1    Basic Techniques - The ?Do?s and Don'ts? of Cell Culture

Given below are a few of the essential "do?s and don?ts" of cell culture. Some of these are mandatory e.g. use of personal protective equipment (PPE). Many of them are common sense and apply to all laboratory areas. However some of them are specific to tissue culture.

The Do?s

1. Use personal protective equipment, (laboratory coat/gown, gloves and eye protection) at all times. In addition, thermally insulated gloves, full-face visor and splash-proof apron should be worn when handling liquid nitrogen.
2. Always use disposable caps to cover hair.
3. Wear dedicated PPE for tissue culture facility and keep separate from PPE worn in the general laboratory environment. The use of different colored gowns or laboratory coats makes this easier to enforce.
4. Keep all work surfaces free of clutter.
5. Correctly label reagents including flasks, medium and ampules with contents and date of preparation.
6. Only handle one cell line at a time. This common-sense point will reduce the possibility of cross contamination by mislabeling etc. It will also reduce the spread of bacteria and mycoplasma by the generation of aerosols across numerous opened media bottles and flasks in the cabinet.
7. Clean the work surfaces with a suitable disinfectant (e.g. 70% ethanol) between operations and allow a minimum of 15 minutes between handling different cell lines.
8. Wherever possible maintain separate bottles of media for each cell line in cultivation.
9. Examine cultures and media daily for evidence of gross bacterial or fungal contamination. This includes medium that has been purchased commercially.
10. Quality Control all media and reagents prior to use.
11. Keep cardboard packaging to a minimum in all cell culture areas.
12. Ensure that incubators, cabinet, centrifuges and microscopes are cleaned and serviced at regular intervals.
13. Test cells for mycoplasma on a regular basis.

The Don?ts

1. Do not continuously use antibiotics in culture medium as this will inevitably lead to the appearance of antibiotic resistant strains and may render a cell line useless for commercial purposes.
2. Don?t allow waste to accumulate particularly within the microbiological safety cabinet or in the incubators.
3. Don't have too many people in the lab at any one time.
4. Don't handle cells from unauthenticated sources in the main cell culture suite. They should be handled in quarantine until quality control checks are complete.
5. Avoid keeping cell lines continually in culture without returning to frozen stock.
6. Avoid cell culture becoming fully confluent. Always sub-culture at 70-80% confluency or as advised on ECACC's cell culture data sheet.
7. Do not allow media to go out of date. Shelf life is only 6 weeks at +4ºC once glutamine and serum is added.
8. Avoid water baths from becoming dirty by using Sigma Clean (Prod. No. S5525).
9. Don?t allow essential equipment to become out of calibration. Ensure microbiological safety cabinets are tested regularly.

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2    Protocol 1 - Aseptic Technique and Good Cell Culture Practice

Aim
To ensure all cell culture procedures are performed to a standard that will prevent contamination from bacteria, fungi and mycoplasma and cross contamination with other cell lines.

Materials

• Chloros / Presept solution (2.5g/l)
• 1% formaldehyde based disinfectant e.g.Virkon,Tegador
• 70% ethanol in water (Prod. No. R8382)

Equipment

• Personal protective equipment (sterile gloves, laboratory coat, safety visor)
• Microbiological safety cabinet at appropriate containment level

Procedure

1. Sanitize the cabinet using 70% ethanol before commencing work.
2. Sanitize gloves by washing them in 70% ethanol and allowing to air dry for 30 seconds before commencing work.
3. Put all materials and equipment into the cabinet prior to starting work after sanitizing the exterior surfaces with 70% ethanol.
4. Whilst working do not contaminate gloves by touching anything outside the cabinet (especially face and hair). If gloves become contaminated re-sanitize with 70% ethanol as above before proceeding.
5. Discard gloves after handling contaminated cultures and at the end of all cell culture procedures.
6. Equipment in the cabinet or that which will be taken into the cabinet during cell culture procedures (media bottles, pipette tip boxes, pipette aids) should be wiped with tissue soaked with 70% ethanol prior to use.
7. Movement within and immediately outside the cabinet must not be rapid. Slow movement will allow the air within the cabinet to circulate properly.
8. Speech, sneezing and coughing must be directed away from the cabinet so as not to disrupt the airflow.
9. After completing work disinfect all equipment and material before removing from the cabinet. Spray the work surfaces inside the cabinet with 70% ethanol and wipe dry with tissue. Dispose of tissue by autoclaving.
10. Cell culture discard in chloros (10,000) ppm must be kept in the cabinet for a minimum of two hours (preferably overnight) prior to discarding down the sink with copious amounts of water.
11. Periodically clean the cabinet surfaces with a disinfectant such as Presept,Tegador or Virkon or fumigate the cabinet according to the manufacturers instructions. However you must ensure that it is safe to fumigate your own laboratory environment due to the generation of gaseous formaldehyde, consult your on-site Health and Safety Advisor.

3    Protocol 2 - Resuscitation of Frozen Cell Lines

Click here for a schematic diagram of "Resuscitation of Frozen Cell Lines"

Aim
Many cultures obtained from a culture collection, such as ECACC, will arrive frozen and in order to use them the cells must be thawed and put into culture. It is vital to thaw cells correctly in order to maintain the viability of the culture and enable the culture to recover more quickly. Some cryoprotectants, such as DMSO (Prod. No. D2650), are toxic above 4ºC therefore it is essential that cultures are thawed quickly and diluted in culture medium to minimize the toxic effects.

Materials

• Media? pre-warmed to the appropriate temperature (refer to the ECACC Cell Line Data Sheet for the correct medium and size of flask to resuscitation into.)
• 70% ethanol in water (Prod. No. R8382)
• DMSO (Prod. No. D2650)

Equipment

• Personal protective equipment (sterile gloves, Laboratory coat, safety visor)
• Waterbath set to appropriate temperature
• Microbiological safety cabinet at appropriate containment level
• CO₂ incubator
• Pre labeled flasks
• Marker Pen
• Pipettes
• Ampule Rack
• Tissue

Procedure

1. Read Technical data sheet to establish specific requirements for your cell line.
2. Prepare the flasks as appropriate (information on technical data sheet). Label with cell line name, passage number and date.
3. Collect ampule of cells from liquid nitrogen storage wearing appropriate protective equipment and transfer to laboratory in a sealed container.
4. Still wearing protective clothing, remove ampule from container and place in a waterbath at an appropriate temperature for your cell line e.g. 37ºC for mammalian cells. Submerge only the lower half of the ampule. Allow to thaw until a small amount of ice remains in the vial - usually 1-2 minutes. Transfer to class II safety cabinet.
5. Wipe the outside of the ampule with a tissue moistened (not excessively) with 70% alcohol hold tissue over ampule to loosen lid.
6. Slowly, dropwise, pipette cells into pre-warmed growth medium to dilute out the DMSO (Prod. No. D2650) (flasks prepared in Step 2).
7. Incubate at the appropriate temperature for species and appropriate concentration of CO₂ in atmosphere.
8. Examine cells microscopically (phase contrast) after 24 hours and sub-culture as necessary.

Key Points

1. Most text books recommend washing the thawed cells in media to remove the cryoprotectant. This is only necessary if the cryoprotectant is known to have an adverse effect on the cells. In such cases the cells should be washed in media before being added to their final culture flasks. See Protocol 7 for further details.
2. Do not use an incubator to thaw cell cultures since the rate of thawing achieved is too slow resulting in a loss of viability.
3. If a CO₂ incubator is not available gas the flasks for 1-2 minutes with 5% CO₂ in 95% air filtered through a 0.25m filter.
4. For some cultures it is necessary to subculture before confluence is reached in order to maintain their characteristics e.g. the contact inhibition of NIH 3T3 (Prod. No. 93061524) cells is lost if they are allowed to reach confluence repeatedly.

4    Protocol 3 - Subculture of Adherent Cell Lines

Click here for a schematic diagram of "Subculture of Adherent Cell Lines"

Aim
Adherent cell lines will grow in vitro until they have covered the surface area available or the medium is depleted of nutrients. At this point the cell lines should be sub-cultured in order to prevent the culture dying. To subculture the cells they need to be brought into suspension. The degree of adhesion varies from cell line to cell line but in the majority of cases proteases, e.g. trypsin, are used to release the cells from the flask. However, this may not be appropriate for some lines where exposure to proteases is harmful or where the enzymes used remove membrane markers/receptors of interest. In these cases cells should be brought into suspension into a small volume of medium mechanically with the aid of cell scrapers.

Materials

• Media? pre-warmed to 37ºC (refer to the ECACC Cell Line Data Sheet for the correct medium)
• 70% ethanol in water (Prod. No. R8382)
• PBS without Ca₂₊/Mg₂₊(Prod. No. D8537)
• 0.25% trypsin/EDTA in HBSS, without Ca₂₊/Mg₂₊(Prod. No. T4049)
• Trypsin (Prod. No. T4424)
• Soybean trypsin Inhibitor(Prod. No. T6414)

Equipment

• Personal protective equipment (sterile gloves, Laboratory coat, safety visor)
• Waterbath set to appropriate temperature
• Microbiological safety cabinet at appropriate containment level
• CO₂ incubator
• Pre-labeled flasks
• Marker Pen
• Pipettes
• Ampule Rack
• Tissue

Procedure

1. View cultures using an inverted microscope to assess the degree of confluency and confirm the absence of bacterial and fungal contaminants.
2. Remove spent medium.
3. Wash the cell monolayer with PBS without Ca₂₊/Mg₂₊(Prod. No. D8537) using a volume equivalent to half the volume of culture medium. Repeat this wash step if the cells are known to adhere strongly.
4. Pipette trypsin/EDTA (Prod. No. T4049) onto the washed cell monolayer using 1ml per 25cm₂ of surface area. Rotate flask to cover the monolayer with trypsin. Decant the excess trypsin.
5. Return flask to the incubator and leave for 2-10 minutes.
6. Examine the cells using an inverted microscope to ensure that all the cells are detached and floating. The side of the flasks may be gently tapped to release any remaining attached cells.
7. Resuspend the cells in a small volume of fresh serum-containing medium to inactivate the trypsin. Remove 100-200uL and perform a cell count (Protocol 6- Cell Quantification).
8. Transfer the required number of cells to a new labeled flask containing pre-warmed medium (refer to ECACC Cell Line Data Sheet for the required seeding density).
9. Incubate as appropriate for the cell line.
10. Repeat this process as demanded by the growth characteristics of the cell line.

Key Points

1. Some cultures whilst growing as attached lines adhere only lightly to the flask, thus it is important to ensure that the culture medium is retained and the flasks are handled with care to prevent the cells detaching prematurely.
2. Although most cells will detach in the presence of trypsin alone the EDTA is added to enhance the activity of the enzyme.
3. Trypsin is inactivated in the presence of serum. Therefore, it is essential to remove all traces of serum from the culture medium by washing the monolayer of cells with PBS without Ca₂₊/Mg₂₊(Prod. No. D8537).
4. Cells should only be exposed to trypsin/EDTA (Prod. No. T4049) long enough to detach cells. Prolonged exposure could damage surface receptors.
5. Trypsin should be neutralized with serum prior to seeding cells into new flasks otherwise cells will not attach.
6. Trypsin may also be neutralized by the addition of soybean trypsin inhibitor (Prod. No. T6414), where an equal volume of inhibitor at a concentration of 1mg/ml is added to the trypsinised cells. The cells are then centrifuged, resuspended in fresh culture medium and counted as above. This is especially necessary for serum-free cell culture.
7. If a CO₂ incubator is not available gas the flasks for 1-2min with 5% CO₂ in 95% air filtered through a 0.25m filter.

5    Protocol 4 - Subculture of Semi-Adherent Cell Lines

Click here for a schematic diagram of "Subculture of Semi-Adherent Cell Lines"

Aim
Some cultures grow as a mixed population (e.g. B95-8 - marmoset) where a proportion of cells do not attach to the tissue culture flask and remain in suspension. Therefore to maintain this heterogeneity both the attached cells and the cells in suspension must be subcultured.

Materials

• Media? pre-warmed to 37ºC (refer to the ECACC Cell Line Data Sheet for the correct medium)
• 70% ethanol in water (Prod. No. R8382)
• PBS without Ca₂₊/Mg₂₊(Prod. No. D8537)
• 0.25% trypsin/EDTA in HBSS, without Ca₂₊/Mg₂₊(Prod. No. T4049)
• Trypsin (Prod. No. T4424)
• Soybean trypsin Inhibitor(Prod. No. T6414)

Equipment

• Personal protective equipment (sterile gloves, laboratory coat, safety visor)
• Waterbath set to 37ºC
• Microbiological safety cabinet at the appropriate containment level
• Centrifuge
• Inverted phase contrast microscope
• CO₂ incubator
• Haemocytometer (Bright-line, Prod. No. Z359629, Improved Neubauer Grid, Camlab CCH.AC1)
• Pre-labeled flasks
• Tissues

Procedure

1. View cultures using an inverted phase contrast microscope to assess the degree of confluency and confirm the absence of bacterial and fungal contaminants. Give the flask a gentle knock first, this may dislodge the cells from the flask and remove the need for a trypsinisation step with the subsequent loss of some cells due to the washings.
2. Decant spent medium into a sterile centrifuge tube and retain.
3. Wash any remaining attached cells with PBS without Ca₂₊/Mg₂₊(Prod. No. D8537) using 1-2ml for each 25cm₂ of surface area. Retain the washings.
4. Pipette trypsin/EDTA (Prod. No. T4049) onto the washed cell monolayer using 1ml per 25cm₂ of surface area. Rotate flask to cover the monolayer with trypsin. Decant the excess trypsin.
5. Return flask to incubator and leave for 2-10 minutes.
6. Examine the cells using an inverted microscope to ensure that all the cells are detached and floating. The side of the flasks may be gently tapped to release any remaining attached cells.
7. Transfer the cells into the centrifuge tube containing the retained spent medium and cells.
8. Centrifuge the remaining cell suspension at 150g for 5 minutes. Also centrifuge the washings from Number 3 above if they contain significant numbers of cells.
9. Decant the supernatants and resuspend the cell pellets in a small volume (10-20mls) of fresh culture medium. Pool the cell suspensions. Count the cells.
10. Pipette the required number of cells to a new labeled flask and dilute to the required volume using fresh medium (refer to ECACC Cell Line Data Sheet for the required seeding density).
11. Repeat this process every 2-3 days as necessary.

Key Points

1. Although most cells will detach in the presence of trypsin alone the inclusion of EDTA is used to enhance the activity of the enzyme.
2. Trypsin is inactivated in the presence of serum. Therefore, it is essential to remove all traces of serum from the culture medium by washing the monolayer of cells with PBS without Ca₂₊/Mg₂₊(Prod. No. D8537). Repeated warming to 37ºC also inactivates trypsin.
3. Cells should only be exposed to trypsin/EDTA (Prod. No. T4049) long enough to detach cells. Prolonged exposure could damage surface receptors. In general a shorter time of exposure to trypsin is required for semi adherent cell lines.
4. Trypsin should be neutralized with serum prior to seeding cells into new flasks otherwise cells will not attach.
5. Trypsin may also be neutralized by the addition of Soybean trypsin Inhibitor (Prod. No. T6414), where an equal volume of inhibitor at a concentration of 1mg/ml is added to the trypsinised cells. The cells are then centrifuged, resuspended in fresh culture medium and counted as above.
6. If a CO₂ incubator is not available gas the flasks for 1-2 minutes with 5% CO₂ in 95% air filtered through a 0.25m filter.

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