Polyproline as a Minimal Antifreeze Protein Mimic That Enhances the Cryopreservation of Cell Monolayers

Abstract

Tissue engineering, gene therapy, drug screening, and emerging regenerative medicine therapies are fundamentally reliant on high-quality adherent cell culture, but current methods to cryopreserve cells in this format can give low cell yields and require large volumes of solvent "antifreezes". Herein, we report polyproline as a minimum (bio)synthetic mimic of antifreeze proteins that is accessible by solution, solid-phase, and recombinant methods. We demonstrate that polyproline has ice recrystallisation inhibition activity linked to its amphipathic helix and that it enhances the DMSO cryopreservation of adherent cell lines. Polyproline may be a versatile additive in the emerging field of macromolecular cryoprotectants.

Keywords: biomaterials; cryopreservation; ice recrystallization inhibitors; monolayers; polymers.

Scheme 1

Condensation polymerisation of proline. The materials were used in stereopure form but both the l‐ and d‐isomers were used, hence no stereocentres are shown.

Figure 1

A) Circular dichroism spectra. B) IRI activity of the polyproline series. C) IRI activity compared to other homopolypeptides. D) Cryomicrograph of a PBS negative control. E) Cryomicrograph of 20 mg mL⁻¹ polyproline. Photographs taken after 30 min at −8 °C. Error bars represent ± standard deviation from a minimum of three replicates. Images shown are 1.2 mm across. MLGS=mean largest grain size.

Figure 2

Hydrophobic surface mapping of A) recombinant type I sculpin AFP, B) PPro₁₀, and C) PGlu₁₀, showing charged hydrophilic surfaces. Hydrophobic regions (red), hydrophilic regions (white).

Figure 3

Cross‐section of ice crystals perpendicular to the temperature gradient: A) ZrAc (positive control), B) PPro₁₉, C) PBS (negative control). The ice crystals expel the dye while growing, appearing in black, while the remaining liquid fluoresces.

Figure 4

A549 cryopreservation. Cell recovery determined by trypan blue assays. Cells were first incubated either in the medium alone or with 200 mm proline for 24 h. They were subsequently cryopreserved by addition of 10 % DMSO with the indicated PPro₁₁ concentration. Error bars ± S.E.M. from n=3 with two nested replicates. # P<0.05 compared to 10 % DMSO treatment; * P<0.05 compared to 200 mm proline exposure with 10 % DMSO treatment.