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Safety

Introduction
Safe Lab Work
Safe project design
Safety laws and regulations

Introduction

Safety was always our top concern, both while working in the lab and during project design.

We divided our safety procedures into 3 general categories: Safety in lab work, Safety in project design and Safety laws and regulations.

Safe Lab Work

All lab work and experiments were done according to the laboratory safety policies at Technion University ^[1]. All team members received in depth safety training, provided by our instructors, which covered all the safety instructions such as lab rules, biosafety levels, biosafety equipment, dangerous chemicals and sterilization while working in the lab. Additionally, we received information about the location of fire alarms, safety showers, eye washing stations, fire extinguishers and fume hoods.

Hazardous substances:

Ethidium bromide - A known carcinogen, used in agarose gel. Therefore, gel preparation was done in designated marked areas and with designated equipment. Addition of EtBr to the mix was done in a hood, and special emphasis was placed on wearing gloves and lab coats and covering our faces when handling gel.

Liquid nitrogen - Can cause frostbite or cryogenic burns if not handled properly. Therefore, when working with liquid nitrogen, we always wore protective clothing, including safety goggles, cryogenic gloves and tongs.

Lab safety was divided into 3 main parts:

Personal safety: All lab workers wore pants, closed toed shoes, lab coats, and gloves while working in the lab.

Keeping the lab environment sterilized: Our work benches were cleaned and sterilized before and after working. When we worked in the biosafety cabinet we sprayed everything that entered the cabinet with ethanol and additionally, at the end of every workday, A UV light inside the biosafety cabinet was turned on. The waste was separated to biologic waste and regular waste.

Different lab spaces with different Safety Levels: Open air benches were designated for regular lab work such as cloning (plasmid purification, restriction analysis, etc.). The fume hood was designated for work with ethidium bromide. Lastly, the biosafety cabinet was designated for work with mammalian cells.

Safe project design

Organisms:

In our project, we dealt with various organisms, but mainly with mammalian cells.

E.coli Top10 from risk group 1 were used for molecular biology (cloning, plasmid amplification, etc.). Mammalian cells from risk group 2 were used as biological chassis. There was no use of organism from risk groups 3 or 4.

We have used two Chassis organisms:

HPC-7 (murine multipotent progenitor cell line)
HEK 293

Besides our chassis organisms, we used two other organisms from risk group 2:

CHO-SCF: A substance called stem cell factor (SCF) needs to be added to the growth medium of the HPC-7 cell line. SCF is very expensive to buy, therefore we will grow CHO (Chinese hamster ovary) cells that secrete the SCF, and then filter it and use it as conditioned medium.

WEHI-231: In the bone marrow, immature B cells that bind to self-epitopes undergo apoptosis. In an attempt to model this process, we will use - WEHI-231 (cell line - immature B cell lymphoma) cells. These cells are known to undergo apoptosis when the IgM presented on their membrane binds to anti-IgM. We want our HPC-7 cells to present anti-IgM (using our display system) so when cultured with the WEHI 231, they will undergo apoptosis due to IgM - Anti-IgM binding.

Our project includes numerous safety risks:

Allergic reaction to the treatment itself is a possibility, and could perhaps even trigger some of the processes we set out to prevent.
Integrating the gene into the genome with viral vectors is known to cause cancer ^[2]. As such, new tools for the safe use of viral vectors are under development and we are considering various other integration techniques (such as CRISPR/Cas9 mediated genome integration) that may be more appropriate for clinical settings if and when our idea becomes clinically relevant.
We designed a kill switch which will allow us to irreversibly disable the expression of antigen on the membrane, so that in case of an adverse reaction or personal dissatisfaction the whole system can be deactivated.

Safety laws and regulations

Since our project deals with a living environment, ideally we would test our theories in animals. Both in Israel^[3] and in iGEM there are strict rules for experiments conducted on animals, and therefore we worked in vitro .

Moreover, our idea includes genetically engineered hematopoietic stem cells, and cord blood is an abundant source of those cells. In order to work with cord blood in Israel, an ethical permit is required (from Helsinki committee that are working in accordance with ICH-GCP ^[4]). Due to time constraints we have decided to use a model cell line (HPC-7 cells), In addition, the use of viruses is needed for our application - working with viruses requires special safety practices and a separate biological hood (which is not available for us at this point), as a result we have decided to work with plasmids and transient transfection methods (Electroporation and Chemical based methods) and thus we can say that in all areas we have encountered so far, we found that there are appropriate rules and regulations which we follow.