Hi! Our lab just starts working with colorectal cancer organoids and breast cancer organoids. And we faced the problem in freezing of organoids.
Currently, we use such protocol: organoid pellet is resuspended in the 1ml of freezing medium, which contains 20% of DMSO and 80% of FBS, then we put the cryovial in Mr. Frosty Freezing Container overnight, and on the next morning we put the cryovial in the liquid Nitrogen. For the unfreezing, we put the cryovial in the incubator (+37 ℃) for a few minutes, while there will leave only a small piece of ice (with size as a pea), then transfer the cryovial to the hood and add the content of cryovial to the cold DMEM by drops. Additionally, we are shaking the tube with a DMEM while adding the organoids. Then we centrifuge for 5 minutes on 450 × g, 4°C, remove the supernatant, and seed the organoids on the Matrigel. Additionally, we are adding the CHIR-99021 and Y-27632 to our medium. However, after few days we are noticing that everything is dead.
What we are doing wrong? Maybe we should change somehow the freezing medium? Could you please recommend a working freezing protocol for organoids?
Thanks in advance!
Hello Anna
Please try using the freezing protocol for organoids mentioned in the link below.
https://www.protocols.io/view/cryopreservation-of-organoid-cultures-bh4ij8ue.pdf
Best Wishes.
20% DMSO seems rather high; I typically cryopreserve cells [suspension, not organoids] in 10% DMSO/90%FBS. I would suggest trying 10% DMSO/90% FBS.
The issue is that DMSO permeates into the cell, and even at 10% is hypertonic [~.375M vs ~.165M for normal saline], so 20% would be even more so. Upon thawing and dilution, your organoid cells may be rupturing from the influx of water due to the DMSO inside the cells.
Upon thawing, I always add my thawed cells into a tube of either HBSS or medium underlayed with 1 ml FBS. This seems to improve recovery.
Hope this helps.
Dear Gary Lee Gilmore ,
Thanks for your answer! We will reduce DMSO concentration!
Could you please give us a little more information about the recovery of the cells with HBSS/ medium+FBS? How do you do it? Do you unfreeze your cells and then immediately add the suspension of cells+freezing medium to the HBSS or you firstly wash off the freezing medium? And do you keep the cells in the HBSS for some time (how long?) or just wash the cells with it?
Thanks in advance!
Hi Anna, in my lab we freeze the organoids (breast cancer, colorectal, brain organoid) using CryoStor CS10 (Stem cell technologies cat #07930). After discarding the old media, usually I dissociate condensed organoids using TrypLE Express and pipetting up & down for 5-10 min. Then, add media (1:1) to stop the reaction and spin down at 400xg for 5 min at RT.
If the organoids are not condensed and too few, I use cold DPBS1X to help melting down the matrigel and incubate for 5 min on ice. Then spin down at 400xg for 5 min at 4*C.
Upon centrifugation, after discarding the supernatant we add cold Cryostor @1 ml or @500ul per cryovial tubes depending on the cell pellet. Then keep the cryotubes in mr.frostie in -80 overnight begore transferring to LiN2 tank
Hi Ariestya Indah Permata Sari , thank you for your answer! It is very helpful!
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