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Cell Freezing Calculator (Cryopreservation)

Plan a cell freeze: the cells needed for your vials at a chosen density, the suspension volume to centrifuge, and the freezing medium with its DMSO.

Formula

Ncells=ρ×Vvial×nvials×(1+extra)N_{\text{cells}} = \rho\times V_{\text{vial}}\times n_{\text{vials}}\times(1 + \text{extra})
Vsuspension=NcellsccellsV_{\text{suspension}} = \dfrac{N_{\text{cells}}}{c_{\text{cells}}}
VDMSO=%DMSO×Vfreezing mediumV_{\mathrm{DMSO}} = \%_{\mathrm{DMSO}} \times V_{\text{freezing medium}}
ρ\rho
cells per mL in each vial
extra\text{extra}
allowance for cells lost in handling

How it works

To freeze a batch of vials, decide the density per vial, then work back: the number of cells needed, the volume of suspension that contains them, and the volume of freezing medium to resuspend the pellet in, which has to fill all the vials. The freezing medium carries the cryoprotectant, most often 10% DMSO in medium or serum.

Cells freeze best when healthy and growing exponentially, at a viability above about 90%, and when cooled slowly. The page checks the viability and warns about large volumes. The details of the medium and the cooling rate depend on the cell line, so follow the protocol for yours.

Worked example

Ten 1 mL vials at 2 × 10⁶ cells/mL with 10% extra, from a suspension of 8 × 10⁵ cells/mL, with 10% DMSO.

  1. Freezing medium = 10 × 1 mL × 1.1 = 11 mL.
  2. Cells needed = 2 × 10⁶ × 11 = 2.2 × 10⁷; suspension = 2.2 × 10⁷ / 8 × 10⁵ = 27.5 mL.
  3. DMSO = 10% of 11 mL = 1.1 mL.

Centrifuge 27.5 mL of suspension and resuspend the pellet in 11 mL of freezing medium containing 1.1 mL of DMSO.

These are the values the calculator opens with, so you can check its output against this example.

Assumptions

  • Cells are counted accurately and all the cells in the suspension are recovered, apart from the allowance.
  • The cryoprotectant percentage is that of the finished freezing medium.

Common mistakes

  • Freezing over-confluent or unhealthy cells, which recover poorly.
  • Leaving the cells in DMSO at room temperature for long, which is toxic.
  • Cooling too quickly, or plunging vials straight into liquid nitrogen.

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