We are committed to designing an advanced engineered human umbilical cord mesenchymal stem cell (hUC-MSC) exosome, specifically targeted for end-stage liver disease, to achieve precision therapy and enhanced therapeutic efficacy. Given that this engineered exosome will operate deep within the human body and directly act on activated hepatic stellate cells, ensuring its safety for patients and the surrounding microenvironment is paramount.

To achieve this goal, we will adopt a phased and systematic safety strategy, focusing on a comprehensive risk assessment of both the "vehicle" (the exosome itself) and the "cargo" (the therapeutic molecules).

We have selected human umbilical cord mesenchymal stem cells (hUC-MSCs) as the source of exosomes due to their low immunogenicity and well-established safety profile. Techniques such as Transmission Electron Microscopy (TEM), Nanoparticle Tracking Analysis (NTA), and Western Blot (WB) will be rigorously employed to characterize exosome morphology, size distribution, and purity, preventing contamination by impurities like cellular debris and ensuring batch-to-batch consistency.

Following thorough characterization of the vehicle, we explored two loading strategies for the therapeutic molecules (microRNAs), with targeted safety optimization and validation for each method. The first utilizes co-incubation, achieving high loading efficiency through controlled electrical pulses. The second employs a gentler method involving direct co-incubation combined with ultrafiltration, which relies on the natural internalization of miRNAs and has minimal impact on membrane integrity. Regardless of the method used, systematic quality checks will be performed post-engineering, including assessments of loading efficiency, bioactivity, and exosome membrane integrity, to ensure the engineering process does not compromise their native structure and function.

The impact of the engineered exosomes on hepatic stellate cells will be evaluated through in vitro experiments. All data will be compared against control groups treated with non-engineered exosomes to accurately attribute any observed effects.

In the process of participating in the igem competition, our team is well aware that the importance of laboratory safety is no less than that of scientific research and innovation itself. In order to create a safe experimental environment, we strictly abide by the laboratory safety regulations, start from the details, and penetrate the safety awareness into every experimental step.

Basic safety training

Prior to the conduct of the wet experiment, members of the experimental team received comprehensive basic safety training to understand and master basic safety knowledge.

Team members are required to receive comprehensive basic safety training before entering the lab, including chemical safety, biosafety, electrical safety, and personal protective equipment (PPE) use. Our training also covers laboratory rules and regulations, emergency evacuation routes, and first aid measures.

For example, chemical safety: Team members are required to have a thorough understanding of the physical and chemical properties of chemicals commonly found in the laboratory and pay attention to the key information in the Chemical Safety Data Sheets (MSDS), paying special attention to their flammable, explosive, toxic, and corrosive properties, so that proper precautions can be taken when handling them. After understanding their properties, we will strictly classify and store them according to their nature, placing them in designated safety cabinets or storage areas to ensure that they are kept away from sources of ignition, heat and incompatible substances. The storage area should be clearly marked with warning signs for easy identification and management.

Special skills training

We train our team members in specialized skills, such as microbiological operations and cell culture, according to the needs of the experimental project. We ensure that each member has the ability to complete the experimental tasks independently, and understand the potential risks and preventive measures, and strictly follow the safety code of biological laboratory.

Photo 1. Silhouette of operating technology training

click to Laboratory Safety Management Measures of Medical Laboratory Center of Lanzhou University-Lanzhou University Medical Laboratory Center (lzu.edu.cn)

Personal protective equipment (PPE)

When performing experimental operations, team members must wear appropriate personal protective equipment (PPE), such as lab coats, gloves, goggles, and masks. Also ensure that the PPE is intact during the experiment, and check and replace it regularly.

Photo 2. Personal protection

division of the experimental area

According to the degree of danger of the experimental objects, the laboratory is divided into different zones, such as low-risk zone, medium-risk zone and high-risk zone. Corresponding safety measures and protective measures are taken in different zones, such as setting up biological safety cabinets and ventilation systems.

Operating Procedures

Before the experiment begins, we develop detailed operating procedures, including steps, safety precautions, and emergency measures. This includes special marking of toxic and harmful reagents used in the experiment to ensure their properties and hazards are understood before use, thereby enabling appropriate protective measures to be taken during the experiment.

Laboratory Waste Disposal and Classification

We commit that all infectious materials generated during the experiment have been cleared of contamination in the laboratory and treated by high-pressure sterilization, disinfection, etc. before being disposed of.

Disinfection of Ultra-clean Bench

Before the experimental operation, clear the ultra-clean bench and disinfect it with ultraviolet light for half an hour; after the operation, clear the ultra-clean bench, wipe it with alcohol, and disinfect the gun with alcohol; then disinfect the ultra-clean bench with ultraviolet light for one hour, and sterilize the consumables used by high-pressure sterilization.

click to Laboratory Safety Management Measures of Medical Laboratory Center of Lanzhou University-Lanzhou University Medical Laboratory Center (lzu.edu.cn)

Photo 3. Laboratory waste collection container

Other security measures

First of all, before the experiment was officially carried out, we checked the physical and mental health of the team members to ensure that they had the ability and conditions to participate in the experiment, and signed a safety responsibility agreement with each member to clarify their duties and obligations in the laboratory and to enhance their safety awareness and sense of responsibility.

Secondly, we have developed an experiment record sheet to record in detail information such as the time of the experiment, the operator, and the content of the experiment, in order to ensure the standardization and traceability of the experimental process.

In addition, to ensure the cleanliness and tidiness of the laboratory environment, our team has established a detailed schedule for regular cleaning, clarified the specific cleaning areas and responsibilities of team members, and incorporated them into the daily laboratory safety management. A clean lab environment not only significantly improves the accuracy and efficiency of experiments, but also reduces potential safety risks.

Photo 4. Our schedule and the cleaned laboratory

Through these efforts, our team has built a strong defense for laboratory safety and provided a strong guarantee for the smooth progress of scientific research.

Artificial mirna mimic

Both miR-455-3P and miR-148a-5p are derived form RIBOBIO.

Photo 5. MIRNA

The exosomes (CTE-052) used in this product are sourced from ECHO AIOTECH, a commercial supplier that adheres to stringent quality standards. Their mature and reliable production system ensures the safety and batch-to-batch consistency of the product from the source. Numerous domestic and international studies have confirmed the therapeutic potential of MSC-derived extracellular vesicles (MSC-EVs). The cell-free nature of MSC-EV therapy offers advantages such as low immunogenicity, high stability, and excellent biocompatibility. These exosomes can deliver a variety of bioactive molecules (e.g., proteins, mRNAs, miRNAs) and improve liver function through mechanisms including modulating hepatic stellate cell activation, promoting hepatocyte proliferation, and ameliorating the liver microenvironment. This approach provides a new research direction for developing safer and more effective therapeutic strategies for end-stage liver disease.

Photo 6. TEM characterization of hUC-MSCs (CTE-052) exosomes

Photo 7. Western blot characterization of hUC-MSCs (CTE-052) exosomes