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.


Ice recrystallisation inhibition (splat) assay
Ice recrystallisation inhibition was measured using a modified splat assay. 1  Photographs were obtained using an Olympus CX 41 microscope with a UIS-2 20x/0.45/∞/0-2/FN22 lens and crossed polarizers (Olympus Ltd, Southend-on-Sea, UK), equipped with a Canon DSLR 500D digital camera. Images were taken of the initial wafer (to ensure that a polycrystalline sample had been obtained) and again after 30 minutes. Image processing was conducting using Image J, which is freely available. In brief, five of the largest ice crystals in the field of view were measured and the single largest length in any axis recorded. The average S3 (mean) of these five measurements was then calculated to find the largest grain dimension along any axis. This was repeated for three individual wafers, and the average (mean) of these three values was calculated to give the mean largest grain size (MLGS). The average value was compared to that of a PBS buffer negative control.

Surface hydrophobicity mapping of proteins
NMR solution phase (AFP Sculpin) and X-Ray crystal structures of proteins and peptides of interest were acquired from the Protein Data Bank and other publically accessible sources, or computationally modelled in-house (PPro 10 and PGlu 10 ). Structures were rendered in PyMOL (Schrödinger LLC, Cambridge, MA), which is freely available for educational use, and surfaces on the structures were displayed. An open source script "color_h" was used to colour the protein surface according to the Eisenberg hydrophobicity scale of its constituent amino acids, from red (hydrophobic) to white (hydrophilic). For the homo-polypeptides where scaling is not possible, aliphatic hydrogen and carbon were defined as hydrophobic whilst oxygen, hydrogen and nitrogen as hydrophilic, utilising the same colour scheme. Due to the lack of hydrogen bond donors in a PPro 10 PPII helix, this was considered representative.

Cell culture
Human Caucasian lung carcinoma cells (A549) were obtained from the European Collection (HyClone, Cramlington, UK). A549 cells were maintained in a humidified atmosphere of 5% CO 2 and 95% air at 37 °C and the culture medium was renewed every 3-4 days. The cells were S4 subcultured every 7 days or before reaching 90% confluency. To subculture, cells were dissociated using 0.25% trypsin plus 1 mM EDTA in balanced salt solution (Gibco) and reseeded at 1.87X10 5 cells per 175 cm 2 cell culture flasks.

Cell solution preparation
Solutions for cell incubation experiments were prepared by dissolving the individual compounds in F-12K supplemented with 10% FBS and 1X PSA (solutions used as freezing buffers did not contain PSA) and sterile filtered prior to use.

Cryopreservation of A549 cell monolayers
Cells to be frozen in the monolayer format were seeded at 0.4X10 6 cells per well in 500 µL of cell culture medium in 24-well plates (Corning Incorporated, Corning, NY). Plates had a total available volume of 3.4 mL with an approximate growth area of 1.9 cm 2 , no coverslips were used and plates were used with the accompanying lid. Cells were allowed to attach to the entire free surface of the bottom of the well and formed a confluent layer not greater in height than one cell. Before experimental treatments, cells were allowed to attach for 2 h to the plates in a humidified atmosphere of 5% CO 2 and 95% air at 37 °C. The medium was exchanged against medium that was or was not supplemented with solutes as indicated in the figure. Control cells received no additional solutes and experimental cells were incubated with 23.1 mg/mL Lproline for 24 h in a humidified atmosphere of 5% CO 2 and 95% air at 37 °C. Following the incubation period, the culture medium was removed and cells were exposed for 10 min at room temperature to different concentrations of solutes dissolved in F-12K supplemented with 10% FBS and 10% DMSO. After 10 min, the freezing solutions were removed and the plates placed inside a CoolCell® MP plate (BioCision, LLC, Larkspur, CA), transferred to a -80 °C freezer and frozen at a rate of 1 °C/min. After 24 h at -80 °C, cells were rapidly thawed by addition of 500 µL cell culture medium warmed to 37 °C. Cells were placed in a humidified atmosphere S5 for 24 h and then dissociated using 0.25% trypsin plus 1 mM EDTA in balanced salt solution.
The number of viable cells was then determined by counting with a haemocytometer (Sigma Aldrich Co Ltd) at room temperature after 1:1 dilution of the sample with 0.4% trypan blue solution (Sigma Aldrich Co Ltd). The initial cell medium was discarded such that any nonattached cells were not included in the assessment. The percentage of recovered cells was calculated by dividing the number of cells with intact membranes after freezing and thawing by the number of cells present prior to freezing (i.e. after application of pre-treatments), multiplied by 100.

Cytotoxicity Measurements
Cells were seeded at 6·10 4 cells per well in 200 µL of cell culture medium with indicated concentrations of polyproline in 96-well plates (ThermoFisher). Cells were incubated for 24 h in a humidified atmosphere of 5% CO 2 and 95% air at 37 °C. Following the incubation period, resazurin sodium salt (Sigma Aldrich) was dissolved in phosphate buffered saline (Sigma Aldrich) and added to wells in an amount of 1/10 th initial well volume. Absorbance was measured at 570/600 nm every 60 minutes until control cells reached ~70% reduction, and the viability reported relative to the control cells.

Haemolysis assay
Samples containing 250 µL ovine red blood cells (RBCs) and 250 µL of PPro solution (at indicated concentration) were incubated at 37 °C for 1 hour. After centrifugation, 10 µL of the supernatant was added to 90 µL of PBS buffer in a 96 well plate. The absorbance was measured at 450 nm and compared against a PBS buffer and deionised water (to lyse cells) controls to determine the % haemolysis relative to the controls.

Statistical analyses
Data were analysed with a one-way analysis of variance (ANOVA) on ranks followed by comparison of experimental groups with the appropriate control group (Holm-Sidak method) followed by Tukey's post hoc test. Excel 2013 (Microsoft, Redmond, WA) and R (R Foundation for Statistical Computing, Vienna, Austria) were used for the analyses. Data sets are presented as mean ± (SEM).

Confocal Microscopy sample preparation and measurements
The solution of 20 mg/mL of poly(proline) in PBS was prepared as previously reported. This solution was stained with 60 µL/mL of 1 mM solution of Sulphorhodamine B in deionized water. The fluorophore absorbs the 552 nm wavelength laser light and emits in a wide band: The fluorescent light was integrated in the 575-625 nm range. Water at the liquid/solid phase transition expels all ions, such that the ice crystals will be constituted by pure water. In this way, we can highlight the ice crystals that upon freezing will grow in the solution as black bodies in the fluorescent liquid. The experimental setup for a similar experiment has been described elsewhere. 3 A thin Hale-Shaw cell was built as follows: on a squared thin glass slide (20 x 20 mm 2 , thickness λ ≈ 170 µm, VWR) a 10 µl drop was deposited by using a precalibrated pipette. The sessile drop was carefully covered with a circular thin glass slide (Ø=15 mm, λ ≈ 170 µm, VWR). The circular contour of such Hale-Shaw cell was sealed with nail polish (L'Oreal). With time, the solvent of the seal eventually evaporates, making the seal porous. The sample was therefore rapidly mounted on the cryostage and frozen only once, while the seal was still effective. The sample was discarded after the experiment.

Confocal Microscopy Measurement
To simulate -to a first approximation -the protocol of splat experiments reported elsewhere, 4 the sample was brought as quickly as possible (a few minutes) at the minimum temperature T min ≈ -22°C that the cryostage can sustain. Nevertheless, this protocol is largely unable to provide the same crystallization feature of the splat experiments owing to the ice crystallization in a fraction of a second being more feasible with the latter. At a temperature below -15°C ice crystals burst from the cold side towards the hot side. The rapid imaging rate of the microscope is able to capture the growing ice crystal and the flow of fluorescent liquid. The cryopreservation method introduced in this work only requires exposure of the IRI active polymer (in this case PPro) for 10 minutes (see experimental, above). After which time the media is removed, and the monolayer frozen, with minimal liquid (which in turn reduces the opportunities for unwanted ice growth). The cells are also thawed by addition of warm media, hence diluting the PPro. Considering this, the cells are only exposed to solutions of poly(proline) for short periods of time, and essentially at dilute conditions. To evaluate the cytotoxicity of polyproline, A549 cells were exposed to PPro (including concentration above that which is optimal for cryopreservation) for 24 hours, and the cell viability determined