Dear all,
Does anyone know or have an experience in freezing (cryopreservation) of the primary Human Hepatic Stellate Cells? How much DMSO is recommended to use? I read that cryopreservation of this cell line is possible, unfortunately, I can't find a good and detailed freezing protocol.
I would be very grateful for receiving any answers. :)
Best regards.
Hi Tomasz,
Found this paper which could help you out from Medical Molecular Morphology, June 2010, Volume 43, Issue 2, pp 107-115.
Here is the text from their methodology:
Cryopreservation
Medium was renewed after 24 h, and cells were further cultivated for another 6–7 h. Before trypsinization, cells were washed carefully two or three times with prewarmed phosphate-buffered saline (PBS). The cells were then detached by treatment with trypsin/EDTA solution (8–10 min) at 37°C. Detached cells were collected and culture dishes were again rinsed with prewarmed DMEM. Both solutions were combined, cooled on ice, and centrifuged at 800 g for 10 min at 4°C. The pellet was resuspended in ice-cold CELLBANKER (Zenoaq, Tokyo, Japan) (3–4 × 106 cell/ml).
Next, cell suspension was placed in 1.8-ml cryovials. First, the cells were frozen overnight in an isopropanol bath freez- ing container (Nihon Freezer Co., Tokyo, Japan) at −80°C, then transferred to liquid nitrogen and stored. For frozen/ thawed cells, the viability was determined by trypan blue8.
https://link.springer.com/article/10.1007%2Fs00795-009-0484-5
Best of Luck!
Hi Tomasz,
You can cryopreserve human hepatic stellate cells using a cryopreservation media that has 10% DMSO. It also helps to have 10% FBS.
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