The ZJUintl-China team believes that safety ensures the proper conduct of researches. Therefore, we put safety first when designing and proceeding experiments. In order to protect the environment and ourselves, all team members follow the safety precautions and Covid-19 regulations. For a detailed overview, please check our safety form.

There are two work places with different biosafety levels in our laboratory. Level 2 space is only used for cell culture, and level 1 space is used for the rest experiments including molecular cloning and bacteria culture. All equipment is placed in defined areas which meet national standards, and contaminated areas are arranged for each laboratory bench, including trash cans and waste liquid pools.

We invited the laboratory seniors and the instructors responsible for the laboratory safety to conduct professional training and guidance for us. A safety meeting was held with full participation to introduce and revise the project design and procedures. In terms of transfection, Prof. He Huang’s lab provided several methods including lentivirus transfection, electroporation, and lipoprotein transfection. Lentivirus are extremely forbidden since it may transfect human to integrate their DNA into human genome, leading to irreversible damage. Considering safety, accessibility and efficiency, we determined to use electroporation to undergo plasmid transfection. In addition, when deciding on animal study (mice), although Prof. He Huang’s lab has been approved by national animal ethics department, none of team members had any experience in animal experiments. In order to prevent accidental harm to both team members and mice in surgery, our team chose to focus on cellular experiments. With regard to experiments on the subject of caffeine-activation of CAR-T cells, the caffeine is considered one of psychotropic drugs which may cause addiction. Instead of pure caffeine, coffee was used to keep team members against caffeine abuse and addiction.

Our team members must have participated in detailed training of laboratory security before starting experiments. We have learned basic laboratory safety rules and regulations, understood how to operate various laboratory instruments and mastered safe practices in bacterial and cellular experiments. Emergency facilities for safety risks in the laboratory are prepared to ensure the safety of laboratory personnel and environment. The safety equipment includes eye showers, emergency showers, fire extinguishers, fire blankets, first aid kits, and electrical emergency stops. In addition, E. coli (DH5-α), Jurkat cells and U-118MG cells were purchased from a life science company, all of which were tested for non-contamination and non-pathogenicity and met national standards.

We strictly abide by the safety regulations of Zhejiang University during the experiments. Ethanol was used to clean the lab bench before and after experiments. As for level 2 space, additional UV disinfection was needed before the experiments. Personal protective clothing such as lab coats and gloves must always be worn.

Some intended experiments had to be accomplished with hazardous reagents. Therefore, team members must follow all rules and guidelines to prevent harm to themselves or the environment, as taught in safety training:

• Ethidium bromide: When performing gel-electrophoresis, ethidium bromide is used to visualize the DNA. However, it may splash on eyes, mouth and face, which could cause cancer. So using it in the fume hood is recommended.
• Antibiotics: The reprogrammed bacteria and cells will be selected by the antibiotics penicillin, streptomycin, ampicillin and puromycin. At high doses, they can be poisonous when inhaled, ingested or in contact with the eyes. Appropriate safety measures were taken when handling antibiotics and disposal protocols were followed.
• DMSO: We need to cryopreserve cells for long-time storage, DMSO in the cryopreserved solution is volatile and may be inhaled or penetrate the skin, which could burn the skin and cause headache, dizziness, and nausea. Therefore, the biosafety cabinet is required in handling DMSO.
• Ethanol: When extracting large amounts of plasmids, it’s possible that ethanol could be inhaled, which could lead to irritation or allergies. A facial mask is needed during the experiment.

Due to the COVID-19 pandemic, masks must be worn at all times in the laboratory. Every three days, team members must perform a nucleic acid test and receive a negative report before continuing the experiments. Besides, the hours worked must be recorded using a punch card machine upon entering the laboratory and before leaving the laboratory.

Our weekly team meetings and regular subgroup meetings were conducted entirely online. In addition, meetings with other teams were also held entirely online to avoid exposing our laboratory members and staff to unnecessary risk factors.