Combining Biomimetic Block Copolymer Worms with an Ice‐Inhibiting Polymer for the Solvent‐Free Cryopreservation of Red Blood Cells

Abstract The first fully synthetic polymer‐based approach for red‐blood‐cell cryopreservation without the need for any (toxic) organic solvents is reported. Highly hydroxylated block copolymer worms are shown to be a suitable replacement for hydroxyethyl starch as a extracellular matrix for red blood cells. When used alone, the worms are not a particularly effective preservative. However, when combined with poly(vinyl alcohol), a known ice‐recrystallization inhibitor, a remarkable additive cryopreservative effect is observed that matches the performance of hydroxyethyl starch. Moreover, these block copolymer worms enable post‐thaw gelation by simply warming to 20 °C. This approach offers a new solution for both the storage and transport of red blood cells and also a convenient matrix for subsequent 3D cell cultures.

Abstract: The first fully synthetic polymer-based approachfor red-blood-cell cryopreservation without the need for any (toxic) organic solvents is reported. Highly hydroxylated blockc opolymer worms are shown to be as uitable replacement for hydroxyethyl starcha saextracellular matrix for red blood cells.When used alone,the worms are not aparticularly effective preservative.H owever,w hen combined with poly-(vinyl alcohol), ak nowni ce-recrystallization inhibitor,ar emarkable additive cryopreservative effect is observed that matches the performance of hydroxyethyl starch.M oreover, these blockc opolymer worms enable post-thaw gelation by simply warming to 20 8 8C. This approach offers anew solution for both the storage and transport of red blood cells and also ac onvenient matrix for subsequent 3D cell cultures.
Donor cells and tissue are essential components of modern medicine.F or example,30million units of blood are annually transfused in the USA, and up to 100 pints (57 liters) of blood are required for asingle trauma victim. [1] Leukemia treatment requires donor bone marrow,a nd emerging regenerative medicines (e.g., stem-cell treatments) require ac onstant supply of cells and the logistical infrastructure to transport them. [2] However,this is complicated by the finite lifetime of isolated cells. [3] Red blood cells can be kept for amaximum of 42 days (but typically for shorter periods), platelets for 8days, and donor organs for just am atter of hours.I np rinciple, cryopreservation (freezing to reduce the rate of cellular degeneration) can be used to enable the storage and transport of cells and tissue. [4] Current state-of-the-art strategies for cryopreservation require the addition of large amounts of water-miscible organic solvents,such as glycerol or DMSO,to promote vitrification (ice-free state) or dehydration. [5] There are several problems with this approach, not least solvent toxicity,and the need (and challenges) of removing all traces of such solvents before transfusion. There are also many cell types (for medicine and basic biosciences) that are challenging to store using current methods. [6] Am ajor cause of damage in cellular cryopreservation is attributed to ice recrystallization (growth) during thawing. Ice-recrystallization inhibitors,such as antifreeze (glyco)proteins (AF(G)Ps), enhance cellular cryopreservation but are challenging to synthesize and have biocompatibility issues. [7] Gibson and co-workers have previously described the use of synthetic polymers as mimics of AF(G)Ps to enhance cell recovery after thawing. [8] However,i nb oth cases (polymer and AF(G)P), it was necessary to add as upplementary extracellular cryoprotectant. Ty pically,h ydroxyethyl starch (HES) is used as anon-toxic alternative to solvents, [9] but this biopolymer does not come as ap ure product:i th as variable degrees of hydroxyethyl moieties and ab road molecularweight distribution. Furthermore,H ES has recently been partially withdrawn from clinical use owing to ap ossible increase in mortality for critically ill patients. [10] To the best of our knowledge,n os ynthetic mimics of HES have been evaluated for cryopreservation. Conversely,t he use of synthetic copolymer gels as mimics of the extracellular matrix for 3D cell cultures is ar apidly developing field. Armes and co-workers have demonstrated that block copolymer worms are potentially useful matrices for cell culture studies as they can be readily switched between fluid and gel phases by ac hange in temperature,e nabling facile sterilization by cold ultrafiltration. [11] Theaim of this study was to investigate the use of diblock copolymer worms as wholly synthetic biomimetic alternatives to HES for cellular cryopreservation and to examine their additive effects when used in combination with polymeric icerecrystallization inhibitors.T he feasibility of thermally triggered hydrogelation after thawing,w hich is highly desirable for tissue-engineering applications,w as also explored.
Ice recrystallization inhibition (IRI) is aunique (and rare) property exhibited by certain macromolecules. [13] To evaluate whether the PGMA 56 -PHPMA 155 worms exhibited IRI behavior, they were assayed using the standard "splat" test and compared to poly(vinyl alcohol) (PVA), which is apotent IRI-active polymer. [14] Briefly,t his assay involves creating a1 0mmt hick wafer of small ice crystals,w hich are then annealed at À6 8 8Cf or 30 min before determining the mean largest grain size (MLGS) of the ice crystals (with smaller ice crystals indicating higher activity). Ther esults of this assay are shown in Figure 2A.T he PGMA 56 -PHPMA 155 worms showed no activity even at 20 mg mL À1 ,w hich is comparable to the negative poly(ethylene glycol) (PEG) control and also HES (see below). In contrast, PVAi sh ighly active even at 1.0 mg mL À1 ,w hich is consistent with our earlier studies. [8a] Differential scanning calorimetry studies confirmed that an aqueous dispersion of PGMA 56 -PHPMA 155 worms does indeed crystallize when cooled (as indicated by the strong exotherm at ca. À20 8 8C; in addition, amelting temperature of around À5 8 8Cw as also observed). This observation is important because many current cryopreservation solutions rely on vitrification by the addition of large quantities of (toxic) organic solvents.Ifvitrification had occurred, amuch weaker (or zero) exotherm would have been observed upon cooling as ar esult of the formation of ag lassy,r ather than acrystalline state.
Theabove data clearly show that the PGMA 56 -PHPMA 155 worms cause neither ice growth nor nucleation. This makes them ag ood candidate to act as wholly synthetic nonpenetrative cryoprotectants like certain biopolymers,such as hydroxyethyl starch, which can form ah ydrated matrix around cells.R ed blood cells were chosen for cryopreservation studies,asthere is an urgent need to improve their longterm storage without recourse to toxic organic solvents.O ur previous studies had demonstrated that the addition of IRIactive PVAincreases cell recovery by minimizing ice-induced damage. [8a] Red-blood-cell recovery can be determined by comparing the relative degree of hemolysis to ap ositive control-this serves as ak ey clinical indicator of their postthaw utility.P reliminary screening studies indicated that the PGMA 56 -PHPMA 155 worms were non-hemolytic towards red blood cells at concentrations up to 20 mg mL À1 .A ccordingly, a5wt %a queous dispersion of PGMA 56 -PHPMA 155 worms was added to the red blood cells (5 10 6 cells mL À1 )i nt he  presence and absence of 1mgmL À1 PVA( this optimal PVA concentration inhibits ice growth without inducing dynamic ice shaping). [8a] These aqueous mixtures were rapidly frozen by immersion in liquid nitrogen and stored above liquid N 2 for three days,followed by slow thawing at 4 8 8C. This thawing protocol was chosen to maximize cell stress,t hus providing as tringent test of the cryopreservative performance of this new PVA/PGMA 56 -PHPMA 155 worm formulation. It is also representative of the environment typically used for largevolume cell freezing for which temperature gradients are known. Moreover,t his thawing temperature ensures that no worm gel formation occurs,a sit is below the CGT of 12 8 8C. Ther esults of these freeze-thaw experiments are shown in Figure 3relative to PBS and PVA-only controls.
TheP GMA 56 -PHPMA 155 worms alone resulted in just 20 %c ell survival after thawing, which is somewhat lower than the optimized 40 %cell survival achieved in the presence of HES and not statistically different from that of PBS alone. Theaddition of PVAtoHES produced asubstantial increase in cell recovery of up to 70 %, which is consistent with the hypothesis that inhibiting ice growth is key to effective cryopreservation. PVAalone only enabled 40 %cell recovery, highlighting the importance of asecondary hydrated component. Remarkably,t he addition of PVAt ot he PGMA 56 -PHPMA 155 worms gave 68 %r ecovery,w hich is statistically indistinguishable to that of the HES/PVAsystem. There was no evidence for any hemagglutination or abnormal cell morphologies.T his is the first demonstration that aw holly synthetic (polymer or otherwise) formulation can be used to achieve efficient cell cryopreservation. Clearly,t here is huge scope for further optimization as well as the incorporation of additional functionality,s uch as cell-adhesion motifs or fluorescent labels,byrational design. Moreover,the potential to achieve in situ aqueous gelation immediately after thawing is highly desirable for tissue-engineering applications (if not for blood itself). Fore xample,F igure 3B shows digital photographs of aw hole blood sample that had been cryopreserved, thawed, and then heated above the CGT of the worms,demonstrating the rapid formation of agel rich in red blood cells directly from the cryopreservation mixture described herein.
In summary,w eh ave demonstrated that diblock copolymer worm gels are the first synthetic alternative to biopolymers (such as hydroxyethyl starch) for the solvent-free cryopreservation of red blood cells,w ith particular efficacy being achieved when combined with an ice-recrystallization inhibitor such as poly(vinyl alcohol). After initial thawing at 4 8 8C, the copolymer worms retained their ability to form freestanding gels upon warming to room temperature,suggesting an attractive one-pot solution for future whole blood cryopreservation and tissue-engineering applications.