I am working on a multi-step long term experiment where I need to cryopreserve my organoids and then thaw them about a month later. I am trying to find the best way to do this.

Answer

Cryopreservation of organoids becomes more and more important to improve organoid-based therapy and for acquiring large numbers of cells. Please find here an article in which the cryopreservation of intestinal organoids either undissociated or dissociated is described: Long-term culture-induced phenotypic difference and efficient cryopreservation of small intestinal organoids by treatment timing of Rho kinase inhibitor.

Interestingly, protocols to freeze can differ when using mouse or human organoids. Here is a protocol handbook that describes organoid culture including those for cryopreservation of human and mouse organoids.

Additionally, we have observed a significant increase in cell survival after thawing of organoids, after being processed as described in the Tuveson lab protocol, step 1 to a small size (between 30-40 µm), organoids are plated in 55% extracellular matrix (ECM) drops for 1-2 days. With this plating step, we let organoids recover from the stress of disruption, but also activate the proliferations state right before freezing. After 1-3 days (depends on the human organoid model, for instance after 1 day some organoids are visually increasing in size (colon), while others need three days (lung or breast). We collect them from the ECM drop and freeze them following the steps indicated in step 3 onwards. We have observed that the presence of some ECM in the pellet does not have a negative impact in the survival of the organoids, so extra washes to remove ECM are not required. We recommend aspirating the ECM free of organoids from the pellet.