Science

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

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