CELL CULTURE (CC) PROCEDURE NUMBER 1: Growing & Maintaining Cells in Culture (feeding cells)

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CC1.1 REAGENTS & MATERIALS

For this basic cell culture procedure, in addition to the normal tissue culture equipment and consumables (glass and plastic-ware), you will also need the materials reagents listed below:

1 * 25 cm2 Culture flask containing apartially confluent cultures of cells (listen carefully in the lectures and practical session to find out what type of cells these are)
1 * 30 ml universal containing DMEM media (see below)
1 * Glass test tube containing 4/5 sterile Pasteur pipettes and sealed with aluminium foil.

NOTES:

• You will be given a culture flask containing the cells and containers full of the appropriate solutions for you to use. Please date and label these with your initials. Always put your name and the date on the sides of any containers of media or cells when you are given them.
  
• The medium used is Dulbecco's Modified Minimum Essential Media (DMEM) containing: Phenol Red (pH indicator); 200 mM L-glutamine; 100mg Streptomycin and 100 Units of penicillin/ml, and 10% Foetal Calf serum (FCS).
• The cell lines used are from the ETCC. (further details of types of cells used will be given out later)
• You will need to label the top of the culture flasks containing the cells and any bottles/tubes of solution with your initials to identify your cells and reagents. You should use only your own solutions and no-one else's. If you loose cells or reagents, or are running out of reagents you should request fresh supplies from the teaching/demonstrating team. DO NOT JUST HELP YOURSELF TO NEW REAGENTS FROM THE FRIDGE, SOMEONE ELSE MAY ALSO NEED THEM!!
• When using the Pasteur pipettes, once inside the cabinet spray the foil and top of the tube carefully, open the foil, remove one pipette for use and then reseal the tube with the foil. Only remove one pipette at a time and then reseal the tube after each removal. Use a fresh tube each day. DO NOT REUSE ONCE THE TUBE HAS BEEN OPENED.
  

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CC1.2 PROCEEDURE

Obeying the general procedures for using the tissue culture laboratory and equipment and uSing aseptic techniques undertake the following procedures:

1. Prepare cabinet as the document on Basic Laboratory Procedures.
2. Remove your 30 ml universal container containing DMEM media from the fridge and allow to come to room temperature on the bench or in a heated water bath (if available).
3. Place the tube containing the media in the test tube rack in the cabinet (see protocol 2.1) and wipe the neck and lid with 70% alcohol.
4. Place the tube containing the Pasteur pipettes inside the cabinet.
5. Remove your culture flask containing your cells from the incubator, taking care to tighten the cap first.
6. Examine the cells under the inverted microscope for health and degree of confluence. Make appropriate notes later in your laboratory notebook. When confluent they will need passaging (see later protocol).
7. If your cells have become contaminated immediately sterilise the flask by addition of hypochlorite or Stericol solution. Then dispose of the solution down the sink and place the empty culture flask into a biohazard bag and seal bag with autoclave tape. Seek assistance from the teaching/demonstrating staff.
8. With your left (or non-dominant hand) lift and partially remove the cap of your culture flask and remove the medium either:

• using a Pasteur pipette attached to a vacuum line and pump or a sterile, disposable Pasteur pipette.
• Or using pipettes and the automatic pipette pump if the vacuum pump is not available
  NOTE: You may find it useful to tilt the flask to pool the medium at one end to enable more efficient removal of the medium.
  

9. Immediately, replace the cap on the culture flask.
10. Loosen the top of the media bottle
11. Well inside the cabinet, holding a plastic, disposable 10 ml graduated pipette at the top and not touching the bottom at all remove the plastic wrapper and firmly insert the end of the pipette into the automatic pipette pump.
12. Insert the pipette into your container of medium (in the test tube rack) and withdraw 6 ml of medium sing the automatic pipette pump. TAKE CARE NOT TO SUCK MEDIUM UP INTO THE BODY OF THE PIPETTE PUMP. If you do this immediately seek advice from the teaching/demonstrating staff.
13. Taking the culture flask with your left (or non-dominant hand) partially remove the cap of each flask and add 5ml of medium to each flask. Immediately replace and tighten the the cap.
14. Wipe around the top of the flask with 70% alcohol and return them to the incubator, remembering to loosen the cap once in the incubator.
15. Clean up the cabinet as given in document on Basic Laboratory Procedures.

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CELL CULTURE (CC) PROCEDURE NUMBER 2: Passaging of Cell Cultures (splitting cells).

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CC2.1 REAGENTS & MATERIALS

For this basic cell culture procedure, in addition to the normal tissue culture equipment and consumables (glass and plastic-ware), you will also need the sterile materials reagents listed below:

1 * 25 cm2 culture flask containing confluent cultures of cells from last week (NOTE THE TYPE!). If these have died other cells will be provided.
1 * 30 ml universal containing DMEM media (see below)
1 * 5ml universal containing 0.25% Trypsin/EDTA solution
1 * Glass test tube containing 4/5 sterile Pasteur pipettes and sealed with aluminium foil.

Notes.

• You will need to label the bottles/tubes of solution with your initials to identify your reagents.
• You should use only your own solutions and no-one else's.
• If you lose cells or reagents, or are running out of reagents, you should request fresh supplies from the teaching/demonstrating team. DO NOT JUST HELP YOURSELF TO NEW REAGENTS FROM THE FRIDGE ETC. SOMEONE ELSE MAY ALSO NEED THEM!!
  

 

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CC2.2 PROCEDURE

Obeying the general procedures for using the tissue culture laboratory and equipment and using aseptic techniques undertake the following procedures:

1. Prepare cabinet as in the Basic Laboratory Procedures document.
2. Remove the appropriate containers (containing the above solutions) from the fridge and allow to come to room temperature on the bench or in a heated water bath (if available).
3. Place the test tubes in the test tube rack in the cabinet (see previous instructions), stand the flask upright in the cabinet and wipe the neck and lid of all three with 70% alcohol.
4. Place the tube containing the Pasteur pipettes inside the cabinet.
5. Remove your culture flask containing your cells from the incubator, taking care to tighten the cap first.
6. Examine the cells under the inverted microscope for health and to confirm their degree of confluency. Make appropriate notes later in your laboratory notebook.
7. If your cells have become contaminated (see other notes) immediately sterilise the flask by addition of hypochlorite, Virkon or Stericol solution. Then dispose of the solution down the sink and place the empty culture flask into a biohazard bag and seal bag with autoclave tape. Seek assistance from the teaching/demonstrating staff. Remove the tissue culture medium as described in Practical Technique No. 3.

8. Wash the cells twice with either PBS or DMEM (without suuplements) as follows.

• Each time add 3-5 ml wash solution, using a plastic disposable pipette and automatic pipette pump, as described in Protocol 3 for feeding the cells.
• Replace the cap and gently rock the flask on the bottom of the cabinet in a circular manner, to allow the solution to move over the cells, for about 30 seconds.
• Draw off the wash solution using a Pasteur pipette (disposable or attached to the pump).
• Repeat once to remove all traces of the medium (the Foetal Calf Serum inactivates the Trypsin).
  

9. Pipette 2.5 ml of the 0.25% Trypsin/ EDTA into the cell container, again gently move the solution over the cells for 30 seconds.
10. Remove the 0.25% Trypsin/ EDTA leaving a thin film to cover the cells, replace the top and place the culture flask in the incubator for 5 minutes (cell culture incubator, set at 37oC, 5% CO2).
11. Remove from incubator and lightly tap the flask on the bench.
12. Check under the invert microscope to make sure cells are rounded up and moving across surface.
13. Add 10 ml of fresh medium to the culture flask.
14. Gently circulate the medium around the cell surface to neutralise the Trypsin for about 30 seconds.
15. Then using a sterile pipette draw up the 6 ml of medium and add 5ml (each) to two new 25cm2 culture flasks (using techniques described previously).Gently circulate the medium around the surface of the containers.

16. Label the new containers with:

• your initials,
• your supervisor's initials
• the cell type,
• the passsge number
• date.

17. Return cells to the incubator.
18. Clean and tidy up the cabinet and your work area as described in previous protocols.

 

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COMPONENTS OF CULTURE MEDIA

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1). BASAL MEDIUM

• In 1955 Eagle investigated the nutritional requirements of two mammalian cultured cell lines, the mouse L cell and the HeLa cell.
• Eagle found that the cell lines could be grown in a defined mixture of amino acids, vitamins, salts and carbohydrate supplemented with a small amount of horse or human serum.
• Specific nutritional deficiencies were noticed with the omission of particular amino acids or vitamins which could be remedied by the replacement of the missing component.
• Twenty seven factors were defined as essential for growth in the presence of supplemented serum and formed the medium known as 'basal medium, Eagle' or BME.
• Thirteen amino acids were to be essential with the remaining six non-essential amino acids being synthesised from other carbon sources. The absence of any one of the seven vitamins led to the development of deficiency symptoms.
• For the healthy growth of cells the basal medium also required frequent changing. Eagle later modified the medium and it was replaced by Eagle's minimum essential medium (MEM) which contained a higher concentration of various components to allow for several days of growth in the culture medium before a change (feed).
• Further modifications have been made by other workers, eg. Dulbecco's modification (DMEM) which contains four times the BME level of amino acids and vitamins with some non-essential amino acids and added ferric nitrate. Other media are all based on Eagle's MEM and have been developed for the improved growth of certain cell lines or strains.

2). SERUM (NORMALLY FETAL CALF OF FETAL BOVINE)

Serum is an essential component of the culture medium and is a source for nutrients and trace minerals, growth factors and hormones, protein and adhesion promoting components necessary for the spreading of cells.

3). L-GLUTAMINE

The most labile component of a standard culture medium. The amino acid, which is required by most mammalian cells has a half life of three weeks. If medium is used for longer than three weeks it should be made without.

4). GLUCOSE

An energy source to allow for normal cell metabolism and an essential ingredient and is present most commercially obtained mediums. Mediums can be purchased with different concentrations of glucose, mainly 1000mg/l or 4500mg/l..

5). ANTIBIOTICS

The use of antibiotics should be restricted to short-term cultures. Routine subculturing of cell lines in the presence of antibiotics can introduce antibiotic-resistant strains of bacteria.

6). OTHER REAGENTS USED IN TISSUE CULTURE MODULE

Phenol red indicator

This is a colour indicator of the pH of the solution. At pH levels below pH 7.0 the solutions containing the indicator will appear orange. At pH 7.2-7.4 the solutions appear red and above pH 7.5 they are reddish-blue nearly purple. Since the health of cells is mainly determined by the condition of the culture medium it is a good indicator of the feeding requirements of the cultured cells.

Trypsin-EDTA solution

Trypsin: Trypsin solutions are normally made up in a saline solution or in saline citrate and used in 0.25% solution. Its function is to release cultured cells from their substratum. Prolonged exposure of cells to trypsin should be avoided as this results in cell damage. To inactivate trypsin the additon of serum (or a medium containing serum) is normally performed which contains a natural trypsin inhibitor.

EDTA: EDTA is a chelating agent which removes divalent ions. The presence of EDTA leads to the dissociation of cell monolayers and the release of cells into suspension without the need for protease action. However, in combination with trypsin the dissociation is often much faster.

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