HANOI UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY

CHAPTER 1

Lecturer: Dr. Nguyen Huu Duc

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Contents
1.5. Preparation area

1.4. Incubation
1.3. Layout of aseptic room or suite

1.2. Construction and services

1.1. Planning
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The major requirement that distinguishes tissue culture from most other laboratory techniques is the need to maintain asepsis. Although it is usually not economically viable to create large sterile areas The laminar-flow hoods has greatly simplified the problem and allows the utilization of unspecialized laboratory accommodation, provided that the location is suitable 3

Several considerations need to be taken into account in planning new accommodation When a new building is contemplated, there is more scope for integrated and innovative design, and facilities may be positioned for ergonomic and energy-saving reasons. The following items should be considered:
Quarantine Access Renovations Accommodation

Ventilation
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1.1.1. Ventilation
Pressure balance
Ideally, a tissue culture laboratory should be at positive pressure relative to surrounding work areas, to avoid any influx of contaminated air from outside

Laminar flow hoods

It is preferable to duct laminar-flow hoods to the exterior to improve air circulation and remove excess heat (300500 W per hood) from the room. This also facilitates decontamination with formaldehyde Venting hoods to the outside will probably provide most of the air extraction required for the room, and it remains only to ensure that the incoming air, from a central plant or an air conditioner

Laminar-flow hoods

Laminar-flow hoods are better left to run continuously, but if they are to be switched off when not in use, then an alternative air extract must be provided and balanced with the extract via the hoods

Staff numbers
These considerations determine how many laminar-flow hoods will be required (based on whether people can share hoods or whether they will require a hood for most of the day) and whether a large area will be needed to handle bioreactors, animal tissue dissections, or large numbers of cultures. As a rough guide, 12 laminar-flow hoods in a communal facility can accommodate 50 people with different

Space
The largest area should be given to the culture operation, which has to accommodate laminar-flow hoods, cell counters, centrifuges, incubators, microscopes, and some stocks of reagents, media, glassware, and plastics. The second largest is for washup, preparation, and sterilization, third is storage, and fourth is incubation. A reasonable estimate is 4:2:1:1, in the order just presented

Hoods

Aseptic area
Ensure tissue culture is reasonably accessible to, but not contiguous with, the animal facility Windows can be a disadvantage in a tissue culture laboratory, leading to heat gain, ultraviolet (UV) denaturation of the medium, and the incursion of microorganisms if they are not properly sealed

The space between hoods should be approximately 500 mm (2 ft), to allow access for maintenance and to minimize interference in airflow between hoods
This space is best filled with a removable cart or trolley, which allows space for bottles, flasks, reagents, and a notebook

What type of incubation will be required in terms of size, temperature, gas phase, and proximity to the work space?

Will regular, nongassed incubators or a hot room suffice, or are CO2 and a humid atmosphere required?

Generally, large numbers of flasks or largevolume flasks that are sealed are best incubated in a hot room, whereas open plates and dishes will require a humid CO2 incubator

Preparation area
Facilities for washing up and for sterilization should be located close to the aseptic area that they service and on an outside wall to allow for the possibility of heat extraction from ovens and steam vents from autoclaves Give your washup, sterilization, and preparation staff a reasonable visual outlook; they usually perform fairly repetitive duties 10

Servicing aseptic areas

Storage
What is the scale of the work contemplated and how much storage space will this require for disposable plastics?
What proportion of the work will be cell line work, with its requirement for storage in liquid nitrogen? 11

Will an elevator be required, or will a ramp suffice?

If a ramp will do, what will be the gradient and the maximum load that you can expect to be carried up that gradient without mechanical help?

If a conversion of existing facilities is contemplated, then there will be significant structural limitations;

choose the location carefully, to avoid space

constraints and awkward

projections into the

room that will limit

flexibility
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Make sure that

doorways are both wide enoughand high enough and that ceilings have sufficient clearance to allow the installation of equipment such as laminar-flow hoods (which may need additional space for ductwork), incubators, and autoclaves doorways and spacing between equipment provide access for maintenance

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What quarantine facilities will be required

Newly introduced cell lines and biopsies need to be screened for mycoplasma before being handled in the same room as general stocks, and some human and primate biopsies and cell lines may carry a biohazard risk that requires containment
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Small Tissue Culture Laboratory

Medium-Sized Tissue Culture Laboratory

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Tissue Culture Lab with Adjacent Prep Room

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These questions will enable you to decide what size of facility you require and what type of accommodation - one or two small rooms or a suite of rooms incorporating washup, sterilization, one or more aseptic areas, an incubation room A separate facility gives better contamination protection, allows tissue culture stocks to be kept separate from regular laboratory reagents and glassware
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The rooms should be supplied with filtered air; adjacent rooms, which lead to the aseptic area, should be regarded as buffer zones and should also receive filtered air but at positive pressure

Furniture should fit tightly to the floor or be suspended from the bench, with a space left underneath for cleaning. Cover the floor with a vinyl or other dustproof finish

If possible it is preferable for the tissue culture lab to be separated from the preparation, washup, and sterilization areas, while still remaining adjacent

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Combustible gas

Vacuum

Power

Compressed air

Carbon dioxide 19

Power is always underestimated, in terms of both the number of outlets and the amperage per outlet. Assess carefully the equipment that will be required

Electricity is cleaner and generally easier to manage from a safety standpoint, but gas may be cheaper and more reliable

Gas is more difficult to judge, as it requires some knowledge of the local provision of power

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Carbon dioxide should be piped into the facility The installation will pay for itself eventually in the cost of cylinders of mixed gases for gassing cultures, and it provides a better supply, which can be protected, for gassing incubators
Compressed air is generally no longer required. It is used to expel cotton plugs from glass pipettes before washing and may be required for some types of glassware washing machine

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A vacuum line can be very useful for evacuating culture flasks, but several precautions are needed to run the line successfully

A collection vessel must be present with an additional trap flask, with a hydrophobic filter between the flasks, in order to prevent fluid, vapor, or some contaminant from entering the vacuum line and pump

Vacuum Line Drawing

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Six main functions need to be accommodated in the laboratory: sterile handling, incubation, preparation, washup, sterilization, and storage If a single room is used, create a sterility gradient; the clean area for sterile handling should be located at one end of the room, farthest from the door, and washup and sterilization facilities should be placed at the other end, with preparation, storage, and incubation in between
The preparation area should be adjacent to the washup and sterilization areas, and storage and incubators should be readily accessible to the sterile working area
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Sterile work should be located in a quiet part of the tissue culture laboratory and should be restricted to tissue culture

Cubicle

Use a separate room or cubicle if laminar-flow hoods are not available

separate room

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The work area should be a plastic laminate-topped bench, preferably plain white or neutral gray, to facilitate the observation of cultures, dissection

The bench should be either freestanding (away from the wall) or sealed to the wall with a plastic sealing strip or mastic Nothing should be stored on the bench and any shelving above should be used only in conjunction with sterile work
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Laminar-flow hoods affords greater control of sterility at a lower cost than providing a separate sterile room. Because only the operators arms enter the sterile area, with sterile air blown onto the work surface
Individual freestanding hoods are preferable (they separate operators and can be moved around), but laminar-flow wall or ceiling units in batteries can be used 26

Select hoods that suite your accommodation freestanding or bench top - and allow plenty of legroom underneath with space for pumps, aspirators, and so forth Laminar-flow hoods should have a lateral separation of at least 500 mm

Chairs should be a suitable height, with adjustable seat height and back angle, and able to be drawn up close enough to the front edge of the hood to allow comfortable working well within it A small cart, trolley, or folding flap (300500 mm minimum) should be provided beside each hood for materials which may be required but are not in immediate use

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Should use a separate room as a quarantine and/or containment room This is a separate aseptic room with its own laminar-flow hood (Class II microbiological safety cabinet), incubators, freezer, refrigerator, centrifuge, supplies, disposal and must be separated by a door or air lock from the rest of the suite

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Newly imported cell lines or biopsies can be handled here until they are shown to be free of contamination, particularly mycoplasma and proscribed pathogens such as HIV or hepatitis B

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1.3.4. Service Bench

It may be convenient to position a bench for a cell counter, microscope, etc., close to the sterile handling area

The service bench should also provide for the storage of sterile glassware, plastics, pipettes, screw caps, syringes..., in drawer units below and open shelves above
It may also be used for other accessory equipment, such as a small centrifuge

service bench

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Hot room

Incubator

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If only one or two are required, incubators are inexpensive and economical in terms of space; but as soon as you require more than two, their cost is more than that of a simple hot room, and their use is less convenient Incubation may be carried out in separate incubators or in a thermostatically controlled hot room

Incubators also lose more heat when they are opened and are slower to recover than a hot room

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If you have the space within the laboratory area or have an adjacent room, it may be possible to convert the area into a hot room
The area need not be specifically constructed as a hot room, but it should be insulated to prevent cold spots being generated on the walls

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The hot room should have a small bench, preferably stainless steel or solid plastic laminate, and the bench should accommodate an inverted microscope, the flasks that you wish to examine, and a notebook. Alternatively, a small laminar-flow hood and a small laminar-flow unit (1218 in) could be located in the room

Wooden furnishings should be avoided as much as possible

Incandescent lighting is preferable to fluorescent, which can cause degradation of the medium

The temperature of the hot room should be controlled within 0.5C at any point

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Heat is best supplied via a fan heater, domestic or industrial, depending on the size of the room. Approximately 23 kW per 20m3 will be required, depending on the insulation
A second fan, positioned on the opposite side of the room and with the airflow opposing that of the fan heater, will ensure maximum circulation. If the room is more than 22m, some form of ducting may be necessary

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Thermostats
should be of the proportional controller type. When the door opens and the room temperature falls, recovery will be rapid; on the other hand, the temperature will not overshoot its mark Ideally, there should be two separate heaters (H1 and H2), each with its own thermostat (HT1 and HT2). One thermostat (HT1) should be located diagonally opposite and behind the opposing fan (F1) and should be set at 37oC

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The other thermostat (HT2) should be located diagonally opposite H2 and behind its opposing fan (F2) and should be set at 36oC. Two safety override cutout thermostats should also be installed and set at 38C

The thermostat sensors should be located in an area of rapid airflow, close to the effluent from the second, circulating, fan for greatest sensitivity

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Overheating
The problem of unwanted heat gain is often forgotten because so much care is taken to provide heat It can arise because of a rise in ambient temperature in the laboratory in hot weather or heat produced from within the hot room by apparatus such as stirrer motors, roller racks, laminar-flow units Try to avoid heat-producing equipment in the hot room
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Access
If a proportional controller, good circulation, and adequate heating are provided, an air lock will not be required. The door should still be well insulated, light, and easily closed preferably, selfclosing

Thermometer
A temperature recorder should be installed and should have a chart that is visible to the people working in the tissue culture room. If possible, one high-level and one lowlevel warning light should be placed beside the chart or at a different, but equally obvious, location

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The need for extensive preparation of media in small laboratories can be avoided if there is a proven source of reliable commercial culture media Smaller laboratories may to purchase readymade media, then need only to prepare reagents, such as salt solutions and ethylenediaminetetraacetic acid (EDTA), bottle these and water, and package screw caps and other small items for sterilization 42

If reliable commercial media are difficult to obtain, the preparation area should be large enough to accommodate a coarse and a fine balance, a pH meter, and, if possible, an osmometer Heat-stable solutions and equipment can be autoclaved or dryheat sterilized at the nonsterile end of the preparation area

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Washup and sterilization facilities are best situated outside the tissue culture lab

If the sterilization facilities must be located in the tissue culture lab, place them nearest the air extract and farthest from the sterile handling area

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The washup area should have plenty of space for soaking glassware and space for an automatic washing machine, should you require one
There should also be plenty of bench space for handling baskets of glassware, sorting pipettes, and packaging and sealing packs for sterilization. You will need spacefor a pipette washer and dryer

Autoclaves, ovens, and distillation apparatus should be located in a separate room if possible, with an efficient extraction fan

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Sinks should be deep enough (450 mm) to allow manual washing and rinsing of your largest items without having to stoop too far to reach into them. They should measure about 900 mm from floor to rim

Sinks should Stainless steel or polypropylene

Each washing sink will require four taps: a single coldwater tap, a combined hot-and-cold mixer, a cold tap for a hose connection for a rinsing device, and a nonmetallic or stainless steel tap for deionized water from a reservoir above the sink

Washing Up Sink and Pipette Washer

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Storage must be provided for the following items ensuring sterile and nonsterile are kept separate and clearly labeled:
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Sterile and nonsterile glassware, including media bottles and pipettes

Screw caps, stoppers, etc., sterile and nonsterile

Sterile liquids, at room temperature (salt solutions, water...), at 4C (media), and at 20C or 70C (serum, trypsin, glutamine...)

Apparatus such as filters, sterile and nonsterile

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Liquid nitrogen to replenish freezers; the liquid nitrogen should be stored in two ways

Cylinder storage for carbon dioxide, in separate cylinders for transferring to the laboratory as required

Gloves, disposal bags, etc

Sterile disposable plastics (culture flasks and Petri dishes, and syringes)

A piped supply of CO2 can be taken to work stations, or else the CO2 supply can be piped from a pressurized tank of CO2 that is replenished regularly

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Safety Note
Adequate ventilation must be provided for the room in which the nitrogen is stored and dispensed, preferably with an alarm to signify when the oxygen tension falls below safe levels The reason for this safety measure is that filling, dispensing, and manipulating freezer stocks are accompanied by the evaporation of nitrogen, which can replace the air in the room

liquid nitrogen

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Refrigerators and freezers should be located toward the nonsterile end of the lab. They require maintenance and periodic defrosting

Remember to allocate sufficient space for storage

freezer

In general, separate 20C freezers are better than a walkin 20C room. They are easier to clean out and maintain, and they provide better backup if one unit fails
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Cold room

Several independent refrigerators will occupy more space than the equivalent volume of cold room, but may be easier to manage and maintain in the event of failure

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