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Cell Culture F.A.Q.'s

Cell Culture F.A.Q.’s:

  1. Can a frozen ampule of cells be put back into liquid nitrogen and stored for any length of time?
     

    In many cases, an ampule shipped in dry ice (-80°C) can be placed back into liquid nitrogen and the population recovered by rapid thawing at a later date. However, the viability may be reduced by such treatment, and for some sensitive cell lines, this may make recovery more difficult. The phenomenon is thought to be due to a change in the ice crystal structure within cells that occurs during the temperature shift. For this reason, we recommend that cells be thawed and placed into culture as soon after receipt as possible. It is best to minimize storage time at -80°C; that is, to use this temperature only for shipping. Celprogen does not warrant the viability of cells stored at -80oC after shipments have been received.
     
     
  2. What safety precautions are necessary for thawing ampules that have been stored in liquid nitrogen?
     

    A glass ampule or plastic vial that has been submerged in liquid nitrogen can explode upon removal if it has not been properly sealed. Resulting glass or plastic fragments fly at high force in all directions creating a hazard. Thus a face guard and protective gloves and clothing must be worn whenever an ampule is removed from liquid nitrogen. Celprogen does not routinely store cells in the liquid phase. For reconstitution, the ampule should be agitated in a covered water bath at 37°C until its contents have thawed completely. To recover the cell suspension from a glass ampule, the neck is nicked with a small file, the ampule is washed with 70% ethanol, wrapped between several folds of a sterile towel or gauze and snapped open. The ampule contents can then be removed with a sterile 1 ml pipette (needles and syringes should be avoided whenever possible).
     
     
  3. Why do you have to store cells in the liquid nitrogen vapor phase rather than in the liquid phase?
     

    The cells when stored in the liquid nitrogen vapor phase they are easily recovered whereas when cells are stored in the liquid phase of the liquid nitrogen if the vial are not properly sealed or if there is a leak the cells come into direct contact with the liquid nitrogen. Thaw the cells the viability and recovery of the cells get comprised in this situation.
     
     
  4. Since we do not have a programmable freezer, how can we achieve the optimum -1°C/min freezing rate when preserving cells?
     

    One can avoid the expense of a programmable freezer by purchasing devices that can be used for cryopreservation in conjunction with mechanical or liquid nitrogen freezers. Alternatively, ampules and vials can be placed in a small Styrofoam box having wall and cover thickness of about 15 to 20 mm. When placed in a mechanical freezer at -70° to -90°C the internal cooling rate will approximate that required. Thermocouples inserted into dummy ampules can be used to check cooling rates if desired.
     
     
  5. Can mechanical freezers be used for cryogenic storage of cultured cells?
     

    Mechanical freezers can be used for storage if temperatures of -135°C or lower can be maintained. Viability will decline at higher temperatures. Backup freezers are needed in case of mechanical failure.
     
     
  6. How do I change the medium in (feed) a suspension culture?
     

    Suspension cell lines can be fed by the simple addition of fresh medium to the culture (if room is available) or by separating the cells from the old medium by centrifugation (100 x g for 5 min) with subsequent resuspension of the cell pellet in fresh medium. However, with most suspension cell lines, a simple addition of medium is the preferred method. In either case it is absolutely necessary to feed the cultures before the cells reach their maximum saturation density. This can range between 3 x 105 to 2x106 depending on the cell line and the culture conditions (stationary versus stirred, oxygenation levels, etc. The cells must be diluted to a lower cell concentration to allow sufficient nutrients for resumption of logarithmic growth. If the medium is simply replaced and the cell density is not decreased cells will rapidly deplete the medium and die. If the cells are diluted below their minimum density they will either, enter into a lag phase and grow very slowly, or they will die. The saturation densities and hence subculture intervals for each suspension line will vary. Therefore, daily cell counts are the best way to monitor suspension cell lines.
     
     
  7. What are the recommended carbon dioxide (CO2 levels need to grow a Cell Culture?
     

    While the levels of carbon dioxide in cell culture systems vary from that in ambient air (about 0.03%) up to 40% in air, generally either no added CO2 or 5% to 10% CO2 in air are the most frequently used. It is very important to adjust the concentration of sodium bicarbonate used in a medium to that required for equilibration with the level of CO2 used in the gas phase. Cells in culture produce CO2 and require small amounts of the compound for growth and survival. If no CO2 is added and mass cell cultures are being propagated, anhydrous sodium bicarbonate at 4 mM (0.34 g/L) can be used. However, the culture vessel should be kept sealed (caps on flasks should be tight). If 5% or 10% CO2 is desired, use 23.5 mM (1.97 g/L) or 47 mM (3.95 g/L) sodium bicarbonate, respectively, for an initial pH of about 7.6 at 37°C. Under these conditions unsealed (loose caps) culture flasks or dishes must be used to allow the gases to equilibrate. For more detail on the gaseous environment for the mammalian cell in culture, consult Chapter 5 by W.F. McLimans in Growth, Nutrition and Metabolism of Cells in Culture, Vol.1 (1972), G.H. Rothblat and V.J. Cristofalo, eds. (Academic Press, New York).
     
     
  8. Why do some cell lines require sodium pyruvate? How much should I add to the medium?
     

    Pyruvate is an intermediary organic acid metabolite in glycolysis and the first of the Embden Myerhoff pathway that can pass readily into or out of the cell. Thus, its addition to tissue culture medium provides both an energy source and a carbon skeleton for anabolic processes. Its addition may help in maintaining certain specialized cells, may help when cloning and may be necessary when the serum concentration is reduced in the medium (Culture of Animal Cells: A Manual of Basic Technique, 3rd edition, (1994) by R. Ian Freshney (Wiley- Liss, Inc., New York). Sodium pyruvate may also help reduced fluorescent light-induced phototoxicity (Spierenberg, G.T., et al. (1984) Phototoxicity of N-2-hydroxyethylpiperazine-N-ethanesulfonic acid-buffered culture media for human leukemic cell lines. Cancer Research 44:2253). Usually sodium pyruvate is added to give a final concentration of 0.1 mM. Sodium pyruvate is commercially available as a 10 mM (100X) stock solution.

     9.   What are the cell densities for tissue culture flasks, plates, and dishes?

         

Seeding Densities for Cells

Flask

Growth Surface Area

Volume

Cell Yield

T25

25cm2

5-10 ml

2.5 x 106

T75

75cm2

15-20 ml

7.5 x 106

T150

150cm2

30-50 ml

15.0 x 106

T175

175cm2

35-60 ml

17.5 x 106

T225

225cm2

45-75 ml

22.5 x 106

Cell yield is cell culture dependent. These approximations are based upon cell densities of

1 x 106 cells/ cm2.

 

Tissue Culture Plates

Growth Surface Area

Volume

Cell Yield

100 mm dish

44 cm2

9 ml

44 x 10(4.4 x 106)

6 Wells

9.40 cm2

2.0 – 3 ml

9.4 x 105

12 Wells

3.83 cm2

1.0 – 2.4 ml

3.83 x 105

24 Wells

1.88 cm2

0.5 – 1.2 ml

1.88 x 105

48 Wells

1 cm2 area/well

0.3 – 0.6 ml

1.0 x 105

96 Wells

0.32 cm2

0.1 – 0.2 ml

0.32 x 105

384 Wells

0.06 cm2

25 – 50 ml

0.06 x 105

1536 Wells

0.015 cm2

5 – 10 ml

0.015 x 105

Cell yield is cell culture dependent.  These approximations are based upon cell densities of

 1 x 105 cells/ cm2.

 Seeding Density for Cells in multi-well plates:

Optimal density at imaging time is usually 7,500-20,000 cells/well on a 96 well plate and 1000-4000 cells/well on a 384 well plate.  Too high cell density causes difficulties in autofocus, especially if the cells are overgrown and on top of each other, and often results in some out of focus cells. Too low cell density, especially when using higher magnification objectives, results in many empty fields and a loss of focus.  Seeding density can be assessed by plating several dilutions of cells across a plate and allow them to grow under experimental conditions (which may either enhance or impede growth).  

10.   Utilizing microscope slide cell culturing pre-coated with ECM:

         1. Remove microscope slide with the appropriate number of wells (8-10) and place in tissue culture hood.

         2.  Please pre-warm media in the tissue culture hood, sterile petri-dish and the 15 ml conical flask.

         3. Transfer the frozen vial of cells and thaw the cells in the dry 37C incubator.

         4. Once the cells are thawed within 2-3 minutes, the thawed vial is tranferred into the tissue culture hood.

         5. The thawed cells transferred into a sterile 15ml conical centrifuge tube and centrifuged for 5 minutes 

              at 100g to obtain a soft cell pellet.

         6.  After centrifugation the cell pellet is transferred into the tissue culture hood.

         7.  The cell pellet is re-constituted in 1ml of fresh pre-warm tissue culture media.

         8. Once the cells are reconstituted in 1ml of media, 20 ul of cell suspension is transferred onto the microscope

             slide.

          9.  The cell suspension is transferred onto the microscope slide and placed in the sterile petri dish.

         10.  Once all the cells are plated onto the microscope slide the petri dish cover is placed on and the cells are

                imaged on inverted microscope.

          11. The cells image is taken on the inverted microscope.

          12. The cells are transferred into the incubator at 37and 5% carbon dioxide humified incubator.

             13. After 24 hours of incubation in the incubator observe the cells under the microscope and then fix the cells for

                IHC or cell based assays.  The glass microscope slide allows one to image the cells at high resolution.