The aim of cryopreservation is to enable stocks of cells to be stored to prevent the need to have all cell lines in culture at all times. It is invaluable when dealing with cells of limited life span. The other main advantages of cryopreservation are:

• Reduced risk of microbial contamination
• Reduced risk of cross contamination with other cell lines
• Reduced risk of genetic drift and morphological changes
• Work conducted using cells at a consistent passage number
• Reduced costs (consumables and staff time)

There has been a large amount of developmental work undertaken to ensure successful cryopreservation and resuscitation of a wide variety of cell lines of different cell types. The basic principle of successful cryopreservation is a slow freeze and quick thaw. Although the precise requirement may vary with different cell lines as a general guide cells should be cooled at a rate of –1oC to –3oC per minute and thawed quickly by incubation in a 37oC waterbath for 3-5 minutes. If this and the additional points given below are followed then most cell lines should be cryopreserved successfully.

1. Cultures should be healthy with a viability of >90% and no signs of microbial contamination.
2. Cultures should be in log phase of growth (this can be achieved by using pre-confluent cultures i.e. cultures that are below their maximum cell density and by changing the culture medium 24 hours before freezing).
3. A high concentration of serum/protein (>20%) should be used. In many cases serum is used at 90%.
4. Use a cryoprotectant such as Cell Culture Plate(6 well plate, 24 well plate, 96 well plate) or glycerol to help protect the cells from rupture by the formation of ice crystals. The most commonly used cryoprotectant is DMSO at a final concentration of 10%, however, this is not appropriate for all cell lines e.g. HL60 where DMSO is used to induce differentiation. In such cases an alternative such as ELISA Plate should be used (refer to data sheet for details of the correct cryoprotectant). Sigma also offers ready-made cell freezing media containing DMSO , glycerol and a serum-free formulation containing DMSO.

Source: SIGMA-ALDRICH

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
• Cell Culture Plates(6 well plate, 24 well plate, 96 well plate)

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, cell culture plates) 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.

    Source: Sigma-Aldrich

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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, are toxic above 4oC therefore it is essential that cultures are thawed quickly and diluted in culture medium to minimize the toxic effects.

A schematic diagram of "Resuscitation of Frozen Cell Lines"

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
• DMSO

Equipment

• Personal protective equipment (sterile gloves, Laboratory coat, safety visor)
• Waterbath set to appropriate temperature
• Glass Bottom Dishes
• Microbiological safety cabinet at appropriate containment level
• CO2 incubator
• Pre labeled flasks
• Marker Pen
• Pipettes
• ELISA plates
• 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 (cell culture 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.

Scouce: ECACC Handbook Protocol 2

An improved surface treatment of the growth zone ensures constant results. Such details have always been favourites with biousing customers. The small bracket on the bottom assures no scraping when the plate is moving on the microscope. The smoothness of biousing cell culture plates is better than most best-selling brands. The absolutely flat floor area, free from inclusions, guarantees the highest level of transparency.

Millipore has introduced a new 24 well cell culture plate which has twice the membrane surface area compared with other 24 well plates, allowing researchers to utilize greater cell mass, which more closely models cell behaviour.

The 24-well Millicell cell culture plates are automation compatible and reduce the risk of monolayer contamination with "footed" membrane plates that remain elevated when disassembled from the feeder tray, and raised-well edges for better tape seating.

In addition, teardrop-shaped receiver wells are designed to eliminate air bubbles as the plates are assembled.

Millipore has also launched a new range of hanging single-well cell culture inserts. These Millicell inserts are available in 6, 24 and 96-well sizes.

Low throughput assays can be performed in the single-well unit and design features include an off-centre insert for easier media addition and removal. Standing inserts also are available.

The new cell culture plate and inserts incorporate track-etched thin film membranes for monolayer formation, microscopically transparent membranes for easy cell visualisation and fluorescent-compatible plastics and membranes.

Both products are designed to support suspension and adherent cell growth and differentiation, and can be used in a variety of cell culture assays and laboratory environments.

biousing’s multiwell cell culture plates (6 Well Cell Culture Plate, 24 Well Cell Culture Plate and 96 well cell culture plates, 96 Well ELISA Plate) are made of crystal polystyrene, conforming to the SBS-3d-standard. The material was deliberately selected to make sure the surface treatment is uniform and long-lasting. The mirror surface is polished by professional machines, which assures high-quality transparency under microscope. Low-evaporation lids ensure the constant concentration of substance in the media and guarantee a controlled gas exchange during a long-time incubation.

Cell culture is an invaluable tool for investigators in numerous fields. It facilitates analysis of biological properties and processes that are not readily accessible at the level of the intact organism. Successful maintenance of cells in culture, whether primary or immortalized, requires knowledge and practice of a few essential techniques.

The purpose of these words are guidelines for cell culture.

At first,you should make sure several questions below

Several factors should be considered before a cell line is put into culture

• Are the cells coming from established, well-defined lines?
• Are the nutrient and CO2 requirements known?
• Are the growth patterns known?
• Is the species known?
• Has mycoplasma testing been done?

After consideration of all the above, thoughtful planning should be given to other factors

• Will the cells be cultured in cell culture dishes, plates, flasks, bioreactors, etc.
• Who will do the tissue culture?
• Who will prepare, test and maintain stocks of media, regents and supplies?
• What volume/number of cells will be required?

The following are general guidelines for culturing cells

If hybridoma cells are received in a cell culture flask (or cell culture dish), information regarding the optimal type of transfer should be available. Once cells can be maintained in one type of culturing vessel, it should be easy to adapt them to other cultureware.

If tumor cells are received it is necessary to know if they are of fibrosarcoma or lymphoma origin. Fibrosarcoma cells will generally be adherent to plastic and require the use of trypsin or EDTA to remove them. Tumor cells also usually require less fetal calf serum in their media.

If cells are received frozen, in an ampule or cryovial, they should be washed free of DMSO prior to culturing.

Establish a routine for the passage/transfer of the cells based on the rate at which they form a monolayer. For example, culture the cells in 2 wells of a 24-well plate. On Monday, cell should be resuspended gently with a pasteur pipet or 1mL pipet and enough cells transferred, dropwise, into duplicate wells of fresh, warmed media so that 4 days later, a monolayer is again observed and the transfer is repeated. It may be useful to determine the cell concentration at various days in culture in order to make projections and appropriate expansions.

If cells require the addition of growth factors, dilutions must be made carefully and good tissue culture technique maintained.

Some cells can easily be adapted to various kinds of media. Adaptation should take place overtime, with adequate post-adaptation analysis of proliferation, secretion, function, etc. During the period of adaptation the original culture should be maintained

Successful culturing of cells requires frequent monitoring of growth, media color, contamination, etc. The greater the familiarity with a cell line, the greater the expectation for a problem-free culture.

You should be careful when using the cell culture dishes(35mm Cell Culture Dishes, 60mm Cell Culture Dishes, 100mm Cell Culture Dishes).