I want to isolate and culture all immune cells from the adipose tissue to isolate EVs. What is the best approach to make sure all cells survive for at least 48h?
Aayush --
Tissue Harvesting and Initial Processing: Harvest adipose tissue using liposuction rather than excision. This method yields a higher number of viable cells and is less damaging to the stromal cell fraction, which includes immune cells. Immediate processing within 60 minutes is ideal, but if delayed, storing the tissue in culture medium at 4°C for up to 24 hours is acceptable without significant loss of cell viability. Cell Isolation Method: (1) Nonenzymatic Isolation: Utilize a nonenzymatic method for isolating cells from adipose tissue. This approach has been shown to be effective in maintaining cell viability and promoting cell migration, proliferation, and confluency, which are crucial for subsequent EV isolation. (2) Chitosan-Based Selection: Consider using a chitosan-based selection system for primary culture. This method reduces the primary culture period and enhances the yield and quality of isolated cells, including immune cells. The chitosan-based system also supports better cell expansion and maintenance of stemness, which may be beneficial for EV production3. Culture Conditions: (1) Scratched Flasks: Culture the isolated cells in scratched cell culture flasks. Scratched surfaces promote better cell migration, proliferation, and confluency, which can help in maintaining cell viability for at least 48 hours. (2) Cryopreservation: If long-term storage is necessary, cryopreserve the adipose tissue. Although initial cell viability may be lower after long-term cryopreservation, continued cell growth can neutralize this effect, ensuring a sufficient number of viable cells for EV isolation.
To isolate and culture all immune cells from adipose tissue to isolate extracellular vesicles (EVs) and ensure cell survival for at least 48 hours, the following approach is recommended:
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