Sourcing of Cell Lines

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

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

 Cell culture is the process by which prokaryotic, eukaryotic or plant cells are grown under controlled conditions. In practice the term “cell culture” has come to refer to the culturing of cells derived from multicellular eukaryotes, especially animal cells. The historical development and methods of cell culture are closely interrelated to those of tissue culture and organ culture.

Animal cell culture became a routine laboratory technique in the 1950s, but the concept of maintaining live cell lines separated from their original tissue source was discovered in the 19th century.

–From wikipeia

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– 35mm Cell Culture Dish
– 60mm Cell Culture Dish
– 100mm Cell Culture Dish
– 35mm Glass Bottom Culture Dish

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