What kind of work can be performed in the Cell Culture Facility?
The Facility is designed to accommodate the culture of mammalian cell lines and other animal cell lines, such as insect cells: Virus- infected and recombinant DNA transfected cells can be cultured for the purpose of isolating viral or recombinant proteins. Both experimental scale and production scale projects can be accommodated.
What type of work is not allowed in the Cell Culture Facility?
The Facility cannot accommodate the culture of bacterial cells, spore-forming yeast, Neurospora or fungi or single-celled microorganisms due to the potential of these cultures to contaminate mammalian cell lines. Projects which do not involve the culture of cells, for instance, samples of animal tissues or blood taken from live animals, (i.e. not cultured) are not appropriate for the Cell Culture Facility. Animal tissues should be presumed to harbor yeast, fungj, mycoplasma, and bacteria that interfere with eucaryotic or insect cell culture. These types of projects are more appropriately handled in the Biomedical Animal Research Facility, which is available for work of this nature. Cell cultures which might reasonably be suspected to harbor contaminants may not be brought into the Facility. New cell lines brought into the Facility should be purchased from the American Type Culture Collection when possible. The ATCC verifies that its cell lines are free of contaminants. Cell lines brought into the Facility from other sources must pass a sterility test before continued culture. The sterility test will consist of growth in antibiotic-free media for a period of 2 weeks without visible growth of contaminants.
What type of permission do I need to conduct work in the Cell Culture Facility?
All work involving human cell lines must first be approved by the University Biosafety Committee. All work involving recombinant DNA must first be approved by the University Recombinant DNA Committee. All work is reviewed for appropriateness of the work for the Facility by the Cell Culture Facility’s oversight committee. The Principal Investigator submits a request to perform work/ proposal of the work to the Facility Manager. If special approvals are needed as described above, these approvals must be submitted at the time of the request to perform work. The Facility manager may request additional information before forwarding the request to the Oversight Committee. The committee, in consultation with the Facility Manager, either approves or denies the request.
What space and equipment are available in the Cell Culture Facility?
Four individual culture rooms are available offering BSL2 containment. An additional room is BSL 2 +. Each room is outfitted with a sterile biosafety cabinet, equipped with a gas flame and a vacuum source, and a water bath. All rooms also contain a hand-washing sink and the bio-safety cabinet is a Class IIA. In the BSL 2.5, the biosafety cabinet is a Class IIB1. Two 43 ft² reach-in incubator, one set at 26 degrees C and one at 37 degrees C and CO2 incubators in 3 culture rooms are available with stir plates for spinner culture and roller apparatus for roller bottle culture. A centrifuge room and a media preparation area contains balance, stir plate and necessary glassware, a dishwashing area, autoclave and two double sliding glass door refrigerators serve all of the culture rooms. A common area contains inverted, phase contrast microscopes for microscopic observations of cells under culture.
Equipment and small devices such as pipettors in the Facility are dedicated, and they are to be used for cell culture only and may not be taken out of the Facility except for repair. Similarly, equipment and small devices used in other laboratories should not be used in the Facility. In cases where this is impossible the item must be thoroughly decontaminated before it is brought into, or out of, the Facility.
What supplies are available in the Cell Culture Facility?
The following glass reusable items are available in the facility:
- glass spinner culture bottles (100 ml, 250 ml, 500 ml and 1, 3 and 6 liter)
- media bottles (100 ml to 1 liter)
- shaker flasks (125 ml, 250 ml, 500 ml, 1 liter, 2 liter and 3 liter)
- measuring and mixing glassware
- pipetting devices are available for each room
Frequently used materials are supplied in the Facility, including
- paper towels
- marking tape
- disinfectants and cleaning agents for surface decontamination and skin cleansing
- microscope slides and cover-slips
- disposal bags for waste
- dishwashing detergent
- cleaning supplies
- standard culture bio-chemicals (buffers, glutamine, other amino acids, salts and nucleosides for preparation of deficient media)
- chemicals for analytical instruments
The following items are available but participating laboratories are asked to participate in a purchase rotation in which all users supply some of the type of items they routinely use:
- flasks for monolayer culture – 225cm2, 75 cm2, 25 cm2 with plug-seal caps
- 850 cm2 roller bottles
- 150mm, 100mm, 60mm, 35mm culture dishes
- 96 well, 24 well, and 6 well plates
- flasks for suspension culture
- cell scrapers
- 1-3 ml graduated disposable transfer pipettes
- 50 ml, 25 ml,10 ml, 1 ml disposable pipettes
- 50 ml and 15 ml conical tubes (disposable)
- culture capsule filters for sterilization of media
- freeze media
- monolayer dissociation agents (trypsin, Accutase)
- micro pipette tips
What supplies does a user provide?
Any specialized supplies which are not expected to be useful for other users must be supplied by the user. However, it is expected that the need for items not available through the Facility will be infrequent. When a needed item is not available, the user should check with the Facility manager to determine whether the item might be added to the Facility inventory. When the manager is unable to supply the user with the item, the user may supply herself. All items to be used in the Facility must be stored in the Facility. If users buy supplies of their own, that portion which they intend to use in the Facility should be stored in the Facility upon purchase. Limited storage space is available for this purpose.
The following items are purchased by the individual user and stored in the facility:
- powdered culture media (or liquid, if desired)
- media supplements (serum)
What services does the Cell Culture Facility provide?
The Cell Culture Facility is a multi-user facility which provides consultation, training, and project supervision. The manager of the Facility is a PhD research scientist and provides consultative and supervisory services on a continuous basis for all projects in the facility. These services include, but are not limited to: advice on cell line selection, advice on growth conditions for cell lines, experimental design in cultured cells, locating or providing experimental protocols, advice and assistance in the development of new protocols, trouble shooting culture problems, training personnel in basic tissue culture techniques and advanced techniques such as DNA transfection, virus infection- growth and purification, biosafety training of all personnel, training in use of specialized equipment.
The Facility can assist with experiments or perform small scale experiments for the user when the user does not have lab personnel available. Training in all aspects is provided to all users. Assistance is expected to be provided, especially, but not exclusively, in the early stages of project development.
What training is provided by the Cell Culture Facility?
All first time users will be trained in the following areas:
- basic sterile technique
- biosafety classifications and requirements
- emergency procedures
- proper use of a biosafety cabinet
- proper disposal of biohazardous wastes
- Good Practice (microbiological techniques)
- proper cleaning of glassware
- preparation and storage of media
- sterility testing
- propagation of cell lines
- assessment of cell growth, including cell proliferation assays
- freezing of and retrieving frozen cell cultures
Training may be on an individual basis or as a workshop presented in conjunction with the University’s Biosafety Officer. Training will include a period of intensive supervision at the end of which the worker must have demonstrated their ability to successfully culture cells independently over an appropriate period of time.
Individuals with prior cell culture experience will not need techniques training but will require training in other areas such as proper waste disposal and glassware cleaning. These individuals will be asked to demonstrate their culture technique and be closely supervised during their initial period of work in the Facility.
What is the cost of using the Cell Culture Facility?
Depending on current funding levels of the Facility, the cost of supplies will be supplemented by the Facility. Users will be asked to replace some of the materials based on their usage of same. This rate is periodically reviewed and is subject to change. Structural Biology and KLB faculty are given preference in this regard.
How is space assigned for projects?
Each project is assigned a biosafety level when it is accepted by the Facility. Biosafety levels determine the degree of containment necessary for that work. These levels are determined by the Biosafety Regulations set forth in the Facility’s biosafety manual following the CDC-NIH guidelines. Each project must be performed in a culture room at its assigned containment level- or if that room is not available, at a higher containment level room. If higher containment rooms are used, all work must be performed in accordance with rules appropriate to the containment level of the room, not the culture.
Incubator space is also assigned based on bio-safety level and availability. Incubator assignments however are temporary and may change with space and temperature demands. One compartment of the CO2 double incubator in room 312B is reserved for virus-infected mammalian cells. The other compartment is available for either spinner or roller bottle or flasks of uninfected cultures. The CO2 incubator in the BSL 2 room (31B) will hold all other BSL2 flask cultures and dish cultures that are incubated at 37°. The priority for the reach-in incubator in the BSL 1 room (312C) is large-scale cultures, which can only be accommodated in this incubator. This includes 6 or more roller bottles, large spinner cultures (5 liters or larger) and large stirred cultures (15 to 30 liters).
A roll-in incubator in room 312F is set at 27° for insect cells. For large scale cultures the zero level containment room (312F) can be used as a 27°.
These policies are subject to change. Any changes will be published in a similar format and distributed to users. The latest version can be viewed at: http://biophysics.fsu.edu/core/pef/pef.html