immediately! Pour a little soap into the cell bottle, replace the cap and place it in the box for special waste.

If you discover that your cells or another person's cells are contaminated, notify the user supervisor so that he/she can take the necessary course of action!

Cleaning rota

Everyone who uses the cell lab must take their share of lab duties. There is a cleaning rota for every cell lab, which rotates every 2 weeks.

Main rule: everyone must clean up after themselves at all times!

Every other week:

• Clean the interior of the LAF benches with mild soapy water. Also clean under the bench plates. Wipe with 70% EtOH.
• Clean the following with soapy water:
  • Benches
  • The exterior of incubator cabinets
  • The tops of refrigerators/freezers
  • The tops of cupboards

Then wipe with 70% EtOH.

The floor is cleaned by the cleaning personnel.

• Ensure that there is enough 70% cleaning alcohol
• Send all lab coats for laundering
• Change the water in water baths
• Autoclave media/cell waste
• Label boxes containing special waste with “INCINERATION” ("FORBRENNING" in Norwegian) or “HEAT DISINFECTION” and place them in the waste room in Level 1.

This file shall be signed and delivered to the user supervisor.

The Safety Laboratory (BSL3)

Person responsible: Bård Sværi (Work tel.: 55 58 68 32 Mobile: 45 25 27 30)

To gain access to the BSL3 laboratory a thorough training in the procedures is required together with trained personnel, as well as approval by Bård Sværi.

Absolute forbidden

1. To enter the safety laboratory without gloves and a change of shoes or shoe protection.
2. To eat (including chewing gum), drink or smoke.
3. To work with hazardous materials outside the LAF benches. Culture flasks and hazardous material containers must only be opened inside LAF benches.
4. In order to reduce the risk of accidents, avoid working with glass equipment and sharp objects. Use plastic materials instead.

Description of the laboratory

The safety laboratory is located on level 6 in the rear/south side of the building. The laboratory in BBB is built in accordance with the current regulations for “Bio-safety Level 3” (BSL-3) which have been drawn up by the National Institute of Health (NIH) in the USA and the Advisory Committee on Dangerous Pathogens (ACDP) in the UK. It has been built in order to permit work to be carried out on highly potent infectious matter.

The guidelines for the practical work are based on many years' experience from Karolinska Instituttet and the Dana-Farber Cancer Institute.

Before you begin working in the safety laboratory, you must read the work regulations carefully and have completed practical training provided by an authorised person. Your safety, and the safety of those who work after you, depends on everyone complying with the safety regulations.

Technical conditions

The laboratory consists of two areas: an inner laboratory area (= the safe area) and an outer area, which is the clean part of the airlock. The airlock thereby serves as the transition area between the clean area and the “safe area”. The airlock doors are equipped with an electronic locking device to ensure that both doors cannot be opened simultaneously.

There is under pressure inside the safe area of approx. 425m³ air per hour in order to prevent air from the safe area escaping to the clean area outside. This under pressure is maintained through a ventilation system. In addition, the windows in the safety laboratory and the instrument room are sealed and cannot be opened. The windows are made of safety glass in order to prevent break-ins.

There are three work benches inside the BSL-3 laboratory, each with their own Laminar Airflow bench (Class 2 LAF bench), where open work with infectious material is carried out. Each LAF bench has two HEPA filters: one which filters the air circulating inside the LAF bench and one which filters the air from the room and exhaust air expelled from the LAF bench. Air from the other parts of the BSL-3 laboratory (the instrument room, the autoclave room and the airlock) are filtered through a HEPA filter in a separate system before being expelled from the roof. The exhaust air from the BSL-3 laboratory is thus divided between two separate systems which are not connected to exhaust air from other parts of the building. The filter systems are doubled so that filters can be changed without interrupting work. For safety reasons, there are also double sets of fans, which are connected to the building’s emergency power system. An acoustic (sound) alarm is connected to the BSL-3 laboratory and to the technical monitoring system. This alarm is triggered if incorrect pressure levels are registered in the BSL-3 area.

The person responsible for BSL-3 must be notified when this alarm is triggered.

BBB Operations, Tel.: 55 58 62 50

Night-time: Bård Sværi, Tel.: 45 25 27 30

The laboratory is equipped with a pass-through autoclave for autoclaving “infected” material before it is disposed of or washed. Water separated out from the autoclave is heated in a spiral system before being washed down the drain. Water from the sink drains directly into the communal drain. If necessary, this water can be collected in a special tank for boiling.

Fault or accident

All accidents must be reported to Bård Sværi.

• The ventilation in the BSL-3 creates underpressure. An alarm will sound if a fault occurs in the ventilation system. Open bottles or test tubes containing infectious material must be closed and placed in an incubator before you leave the laboratory.
• If a small fire starts, extinguish it using a CO2-extinguisher.
• In the event of a larger fire, leave the laboratory immediately after you have closed open flasks and test tubes and checked that the CO2 incubator and freezer are shut.
• If infectious material is spilled inside or outside the LAF bench, dry it with paper towels soaked in Jodosan and dispose of them in the autoclave sack in the stainless steel container in the LAF bench. Pour plenty of Jodosan over the area and leave it to work for 10 minutes. Finally, clear up the spill and throw the paper in the same container.
• In the event of needle injuries or cuts, or direct spills on skin, rinse immediately with running water and wash with Jodosan. In the event of bleeding, allow the blood to run for a while under running water.

Rules for service personnel/fire-fighters in BSL-3

• Before entering the BLS-3 area, protective footwear and gloves must be put on in the airlock.
• In the event of fire, the CO₂ incubators or the -80 °C freezer must not be opened.

Procedures for working in the safety laboratory

Entrance to the safety laboratory

1. Enter the first part of the airlock and close the door.
2. Remove your lab coat and shoes (if relevant, put on plastic shoe protection).
3. Put on gloves and, if relevant, a face mask.
4. Open the door to the “infected” side of the airlock (the outer door is now electronically closed).
5. Enter the part of the floor marked yellow, which is the “clean” area, and put on clogs which are designated for use inside the BSL-3 area only.
6. Shut the door. You are now inside the BSL-3 area where autoclaving is carried out.
7. Put on the yellow lab coat and pull the gloves up over the sleeves so that they are sealed.
8. Enter the door to the BSL-3 safety laboratory where work with extremely hazardous material is conducted.

Working inside the BSL-3 laboratory

1. Switch on the LAF bench. The bench must be left on for 15 minutes before you can start working (see the instructions for LAF benches).
2. Cover the work surface with absorbent paper. (NOTE: The ventilation holes in the bench plate must not be covered. Place an autoclave sack in the metal container in the LAF bench for collecting disposable materials for autoclaving. Keep a small beaker or similar of Jodosan in the LAF bench for rinsing pipettes, sharps, test tubes, flasks etc. before placing them in the autoclave sack in the metal container. The Jodosan beaker must be poured into the flask marked ”waste”, together with sharps etc. once the work is finished. Screw the cap onto the flask and, when it is half full, place it in the autoclave sack in the metal container. Large amounts of medium, PBS etc., must be collected in yellow buckets containing approx. 100 ml of liquid disinfectant. When the bucket is three-quarters full, it must be placed in a metal bucket for autoclaving.
3. Put on arm protectors, extra plastic gloves and a plastic apron before starting work at a LAF bench.
4. Double sets of gloves are only used when working with extremely hazardous material.
5. All material removed from the LAF bench on completion of work must be sprayed with 70% ethanol.

When work in the LAF bench is completed

1. If the paper covering has been exposed to spills or used for several days, it must be placed together with all disposable materials in the metal container.
2. Wash the LAF bench with 70% ethanol.
3. Remove outer gloves and place them in the metal container. Plastic aprons and arm protectors can be reused. These can be hung on hooks for later use. They can be discarded in the cardboard box on the floor when they are no longer to be used.
4. Put the lid on the metal container if it is not full. When the autoclave sack is full, it must be folded and the lid must be put on the metal container. The container can then be placed under the incubator cabinet, ready for autoclaving. Only autoclave sacks must be sent for autoclaving.
5. Switch off the LAF bench in accordance with the instructions.

NB! It is not permitted to work outside the LAF bench while wearing double sets of gloves which have been used to work inside the LAF bench. Spray the inner pair of gloves with 70% ethanol when you move to another part of the room; you can continue wearing the arm protectors and plastic apron.

When you leave the BSL-3 laboratory

1. Remove your lab coat and place your gloves in the cardboard box in the autoclave room.
2. Open the door to the clean area (outer airlock). The door handle is designated as a clean area and it is forbidden to touch it with “infected” gloves.
3. Take off your clogs and proceed to the clean side (inside the yellow stripe) and shut the door.
4. Wash your hands before leaving the outer airlock.

Transport from the BSL-3 area

• If viruses or infected cells are to be removed from the laboratory (e.g. for transport to other laboratories), all flasks and test tubes must be screwed tight and the caps must be sealed with parafilm. Flasks must be placed in a sealed plastic sack or other container (e.g. a styrofoam box).
• Spray the outside of the sack/container with 70% ethanol before it leaves the BSL-3 area.
• Samples from p24 antigen or reverse transcriptase tests shall be inactivated using Triton X-100 before being removed from the room. All test tubes/plates that leave the BSL-3 area must be sprayed with 70% ethanol.
• Instruments which are to be sent for repair must be sprayed with 70% ethanol and, if possible, placed in a clean plastic sack.

Waste handling BSL3

Non-infectious material

Paper, plastic, cartons and packaging (e.g. packaging for disposable pipettes, bottles and microplates) which have not been in contact with infectious material are collected in an autoclavable plastic sack in cartons which are placed beside each LAF bench. When full, the autoclave sack is autoclaved and thrown away.

Yellow lab coats are collected in an autoclave-able plastic sack and are autoclaved before being sent for laundering.

Infectious material

All material which has been in contact with infectious agents must be placed in the stainless steel container (in the LAF bench) for autoclaving. This applies to plastic material, test tubes that have contained blood, absorbent bench paper and the extra pair of plastic gloves. Pipettes, sharps, test tubes, flasks etc. must be rinsed with Jodosan or Virkon before being placed in the container. Small amounts of liquid must be poured into empty medium flasks containing approx. 100 ml 5% SDS or Virkon. Replace the flasks when half full (empty flasks are kept in the inner airlock), screw on the cap and place in the autoclave sack in the metal container. Large amounts of liquid must be poured into the yellow bucket which contains approx. 100 ml 5% SDS or Virkon. When the bucket is three-quarters full, it must be placed in a metal bucket for autoclaving.

LAF benches in cell laboratories

Main types of LAF benches:

1. Sterile benches

A. Horizontal

B. Vertikal - Due to air flow

2. Safety benches

Class I	Operator protection, turbulent air, risk of cross contamination of the product
Class II	Product and operator protection, laminar air flow, recirculation of air
Class III	Operator protection, glove box

Class II safety benches

The Department of Biomedicine has class II safety benches, Maxi Safe 2010 supplied by Medinor.

The P3 laboratory contains both safety benches with two filters used for "ordinary" cell culture and benches with three filters that are specially designed for working with particularly hazardous micro-organisms. This extra HEPA filter is located directly below the work surface, while the other two filters are located on top. The bench’s innermost intake provides a uniform air flow.

The HEPA filter removes 99.9997% of particles of 0.3 µ in diameter.

Filters must be changed by a professional. Filters last 7-8 years but should be tested annually by a professional.

Air is drawn in at the front of the bench and down through the first HEPA filter. The air passes through the bench and down through another HEPA filter in the roof of the bench panel. The air is blown from top to bottom in a laminar air flow, which requires the same gradient on the back panel as at the front; the air intake speed is 0.4 m/s. The air flows down to the innermost corner in the bench and to the front. Here, the air (from both outside and inside the bench) is drawn down and passes through the first filter again. 25% of the air is exhaust air and 75% is recycled.

The bench has a negative pressure chamber (under pressure) which prevents leakage to the surroundings.

Cleaning

The bench must be cleaned regularly (once a week) using mild soapy water (washing-up liquid). Use clean room wipes (Aet, Puls) to avoid extra particles. The bench panels can easily be lifted up for access below.

The bench must then be disinfected with 70% alcohol. Bench panels can be autoclaved.

NB! The HEPA filter in the roof and the HEPA filter under the table must not become moist.

The HEPA filter at the bottom can tolerate some moisture but be careful with spills.

UV-light

UV lights are positioned in the LAF bench close to the surface. They emit UVC rays with a wavelength of 254 nm and only 15-20 minutes exposure is required. The glass in the front window stops UV rays and must be completely closed before the light is switched on.

Work procedures

1. The bench must be switched on for at least ten minutes before starting work.
2. Place everything you need for carrying out sterile work in the bench on start-up, however, try to keep the amount of objects to a minimum (you must assess the number of objects in relation to the amount of handling in and out of the bench).
3. Hand and arm movements must be slow.
4. Your hands must be placed between the air flow and the sterile object, and arms must not cover air holes (perforations).
5. The front window must be in the working position during work (otherwise the alarm is triggered). Never lift the front window manually!
6. The air nearest the opening is non-laminar (turbulence) and work must therefore be carried out at least 15 cm inside the bench (inside the perforations).
7. The work must always be carried out with the bench set at full speed. Beware of turbulence behind larger objects inside the bench.
8. Avoid walking back and forth behind people working at LAF benches, and keep windows shut, since open windows create extra turbulence.

NB! Turbulence is formed behind everything which blocks the air flow. Avoid placing sterile samples in turbulence zones. Place objects at the sides in the exhaust duct and remember to flip down the UV light switch.

Note! Do not use burners! If burners must be used, use safety burners.

Instructions

1. Set the front window to the work position (up) (10) (hold the button in)
2. Switch on the light (4)
3. Switch on the fan and set it to high (1), place your equipment (NB! sterile objects) inside the bench and leave the fan running for 15 minutes before you start sterile work.
4. Once sterile work is finished, remove everything from the bench. Clear up any spills and clean the bench with 70% alcohol. Leave the fan on for 10 minutes after cleaning.
5. Thereafter, switch off the fan (1), switch off the light (4), open the UV light hatch and lower the front window completely (11) (hold the button in for a while)
6. Switch on the UV light (3). The UV light is switched off automatically after 20 min. (using an adjustable timer).

The HEPA filter in the top of the bench does not tolerate moisture, knocks or heat. Be careful!

Avoid spills in the HEPA filter under the bench.