System converts a cell suspension into droplets and vitrifies it at a throughput starting at 100 ml per hour

System converts a cell suspension into droplets and vitrifies it at a throughput starting at 100 ml per hour

On July 27, a group at the University of Minnesota posted a preprint on cryoaerosolization. A vibrating nozzle breaks a cell suspension into microdroplets, which are then frozen by a stream of liquid nitrogen. After thawing, about 90% of the human fibroblasts and induced pluripotent stem cells remained viable.

Cells intended for therapy are first grown, then stored and transported. During freezing, water forms ice crystals that damage cell membranes and internal structures. Vitrification protects cells from ice formation if the solution is cooled and rewarmed rapidly.

This is easier to achieve with small volumes. A microdroplet loses heat quickly because it has a large surface area relative to its volume. A cell dose can occupy tens or hundreds of milliliters, and a large volume cools more slowly. Cryoprotectants suppress ice formation, but high concentrations can harm cells.

In this system, the nozzle produces droplets about 200 micrometers in diameter. A stream of liquid nitrogen meets them in flight, and the droplets fall into a collection vessel for storage. According to the authors’ thermal model, contact with nitrogen accelerates heat removal and prevents a droplet from remaining suspended above the nitrogen on a layer of vapor. In the experiment, the system processed from 100 ml of cell suspension per hour, with a penetrating cryoprotectant concentration of 19–25%.

Instead of freezing one large volume, the system freezes many microdroplets. The authors measured an average cooling rate of up to 210 000 degrees per minute, while their rewarming model predicted about one million degrees per minute. After one cycle, about 90% of the fibroblasts and induced pluripotent stem cells remained viable, while pig erythrocytes showed 94% recovery. For the induced pluripotent stem cells, the team also assessed colony formation after replating.

A review of cell therapy cryopreservation describes cryobags with volumes ranging from 10 to more than 100 ml. Cryoaerosolization allows such material to be processed continuously while preserving rapid heat transfer in each individual droplet.