Rate-Controlled Freezing Container

Description:

This system allows you to freeze mammalian cells at a controlled rate, greatly improving long-term cell viability during cryopreservation. By filling a 250 mL glass beaker with ethanol and placing the assembly into a –80 °C freezer, the ethanol acts as a thermal buffer. As a result, samples cool at approximately –1 °C per minute, which is the ideal rate for most cultured cell lines.

This design replicates the function of the commercially available Thermo Scientific™ Mr. Frosty™ Freezing Container (link) but at a fraction of the cost. Both systems rely on the same scientific principle: ethanol’s high heat capacity slows heat transfer, preventing cells from freezing too quickly. Rapid freezing causes intracellular ice crystals, which rupture membranes. A gradual, controlled cooling rate allows water to exit the cell before freezing, minimizing ice formation and increasing post-thaw survival.

This printable setup suspends cryovials inside the ethanol bath while preventing floating or bobbing. A weighted metal beam applies gentle downward pressure through the spacer and collar system, ensuring the cryovials stay at the proper depth throughout the cooling process.

Scientific Principle (What Mr. Frosty Does):

Ethanol cools more slowly than air, providing a thermal buffer in the freezer.

This produces a near-linear cooling rate of ~1 °C/min.

Slow cooling allows osmotic dehydration of cells before freezing.

Reduced intracellular ice formation leads to significantly higher post-thaw viability.

This method is standard for cryopreservation of mammalian cells using DMSO-containing freezing media.

Notes:

Print all components in PETG to tolerate ethanol exposure.

Supports are required for the lids.

The steel or aluminum brick used as a weight should measure 20 × 20 × ≥80 mm.

Its job is to keep the cryovial assembly properly submerged and prevent movement.

The spacer fits over the vial collar so that the suspended metal beam presses directly onto the vial top.

M3 square nuts insert into the collar, allowing the screws to clamp the collar onto the cryovial lid securely.

Bill of Materials (BOM):

2× M3 square nuts (for collar)

Dimensions: thickness 2.2 mm; width across flats 5.3 × 5.3 mm

2× M3 × 8 mm screws

Optional metal beam (steel or aluminum) for weighting

250 mL glass beaker

Printed components (collar, lid, spacer, etc.)