Methods and formulation to improve cellular survival of thawing after cryopreservation

Unmet Need

Cryopreservation is the main method of long-term storage and transport of living cells, and it is used for cell lines and patient samples such as stem cells, CAR-T cells, bone marrow, cord blood, and embryos. During the cryopreservation process, cells or samples are suspended in a freezing medium, stored at sub-zero temperatures, and then thawed and cultured when they are needed again. However, significant cell death can occur during the freezing and thawing process, reducing the number of cells available after storage and causing other side effects such as genetic drift. While there are protective agents that can be added to the freezing media to protect cells from the stress of cold temperatures, significant cell loss also occurs during the thawing process through a separate mechanism called ferroptosis. Ferroptosis causes cell death via lipid peroxidation and oxidative stress. There is a need for a reagent to add to cell culture medium during the thawing and culture process that would improve cell viability and the number of cells available to use after cryopreservation.

Technology

Duke inventors have developed a protective media supplement that improves cell survival after cryostorage. Specifically, this is intended to be used as an additive to a cell culture medium to protect against ferroptosis. This technology inhibits the process of ferroptosis to improve cell viability during the thawing process. Previously developed, synthetic ferroptosis inhibitors often have off-target effects, leading to cell toxicity and epigenetic changes. However, this technology is derived from naturally occurring nutrients that robustly inhibit ferroptosis with fewer side effects. This technology has been demonstrated to inhibit ferroptosis and improve cell viability after cryopreservation of mammalian cells.

Other Applications

This technology could be used across a wide variety of clinical, academic, and industry applications related to cryostorage of cells. Specifically, this would greatly benefit cryostorage of patient samples with a limited number of cells, to help prevent damage to precious samples.

Advantages

• No changes required to current cryopreservation protocols, since the reagent is added to culture medium during the thawing process
• Improves yield of cells collected from cryopreserved patient samples
• Decreases the number of cells needed per cryovial to maintain the same density of viable cells, leading to a higher potential number of stored samples from a single cellular source