ICLAC: Cell Banking

Reliable Storage of Authentic Cell Lines

  • Fig. 1: Evolution of authentication testing methods for human cells. Methods represented here include karyotyping (A); isoenzyme analysis (B); and STR profiling (C). All of these methods are effective ways to determine cell line identity. The evolution of test methods has enabled standardization and direct comparison of results between Culture Collections and testing centers worldwide.Fig. 1: Evolution of authentication testing methods for human cells. Methods represented here include karyotyping (A); isoenzyme analysis (B); and STR profiling (C). All of these methods are effective ways to determine cell line identity. The evolution of test methods has enabled standardization and direct comparison of results between Culture Collections and testing centers worldwide.

This article is the third from members and colleagues of the International Cell Line Authentication Committee (ICLAC). In the first article, the ICLAC discussed why quality is important for cell lines used in research laboratories (take a look at the first part: http://bit.ly/ICLAC-1). The second article focused on the advantages of obtaining cell lines from a cell repository or culture collection, and the authentication testing that repositories perform (part 2 at http://bit.ly/ICLAC-2). But how do such collections handle their own cell lines? This article deals with the challenges to achieving good cell culture quality and describe how cell lines are handled and shipped.

A. Acquiring Cell Lines - Retentive Authors Hamper Authentication
While most accept that cell lines sourced from colleagues or collaborators should be authenticated before use, fewer perhaps realize that this duty applies equally to cell lines obtained from originators.

When the DSMZ first quantified cross-contamination 15 years ago among originator supplied material, we were shocked to find that approximately 17% were false. Now that all major cell repositories routinely perform STR profiling, false cell lines are identified via their shared DNA profiles and rapidly weeded out. Hence, examples sourced from public Culture Collections in Germany (DSMZ), Japan (JCRB, Riken), United Kingdom (ECACC) and USA (ATCC) have been authenticated.

Alas, only a minority of existing cell lines are held by public cell repositories. Most new examples are retained by their originators for private research, pecuniary gain (seldom realized), bargaining against co-authorships, or plain old miserly gloating [1]. Originators who retain their cell lines may find it harder to perceive their shortcomings than outsiders; as the old proverb tells us, "in the eyes of the mother crow all her chicks are white". In bypassing cell repositories, "private" cell lines also circumvent rigorous authentication. Unsurprisingly, retentiveness blights novel cell lines the most (e.g., those modelling rare or low grade tumors which may be difficult to establish).

It is high time, therefore, that journals and funding agencies mandate public reposition.

B. Additional Problems Confronting Culture Collections
Whether a cell line is cultured by its originator, their colleagues, or a cell repository, the same rules apply regarding good cell culture practice. Cell lines have common problems that may affect anyone who handles or stores cell lines routinely. Culture Collections see all of these problems first hand, and have experience with how to respond to each problem and the relevant best practice guidelines that will help to avoid that problem in future.
The challenges commonly encountered by collections are listed below. More information on each of these challenges can be found in the online version of this article at http://bit.ly/ICLAC-3B.

1. Law, ethics and traceability requirements
2. Recovering frozen vials of good quality (including Fig. 1, see online at http://bit.ly/ICLAC-3B)
3. Problems with misidentified cell lines
4. Mycoplasma contaminated cell lines and the need to quarantine
5. Misuse of antibiotics
6. Lack of adequate segregation

C. What Happens between Receipt and Shipment?
Whether a cell line is acquired by a repository organization or a commercial or research entity, certain steps should be taken as soon as possible after receipt. Quality control testing should be performed, with authentication to confirm correspondence to donor tissue and/or to exclude misidentification; phenotypic characterization; and assurance of purity. This is followed by expansion of the cells to generate a master cell bank, and confirmation of the attributes of the master cell bank. Additional (working) cell banks may be prepared, depending on the nature of the cells and the needs of the organization.

1. Quality Control Testing
The first steps of quality control testing can be performed during the early stages of cell expansion. There is little sense in expending effort on a cell line unless it is reliably identified and found to be pure.

For human cells, authentication is best achieved through short tandem repeat (STR) profiling [8] to establish donor-level identification (Fig. 2). In the absence of reference donor material, "uniqueness" (among known sample STR profiles listed by cell banks) remains the key criterion. For animal cells (including insects), species-level identification through isoenzyme analysis or sequence-based barcoding [9] methods may be the best that can be done at this time. Alternately, for human cancer cell lines cytogenetic analysis may be used to augment STR profiling with reference to published karyotypes, while providing rapid species identification for animal cells. Phenotypic characterization may be necessary to assure that the cell line "performs" as expected in the hands of its new owner. This may include evaluation of attributes such as cell morphology, doubling time, receptor status, protein secretion, etc. Such phenotypic analysis is separate from authentication testing, which focuses on genotype and compares the results to donor material or other cell lines that may act as contaminants.

The final quality check for all incoming cells is the assurance of purity. For a cell line, establishing purity means demonstrating that the culture is free of cross-contaminating cells, and that the cells are free of adventitious agents (primarily mycoplasma, bacteria, and viruses). Freedom from viruses is especially important for a primary human cell line but is often not assessed. To help assure the safety of those handling the cell line, it should be demonstrated that the cell line is free from human immunodeficiency virus types 1 and 2, human T-lymphotropic virus types 1 and 2, and hepatitis A, B, and C viruses. These potential viral contaminants may be detected using PCR, so testing can be performed on DNA extracted from cells prior to routine culturing.
Prior to acquiring a cell line, evidence of the safety testing mentioned above should be requested from the originator. If such evidence cannot be provided, the cell line should be handled under appropriate biosafety containment conditions until the viral safety profile can be established. It should also be noted that cell lines can acquire adventitious agents during the culture process, from reagents or from other cell lines. There will always be a risk of introducing new viruses into cell cultures as long as we continue to use animal-derived sera and other components, and animal-derived trypsin and other enzymes to subculture cells [5].

2. Preparation of a Master Cell Bank and Working Cell Bank
Provided that the initial characterization of the incoming cell line is satisfactory, the amplified cells can be used to prepare a master cell bank. Depending on the expected use of the cell line, and on the nature of the cell line itself (finite vs. continuous), cells from the master cell bank may be used to generate additional (working) cell banks. Some cell lines, notably those with normal karyotypes, are passage-limited and the cell amplification required for cell banking may allow only for generation of a master cell bank.

The use of a master cell bank and (where required) a working cell bank allows for repeated restarts of a cell line at a relatively low passage level, without having to go back to the cell line originator. It also minimizes the need for repeated quality control testing. Rather than continuing to perform quality control testing at regular intervals, a representative cryovial from each cell bank can be tested, and results will apply to the entire series of vials from that bank. Each cell bank should, at a minimum, be checked for post-thaw viability, authenticity, and mycoplasma contamination.

D. Shipping and Distribution of Cell Lines
Completion of the cell banking process means that pure, authentic stocks of that cell line will be reliably available for future use. A Culture Collection will distribute those stocks to many laboratories, and staff members have experience with the problems that may arise during the shipping process.
The points that should be considered for effective shipment are listed below. More information on each of these points can be found in the online version of this article at: http://bit.ly/ICLAC-3D.

1. Preparation before commencement
2. Shipping frozen cryovials
3. Shipping growing cultures
4. Shipment across international borders
5. Cites (the Convention on International Trade in Endangered Species of Wild Fauna and Flora)
6. Biosafety considerations
7. On delivery

It is important to consider all of these points to minimize the risks associated with cell line shipment. Attention to detail during shipment, thawing, and subsequent handling will help to make experimental work with cultured cells more reliable and reproducible.

Acknowledgements
Thanks to Deborah Beer, Lisa Purvis and Marzena Humphreys of the Culture Collections of Public Health England for advice on shipping cells, and to George Theodosopoulos of CellBank Australia for the STR profile in Figure 2C.

References
1. MacLeod R.A.F. & Drexler H.G.: Nature 439, 912 (2006).
2. Stacey G. N. et al.: Animal Cell Culture: Essential Methods (Davis, J. M. ed.) 185-203 (John Wiley & Sons, Ltd, 2011).
3. Capaccioli S. & Ngai K. L.: J. Chem. Phys. 135, 104504 (2011).
4. Mazur P.: Am. J. Physiol. 247, C125-C142 (1984).
5. Geraghty R.J. et al.: Br. J. Cancer, in press, DOI 10.1038/bjc.2014.166.
6. Coecke, S. et al.: Altern. Lab. Anim. 33, 261-287 (2005).
7. Freshney R. I.: in Culture of Animal Cells, 317-334 (John Wiley & Sons, Inc., 6th edition, 2010).
8. ANSI/ATCC ASN-0002-2011: Authentication of Human Cell Lines; Standardisation of STR Profiling. ANSI eStandards Store (2012).
9. Nims R. W. et al.: In Vitro Cell. Dev. Biol. Anim. 46, 811-819 (2010).
10. Begley C. G. & Ellis L. M.: Nature 483, 531-533 (2012).

Authors
Jim Cooper1, Ed Burnett1, Roderick A.F. MacLeod2, Ray Nims3, Elsa Moy4, Amanda Capes-Davis4

1Culture Collections Public Health England, Porton Down, UK

2Leibniz-Institut, Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany

3RMC Pharmaceutical Solutions, Inc., Longmont, CO, USA

4CellBank Australia, Children's Medical Research Institute (CMRI), Westmead, Australia

 

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