ICLAC: Shipping and Distribution of Cell Lines
Content to Accompany Section D
ICLAC - Additional Content to Accompany Section D. 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, 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 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 at shipment of cell lines in detail.
Read the full article on Cell Banking - part 3 here:
1. Before Commencement
Before shipping a cell line to another organization, whether locally or geographically distant, it is essential that all of the various legal requirements for import and export have been considered [5,6]. Legal requirements may relate to ethical issues, disease control, endangered species permits, and bioterrorism regulations.
All necessary documentation for transit and receipt should be placed securely on the outer packaging so it can be consulted before opening. The package must be appropriately labeled with United Nations (UN) hazard category and the correct accompanying customs documents to avoid unwanted hold-ups.
In recent times, there have been concerns about reproducibility in Life Science Research . Distribution of cells to another lab should be considered as technology transfer; adequate technical data and information is critical to accompany the cells, improving scientific reproducibility .
Accompanying information may include when the cells will arrive and by what method; what to do with the material on arrival; advised storage conditions; growth conditions including medium composition, serum specification and additives; and how to subculture the cells, including advised split ratios and seeding densities. Inclusion of an expected growth profile and reference photomicrographs could be extremely helpful to manage expectations.
2. Shipping Frozen Cryovials
Frozen cryovials should be shipped either on solid carbon dioxide (below -50 °C), or in a liquid nitrogen dry shipper. There are advantages to both processes, but regardless of the shipment method, the transit period should be as short as possible.
If solid carbon dioxide (dry ice) is used, then there must be an adequate mass to survive the shipping period with some contingency for shipping delays. Any mass calculations should include careful consideration of the ambient temperature (season and climate of the destination country). An insulated container (e.g., polystyrene box) with adequate volume and wall thickness should be used (Table 1), with an exit point to allow carbon dioxide gas to escape during transit.The main disadvantage of using dry ice is that its temperature (-78.5 °C) exceeds the glass transition point (GTP) of water [3,4], generally accepted to be -136 °C (3). In theory, any excursions in temperature above this point will lead to sample deterioration and loss of viability. Although this deterioration is likely to be negligible in dry ice for the relatively short period of 2-5 days required for most shipments, the damage is likely to be cumulative and irreversible, so repeated, uncontrolled shipments of the same cryovials on dry ice should be avoided.
Small-scale simulation trials of potentially sensitive materials should be performed to ensure they do not significantly deteriorate in transit, prior to shipping large batches of cells. In addition to assessing cell viability, plating efficiencies and growth characteristics should also be evaluated after transit. At ECACC, we have found that Automated Live Cell Microscopy is particularly useful in assessing the plating characteristics of cryopreserved cells, to determine if there has been significant insult to the vials.
Despite this disadvantage, there are some major advantages of dry ice shipping. Dry ice shipments present a relatively low price, and most couriers will have access to dry ice supplies, allowing the "re-icing" of packages. If properly packaged in a polystyrene-insulated box with a robust outer box, the risk of loss of insulation due to mishandling and subsequent rapid evaporation of cryogen is relatively low.
The second commonly used method for shipping frozen cryovials is a "dry shipper". Typically, this is a vacuum-insulated Dewar style container with an internal matrix designed to absorb liquid nitrogen. Absorbance of the liquid nitrogen by the internal matrix means there is no free liquid in the container, removing any risk of nitrogen spill. Vials are placed, usually on canes, into a cavity within the matrix.
Although costly, dry shippers maintain the cells below the GTP of water, and from a thermal perspective, are the preferred solution for cells that might be particularly sensitive to excursions above the GTP. The major disadvantage of the dry shipper is its reliance on a vacuum to provide the insulation. If the vessel's vacuum fails in transit due to mishandling or general wear and tear, the loss of insulation is rapid, resulting in a catastrophic temperature rise and (usually) loss of all samples. If shipping high value or rare cell lines, the shipment should always be split and sent on two different occasions to spread the risk.
A lab can purchase its own dry shipper and arrange carriage thereof, but there are specialized courier companies who supply a comprehensive service.
3. Shipping Growing Cultures
If the recipient's lab does not have liquid nitrogen capability, or if frozen shipment is not an option for other reasons, growing cultures can be shipped at room temperature.
Cells should be in mid log-phase growth before shipping to avoid medium exhaustion, and in the case of attached cultures, to help avoid the monolayer becoming detached in transit. The flask should be filled to the top with culture medium, to minimize potential shear damage to the cells and foaming due to medium sloshing around in the flask. Avoid using vented caps, and ensure there is adequate absorbent material within the packaging to soak up all the liquid inside if a spill occurs.
Ensure the package is clearly labeled as fragile, conforms to UN packaging and labeling requirements, and is clearly labeled "do not freeze".
4. Shipment across International Borders
Shipping of cell lines across international borders can be problematic and very often requires careful thought and planning, months in advance of the delivery. If regular international shipping is likely then it is important to establish a network and rapport with the relevant government departments. It is also helpful to develop a relationship with a courier company with expertise in international shipping using dry ice or dry shippers. A courier company that is willing to top up dry ice in transit can make all the difference between safe arrival of a frozen shipment, and thawing of cryovials on the dock before they reach the lab.
It is essential that packages are shipped with a dispatch note, a commercial invoice, and the appropriate licenses and official documents to satisfy customs and local and international regulatory bodies. These will vary depending on the country of origin, the destination, the biosafety level, and the species of the cell line. In all cases a Material Safety Data Sheet (MSDS) is essential. The outer packaging must be labeled with the appropriate UN Classification Code to advise the safe handling of the cultures during transit.
Often, recipient territories require a statement on letterhead stationery from the originating institute that the cells were obtained ethically.
The sheer size of some countries can be an issue, as can language barriers where English is not necessarily the universal language of logistics. Local and subjective interpretations of the regulations (even in the same port) can mean that similarly documented shipments may get through on one day but not another.
Examples of territory-specific regulators and the documents required are the United States Department of Agriculture (USDA) "Questions and Answers"; United States Food and Drug Administration (FDA) "Declaration"; Department of Agriculture, Fisheries and Forestry (DAFF); and Health Canada.
For non-human cell lines, all recipient countries will require an Export Health Certificate (sometimes referred to as a Veterinary Permit) that certifies the cells are free from specific pathogens, and will usually require the signature of a veterinary surgeon. Export Health Certificates are not required for shipping between European Union (EU) member states.
When shipping to a non-EU member from the UK, it is important to check that there is a pre-existing Health certificate template for the recipient country held by the Department for Environment, Food and Rural Affairs (DEFRA). If the recipient country does not have a template, then the DEFRA Policy Unit has to contact the recipient county for the drafting and agreeing of a new template, in an iterative process that can take a year or more.
In the UK, DEFRA are especially helpful in outlining the requirements of recipient countries. In most cases the essential information to be listed are: the species the cell line was sourced from; the storage fluid; a descriptive statement of the culture, including the origin of the serum; the history and origin of the cells; their intended use; the country of origin; storage temperature; and full details of the sender and recipient.
Shipping to Russia and Eastern Europe can be facilitated by using a geographically close distribution hub. For example, ECACC uses a redistribution node in Germany to act as a stepping stone to Russia. These distribution hubs can also help in countries where local politics and corruption can impede delivery.
China requires a Safety Declaration certifying the cells are non-hazardous and outlining their storage conditions.
To facilitate delivery to more difficult territories, Global Freight Forwarding (Airfreight) allows delivery to the nearest airport with pre-arranged customs clearance. For these territories, the choice of courier is essential. Specialized couriers have expert compliance departments and will be aware of specific issues in certain countries. They will be able to advise on the required documentation and the best course of action. Shipping to countries with known terrorist affiliations, such as Iran or Iraq, is especially difficult and within these countries there may be regions that will be forbidden to ship to.
Sending materials as "Medical Supplies" tends to avoid many customs issues in difficult territories.
5. Cites (the Convention on International Trade in Endangered Species of Wild Fauna and Flora)
This internationally recognised treaty came into force in 1973. CITES is designed to control the trade in materials that are derived from species in danger of extinction. All products, including cell lines, that are manufactured from an endangered species require a CITES license to cross international boundaries. Thus, any non-human cell lines generated after 1973 should be checked against the current CITES species list and a permit obtained if necessary.
Note that there is only one original copy of a CITES permit and this original must accompany the goods at all times. Often there is confusion about this during transit, when officials are unaware of which copy is the original, and transit will stop until the confusion is resolved. Customs Clearance Agents are useful in this regard and will clear transit in advance if they are forwarded an electronic version of the permit.
There is a difference between pre-convention and post-convention CITES permits. If a cell line was generated before 1973 (such as the African green monkey cell line "Vero"), it is considered "pre-convention" and although it is still subject to CITES, it will be easier to obtain a permit. However, if the cell line was established post-convention, a permit will be more difficult to obtain. Primary animal tissues from species that are subject to CITES are the most difficult to obtain permits for.
Other information that might be required by the recipient, organization or territory include Certificates of Analysis, and less commonly, Certificates of Origin.
6. Biosafety Considerations
There are international concerns regarding bovine spongiform encephalitis (BSE), which may be present in fetal bovine serum. This may make import of cells that have been cultivated in serum more difficult for many countries. It may be necessary to have a separate import permit for the serum, which in turn will require certificates from the abattoir that harvested the serum. This issue highlights the need to record and be fully aware of the provenance of all reagents that are used in the production of a cell bank.
Since September 11, 2001, the international shipping of frozen cultures is easier than growing cultures, as there is a general perception that "live" or growing cells pose a greater biosafety and biosecurity risk. However, any cell lines containing Category 3 pathogenic viruses or high containment Genetically Modified Organisms (GMOs) may be subject to Select Agent status, particularly in the USA and Australia, where strict import restrictions apply.
7. On Delivery
Recipients of cell cultures should be given specific detailed instructions on how to handle, store and culture the cells in their shipment. However, there are some general considerations to be aware of.
When transferring frozen cryovials to and from liquid nitrogen, face and eye protection should be worn to counter the danger of explosive decompression from potential egress of liquid nitrogen into the vial.
During recovery of frozen cryovials, the rate of thawing is critically important. Optimal thawing is usually achieved through rapid warming of the vial in a waterbath set to 37 °C; slow rates of thawing can critically affect recovery. Care should be taken to avoid contaminating the vial contents, and any protective coatings (e.g., Cryoflex) should be removed prior to warming the vial.
Concerns may be expressed surrounding the cryoprotectant used and its potential detrimental effects (e.g., the risk of differentiation in response to DMSO). If there are concerns, the cryoprotectant should be immediately removed after thawing, by centrifugation. However, some fragile cell lines may be damaged by the centrifugation process, in which case a medium exchange should be carried out as soon as possible.
If our advice could be condensed into two take-home messages, they would be as follows. Shipping of cell cultures is something that needs to be well thought out in advance; and shipping across borders, particularly to more sensitive territories, may be best achieved with the help and advice of a courier that specializes in this area and can give you the benefits of their experience.
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).
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