A highly active mineral-based ice nucleating agent supports in situ cell cryopreservation in a high throughput format.

Cryopreservation of biological matter in microlitre scale volumes of liquid would be useful for a range of applications. At present, it is challenging because small volumes of water tend to supercool, and deep supercooling is known to lead to poor post-thaw cell viability. Here, we show that a mineral ice nucleator can almost eliminate supercooling in 100 µl liquid volumes during cryopreservation. This strategy of eliminating supercooling greatly enhances cell viability relative to cryopreservation protocols with uncontrolled ice nucleation. Using infrared thermography, we demonstrate a direct relationship between the extent of supercooling and post-thaw cell viability. Using a mineral nucleator delivery system, we open the door to the routine cryopreservation of mammalian cells in multiwell plates for applications such as high throughput toxicology testing of pharmaceutical products and regenerative medicine.

Cryopreservation of primary cultures of mammalian somatic cells in 96-well plates benefits from control of ice nucleation.

Cryopreservation of mammalian cells has to date typically been conducted in cryovials, but there are applications where cryopreservation of primary cells in multiwell plates would be advantageous. However excessive supercooling in the small volumes of liquid in each well of the multiwell plates is inevitable without intervention and tends to result in high and variable cell mortality. Here, we describe a technique for cryopreservation of adhered primary bovine granulosa cells in 96-well plates by controlled rate freezing using controlled ice nucleation. Inducing ice nucleation at warm supercooled temperatures (less than 5 °C below the melting point) during cryopreservation using a manual seeding technique significantly improved post-thaw recovery from 29.6% (SD = 8.3%) where nucleation was left uncontrolled to 57.7% (9.3%) when averaged over 8 replicate cultures (p < 0.001). Detachment of thawed cells was qualitatively observed to be more prevalent in wells which did not have ice nucleation control which suggests cryopreserved cell monolayer detachment may be a consequence of deep supercooling. Using an infra-red thermography technique we showed that many aliquots of cryoprotectant solution in 96-well plates can supercool to temperatures below -20 °C when nucleation is not controlled, and also that the freezing temperatures observed are highly variable despite stringent attempts to remove contaminants acting as nucleation sites. We conclude that successful cryopreservation of cells in 96-well plates, or any small volume format, requires control of ice nucleation.