An Oregon State University team built a robot that measures the vitrification threshold of hundreds of cryoprotectant mixtures in one week
An Oregon State University team built a robot that measures the vitrification threshold of hundreds of cryoprotectant mixtures in one week
On 22 July, the team published an article describing a system that uses three 384-well plates. For each cryoprotectant mixture, it identifies the lowest concentration at which the solution forms a glass rather than ice during cooling.
When water freezes, it forms ice crystals that damage cells. Cryoprotectants prevent water molecules from assembling into crystals, causing the solution to become glassy. For each mixture, researchers must find a concentration that suppresses ice formation without making the solution too toxic to cells.
Researchers usually conduct this search manually. They prepare a mixture, cool it, check for ice, and adjust the concentration. The authors transferred this cycle to three 384-well plates. A robot prepares the mixtures, a camera distinguishes transparent wells containing glass from wells containing ice, and software uses a binary search to select the next concentration. A single run produces 1 152 measurements. After five steps, the system locates the boundary to within 1% mass concentration.
In one week, the system collected about 26 thousand measurements and about 400 vitrification threshold values. The authors estimate that its throughput is approximately 50 times higher than that of test tube experiments. The threshold depends not only on the composition of the solution, but also on the experimental conditions. In all 11 mixtures tested, placing a silicone mat over the plate lowered the threshold. The authors attribute this result to the way ice forms at the interface between the solution and the air. The same formulation may therefore require different concentrations in an open well and a covered well.
Using these measurements, the authors developed a model for mixtures containing up to seven components. It predicts thresholds close to the measured values (R² no lower than 0.93) and allows them to be compared with published toxicity data. A review of cryoprotectant development describes the same problem: a solution must suppress ice formation while remaining tolerable to cells.
A system with a linear cryoprotectant gradient gradually brings cells, oocytes, and two-millimeter skin samples to the selected concentration to reduce osmotic damage. The new system selects the formulation and target concentration. The gradient system introduces the solution into the sample.