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Caring for Cell Cultures

The lab manager's role in preventing and detecting of misidentification and cross-contamination of cell lines.

by Roland M. Nardone, Ph.D.
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The rampant expansion of misidentification and cross-contamination of cultured cell lines presents a unique challenge for managers overseeing research and production units using cultured cells as research tools or as substrates for the production of products such as vaccines and secreted proteins. The challenge stems from the fact that cell culture technology is highly specialized with many subtleties, nuances and alternative experimental approaches that may be alien to the manager who has oversight responsibilities for protocols that can be rendered counterproductive if not executed in a very faithful fashion. The challenge is further magnified by a broad range of individuality on the part of the cell line being cultured as well as the worker at the bench. 

Causes, prevention and the lab manager
The oversight responsibilities of the lab manager include the proper maintenance of a cell bank for the cell lines and the eventual use of the cells in the bank for experiments or production. The cell banks assure that an adequate supply of cells will be available over the undetermined or determined expected lifespan of experimental need. Indeed, uncertainties regarding future use compel some persons to set up their bank with sufficient ampules of cells to accommodate needs almost into perpetuity. Cell banking also provides sufficient cells for extensive characterization and testing for adventitious organisms as well as providing insurance against laboratory/experimental catastrophes such as contamination and power outages. The usual cell bank system consists of two tiers: a master cell bank (MCB) and a working cell bank (WCB).
The MCB represents a collection of cryopreserved ampules of cells of one type having been derived from a single source and prepared under defined culture conditions. The source of the WCB are the ampules of cells in the MCB. One or more vials (pooled) provide a culture(s) to be inoculated into flasks or other containers for expansion by replication followed by subculturing, and if needed, additional rounds of proliferation and subculturing. After expansion, the cells are pooled, aliquoted into cryotubes, and transferred into liquid nitrogen and designated as the WCB. Authentication of the cells and other characterization is usually done on the cells in the MCB, although some circumstances, such as protracted growth and subculturing of the “working” cells may warrant authentication/
characterization of the cells of the WCB, sometimes in a more limited way.
We are fortunate that we have good information on the causes and appropriate remedial steps to take to prevent cross-contamination and misidentification of cell lines in the cell banks as well as those that are growing. The laboratory manager must insure, directly or indirectly, that all personnel involved in cell culture work understand the unpredictable and costly consequences of breaches or deviations from an effective protocol predicated on rigorous quality control principles. Actual demonstration of pertinent knowledge of the causes and prevention
of cross-contamination and misidentification should be a key factor in deciding to allow a worker to be involved in the handling of cultured cells, especially those in the banks.
The need for this caution stems from the fact that many otherwise competent investigators and technicians have been lured by the potential rewards of cell culture technology but do not have adequate training regarding rigorous quality control; nor do they have sufficient understanding of the origin and prevention of misidentification and cross-contamination. Hence, careful scrutiny of the worker‘s qualification in this particular area must replace the “free pass” that is often given to persons with very good credentials.
This statement is based on 40 years of research experience with cultured cells and as a sponsor of cell culture training courses offered at the National Institutes of Health and elsewhere. More importantly, it may explain, at least in part, why cross-contamination continues to be rampant. Also contributing to the existing chaos are situations where several persons may share in an unspecified and uncontrolled manner responsibilities for one cell line. Whenever possible, limit the handling of a specific cell line to one person and a back-up. The back-up person will have familiarity with the cell line and the protocol, thereby will be ready to step in when an emergency arises.
Adherence to the following guidelines will minimize the probability of misidentification and cross contamination of cell cultures.
  1. 1. Handle cell cultures at a time of day when distractions and fatigue will be minimal
  2. Work in a certified vertical laminar flow hood. Before and after working with cultures thoroughly wipe the inside of the hood with a disinfectant.
  3. Work with only one cell line or lineage in the hood at one time. Limit the number of flasks you remove from the incubator at one time lest they experience a severe temperature and pH change.
  1. Disinfect the hood prior to and after each cell line is fed, sub-cultured, or handled for other purposes.
  2. Each cell line or lineage must be fed, rinsed, or trypsinized , etc. with reagent specifically dedicated for that line. In other words, never share reagents among different cell lines, even if the cell lines are maintained in the same medium formulation. A dedicated set of reagents and supplies should be assigned for each cell line
  3. Plan ahead to ensure that an adequate supply of reagents and other materials will be available. If you find your supply lacking at an inopportune time you may be tempted to break the “no sharing” cardinal rule.
  4. A pipette should be used only once. Fill and dispense only once. Never reinsert a used pipette into a bottle of reagent.
  5. Examine and evaluate the cultures before they are brought to the hood. Check the cultures for growth and morphological characteristics. Become familiar with alterations in morphology due to crowding or changes in medium formulation. Establish a collection of reference slides and/or photographs for future use.
  6. Whenever possible, use authenticated cell lines obtained from a repository with high standards for certification and quality control. Establish and adhere to a logical schedule for reevaluation of authenticity. Do not distribute cell lines if those lines can be procured from a repository. Do not distribute cell lines unless they have been authenticated.

A review of a variety of cell line characterization methods

Misidentification and cross-contamination of cell lines surfaced almost fifty years ago, yet this problem continues unabated. Thus, millions of dollars are wasted and the published literature continues to be corrupted, perhaps at a rate in excess of 20%. The causes of misidentification and cross-contamination are known and can be minimized or prevented. Robust methods exist to detect interspecies and intraspecies problems. These include DNA profiling, chromosome analysis, and isoenzyme profiling. DNA profiling by analysis of short tandem repeats has become the method of choice by major repositories and is being adopted by researchers to identify the individual donor. The laboratory manager must ensure that employees trusted with the care of cell cultures are more than dexterous. They must also be cognizant of and sensitive to causes, prevention and consequences of misidentification and cross-contamination.