Cell Culture

The Responsibilities of Tissue and Cell Culture Managers in the Tissue and Cell Culture Laboratories.

1. Oversees all culture facilities.
2. Standardize all procedures for each tissue and cell culture laboratory.
3. Assist each group by offering a brief introduction to the theory and general sterile techniques for each new staff/student, which include: general rules, standard practices for cell culture, cleaning and waste disposal, use of tissue culture laboratories, use of equipment, including laminar flow hoods, incubators and inverted microscopes, etc.
4. Schedule and perform all maintenance by organizing the monthly rotation of regular cleaning and maintenance of the tissue culture rooms, and make sure the following duties have been done by each research group:
   • Clean the open benches every week (monthly rotation)
   • Clean the water bath (monthly rotation)
   • Clean the incubators every four months (each group is responsible for cleaning their own incubators).
   • Clean biosafety cabinets (BSC) every four months (each group is responsible for cleaning BSC they are assigned to)
5. Design and perform examinations to test the examinee’s general principles and techniques of good tissue and cell culture laboratory practice and to make sure all the rules and protocols are followed properly.
6. Provide clean yellow gowns for the main culture rooms’ daily needs.
7. Design cell culture database for the cell lines currently used in the James Hogg Research Centre.
8. Organize regular meetings for tissue culture room cleaning, safety and administration.

Protocols for Cleaning Tissue and Cell Culture Rooms and Equipment.

Protocol 1 – Cleaning Incubators

• Remove all cultures to another CO2 incubator.
• Remove all the shelves, the water tray, and any removable panels from the incubator.
• Wipe the shelves and panels with Conflikt, wash them in large sink using 7X detergent, rinse them in distilled water and place in dry cupboard until dry. Once dry, wrap in green surgical drapes and autoclave.
• Wash the inside of the incubator with Conflikt, try to reach all corners and crevices.
• Rinse with distilled water.
• Return the autoclaved shelves and panels to the incubator.
• Replace the water tray and fill it with distilled water containing 2% Roccall.
• Check the temperature and CO2 concentration. When both temperature and CO2 concentration have stabilized (overnight), return the cultures to the incubator.

Protocol 2 – Cleaning Laminar Flow Hoods

• Remove all of the removable parts.
• Wash these parts with Conflikt, rinse them in distilled water.
• Wash the inside of the hood (underneath and sides) with Conflikt, try to reach all corners and crevices.
• Rinse with distilled water.
• Return the removable parts to the hood.

Protocol 3 – Cleaning Water Bath

• Turn off the power and unplug.
• Wash water bath with Conflikt, then rinse them with distilled water.
• Place fresh distilled water into the water bath and fill it with water containing “Clean Bath Algicide” (15 drops).
• Turn on the power and keep the temperature at 37C.

Protocol 4 – Cleaning the Open Benches

• Wash the open benches with Conflikt.
• Wipe the open benches with distilled water.

• User Guide
• Liquid Nitrogen FAQ

• Part 1: PPE, Tank Labels, Liquid Levels & Indicator Gauge
• Part 2: Dispensing Liquid Nitrogen
• Part 3: Troubleshooting & Other Tips

It is vital that summer students who must work in the sterile areas receive adequate training in techniques and procedures. In Chapter 10 (Training in tissue culture techniques), we mentioned that “Before new staff/students are allowed to work independently, they should be apprenticed to a skilled operator for a period of up to three months; they should not learn from the last recruit to come in”. However, some summer students stay here only three months. To ensure the summer students work appropriately in the tissue culture laboratories, special rules for training of summer students include:

1. The principle investigator in each research group must limit the number of summer students doing tissue culture experiments. A maximum of 3 summer students per research group will be allowed into the culture facilities during a given summer.
2. The tissue culture administrative group will assist each group by offering a brief introduction to the theory and general sterile techniques for each new summer student, which include:
   • General Rules (chapter 1 and chapter 2)
   • Standard Practices for Cell Culture (chapter 3)
   • Cleaning and Waste Disposal (chapter 4)
   • Use of Tissue Culture Laboratories (chapter 5)
   • Use of Equipment, including Laminar flow hoods, Incubators and Inverted microscopes (chapter 6)
   • Safety (chapter 9)
3. Each research group is responsible for supervising their new summer students for a period of time (two weeks to two months, individually dependent), with emphasis on training the new summer student’s practical skills.
4. After the new summer students have been apprenticed to a skilled operator for a period of time, they should be sent back to the administrative group to take an examination. The examination has been designed to test the examinee’s knowledge of the general principles and techniques of good tissue and cell culture laboratory practice, and to make sure all the rules and protocols are followed properly.
5. After passing the examination, the new summer students are allowed to work independently in the tissue and cell culture laboratories of the JHRC.