FAQs

For short-term storage, exosomes can be kept at 4 °C for up to one week. For long-term storage, they can be stored at −20 °C or −80 °C. When storing exosomes long-term, it is important to consider whether multiple thawing events will be needed. If repeated use is required, we recommend aliquoting the exosome solution into multiple tubes so that each tube undergoes only a single freeze–thaw cycle. Multiple freeze–thaw cycles have been found to damage exosomes and reduce their quantity.

Isolating exosomes from cell culture media or bodily fluids is a complex and challenging process. It typically involves ultracentrifugation combined with sucrose density gradient or sucrose cushion centrifugation, allowing low-density exosomes to be separated from other vesicles and particles. These procedures can take up to 30 hours and require an ultracentrifuge as well as extensive training. Despite these drawbacks, this remains the most commonly used method for exosome isolation.

Nanoparticle tracking analysis (NTA) is currently one of the most commonly used methods to determine the average particle size and concentration in solution. However, this technique cannot distinguish vesicles from protein aggregates of similar size. Therefore, we recommend combining NTA with microscopy techniques to verify the presence of membrane-bound vesicles. We place great emphasis on consistent cell culture and exosome collection conditions to ensure comparable exosome yields between preparations, which we consider more important than simply estimating the total protein concentration of the preparation.

Depending on their origin and function, exosomes can contain a variety of components, such as immune factors, growth factors, microRNAs, and mRNAs. The applications of exosomes depend on the specific substances they carry.

At present, the definition of exosomes is relatively complex, and there is no absolute consensus in the field. Exosomes are commonly defined as vesicles with a density of approximately 1.13–1.19 g/mL that float in a sucrose gradient during ultracentrifugation, with an expected size of 30–150 nm based on electron microscopy analysis. Exosomes can also be defined and identified by their surface protein markers, including tetraspanins (CD63, CD81, CD9) and other markers such as ALIX.

In theory, earlier passages retain stronger stemness. However, during in vitro culture, stem cells undergo adaptation from their tissue microenvironment to the culture conditions, and cells that fail to adapt are eliminated. In the first two to three passages, the genome may be unstable, making these cells less suitable for clinical use. Studies have shown a higher proportion of abnormal karyotypes in early passages. For example, for mesenchymal stem cells (MSCs), the optimal passage range for clinical applications is generally between passages 4 and 6.

Maintain appropriate culture conditions: Ensure that cells remain in suitable environments after passaging, including medium composition, temperature, and CO₂ concentration.

Optimize culture medium: Use validated stem cell culture media and supplements to help maintain pluripotency.

Avoid excessive passaging: Over-passaging can compromise pluripotency and stability, so passage cells according to their growth characteristics.

Chromosomal abnormalities, such as gains, losses, or partial translocations, are common during cell culture. Ensuring that HiPSCs have a normal karyotype is critical for the reliability of cell behavior, observed phenotypes, or drug testing results, rather than effects caused by abnormal karyotypes. Due to the rapid proliferation of HiPSCs, they are particularly prone to such abnormalities. To minimize the risk, experimental passages of HiPSCs should be kept below 40, and karyotype analysis is recommended every 20 passages.

ROCK stands for Rho-associated protein kinase. Y-27632 is a small-molecule inhibitor that selectively targets ROCK1. Inhibiting ROCK1 is known to enhance the survival and cloning efficiency of stem cells without affecting their pluripotency.