ICLAC: Additional Problems Confronting Culture Collections

Content to Accompany Section B

  • Fig. 1: Temperature changes during cryopreservation. Temperature data collected from a sample cryovial containing cryoprotectant (90% fetal bovine serum, 10% DMSO). Left panel, sample cooled at -1 oC/minute in a rate-controlled freezer, with no compensation for changes within the sample. The sudden increase in temperature at around 45 minutes is caused by release of heat during ice crystal formation. Right panel, sample cooled in a foam box used as a simple rate-controlled freezing apparatus. The heat released during ice crystal formation is absorbed, minimizing its impact on cell viability.Fig. 1: Temperature changes during cryopreservation. Temperature data collected from a sample cryovial containing cryoprotectant (90% fetal bovine serum, 10% DMSO). Left panel, sample cooled at -1 oC/minute in a rate-controlled freezer, with no compensation for changes within the sample. The sudden increase in temperature at around 45 minutes is caused by release of heat during ice crystal formation. Right panel, sample cooled in a foam box used as a simple rate-controlled freezing apparatus. The heat released during ice crystal formation is absorbed, minimizing its impact on cell viability.

ICLAC - Additional Content to Accompany Section B. The information belongs to an article, which is the third part in a series from members and colleagues of the International Cell Line Authentication Committee (ICLAC). In the first article, 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 third article deals with the challenges to achieving good cell culture quality and describe how cell lines are handled and shipped. Here, online content looks in detail at common cell culture problems that are seen by culture collections.

Read the full article on Cell Banking - part 3 here:
http://bit.ly/ICLAC-3

1. Law, Ethics and Traceability Requirements
It is important for Culture Collections to determine that cell lines have been sourced ethically and with appropriate patient consent, and that the ownership of a cell line is clear considering all potential stakeholders (donor, family, institution, funding agency).
The provenance of the cell line is vital. This not only includes the identity of the cell line, but also any clinical diagnostic and phenotypic information (so called "metadata"). Information should be examined to ensure it is accurate and untraceable to the donor.
Culture Collections must be exemplary in their record keeping and documentation and ensure that cell line provenance is recorded.
The designation, or name, of the cell line is important and Culture Collections should ensure that every cell line is uniquely identified. Hence, the cell line should be stored together with a unique reference ID number (accession number).

2. Recovering Frozen Vials of Good Quality
Production of a cryopreserved cell bank is the most secure way to stabilize and store a cell line. However, it is not an antidote to poor cell culture practices. Cryopreservation simply provides an indefinite hold point where the cells can be stored in stasis, and because of this, poor recovery of cells from frozen cryovials is one of the most difficult issues in cell culture to trouble-shoot.

Low viability or poor plating efficiencies of cell banks may be due to: poor upstream cell technique (often years or decades previously), suboptimal storage or shipping (Fig. 1), or poor resuscitation technique. Technical enquiries regarding revival of cells from frozen storage are perhaps the most commonly reported issues from end users of Culture Collections.

For good cryopreservation, it is vitally important that cell culturing upstream of cell banking is well controlled and optimized. Any situation that is stressful for the cells may affect cell viability, and place a selective pressure resulting in the development of a subpopulation that no longer represents the original culture.

For most continuous cell lines, the growth characteristics of the cells should be ascertained (e.g., by determining a growth profile), and the cells maintained in log-phase growth. Anchorage-dependent cultures that are over-confluent and differentiated may be difficult to remove from the substrate by trypsinization, producing cultures that recover poorly from cryopreservation [2].

Unexpected changes in behaviour after thawing (e.g., unexpected morphology or growth rate) may be symptomatic of a cross-contamination or mislabeling event prior to cryopreservation. If unexpected changes are noted, the cell line's identity should be checked.

Cells are generally mixed with a cryoprotectant such as dimethylsulfoxide (DMSO) before freezing. A typical cryoprotectant solution consists of a 90% (v/v mix) of culture medium or fetal bovine serum (FBS), with 10% (v/v) DMSO. Despite its common use, DMSO may not always be suitable. DMSO can induce differentiation in cultures, particularly leucocyte-derived cell lines or stem cell cultures. In such cases, DMSO should be used at lower concentrations, or an alternative cryoprotectant such as glycerol should be used. Although 10% DMSO is quoted as the norm in many cell culture protocols, this concentration may not be optimal for all cell lines, and often 5% DMSO or even lower may be adequate.

To minimize toxicity, chilled (4 °C) reagents and cryovials should be used. Log-phase cultures of cells should be pelleted by gentle centrifugation, resuspended at a cell density of 1-20 million cells/ml, and aliquoted into vials, usually at a volume of 1 ml/vial. Once filled, the vials should be immediately transferred to the rate-controlled freezing apparatus.

A rate-controlled freezing apparatus may be something as simple as an expanded polystyrene box, or a "Mr Frosty" style container, placed overnight in a -80 °C freezer; or a specialized programmable rate-controlled freezer. Whatever the method selected, it is critical that the rate of freezing is slow, in the order of 1-2 °C/minute, to a temperature of -70 °C or lower. If using a rate-controlled freezer, it is important to compensate for the release of heat during ice crystal formation, which can impair viability if not dissipated effectively (Fig. 1).

Once the cells reach -70 °C, they should be immediately transferred to liquid nitrogen storage. Temperatures should be kept below the glass transition point (GTP) of water, generally agreed to be -136 °C [3]. This is the point at which liquid water becomes static, putting biological systems into stasis [4]; theoretically, below this point, cells can be stored for millennia. Any excursions above the GTP will result in the reactivation of biological processes and subsequent deterioration of the samples. There is evidence that storage of cells at -80 °C can result in reduced viability.

Once frozen and stored below the GTP, at least one cryovial from the cell bank should be thawed, and its viability and any other key characteristics checked. Vials should be stored in at least two separate freezers to mitigate the risk of losing the entire cell bank due to mechanical or power failure.

3. Problems with Misidentified Cell Lines
The use of misidentified cell lines in life science research is a chronic problem that can impact Culture Collections and researchers in several ways.

Problem A. Using a known misidentified cell line as a model for the original tissue type or disease state
Misuse of cell lines that have been proven to be misidentified is wholly inexcusable, yet easy to find in literature searches. Although a serious issue in life science in general, it does not seriously impact on Cell Collections, which can easily identify rogue cell lines through ICLAC's misidentified cell line database (www.iclac.org). However, the problem is compounded by the fact that many misidentified cell lines, particularly HeLa derivatives such as HEp-2 and Chang Liver, are now phenotypically dissimilar to the parental HeLa cell line. This may make certain misidentified cell lines useful in their own right for specific applications. It is important that collections describe these "variants" accurately so as not to mislead end users. All misidentified cell lines should be clearly linked to the contaminant, which quickly outgrows and replaces the original culture. This link should be clearly stated for the end user, preferably as part of the cell line name, and the correct tissue type and disease state (that of the contaminant) should be listed.

Problem B. Using misidentified stocks of a cell line that is known to be authentic
Scientists may be working with misidentified stocks of a known "authentic" cell line due to a historic mix-up in their own lab, or through the sourcing of non-authenticated stocks from a colleague. This problem is a major concern and estimated to affect up to 20% of researchers without their knowledge. In these cases, Culture Collections can only advise on best practice and work as closely as possible with end users to try to ensure that authentic materials are used.

Ultimately, this problem can only be controlled by researchers themselves. The risk can be minimized by performing authentication testing of cell banks; sourcing authentic materials from recognized Culture Collections; working to best practice guidelines; and ensuring that staff are adequately trained in the theory, dangers and safety measures required for Good Cell Culture Practice [5,6]. All cell culture processes should be supported by Standard Operational Procedures (SOPs), robust documentation, and policies that prevent the sharing of reagents between cell lines.

Problem C. Failure to authenticate newly established cell lines prior to publication
The third way misidentification can strike is one of the easiest to detect by a Culture Collection, but has the most serious impact on the researcher and potentially the cell repository itself. In this case, a researcher or research team will have developed a novel human cell line or panel of cell lines. Often novel cell lines will be well characterized in terms of phenotype, but will lack basic identity testing. Results from these cells may have been published, and the cells may be in use in several other collaborating laboratories, before they are deposited. It is not unusual for a Culture Collection to discover, through its routine quality control testing, that these cell lines are mixed up across the panel and/or cross-contaminated with other authentic cell lines.

This issue has affected many researchers during the development of cancer cell line panels. Currently, the risk applies to labs across the world that are generating large panels of Induced Pluripotent Stem Cells (iPSCs). Like many other cell types, iPSCs can be easily mixed up or contaminated during initiation. The point at which cultures became mixed up may be difficult or impossible to determine.

The experiences of many research labs highlight the importance of incorporating a cell line authentication strategy into the cell line development process; the benefits of bio-banking samples of original tissue for future identity verification; and the need to submit cultures to a cell repository as soon as possible to preserve cell line integrity [7].

When a Culture Collection uncovers a new misidentified cell line in this way, the issue has to be managed sensitively and carefully, as it could lead to retractions of papers from journals and damage to the originating scientists' reputations.

Problem D. Failure to interpret authentication test results correctly in publications
The final effect of misidentification comes from publications where scientists have published authentication data, but neither the authors nor the peer reviewers have thoroughly checked the data against available online datasets (e.g., the DSMZ's interactive database of STR profiles (https://www.dsmz.de/services/services-human-and-animal-cell-lines/online-str-analysis.html)). This lack of comparison results in potentially invalid papers passing peer review and entering the literature. Culture Collections can play a part, along with ICLAC, by identifying these cases and approaching the original authors and journal editorial boards in an attempt to rectify these erroneous articles.

4. Mycoplasma-Contaminated Cell Lines and the Need to Quarantine
Any cell line supplied by a cell repository should be free from microbial contamination; every cell bank produced will have been tested for microbial contamination.

Cryptic mycoplasma infection of cell lines can only be detected through testing and is a common cell culture problem. It is important that Culture Collections keep any potentially contaminated cultures isolated from their "clean" banking operations. Newly deposited cell lines must be maintained in a dedicated quarantine laboratory until mycoplasma testing has been performed using at least two complementary tests (e.g., PCR, direct culture isolation, or DNA fluorochrome (Hoechst) staining).

As with the reporting of cell line misidentification, the reporting of a positive mycoplasma result to a cell line depositor can be devastating news. Mycoplasma treatment can be undertaken using an appropriate antimicrobial. However, attempts to eradicate the infection can have a detrimental, irreversible effect on the cells' phenotype. It is important to quarantine the cell line during treatment and for a prolonged period thereafter, lest the infection return. Repeated testing is also important. All test methods have a limit of detection, and the mycoplasma titre may fall to below that level in response to treatment. Ongoing testing will detect incomplete eradication or subsequent re-contamination.

5. Misuse of Antibiotics
The need for cell lines to be free from contamination with other microbes (bacteria and fungi) is often confounded by adding prophylactic antibiotics such as penicillin and streptomycin. Unless there is a specific requirement for antibiotics, for example to maintain the expression of a transfected gene, Culture Collections will always work "antibiotic-free".

In general, overuse of antibiotics relates to handling cell lines that are already contaminated, or as an excuse for poor technique. An infection will often manifest itself after deposit, in a cell line that has been assumed by the depositor to be pure and free from infection. Again this can be difficult news to report, particularly if the previous prolonged use of antibiotics has resulted in a multi-resistant bacterial infection that cannot be eradicated.

6. Lack of Adequate Segregation
Segregated cell culture and banking is important in all laboratories, but especially so in a Culture Collection. Only one cell line should be handled in a biological safety cabinet at any time, and all culture media and reagents should be dedicated to individual cell lines, to prevent any chance of cross-contamination.
This is particularly important in the cultivation of species where there are no readily available tests for individual identity. Forensic STR testing approaches can be used to detect cross-contamination between individual human cell lines in post-production analysis. A cross-contamination event affecting two or more cell lines from the same non-human species may go undetected, due to the limitations of the testing technologies. Careful planning is needed for scheduling and segregation of non-human cell lines to avoid overlaps during cell banking in the lab.

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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