Pathogen Screening of Biological Materials Introduced into Rodents

Introduction

Administration of human or animal tissues (e.g., tumors, cell lines) or other biological materials into animals, especially rodents, is a common research practice. These tissues and biological materials may be infected with a variety of agents that may be infectious to humans or animals and potentially jeopardize the health of both. Additionally, contaminating pathogens may act as a confounding variable on research results.

Because collaborative research is the rule rather than the exception in today’s research environment, and rodents (especially mice) and biological materials (including cell lines) are shared and sent between investigators within and between institutions, ensuring pathogen-free animals and biological samples greatly facilitates collaborative research.

The BU IACUC and BU IBC work together to assure human and animal health. Any biological materials of human origin require handling at the BSL-2 level (including animal housing at ABSL-2 containment) and observation of standard operating procedures for universal precautions and blood-borne pathogens. Therefore, testing cell lines of human origin for human pathogens is NOT required.

Purpose

The three main reasons for testing are as follows:
A. To protect human staff from zoonotic agents.
B. To protect research and research animals.
Many rodent research models, especially those involving immunology and cancer research, are vulnerable to   interference if the animal is host to an adventitious murine virus (a virus that does not result in clinical disease but nevertheless affects immune function).
C. To identify and eliminate any sources of mouse parvovirus (MPV), which has been endemic in the BUMC institutional mouse colonies.

It has been reported that approximately 1% of biological screens tested come up positive for pathogens. 70% of these consist of MPV and lactate dehydrogenase virus (LDV). The rest include polyomavirus (POLY), reovirus (REO), mouse adenovirus (MAV), and others.13 Mycoplasma is also a frequent contaminant of cell lines.

Policy

A. Principal Investigators (PIs) are responsible for testing all cell lines and biological materials before administering into any rodent as needed. Mandatory testing or retesting is required every five years. Frequency of testing also depends on:
1. Origin of material.
2. Previous pathogen testing of material and information about results.
3. Quality of storage and use of cell line(s) and material(s) in the laboratory.
B. When any pathogen is diagnosed as a result of sentinel surveillance, biological materials used in the rodents in the affected room must be tested for that pathogen.
C. The Intent to Administer Biological Material into Animals form must be completed and submitted to BU ASC before administering the materials into rodents. This form, which must be reviewed and approved by BU ASC staff before administration of the biological material, is available on RIMS. The ASC will provide pricing upon request.
D. PIs currently using biological materials in their animals or anticipating administration of biological materials must:
1. List the biological materials in their IACUC protocol.
2. If the material is of human, nonhuman primate, or sheep origin, or rDNA or an infectious agent, complete an IBC application.
E. Biological materials that may require testing: “Biologic materials that must be tested include any that have originated from rodents or which have been exposed to rodents directly (in vivo passage) or indirectly (e.g., via tissue culture media additives).”12

Cell lines which have originated in rodents or which have been exposed to rodents directly or indirectly:
• Embryonic stem cells
• Cell lines of human origin
• Cell lines of animal origin

Viruses which have originated in rodents or which have been exposed to rodents directly or indirectly:
• Lentiviruses which fulfill above criteria
• Adenoviruses which fulfill above criteria
• Any virus used to transform cell lines which fulfills above criteria

EXEMPT:

Viruses or viral vectors which have been synthesized without any exposure to murine cell lines or tissues.

Tissues which have originated in rodents or which have been exposed to rodents directly or indirectly.

Transplantable tumors which have originated in rodents or which have been exposed to rodents directly or indirectly.

Subcellular components which have originated in rodents or which have been exposed to rodents directly or indirectly:
• Myelomas
• Hybridomas
• Spermatozoa
• Ascites fluid (not if harvested; only if being passaged into new host)

Purified proteins including antibodies

Cell culture media and components which have originated in rodents or which have been exposed to rodents directly or indirectly:
• Any rodent serum*
• Basement membrane matrix (Matrigel®)11
• Any biologic material used in the administered material or for cell or tissue culture
*This excludes sera from nonrodent species such as fetal calf serum.
F. Biological materials exempt from testing: Exemption is based upon review and approval by BU ASC of the Intent to Administer Biological Material into Rodents form (access form in RIMS). Rodent fresh cells or tissues harvested from within a Specific Pathogen Free (SPF) BU ASC rodent colony, and being used for a single passage in rodents, do not require pathogen testing. This exemption assumes that the health status of the rodent colony is confirmed by BU ASC sentinel testing results and is SPF for at least the previous six months or longer including the one large test panel (CRL Assessment Plus or Rat Comprehensive Panel) completed annually.
G. Sources of biological materials and testing requirements:
1. Boston University
Murine cells or human cells which have been passed through rodents: Testing or documentation of SPF test results.
Other biological materials of unknown pathogen status: Testing or documentation of SPF test results.
Primary human cell lines or tissues: Exempt.
2. Other Institution
Murine cells or human cells which have been exposed to rodents directly or indirectly: Testing required.
Other biological materials of unknown pathogen status (biological materials from colleague at any other institution): Testing required.
3. Primary Human Cell Lines or Tissues
Complete the Intent to Administer Biological Materials into Animals form (access via RIMS) in the IACUC protocol and complete the IBC application.
4. ATCC (American Type Culture Collection) or Other Commercial Source
Testing required. All cells and other biological materials obtained from ATCC or other commercial source require testing for murine viruses prior to administration into laboratory rodents.
5. It is RECOMMENDED that human-origin or nonmurine-origin tumors and cell lines be tested for murine pathogens unless credible documentation is available to show they have never been passaged through or exposed to rodents directly or indirectly. They also must be tested for Mycoplasma. Therefore, it is required that human cell lines and transplantable tumors obtained from ATCC or other cell culture collections be tested because complete documentation of their handling prior to deposition is not available.
Background
A. Murine viruses have been shown to be transmitted both by cell culture products and additives and by the cells themselves.
B. If cell lines are to be administered to immunodeficient mice, the PI must bear in mind that murine pathogens that will not make an immunocompetent rodent sick may very well cause disease in an immunocompromised rodent.
C. Cells may be tested before or after they have been cultured in the medium used. If tested after culture, the test would pick up both possible pathogens from the medium components as well as from the cell line, tissue, or subcellular components.
D. Factors to evaluate:
1. The origin and available test results for this cell line.
2. There is a greater risk of contamination for cell lines obtained from rodents housed in conventional colonies than from those housed in SPF colonies.
3. It is harder to predict what viruses may be present in cultured cells and culture media compared to cells obtained from known mouse colonies under sentinel surveillance. We know pretty well what viruses are common in US mouse colonies. These include MNV, MPV, MVM, MHV, EDIM, and TMEV. Less frequently seen are REO3, LCMV, and Sendai virus. LDV is found in cell cultures with some frequency13. Some are hardly seen, including Ectromelia, Hantaan, MAV, PVM, and polyomavirus. Some pathogens are more common in cell cultures than in mouse colonies, for example Polyoma, Ectromelia, and Hantavirus.
E. About six years ago, mouse Ectromelia virus was detected at Cornell’s Sloan-Kettering from a mouse cell culture media (serum) which was shown to come from China5.
F. Mycoplasma is a major contaminant of cell lines. Only M. pulmonis infects rodents, but other Mycoplasma species may contaminate cell lines.
G. The SPF profile adapted for BU mouse colonies as listed in the CRL Assessment Plus or Rat Comprehensive Panel and tested for annually include all these pathogens plus a few more. These are the pathogens we want to exclude from our rodent facilities.
H. Once your cells and your culture media have been tested for all of the above, they do not need to be tested again for five years as long as they stay in the PI’s hands, under the PI’s control, and the PI maintains the cells according to the recommendations below to ensure SPF status.
I. Repeat testing is required every five years.
Procedures Prior to Testing Cell Lines
Prior to testing, it is imperative to build up a cell line to anticipated volume and concentration required for the next several years. The aliquots are frozen at −80oC. One aliquot is submitted for pathogen testing and the rest, and provided this tests SPF, are thawed and used as needed.
Decontamination of Mycoplasma or Virus-Infected Cell Lines
“Cells can be treated with antibiotics to eliminate Mycoplasma, but a virus-infected cell line cannot be cured. On the other hand, transplantable cell lines (i.e., those maintained by passage in animals) can be virus-contaminated without actually being infectious. For example, lactate dehydrogenase elevating virus (LDV) has generally be shown to be the most common contaminant of tumor lines maintained by transplantation in mice (probably because LDV causes a persistent, high-level viremia). As LDV has a very narrow host range (i.e., I believe it replicates only in mouse macrophages), however, in most if not all cases the LDV-contaminated transplantable cell line would not be LDV-infected. In this situation, it may be possible to eliminate the LDV contamination by passaging the cells in vitro or in rats, which are not susceptible to LDV infection.”16
Recommended Procedures for Maintaining Pathogen-Free Cell Lines in the Laboratory12
A. Preventing the Introduction of Adventitious Agents
“As procedures to inactivate or remove adventitious agents may also do the same to a biologic product, it is preferable to prevent microbial contamination in the first place by controlling the risk factors associated with its sources.
This is accomplished by:
1. Selecting high-quality raw materials, including original cells, tissues or animals, and microbial inocula that are from a reliable supplier and have undergone the appropriate quality control testing.
2. Banking cell substrates and microbial inocula.
3. Following microbiologic and manufacturing practices that prevent operator-induced contamination.”
1. Selecting Raw Materials
“Biologic raw materials should also be screened for microbial contaminants. Whenever possible, raw materials should be obtained from suppliers that provide material traceability and lot analysis information. The supplier’s document, showing that a lot of material has been tested and met certain specifications, is referred to generally as ‘Certificate of Analysis.’
It is preferable whenever possible to use raw materials that can withstand conditions known to remove or inactivate viruses. Preparing or acquiring large batches of raw materials is crucial to consistent production of biologics free of extraneous infectious agents; this minimizes the adverse effects of batch-to-batch variation and makes comprehensive quality control more economical and efficient, provided materials are stored and handled in a manner that prevents their degradation or contamination.
Biologic raw materials, such as cells or serum, that originate from animals or humans should be generated from sources that are likely to be pathogen-free.”
2. Banking Cells and Inocula
“It is well established that biologic product quality and safety are in large part determined by the cell substrate. The first step for controlling cell substrate quality is to document its source (or parental cell line), method of preparation, and cultivation history.
Most modern biologicals are produced in serially cultivated animal cell lines. The chief advantage of a cell line is that it can be preserved as a cell bank, which provides a well-characterized and standardized starting point for production. A cell bank is a uniform pool of cells that is typically suspended in a medium containing cryoprotectants, divided into small aliquots in sealed containers and cryopreserved in the vapor or liquid phase of liquid nitrogen or at equivalent ultra-low temperatures. The two-tiered banking system, consisting of a master cell bank (MCB), and a working cell bank (WCB) created from the MCB, has become the generally accepted approach for ensuring a consistent, uninterrupted supply of cells for production of biologic products.”14,15
Testing Procedures
A. Sample Submission Procedures for Charles River Laboratories (CRL)
1. For biological materials that have been exposed to mice directly or indirectly, the CR Mouse Essential Panel is REQUIRED.
2. For biological materials that have been exposed to rats directly or indirectly, the BUMC Rat Small Panel is REQUIRED.
3. Boston University Animal Science Center (BU ASC) will send out samples and the courier service is free.
4. Complete the Intent to Administer Biological Materials into Rodents form through RIMS. BU ASC will assist with packing samples. Shipping materials are supplied free-of-charge by CRL. The PI brings the sample(s) for pickup according to the following schedule:
BU Medical Campus
a. Tuesdays and Thursdays by 9 a.m. Courier pickup 10 a.m. Additional weekdays may be scheduled as needed.
b. Packing and pickup as scheduled with the BU ASC office.
Charles River Campus
The PI brings sample(s) to BU ASC for packing and pickup after establishing a pickup date and time with BU ASC.
B. Sample Preparation CRL
1. Samples should be representative aliquots of the larger (master cell bank) volume of material proposed to be utilized in the project. Collection of material for PCR pathogen testing should be performed ASEPTICALLY to prevent inadvertent contamination of the samples.
2. Cell Cultures
1. Submit two undiluted cell culture aliquots of at least 200 microliters each (to allow CRL to perform confirmatory testing) and ship frozen files on dry ice.
2. Cell number is not critical; however, please note on the submission form if there are more than 5 x 10^7 cells/ml.
3. If possible, samples should be passaged without antibiotics prior to submission. CRL recommends a minimum of two passages if the cells are washed twice between passages. If cells are cryopreserved without antibiotics, samples may be submitted as is.
3. Other Biological Materials
1. Serum, ascites, or other liquid: 2 vials of at least 0.2ml/vial shipped on dry ice.
2. Antibodies: It may not be possible to send this sample amount for expensive materials such as antibodies. In this case, a single undiluted aliquot of a reasonable volume can be submitted.
3. Reducing sample volume will reduce analytical sensitivity. Do not dilute sample to reach a specified sample volume.
CRL recommends that for pooling samples, add 200 microliters of each sample (media/cells) to two vials. Please note: it is important that the media is included as the virus may be associated with the media and not necessarily the cells.
Up to four samples may be pooled to save money. If results are negative, then the tested samples are SPF. If results indicate contamination, individual vials representing each of the pooled submissions will need to be submitted and each sample must be tested separately for the pathogen detected at a cost of about $70 per agent (please contact ASC for current pricing).

References

1. Massachusetts Institute of Technology Policy on Biological Materials
2. UCSF IACUC Policy on Testing of Biological Materials
3. http://www.criver.com/flex_content_area/documents/rm_ld_c_research_animal_diagnostic_services.pdf
Published Papers Documenting Biological Materials Contamination of Cells, Tissues, and Biological Materials
1. Niklas, W., V. Kraft, and B. Meyer. 1993. Lab Anim Sci 43:296–300. “Contamination of transplantable tumors, cell lines, and monoclonal antibodies with rodent viruses.”
2. Ohnishi, Y., M. Yoshimura and Ueyama, Y. 1995. J Nat Cancer Inst 87(7):538–9. “Lactic dehydrogenase virus (LDHV) contamination in human tumor xenographs and its elimination.”
3. Percy, D. H., and S. W. Barthold. Pathology of Laboratory Rodents and Rabbits, Iowa State University Press, Ames, IA. 1993.
4. Bhatt, P. N., R. O. Jacoby, S. W. Barthold. 1986. Lab Anim Sci 36:136–139.
5. Lipman, N. S., S. Perkins, H. Nguyen, et al. 2000. Comp Med 50:426–435. “Mousepox resulting from use of ectromelia-imported mouse serum”.
6. Dick, E. J., Jr., C. L. Kittell, H. Meyer, et al. 1996. Lab Anim Sci 46:602–611. “Mousepox outbreak in a laboratory mouse colony”.
7. Dykewicz, C. A., V. M. Dato, S. P. Fisher-Hoch, et al. 1992. JAMA 267:1349–1353. “Lymphocytic choreomeningitis outbreak associated with nude mice in a research institute.”
8. Biggar, R. J., T. J. Schmidt, J. P. Woodall, 1977. J Am Vet Med Assoc 171:829–832. “Lymphocytic choreomeningitis in laboratory personnel exposed to hamsters inadvertently infected with LCM virus.”
9. Labelle, P., Fraser, J.K., Kendall, L.V., et al. 2004. Contemp. Top. Lab. Anim. Sci.43:47.
10. McKisic, MD, Lancki, DW, Otto, G, et al 1993. J Immunol 419–428. “Identification and propagation of a putative immunosuppressive orphan parvovirus in cloned T cells.”
11. Carlson, J., Garg, R., Compton, S.R., Zeiss, C. and Uchio, E. 2008. JAALAS 47(5): PS 35: Poliomyelitis in SCID Mice Following Injection of Basement Membrane Matrix Contaminated with Lactate Dehydrogenase-elevating Virus.”
12. Shek, William R. Quality Control Testing of Biologics. in The Mouse in Biomedical Research. Vol. III. 2nd Fox, J.G., et. al. Eds. Academic Press, 2007.
13. Personal Communication. Charles River Laboratories. January 27, 2009.
14. Hayflick, L. 1989. History of cell substrates used for human biologicals. Dev. Biol. Stand. 70, 11–26.
15. Wieve, M.E. and May, L.H. 1990. Cell banking. Bioprocess. Technol. 10:147–160.
16. Dr. Willam Shek, Charles River Laboratories. Personal Communication.
17. With thanks to Dr. Ken Henderson, Charles River Laboratories, for assistance in reviewing and assuring accuracy of this document.

BU IACUC Approved June 2009, Revised January 2014