IBC Policy on Use of Human Cell Lines

(Adopted 5/24/07 - Updated 3/22/2012)

Introduction

Human cell lines are often utilized in research; however, the appropriate level of containment for handling human cell lines is not clear and is not adequately addressed in regulatory guidelines. To facilitate the University of Missouri's position on the use and handling of human cell lines, the Institutional Biosafety Committee has approved this document and accompanying policy statement.

Background

In 1991, the Occupational Safety and Health Administration (OSHA) released the Bloodborne Pathogens (BBP) Standard. The intention of the standard is to protect employees who may have occupational exposure to human blood or other potentially infectious materials. Human cell lines that are derived from human clinical specimens were not defined anywhere in this standard and it was not clear of the regulatory applicability of this particular material versus the clearly defined use of blood and other potentially infectious materials.

In 1994, OSHA issued a statement of the applicability of the Bloodborne Pathogen Standard towards human cell lines in response to an inquiry from the American Biological Safety Association (ABSA) (Enclosure 1). All primary human cell explants from tissues and subsequent in vitro passages of human tissue explant cultures (human cell "strains") must be regarded as containing potential bloodborne pathogens and should be handled in accordance with the Bloodborne Pathogen Standard. The exception for any human cell line would be if the specific cell line has been screened or otherwise characterized to be free of hepatitis viruses, the Human Immunodeficiency Virus, Epstein-Barr virus, and other recognized bloodborne pathogens.

The American Type Culture Collection (ATCC) in its Frequently Asked Questions reference on its web page stated, we recommend that all human cell lines be accorded the same level of biosafety consideration as a line known to carry HIV. (Enclosure 2). In addition, ATCC stated “As of January 1, 2010, all human cell lines accessioned into the ATCC general collection are tested for the adventitious agents HIV, Hepatitis B, Hepatitis C, HPV, EBV, and CMV. Testing is performed on each lot when it is prepared, and the test results are reported on the Certificate of Analysis for the specific lot.” In February 2007, the Fifth Edition of the Centers for Disease Control and Prevention publication, Biosafety in Microbiological and Biomedical Laboratories (BMBL), Appendix H (Enclosure 3), recommends that human and other primate cells should be handled using BSL-2 practices and containment and that OSHA has developed a bloodborne pathogens standard that should be applied to all work in the laboratory with human blood, tissues, body fluids and primary cell lines.

Using the information contained in the aforementioned three references, the University of Missouri Institutional Biosafety Committee has adopted the following policy with regard to the laboratory usage of human cell lines: All cell and organ cultures of human origin, including well established cell lines, shall be handled in accordance with the OSHA Bloodborne Pathogens Standard and under Biosafety Level 2 (BSL2) containment. Any injections with human cell lines into animals will be conducted in a biological safety cabinet and handled at Animal Biosafety Level 2 (ABSL-2).  After injection, the site will be sterilized and swabbed with an alcohol pad to eliminate any residual Bloodborne Pathogens material.  Appropriate biohazard signs will need to be posted during injections with the animals while Bloodborne Pathogens are present and taken down once the procedure is complete.

Enclosure 1:
Reference
U.S. Department of Labor Occupational Safety & Health Administration www.osha.gov

Standard Interpretations 06/21/1994 - Applicability of 1910.1030 to establish human cell lines

June 21, 1994

Dr. Diane Fleming
President
University of South Alabama
College of Medicine
CSAB 170
Mobile, Alabama 36688

Dear Dr. Fleming:

This is in response to a September 23, 1993 letter from Joseph H. Coggin, an American Biological Safety Association member, requesting clarification of our August 3, 1993 letter of interpretation to the former ABSA President Dr. Jerome P. Schmidt. That letter attempted to explain the applicability of the Occupational Safety and Health Administration's (OSHA) standard 29 CFR 1910.1030, "Occupational Exposure to Bloodborne Pathogens," to establish human cell lines.

Dr. Coggin informed us that our August 3, 1993 letter may be more confusing rather than enlightening to biological safety professionals.

We have reconsidered our earlier comments and are providing a more detailed letter of interpretation. We regret any misunderstanding our earlier response may have caused.

As you know, the Bloodborne Pathogens standard (BPS) provides protection to employees who have occupational exposure to human blood or other potentially infectious materials (OPIM). Established human cell lines* (see attachment) which are characterized** (see attachment) to be free of contamination from human hepatitis viruses, human immunodeficiency viruses, and other recognized bloodborne pathogens, are not considered to be OPIM and are not covered by BPS. Established human or other animal cell lines which are known to be or likely infected/contaminated with human microbes or agents classed as bloodborne pathogens, especially hepatitis viruses and human immunodeficiency viruses are covered by the BPS. The final judgement for making the determination that human or other animal cell lines in culture are free of bloodborne pathogens must be made by a Bio-safety Professional or other qualified scientist with the background and experience to review such potential contamination and risk, in accordance with the requirements of the BPS. Documentation that such cell lines are not OPIM should be a matter of written record and on file with the employer for OSHA review.

All primary human cell explants from tissues and subsequent in vitro passages of human tissue explant cultures (human cell "strains" ***, see attachment) must be regarded as containing potential bloodborne pathogens and should be handled in accordance with the BPS. Non-transformed, human cell "strains", characterized by documented, reasonable laboratory testing as described in the attachment, to be free of human immunodeficiency virus, hepatitis viruses, or other bloodborne pathogens may be exempted from the standard's requirements. However, if such tissue explants or subsequent cultures are derived from human subjects known to carry bloodborne pathogens, such as hepatitis viruses or human immunodeficiency viruses or are deliberately infected with bloodborne pathogens, they must be handled in accordance with the precautions noted in the BPS. Likewise, animal tissues, explants or cell cultures known to be contaminated by deliberate infection with human immunodeficiency virus or Hepatitis B virus are also subject to the BPS.

All laboratory work with primary human tissues or body fluids is covered by the BPS.

We hope this information is responsive to your concerns and thank you for your interest in worker safety and health.

Sincerely,

 

Ruth E. McCully, Director
Office of Health Compliance Assistance

Enclosure

DEFINITIONS

* A Human Cell LINE is defined as in vitro or animal passaged (e.g., nude mouse) cultures or human cells that fulfill traditional requirements of a cell line designation. That is, the cells are immortalized cells, transformed by spontaneous mutation or natural or laboratory infection with an immortalizating agent such as Epstein-Barr virus (EBV). EBV is a bloodborne pathogen. It should be noted that human cervical carcinoma cells or other transformed human cell lines like HeLa cells are sometimes adulterated with laboratory pathogens accidentally introduced by cultivation with other cell cultures, or physically contaminated by other cell cultures handled in the same lab. In order to handle human HeLa cells, without having to comply with the requirements of the bloodborne pathogens standard (BPS), human HeLa cells should be documented to be pure HeLa cells and shown to be free of bloodborne pathogens by testing.

**Characterization of human cells, for inclusion or exclusion from compliance with the BPS, would include screening of the cells lines or "strains" for viruses characterized as bloodborne pathogens by the Standard, including human immunodeficiency viruses, hepatitis viruses or EBV, if the cells are capable of propagating such viruses. Most cell lines are screened for human mycoplasmas and are free of bacterial and mycotic contaminants. Testing may include antigenic screening for viral or agent markers, co-cultivation with various indicator cells that allow contaminants to grow, or using molecular technology (polymerase chain reaction or nucleic acid hybridization) to identify latent viruses capable of infecting humans such as Herpesviruses(e.g., EBV), or papilloma members of the Papovavirus group, etc. Cell lines that are procured from commercial vendors or other sources with documented testing to be free of human bloodborne pathogens and which have been protected by the employer from environmental contamination may be excluded from the BPS.

*** Human cell STRAINS are defined as cells propagated in vitro from primary explants of human tissue or body fluids which have finite lifetime (non-transformed) in tissue culture for 20-70 passages. Human cell "strains" must be handled as potential biohazards unless characterized by testing to be free of bloodborne pathogens (i.e., WI-38 cells are often so documented).

 

September 23, 1993

Dr. Roger A. Clark, Director
Directorate of Compliance Programs
Occupational Safety and Health Administration
Washington, DC 20210

Dear Dr. Clark:

The American Biological Safety Association [ABSA], of which I am a member, recently contacted your office concerning the inclusion of "well established human cell lines" under the OSHA 29 CFR 1910.1030. I have a copy of your response letter dated August 3, 1993 to Dr. Jerome Schmidt, President of ABSA. Dr. Schmidt had submitted the inquiry letter at the request of ABSA's Technical Review Committee. ABSA was seeking exclusion for the use of well characterized human cells lines from the Standard when the lines have been proven virus/agent free by rigorous techniques. Dr. Schmidt's letter to you of March 25, 1993 acknowledges that "primary cultures" of human cells are potentially risky and require Universal Precautions. Well characterized human cells referenced in the ABSA inquiry means, I believe, transformed lines of human cells that have been tested with rigorous methods [e.g., culture, viral or agent antigen or markers, PCR in the case of human lymphocytes or epithelial cells for HIV or HBV, respectively].

Two statements in your response cause me grave concerns as a biological safety professional. First, your statements go much further than ABSA members ever expected when you included, by implication, that "protected" established cell lines, "primary cell lines" [Strains?] as well as secondary or higher passaged human cells were excluded from the Standard. According to your letter, cell strains cultured from primary explants or subcultures after passage 1 would not be covered by the BBP Standard. Most virologists recognize that many such human subcultures of primary cells, endogenously infected in the donor with silent HTLV viruses, papilloma, JC, BK, CJ, herpes, hepatitis and other viruses, as well as possible intracellular bacterial pathogens may represent a real and present source for human infection. A person receiving secondary or subsequent cultures of human lymphocytes, fetal cell mixtures, or hepatocytes from a vendor or laboratory may be obtaining human cells that contain a myriad of human viruses including hepatitis viruses and even HIV without any knowledge that the agents are present. Recall that 1 in every 250 American donors of tissue today may have HIV and that many more persons may harbor HBV. Such human cell "strains" would not require careful testing to determine their status as infectious agent free cultures so long as they are not "primary cultures" or deliberately infected with HIV. According to your recommendation, these passages of cells can now be handled by personnel without compliance with 29 CFR 1919.1030. Rest assured, if this door is left open, many will use your statement in this way, even though I do not believe that is what you and OSHA meant to happen. All human cell primary explants, derived cell strains from these explants, at any passage, and established human cell lines should be included under the standard unless well characterized by rigorous techniques and shown to be free of the BBP agents.

The second statement of concern in your letter is that "Established cell lines, which are protected from contamination with environmental organisms to ensure their integrity for research purposes, are not considered OPIM and, are therefore not covered under the Bloodborne Pathogen Standard". You then clarify this statement implying that if HIV is [deliberately] cultured in the cells, the established cell lines are included under the Standard. It is my considered opinion that your official interpretation will now cause great confusion. Human cell lines from the American Type Culture Collection [ATCC] and other sources bear clear warning that they may contain BBP. ATCC recommends that these cells must be handled at BL-2 and in compliance with the BBP Standard. It is clear that some BBPs, especially endogenous human retroviruses can be harbored in established cells. If taken literally, your statement says that these cells may be considered excluded from the BBP Standard as long as they are kept protected from contamination in the laboratory handling them. In fact, they may already be contaminated with a spectrum of viruses, some of which can only be detected with nucleic acid blotting techniques that are not used routinely to screen for common viruses. So long as the receiving lab protects them from contamination with environmental pathogens in that lab, handling them does not require compliance with the BBP Standard. This is a potentially dangerous precedent that will almost surely lead to a laboratory exposure to BBP in the American work place. Such established cells showing no active viral replication, may be induced by a variety of agents to replicate endogenous viruses that are capable of infecting humans, especially if a worker is cut handling the cultures. I know you meant to be helpful in making the statement; however, many lab workers and especially their supervisors are more interested in getting around having to comply with the Standard than in seriously considering the true risk. They will contend that they did not expose the cells to environmental pathogens in their handling and this may be true, but not relevant, if the cultures are already contaminated upon receipt in the lab. Many labs do not have knowledgeable biosafety professionals with real expertise to correctly advise them about the requirements for characterization of established cell lines to reasonably establish the lines are likely to be viral or agent free. Now these labs will have license to do so without fear of regulation so long as they do not culture the cells with other cultures of BBPs.

ABSA was only asking for permission to exclude only well characterized human cell lines. Your letter gives authorization to exclude any human cell line, including secondary explants, so long as it is protected from environmental contamination with BBP in the recipient laboratory. Again, the cell line may already harbor BBP when received, but ignorance in this case would be adequate excuse to avoid compliance with the BBP Standard.

Please reconsider these two statements in your letter very carefully. I support ABSA's request for excluding rigorously characterized human cell lines, proven to contain no BBPs by stringent techniques [PCR, sensitive antigen detection, stimulation and co-culture assays, enzyme analysis, etc], but the wording of your letter will generate great confusion when I know that you were attempting to be helpful and cooperative.

Sincerely yours,

 

Joseph H. Coggin, Jr. Ph.D.
Professor and Chair, Microbiology and
Immunology, Professor of Pathology, and Associate Dean

 

November 10, 1993

Dr. Jessica Sandler
OSHA
Office of Compliance Programs
Occupational Safety and Health Administration
Washington, DC 20210

Dear Dr Sandler:

Thank you for your phone call regarding my letter of September 23, 1993 to Dr. Roger Clark, Director of The Directorate of Compliance Programs of OSHA. A copy of his response to Dr. Schmidt of ABSA is enclosed for your reference, along with a suggested redraft that I composed to deal with the issues of concern raised in my letter to Dr. Clark. As you can see I kept to the theme of his letter, but believe I used more traditionally accepted definitions of terms used to refer to tissue cultures.

I hope that these changes will be specific enough to be clarifying and faithful to the classic, widely accepted definitions of the terms "cell line" and "cell strain". The draft I enclose, hopefully will avoid the confusion I noted in the letter from Dr. Clark. I also defined the term "Characterization" to provide employers with a clear indication of the general laboratory testing criteria which should be used to establish human cell lines and strains as safe from the most problematic, non-treatable human blood borne pathogens.

Thank you for this opportunity to be of service.

Sincerely,

 

Joseph H. Coggin, Jr. Ph.D. Professor and Chair and Professor of Pathology

 

August 3, 1993

Mr. Jerome P. Schmidt
President
American Biological Safety Association
1202 Allanson Road
Mundelein, IL 60060

Dear Mr. Schmidt:

This is in response to your letter of March 25, requesting an interpretation of the Occupational Safety and Health Administration (OSHA) standard 29 CFR 1910.1030, "Occupational Exposure to Bloodborne Pathogens." Specifically, you requested information as to the applicability of established human cell lines to the bloodborne pathogens standard.

As you know, the standard provides protections to employees who have occupational exposure to blood or other potentially infectious materials (OPIM). Established cell lines, which are protected from contamination with environmental organisms to ensure their integrity for research purposed, are not considered to be OPIM, and are therefore not covered under the bloodborne pathogens standard. However, please bear in mind that established cell lines containing the human immunodeficiency virus (HIV) are covered by the standard.

Primary cell lines, except those containing HIV, are also not covered by the standard. However, employees who initially handle the tissue from which any human cell lines are derived and do the initial steps in the culture of the cells are covered by the standard because of their reasonably anticipated exposure to unfixed tissues and blood.

We hope this information is responsive to your concerns. Thank you for your interest in employee safety and health.

Sincerely,

Roger A. Clark, Director
Directorate of Compliance Programs

Enclosure 2

American Type Cell Culture FAQ:

Are ATCC human cell lines tested for viruses such as Epstein-Barr (EBV) virus, human immunodeficiency virus (HIV, AIDS virus), human T cell leukemia (HTLV), and hepatitis B virus? Are ATCC cell lines tested for bovine viral diarrhea virus (BVDV)?

Answer: Some of our human cell lines are known to produce, EBV, HTLV, or hepatitis virus, and this information is given in the catalog description and on product sheets. In addition, the human lung cell lines in our CCL collection have been screened and found negative for viruses by procedures that are detailed in our quality control manual (egg inoculation, hemadsorption, and co-cultivation with indicator cells). At this time, ATCC is distributing the HIV-positive line H9/HTLV-IIB (ATCC CRL-8543). However, some of our other patent deposits have been derived from AIDS patients and may carry HIV.

Since it is not possible for us to test every cell lines for every possible virus, we rely on the tests performed by the depositor. We recommend that all human cell lines be accorded the same level of biosafety consideration as a line known to carry HIV. With infectious virus assays or viral antigen assays, even a negative test result may leave open the possible existance of a latent viral genome. Thus it is best to use caution when handling any human cell lines. Concerning BVDV, the virus is present in most serum samples, often at very low levels. Hence, it is probably present in all cell lines in which it can replicate unless the cultures have been grown in rigidly tested sera or sera of non-bovine origins. A paper describing tests of some ATCC lines was published in 1994 [S.R. Bolin et all. (1994) Survey of cell lines in the American Type Culture Collection of bovine viral diarrhea virus. J. Virol. Methods 48:211]. Lines that are positive for BVDV are so described in the ATCC catalog descriptions.

Enclosure 3

Biosafety in Microbiological and Biomedical Laboratories (BMBL)
Appendix H
Working with Human, NHP and Other Mammalian Cells and Tissues

Although risk of laboratory infection from working with cell cultures in general is low, risk increases when working with human and other primate cells, and primary cells from other mammalian species. There are reports of infection of laboratory workers handling primary rhesus monkey kidney cells, 1 and the bloodborne pathogen risks from working with primary human cells, tissues and body fluids are widely recognized. 2,3 OSHA has developed a bloodborne pathogens standard that should be applied to all work in the laboratory with human blood, tissues, body fluids and primary cell lines. 4 Procedures have also been published to reduce contamination of cell cultures with microorganisms. 5,6

Potential Laboratory Hazards

Potential laboratory hazards associated with human cells and tissues include the bloodborne pathogens HBV, HIV, HCV, HTLV, EBV, HPV and CMV as well as agents such as Mycobacterium tuberculosis that may be present in human lung tissue. Other primate cells and tissues also present risks to laboratory workers. 7 Cells immortalized with viral agents such as SV-40, EBV adenovirus or HPV, as well as cells carrying viral genomic material also present potential hazards to laboratory workers. Tumorigenic human cells also are potential hazards as a result of self-inoculation. 8 There has been one reported case of development of a tumor from an accidental needle-stick. 9 Laboratory workers should never handle autologous cells or tissues. 1 NHP cells, blood, lymphoid and neural tissues should always be considered potentially hazardous.

Recommended Practices

Each institution should conduct a risk assessment based on the origin of the cells or tissues (species and tissue type), as well as the source (recently isolated or well-characterized). Human and other primate cells should be handled using BSL-2 practices and containment. All work should be performed in a BSC, and all material decontaminated by autoclaving or disinfection before discarding. 6,10,11,12 BSL-2 recommendations for personnel protective equipment such as laboratory coats, gloves and eye protection should be rigorously followed. All laboratory staff working with human cells and tissues should be enrolled in an occupational medicine program specific for bloodborne pathogens and should work under the policies and guidelines established by the institution’s Exposure Control Plan. 4 Laboratory staff working with human cells and tissues should provide a baseline serum sample, be offered hepatitis B immunization, and be evaluated by a health care professional following an exposure incident. Similar programs should be considered for work with NHP blood, body fluids, and other tissues.

REFERENCES

  1. Doblhoff-Dier O, Stacey G. Cell lines: applications and biosafety. In: Fleming D, Hunt D, editors. Biological safety: principles and practices. Washington, DC: ASM Press; 2000. p. 221-39.
  2. Centers for Disease Control and Prevention. Update: universal precautions for prevention of transmission of human immunodeficiency virus, hepatitis B virus and other bloodborne pathogens in healthcare settings. MMWR Morb Mortal Wkly Rep. 1988;37:377-82, 387-8.
  3. Centers for Disease Control and Prevention. Guidelines for prevention of transmission of human immunodeficiency virus and hepatitis B virus to healthcare and public safety workers. MMWR Morb Mortal Wkly Rep. 1989;38;No.SU-06.
  4. Occupational exposure to bloodborne pathogens. Final Rule. Standard interpretations: applicability of 1910.1030 to established human cell lines, 29 C.F.R. Sect. 1910.1030 (1991).
  5. McGarrity GJ, Coriell LL. Procedures to reduce contamination of cell cultures. In Vitro. 1971;6:257-65.
  6. McGarrity GJ. Spread and control of mycoplasmal infection of cell culture. In Vitro. 1976;12:643-8.
  7. Caputo JL. Safety procedures. In: Freshney RI Freshney MG, editors. Culture of immortalized cells. New York: Wiley-Liss; 1996.
  8. Weiss RA. Why cell biologists should be aware of genetically transmitted viruses. Natl Cancer Inst Monogr.1978;48:183-9.
  9. Gugel EA, Sanders ME. Needle-stick transmission of human colonic adenocarcinoma (letter). N Engl J Med. 1986;315:1487.
  10. Barkley WE. Safety considerations in the cell culture laboratory. Methods Enzymol. 1979;58:36-43.
  11. Grizzle WE, Polt S. Guidelines to avoid personnel contamination by infective agents in research laboratories that use human tissues. J Tissue Cult Methods. 1988;11:191-9.
  12. Caputo JL. Biosafety procedures in cell culture. J of Tissue Cult Methods. 1988;11:233-7.