Dear all,
I have 2 questions:
1. how to dissociate organoid in general to process single cell sequencing experiment?
2. can I cryopreserve organoid and thaw to process single cell sequencing experiment?
Thanks!
Best,
Lux
Dear Xiang Lu
1.In brief, collected organoids were incubated with pre-warmed (37 °C) TrypLE for a total of 8–30 min (depending on the organoid sizes and densities) in a 37 °C incubator on a shaker at 65 rpm for a gentle swirl. During the incubation, the dissociation mixture was agitated every 3 to 10 min with gentle pipetting using wide-orifice p1000 and p200 tips (Mettler-Toledo). By 8–30 min of incubation, the tissue structure dissociated into a single-cell suspension with no visible cell aggregation. Cold 3% bovine serum albumin (BSA) solution (Sigma-Aldrich) was added to the dissociated cell suspension to inactivate TrypLE enzymatic activity. The suspension was filtered through a 40-μm Flowmi cell strainer (Bel-Art) to eliminate any debris. An additional three washes with cold 3% BSA solution were performed. Cells were resuspended in 3% BSA and filtered through a 40-μm Flowmi cell strainer. Viability (live cell percentage) and live cell count was determined using Trypan blue (used at a 1:1 ratio) and Countess II.
The final cell concentration was around 1,000 cells /μl, with a cell viability greater than 90.
You can refer to the following article: https://www.nature.com/articles/s41586-020-2352-3
Follow Hui Sun's method. You can not isolate single cells from frozen organoids. Trypsin will not work to isolate single cells
I did use TrypLE, the purpose is to separate cells from each other. TrypLE™ Express is free of animal-derived components.
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