Dear flow listers, I want to thank all of you who responded to my inquiry about biosafety and the sorting of human cell lines. It has been quite helpful to me in addressing the Caltech Biosafety Committee discussions. Below you will find a summary of the responses that I received. Best wishes to all of you. Rochelle (Shelley) Diamond Caltech Flow Cytometry Cell Sorting Facility Pasadena, CA ----------------------- Risk assessment is based on pathogenicity of the infectious or suspected infectious agent and its route of transmission. It is prudent to consider the potential for aerosol transmission when working with a relatively uncharacterized agent. Greater aerosol potential means greater risk. Origin of the potential infectious material pertaining to a geographic location or a host is also critical for assessing the risk of performing a given experiment. Other factors include agent stability, concentration, infectious dose, data from animal studies, and availability of effective prophylaxis or therapy. Clinical samples may contain unknown pathogens; in these cases in the absence of hard data a cautious approach and adaptation of a higher biosafety level is advisable. Risk assessment is likely to be most difficult for samples containing recombinant DNA molecules. In recent years technologies have evolved that lead to the generation of modified viruses, bacteria, yeast, and other microorganisms. Common recombinant viruses include adenoviruses, alphaviruses, retroviruses, vaccinia, and herpesviruses designed to express heterologous gene products. The challenge faced when selecting the appropriate biosafety level for such work begins by establishing the classification of the non-modified virus and then proceeds to an evaluation for a possible increase in hazard potential associated with a given genetic alteration. Of particular concern are modifications that result in expression of a toxin or a known oncogene, or of sequences that alter the host range or cell tropism, or allow the virus to integrate into the host genome. If needed, advice from a virologist should be sought to determine the proper BSL for planned flow cytometric experiments. It is therefore recommended that viable, unfixed samples that are potentially infectious be sorted at a minimum in a BSL-2 facility (for details refer to the Environmental Control section of the document) using BSL-3 work practices and BSL-3 personal protective equipment. However, because of the increased hazard for a quick release of large amounts of fluid or aerosols into the environment, it is highly recommended that high-speed sorting be performed in a BSL-3 laboratory facility under complete BSL-3 containment. Handling of all unfixed human specimens and primary cell cultures as if infectious is recommended. This practice also applies to established cell lines that are in vitro or animal-passaged human explanted tissues transformed by spontaneous mutation or a natural or laboratory infection with an immortalization agent, e.g., Epstein Barr virus. In fact, cell lines from the American Type Culture Collection (ATCC) and other sources bear warnings that they may contain bloodborne pathogens, and ATCC recommends they be accorded the same biosafety level as the ones known to be infected with HIV. Likewise specimens from non-human primates and animal tissues, explants, or cell cultures known to be deliberately infected with human pathogens are subject to safety procedures as outlined in the Bloodborne Pathogen Standard. Only rigorously characterized human cell lines that have been proven by stringent techniques such as PCR, sensitive antigen detection, stimulation and co-culture assays, enzyme analysis etc. to be completely devoid of bloodborne pathogens could be excluded. However, as most laboratories are not able to provide reliable confirmation of samples before they are subjected to cell sorting using biosafety precautions as outlined in the standard are advisable. ----------------------------------------- For all new cell lines brought into the cell biology facility here I only accept primary providers (ATCC, ECACC) rather than seed stocks which have been through two, three or more hands (as is usual in academia), unless there is no alternative for a specific line (say, transfectant derivatives etc.). You can readily get biosafety level (BSL) data from these providers - it's standard on all cell line info sheets these days. Keep in mind these sheets only reflect the "baseline" cell. Introducing genes using i.e. certain viral systems may jack up the level. Although levels of individual and/or institutional paranoia vary widely, I personally only get really worried about BSL2 and up; you can pretty much eat BSL1's for all practical purposes. Happily enough, many types of experiments can be done using only BSL1 cells. When analyzing the risks involved, also keep in mind it also makes a difference if you're sorting (flow in air, intense droplet formation) or analysing in a cuvette based closed system as well (much less aerosol formation, but don't assume it's nonexistent!) ---------------------------------------------- CDC guidelines list HeLa and HEK293 as needing BSL-2 level containment. Whatever your state quidelines require for that level of containment would be your legal obligation. If cells are fixed, there is not much of a worry. If you are running lots of live cells through the flow cytometer, it would make sense to take precautions. The cell lines are known to be infected with papavovirus (?HPV) and adenovirus, respectively. BSL-2: Associated with human disease, hazard = percutaneous injury, ingestion, mucous membrane exposure ---------------------------------------------------------------- We work with HeLas and since these are human cell lines, always use Universal Precautions. Remember that not all human cell lines are screened for nasty things like Hep B/C, Herpes viruses, or Eppstein Barr virus (mononucleosis). You don't necessarily need to completely gown up as in a BSL3, but do work in a BSL2 laboratory and wear gloves. You should be fine! ------------------------------------------------------------------ We run Hela cells all the time on our Moflos and don't think twice about them. Unless I am pre-warned about anything specific, we consider our cells "clean". One thing to note is that it doesn't mean it's okay, if you have sick people or immunodeficient people around, we always caution ourselves. Since working with Hela cells in culture does not require any extra precautions we treat them the same as mouse lines. ---------------------------------------------------------- .when I worked for a biotech that developed anti-cancer viruses, we treated all of the tumor cell lines we worked with as biohazardous - whether infected experimentally or not. We used bleach to disinfect all spent cultures, media, pipettes, etc, and treated solid waste before disposal...... --------------------------------------------------------------- You could check their biosafety level rating at ATCC. Both cell lines you mentioned are BS level 2. Here is the reference for HeLa: http://www.atcc.org/catalog/numSearch/numResults.cfm?atccNum=CCL-2 And the reference for 293 http://www.atcc.org/common/catalog/numSearch/numResults.cfm?atccNum=CRL-1573 ---------------------------------------------------------------------- I have been working with Hela cells for twenty five years. They do carry HPV genes but as far as I know do not produce the virus. In any case, no one has ever been documented to have received an infection from Hela. In my view, all animal biomass should be treated with some respect, but the risk is exceptionally low if not effervescent. I would worry much more about eating rare or poorly cooked beef, pork, tuna, etc. Mouse cells can be infected with retroviruses that will infect humans but the ability to get infected must be very low as there have been no documented infections that I know of. We use BIosafety II for culture and sort live cells all the time. We have an Aria and EPICS Elite. Both would qualify as ~Biosafety II. --------------------------------------------------------------------------- Here is our policy: 1. All KNOWN infected material (HIV, Hepatitis, TB, Ebola, etc.) goes to a BL3 level sorter on campus. This also includes any live monkey tissues and most brain and lung tissue, even from non-infected humans. 2. Everything else, inlcuding human blood from normal donors, transplant patients who are screened for every disease known to man, or cancer patients who cannot receive chemo until they are also screened comes here as a BSL2 or lower sort. We use negative pressure, universal precautions, and have biocontainment on the sorters. 3. We have an on-line scheduling system that asks for specific information every time someone schedules a sort, including the biological materials, like transfection vectors, they are using, as well as their biosafety protocol number. 4. Our biosafety committee reviews all protocols now specifically for sorting if the investigator wants to do it. (Some BSL2 agents become BSL3 when aerosolized, but the committee checks on this) If we have any concerns they review the agents being used and tell us the biosafety level involved. If it qualifies as BSL2 or lower (as an aerosol) we sort it. Personally, I do not want to wear a suit and I know my staff feels the same way. In fact, I think being overly cautious can be counter-productive to research. To say nothing of retaining staff or running your facility efficiently. I believe every facility has the right to refuse samples they feel are unsafe regardless of the BSL classification. So we also reserve the right to sort things we feel pose an almost zero risk without wearing a space suit. So even though some in the community poo-poo the fact that there has never been a documented case of aerosol infection from a sorter, there is some validity to claims that sorting is still pretty safe. In fact after some got over the horror that most do not suit up in spite of the "dangers", I think this no infection thing is pretty important. So--all of these cells would be sorted here as long as the investigator was not using some deadly virus or something else knowingly inserted. Of course, you will never be 100% safe from anything, including stray asteroids. You can set your own policies, but I say sort on. --------------------------------------------------------------------- Someone else essentially said it in a general posting. These lines should be well tested and failry benign, except that they are very very very often the subject of genetic modification, which ranges from minor to producing infectious viral particles of various types. 293 are very common packaging lines for lenti and retroviruses. So I'd say it depends on what is being run. At Gladstone I ran a lot of HIV (and CMV and HCV and God only knows what else) positive blood products. We had one sorter in a negative pressure room and I would gown for "BSL3" sorts, but labcoat only for others. At MSKCC we treat everything on the MoFlos as potentially hazardous and they have containment hoods, but the DiVa sits out in the open, Naked to the world until I get a primary human sort and on goes the Cytek blower. ---------------------------------------------------------------------------------- I work at an Army Medical Research lab and co-teach the BloodBorne Pathogens course with our Safety Manager. Our Institute policy is that the use of all human materials NOT specifically accompanied by a Certificate of Analysis assuring negative status for HIV and HBV require enrollment in our BloodBorne Pathogens program with training, knowledge of the Exposure Control Plan, containment of samples, and institute-provided HBV Vaccine. ATCC does not provide such Certificates so we are currently looking into third party testing of the HIV/HBV status for their cells. Most of our human cultures come from Cambrex and Cascade and are accompanied by appropriate CoA. I would think that you organization's Safety department already has a program in place for compliance with OSHA BloodBorne Pathogens regulations. We also use the CDC-NIH Biosafety in Microbiological and Biomedical Laboratories guide (I have the 4th edition, 1999). Appendix D addresses use of human cells and tissues. Fell free to call if you have questions. ------------------------------------------------------------------Received on Wed Jul 12 11:38:00 2006
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