Safety is a very important aspect to consider, especially as this is a new platform that is being introduced into the competition.
For a better insight, the safety section has been split in two: one based on the particularities of our project and the design we developed to overcome the safety issues, and the other section discusses details about the lab and measures we took to ensure the safety against hazardous situations.
We believe that these precautions, along with good practices before, during and after the execution of the project, will work and help maintain and highlight safety in our lab and our society.
The project was carried out in its entirety in the facilities of the “FEMSA - Biotechnology Center” inside our institution, ITESM Monterrey. The majority of it was worked in the Genetic Manipulation and Molecular Diagnostics Laboratory, and certain labours such as Insect cell handling, was performed in the 5th floor, where most of the researchers in biotechnology work, and where the vast variety of specialized devices are. As part of the requisites of working in the Center for Biotechnology, our team received a general training on lab safety and rules, such as adequate use of equipment, correct waste disposal, emergency protocols, and Clean in Place practices (CiP). We received, as well, approval and supervision by the equivalent group of an Institutional Biosafety Committee at our institution, which is formed by Dr. Silverio García Lara, Dr. Rocio Diaz and Dr. Guy A. Cardineau, who is one of the advisors of the team.
Since the insect cell line Sf9 has not been used by any laboratory inside the Center for Biotechnology, our advisors could only gíve us general guidance on their use. Nevertheless, many of the insect cell protocols, such as cell passages, transfection, and DNA extraction, are similar to those used for mammalian lines (which are worked in our Center) and we could receive some useful tips on the aseptic technique specific to eukaryotes and the correct cell handling for transfection.
Since the aseptic technique is of special relevance for the project, we carefully reviewed the cautions to avoid any bacterial or fungal contaminations, since they are known to occur commonly when working with the medium that we handled, SF-900. For this, we based our work on the “Cell Culture Basics Handbook” from ThermoFisher Scientific, as well as on the advice of the researchers that were supervising us. This company suggests a Biosafety level 2, which we have the requirements needed to work with in the Genetic Manipulation and Molecular Diagnostics lab. (Life Technologies, 2015)
Risks to the people working in the lab
The Biosafety Level 2 (BSL-2), which is to be followed when working with Sf9 cells, is given the following description in the ThermoFisher Handbook mentioned before: “BSL-2 is appropriate for moderate-risk agents known to cause human disease of varying severity by ingestion or through percutaneous or mucous membrane exposure.”
Considering this, the appropriate precautions must be taken. A standard protocol of 70% ethanol washing of and 30 min UV was used before and after each manipulation of the cells in the hood. Every object used for cell handling was washed with 70% ethanol when put into the hood and when taken out. Important to mention is that this manipulation was performed always in a laminar flow biosafety hood, located in a different laboratory than the lab that we used for E. coli manipulation, to avoid cross contamination and also to avoid spread of biological insect material that could cause an immune response in any person around the area.
Environment and waste disposal
To reduce these kind of risks, a special separated container was allocated for insect media disposal, as well as for pipettes and general labware that was used to handle the cells. This container was then disposed of by the administration of the Biotechnology Center, that counts with specific and curated protocols and guidelines to proceed with different biological materials.
Safety of the Project
The overall nature of our project involved two main working lines for the establishment of a characterized biological platform of high value protein production in insect cells.
The first one is the Baculovirus Expression Vector System or BEVS in which we used commercial DH10bac E. coli cells for the homologous recombination of our constructs for further transfection in our next project stage. The second working line was the insect cell line Spodoptera frugiperda Sf9, which was to be genetically modified by infection with baculovirus. Several considerations were established to ensure a proper handling of the biological material in each case.
The first consideration was the homologous recombination carried out in the baculovirus expression system for each of our constructs. When considering if handling this type of technology represented a risk for humans or mammals, investigation was carried out to ensure the safety of the overall use of the BEVS technology. As reported by Invitrogen Life Technologies’ Guide to Baculovirus Expression Vector Systems (BEVS) and Cell Culture Technique, the history of BEVS technology use, from eukaryotic vector systems to recombinant protein expression, has gained experience over the years since its beginnings in 1983. One of the advantages, is the fact that baculoviruses are “essentially nonpathogenic to mammals and plants.” (Life Technologies) Their host range is restricted to specific invertebrate species, exclusively to arthropods (McWilliams) and besides, no helper cell lines or viruses are required due to the fact that baculovirus genome has all the genetic information and minimal containment conditions for caring. Extensive testing for the safety of more than 30 baculoviruses has been made over the past 40 years, resulting in a long and complete safety record with no adverse effect on human health, indicating that this technology does not cause any health hazard.
Secondly, the risks of liberating biological material of baculovirus outside the laboratory facilities were considered. As reported by the University of Oregon, “polyhedrin negative baculovirus expression system is susceptible to desiccation and UV light; survival time is limited to hours and wild-type and AcNPV baculovirus with normal polyhedron genes can survive for days or weeks in the environment.”
Thirdly, the transformed insect cells Sf9’s safety issues were assessed. We wanted to characterize different promoters, proteins, reporters, and signal peptides. These included polyhedrin promoter, OpIE2, red fluorescent protein, honeybee melittin, Chicken Lysozyme, Nanoluc, HisTag, and others mentioned in our project’s description and none of which represent a threat to the environment or human nature. Since these were to be recombined in the BEVS system prior to the insect cell infection, the only organisms prone to infection were Sf9 cells. Therefore, the appropriate management of the insect cell line as described in the safety protocols was followed avoiding as much cross contamination as possible and creating greater aseptic working areas to ensure a safe laboratory environment.
Finally, it is important to mention that due to the standardized technology being used in the project, the feasibility to scale up the process after considering safety issues, as one of the main barriers in many biotechnological investigations, is enhanced and therefore biochemical evidence would be the key to implement the obtained results regarding the new platform for high value protein synthesis.
- Life Technologies. (2015). Cell Culture Basics Handbook. Thermo Fisher Scientific Inc.
- Life Technologies. (n.d.) Guide to Baculovirus Expression Vector Systems (BEVS) and Insect Cell Culture Techniques. Retrieved from Thermofisher Manuals
- McWilliam, Andrew. (n.d.) Environmental Impact of Baculoviruses. Food and Agriculture Organization. Retrieved from Agrotech documentation