Proline pre-conditioning of cell monolayers increases post-thaw recovery and viability by distinct mechanisms to other osmolytes†‡

Cell cryopreservation is an essential tool for drug toxicity/function screening and transporting cell-based therapies, and is essential in most areas of biotechnology. There is a challenge, however, associated with the cryopreservation of cells in monolayer format (attached to tissue culture substrates) which gives far lower cell yields (<20% typically) compared to suspension freezing. Here we investigate the mechanisms by which the protective osmolyte l-proline enhances cell-monolayer cryopreservation. Pre-incubating A549 cells with proline, prior to cryopreservation in monolayers, increased post-thaw cell yields two-fold, and the recovered cells grow faster compared to cells cryopreserved using DMSO alone. Further increases in yield were achieved by adding polymeric ice recrystallization inhibitors, which gave limited benefit in the absence of proline. Mechanistic studies demonstrated a biochemical, rather than biophysical (i.e. not affecting ice growth) mode of action. It was observed that incubating cells with proline (before freezing) transiently reduced the growth rate of the cells, which was not seen with other osmolytes (betaine and alanine). Removal of proline led to rapid growth recovery, suggesting that proline pre-conditions the cells for cold stress, but with no impact on downstream cell function. Whole cell proteomics did not reveal a single pathway or protein target but rather cells appeared to be primed for a stress response in multiple directions, which together prepare the cells for freezing. These results support the use of proline alongside standard conditions to improve post-thaw recovery of cell monolayers, which is currently considered impractical. It also demonstrates that a chemical biology approach to discovering small molecule biochemical modulators of cryopreservation may be possible, to be used alongside traditional (solvent) based cryoprotectants.

Monolayer plate collagen coating. As indicated in the discussion, to promote attachment of cells, collagen I from rat tail (Sigma-Aldrich) was diluted to 50 µg•mL -1 in 200 mM acetic acid (Sigma) and added to each well of a 24-well cell culture plate at 5 µg collagen•cm -2 . Plates were incubated with the dissolved collagen for 1 h at room temperature. After this incubation period the collagen solution was removed and the plates were rinsed three times with 200 µL Dulbecco's phosphate buffered saline (Thermo Fisher) to remove any residual acetic acid solution. The collagen treated plates were allowed to dry for 1 h in a laminar flow hood and stored for less than 1 week at 4 °C prior to use.

Tandem mass tagging proteomics experiments
Sample dissolution and protein digestion. Cell were lysed in 4% SDS, 0.1M Tris-HCl pH7.4, 10 mM TCEP and 40 mM CAA followed by incubation in a preheated thermal block at 95 o C for 5 min and bath sonication for 15 min at room temperature. Protein concentration was measured according to manufacturer's instructions using the fluorometric Qubit Protein assay kit (ThermoFisher), measurements were performed using the Qubit Fluorometer (Invitrogen).
Aliquots of 300 µg total protein per sample were precipitated methanol-chloroform method. 1 Briefly, experiments were performed at room temperature. Four volumes of methanol were added to one volume of the protein sample, and the mixture was vortexed. One volume of chloroform was then added, and the mixture was vortexed. Three volumes of water was added, and the mixture was vortexed. The sample was centrifuged at 10,000xg for 5 min and the aqueous methanol layer was removed from the top of the sample. The proteins remained at the phase boundary between the aqueous methanol layer and the chloroform layer. Four volumes of methanol were added, and the mixture was vortexed. The sample was spun at 10,000xg for 15 min. The supernatant was removed without disturbing the pellet, and the pellet was air dried.
For protein digestion 50 µl of 50 mM HEPES pH8, 60 ng/µl of trypsin (Promega) and 20 ng/µl of LysC (Promega) was added to each sample for overnight proteolysis.  After mixing, the samples were incubated for 2 h at room temperature and 1 µg of peptides from each condition was mixed and ran during 2 hours gradient in the mass spectrometer, as described below, to check the labelling efficiency and calculate the coefficient of variance. 2 Samples were kept at -20°C until next step. After, the reaction was quenched with 1 μl of 5% hydroxylamine and 15 µg of peptides per sample were combined for fractionation using High pH Reversed-Phase Peptide Fractionation Kit (Pierce, ThermoFisher) following the manufacturer instructions. All fractions were dried in a vacuum concentrator (Eppendorf) and dissolved in 50 µl of 2% acetonitrile and 0.1% TFA in sonication bath during 5 min.
Protein groups with significant intensity regulation were determined according to Student's Ttest using the Perseus proteomics data analysis tool. 4 Significant hits were filtered using permutation-based FDR <5%. The background proteomics data is available from wrap.warwick.ac.uk.

Additional Data
Phase transitions of individual osmolyte solutions in both PBS, followed by 5 mg•mL -1 PVA, and then the combination of PVA + DMSO ( Figure S1). This data shows that with the addition of osmolytes or PVA, our solutions are freezing and not vitrifying (i.e. ice formation).

S8
From the heating curves, we can see that we do not have large peak shifts with the addition of osmolytes or PVA ( Figure S2).

S9
We investigated PVA toxicity on A549 cells for 10 min and 24 h and compared these results to cells exposed to 10% DMSO. Normalized alamarBlue reduction for all concentrations of PVA was not significantly different from control cells (0 mM) after a 10 min incubation, however, 10% DMSO did show significantly less reduction following 10 min exposure ( Figure   S3, n ≥ 3, P = 0.0000000001). For our 24 h incubation, PVA led to reduced alamarBlue reduction when treated at 1, 2, and 10 mg•mL -1 . However, this 24 h PVA reduction was not extreme and was comparable to cells treated with 10% DMSO for 10 min. Cells exposed for 24 h to 10% DMSO had a significantly lower alamarBlue reduction of only 31.9%. This correlates with previous data showing that PVA is non-toxic at up to 20 mg•mL -1 concentrations 5 as PVA is regularly utilized in eye drops and is FDA approved. We have shown that PVA was non-toxic for the exposure time of a CPA (10 min) and only minimally reduced metabolic activity when incubated for 24 h (an extreme exposure time).

AlamarBlue reduction of A549 cells incubated for 24 h with varying concentrations of
osmolytes showed no significant difference in reduction compared to control (F-12K 0 mM) ( Figure S4, n = 3). These results were somewhat unsurprising, as compatible solutes are highly soluble organic compounds of low molecular weight that maintain osmotic balance (osmoregulation) without interfering with cell metabolism. 6 We have shown that incubation with osmolytes for 24 h did not alter the metabolic capabilities of the cells compared to control and were non-toxic to our cell monolayers.