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Serum is a complex mix of albumins, growth factors and growth inhibitors and is probably one of the most important components of cell culture medium. The most commonly used serum is fetal bovine serum. Other types of serum are available including newborn calf serum and horse serum. The quality, type and concentration of serum can all affect the growth of cells and it is therefore important to screen batches of serum for their ability to support the growth of cells(take especial care of 96 well plate). In addition there are other tests that may be used to aid the selection of a batch of serum including cloning efficiency, plating efficiency and the preservation of cell characteristics.

Fetal bovine serum (FBS) has been used to prepare a number of biological and has an excellent record of safety. The recognition of Bovine spongiform encepalopathy (BSE) in 1986 and it’s subsequent spread into continental Europe along side the announcement of the probable link between BSE and a new variant of Creutzfeldt Jacob disease in Humans, stimulated an increased concern about safe sourcing of all bovine materials. In 1993 the Food and Drug Administration (FDA) "recommended against the use of bovine derived materials from cattle which have resided in, or originated from countries where BSE has been diagnosed. The current (European Union) EU guidelines on viral safety focus on sourcing, testing and paying particular attention to the potential risk of cross contamination during slaughtering or collection of the starting tissue. As far as BSE is concerned, the EU guidelines on minimizing the risk of BSE transmission via medicinal products, CPMP/BWP/877/96, recommends the main measures to be implemented in order to establish the safety of bovine material versus the BSE risk. Again, similarly the focus is on geographical origin, the age of the animals, the breeding and slaughtering conditions, the tissue to be used and the conditions of it’s processing.

The use of FBS in production processes of medicinal products is acceptable provided good documentation on sourcing, age of the animals and testing for the absence of adventitious agents is submitted. All responsible suppliers of FBS for bio-pharmaceutical applications will provide such documentation.

Recent regulatory requirements in Europe stress the importance of justifying the use of material of bovine, caprine or ovine origin in the production of pharmaceutical products. Thus, although FBS has been used for many years in the production process of many medicinal products such as viral vaccines , recombinant DNA products and ELISA Plate, at present there is a justified trend to remove all material of animal origin from manufacturing processes. Sigma-Aldrich has recognized this growing trend and works closely with customers to optimize animal free media formulations to meet each customer’s cell culture requirements.

Similarly the FDA has similar guidelines when accepting regulatory submissions. The FDA regulates all medicinal products for Human use, such as therapeutics, vaccines ,diagnostics and Cell Culture Plates, and, usually, the United States Department Agriculture (USDA) are not involved.

More infomation from Sigma-Aldrich

Large numbers of cell lines look identical. Cell lines with very different origins and biological characteristics typically cannot be separated on grounds of morphology or culture characteristics. Infection or contamination of a cell line with an adventitious virus or mycoplasma may significantly change the characteristics of the cells but again such contamination will be inapparent. Cell lines will also change with time in culture(even in glass bottom dishes/elisa plate), and to add to all these natural hazards it is all too easy to mis-label or cross-contaminate different cell lines in a busy cell culture laboratory.

The opportunities for inadvertently introducing error into a cell line are limitless and ever present. It is in the nature of the science that, once introduced, an error will be propagated, compounded, consolidated and disseminated.

The integrity and biological characteristics of a cell line have to be actively maintained by a well-organized system of “husbandry” based on systematic cell banking supported by testing regimens in a structured quality assured environment. Such a controlled environment will only prevail in a dedicated professionally organized cell culture laboratory or cell bank. A small research laboratory with a high throughput of short-term research students, a minimum of permanent laboratory staff and no formal quality management program will find it difficult to maintain its cell lines unchanged over many years.

For all these reasons it is strongly recommended that new cell lines should only be acquired from a specialist, reputable culture collection such as ECACC. Moreover, if a laboratory believes it already has a certain cell line in its liquid nitrogen store, the identity and purity of such a cell line should be questioned in the absence of a well-recorded culture history and recent test data. If there is a doubt, it is straightforward and cost effective to replace such cell stocks with authenticated material from a Culture Collection.

When a Cell Culture Collection “accessions” a new cell line it will characterize the cell line using techniques such as isoenzyme analysis and DNA profiling so that the identity of the cell line can subsequently be verified. The Collection will then establish a hierarchy of Master and Working cell banks, cryopreserved in liquid nitrogen, that are demonstrated free from microbial contamination including mycoplasma. Customers are supplied from these authenticated Working Cell Banks (WCB). Replacement WCB's are manufactured from the original Master Cell Bank (MCB) and the new WCB will again be fully tested.

ECACC supplies its cell lines together with advice on how to maintain the line. A Technical Support team will subsequently assist with any difficulties and can often provide additional technical information about the cell line. Culture Collections exist to ensure that animal cell research is conducted using standardized, authenticated material that ensures the work can be reproduced(such as Glass Bottom Cell Culture Dishes, 96 well plate etc). An authenticated cell line of known provenance is the very "bed rock" of any cell based project.

Source: ECACC Handbook-SIGMA

Disinfection

Methods designed for the disinfection/decontamination of culture waste, work surfaces and equipment represent important means for minimizing the risk of harm.

The major disinfectants fall into four groups and their relative merits can be summarized as follows:

Hypochlorites (e.g. Chloros, Presept)

• Good general purpose disinfectant
• Active against viruses
• Corrosive against metals and therefore should not be used on metal surfaces e.g. centrifuges
• Readily inactivated by organic matter and therefore should be made fresh daily
• Should be used at 1000ppm for general use surface disinfection, 2500ppm in discard waste pots for washing pipettes, and 10,000ppm for tissue culture waste and spillage

Phenolics (e.g. Sudol, Hycolin)

• Not active against viruses
• Remains active in the presence of organic matter

Alcohol (e.g. ethanol, isopropanol)

• Effective concentrations 70% for ethanol, 60-70% for isopropanol
• Their mode of activity is by dehydration and fixation
• Effective against bacteria. Ethanol is effective against most viruses but not nonenveloped viruses
• Isopropanol is not effective against viruses

Aldehydes (e.g. glutaraldehyde, formaldehyde)

• Aldehydes are irritants and their use should be limited due to problems of sensitization
• Glutaraldehyde may be used in situations where the use of hypochlorites is not suitable e.g. cleaning of centrifuge bowls or materials constructed of stainless steel that may be attacked or corroded by using hypochlorite solutions.

Waste Disposal

Any employer has a ‘duty of care’ to dispose of all biological waste safely in accordance with national legislative requirements. Given below is a list of ways in which tissue culture waste can be decontaminated and disposed of safely(especially the solid waste, such as flasks, centrifuge tubes, contaminated golves etc). One of the most important aspects of the management of all laboratory-generated waste is to dispose of waste regularly and not to allow the amounts to build up. The best approach is ‘little and often’. Different forms of waste require different treatment.

• Tissue culture waste (culture medium) - Inactivate overnight in a solution of hypochlorite (10,000ppm) prior to disposal to drain with an excess of water
• Contaminated pipettes should be placed in hypochlorite solution (2500ppm) overnight before disposal by autoclaving and incineration
• Solid waste, such as flasks, centrifuge tubes(such as 15ml Centrifuge Tube, 50ml Centrifuge Tube), contaminated gloves, tissues etc. should be placed inside heavy duty sacks for contaminated waste and autoclaved prior to incineration. These bags are available from Bibby Sterilin and Greiner.
• If at all possible waste should be incinerated rather than autoclaved

Source: Sigma-Aldrich

The main aim of risk assessment is to prevent injury, protect property and avoid harm to individuals and the environment. The performance of risk assessment is a legal requirement under the Health and Safety at Work Act, UK. There are other EC directives covering Health and Safety at Work, you can visit the European Agency for Safety and Health at Work website www.europe.osha.eu.int for information on legislation and standards, or you should contact your on-site representative. Consequently risk assessments must be undertaken prior to starting any activity. The assessment consists of 2 elements:

1. Identifying and evaluating the risks.
2. Defining ways of minimizing or avoiding the risk.

For animal cell culture the level of risk is dependent upon the cell line to be used and is based on whether the cell line is likely to cause harm to humans. The different classifications are given below:

• Non human/non primate continuous cell lines and some well characterized human diploid lines of finite lifespan (e.g. MRC-5).

• Poorly characterized mammalian cell lines.

• Cell lines derived from human/primate tissue or blood.
• Cell lines with endogenous pathogens (the precise categorization is dependent upon the pathogen) – refer to ACDP guidelines, 1985, for details.
• Low quality Cell Culture Dishs, Flasks adn Plates.
• Cell lines used following experimental infection where the categorization is dependent upon the infecting agent - refer to ACDP guidelines, 1985, for details*.

*Advisory Committee on Dangerous Pathogens (1985) Categorization of Biological Agents According to Hazard and Categories of Containment, 4th edition, HSE books, Sudbury, UK

A culture collection, such as ECACC will recommend a minimum the containment level required for a given cell line based upon its risk assessment. For most cell lines the appropriate level of containment is Category 2. However, this may need to be increased to Category 3 depending upon the type of manipulations to be carried out and whether large culture volumes are envisaged. For cell lines derived from patients with HIV or HTLV Category 3 containment is required.

Containment is the most obvious means of reducing risk. Other less obvious measures include restricting the movement of staff and equipment into and out of laboratories, especially the Cell Culture Dish(35mm Cell Culture Dish, 60mm Cell Culture Dish, 100mm Cell Culture Dish). Good laboratory practice and good bench techniques such as ensuring work areas are uncluttered, reagents are correctly labeled and stored, are also important for reducing risk and making the laboratory a safe environment in which to work. Staff training and the use of written standard operating procedures and risk assessments will also reduce the potential for harm. Training courses covering the basics of tissue culture safety are offered by ECACC.

Source: ECACC Handbook

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.

Schematic diagram of "Subculture of Adherent Cell Lines"

Materials

• Media– pre-warmed to 37oC (refer to the ECACC Cell Line Data Sheet for the correct medium)
• 70% ethanol in water
• PBS without Ca2+/Mg2+
• 0.25% trypsin/EDTA in HBSS, without Ca2+/Mg2+
• Trypsin
• ELISA Plate
• Soybean trypsin Inhibitor

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
• Cell Culture Plates(96-well plate)
• 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+ 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 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.
8. Transfer the required number of cells to a new labeled flask containing pre-warmed medium.
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 and 96 well plate, 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+.
4. Cells should only be exposed to trypsin/EDTA 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, 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 plates.
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.

Source: Sigmaaldrich

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

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

The "Do's and Don'ts" of Cell Culture

Since it was established in 1981, the Cell Culture Facility has made it easier, more economical and more effective to use mammalian cell cultures in research at Fox Chase. Cell culture procedures provide the cellular materials for many of the molecular methods used to study normal and cancer cell proliferation, cellular regulatory mechanisms, and normal and abnormal development. As a result, cell culture has become a core technique in current molecular and cell biological research.

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 e.g. cell culture plate. 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.
10. Anyway, Don't break the Cell Culture Plates(6 well cell culture plate, 24 well cell culture plate, 96 well cell culture plate).