Synthetic zwitterions as efficient non-permeable cryoprotectants

The study addresses the challenge that some cell lines remain difficult to cryopreserve effectively using conventional, often DMSO-based, commercial cryoprotectants. Prior work showed that low-toxicity synthetic zwitterion aqueous solutions can act as cryoprotectants, but performance was inadequate for certain cells (e.g., BOSC). The research question is to elucidate the cryoprotective mechanisms of synthetic zwitterions, identify the physicochemical factors governing their efficacy, and optimize formulations—potentially by combining non-permeable zwitterions with cell-permeable agents—to achieve broad, efficient cryopreservation across diverse cell types.

Background highlights include: (i) DMSO is a widely used cell-permeable cryoprotectant but has toxicity concerns and requires non-permeating solutes to regulate osmotic balance during freezing; (ii) Non-permeable components like salts modulate osmotic pressure, affecting cell volume changes during cooling and thawing; (iii) Prior studies by the authors reported that specific synthetic zwitterions (e.g., OE2imC3C) exhibit low cytotoxicity, have limited cell permeability, and can serve as alternative solvents or media components; (iv) Literature suggests various materials and strategies to inhibit ice formation and protect membranes (e.g., sugars, polyampholytes) and notes the importance of both extra- and intracellular ice inhibition. The study builds on these by synthesizing a series of zwitterions, probing mixture properties (osmotic pressure, glass transition, unfrozen water), and evaluating synergy with permeable agents like DMSO.

• Materials and zwitterions: Eighteen zwitterions were synthesized following prior reports with minor modifications, including OE2imC3C, OE2imC3S, C1imC3S, A1imC3S, A1imC4S, V1imC3S, V1imC4S, C1imC2C, A1imC3C, V1imC3C, PyC3C, PyrrC3C, N14C3C, etc. Commercial additives included DMSO, glycerol, sucrose, trimethylglycine, and L-carnitine.
• Cell lines: hNF, mNF, BOSC, WM266.4 (WM), MDA-MB-231 (MDA), PC9, B16F10, 4T1, MDCK, HL-60, K562, Vn1919, and OVMANA. Culture conditions followed standard protocols (DMEM or RPMI with 10% FBS and antibiotics, 37 °C, 5% CO2). Monolayer vs floating cultures as appropriate.
• Cryopreservation protocol: Zwitterion and zwitterion/DMSO solutions prepared in ultrapure water. Cells (1×10^6) were pelleted (100×g, 5 min, RT), supernatant removed, and 100 µL of cryoprotectant added. Samples were frozen in a controlled-rate freezing box (Mr. Frosty) in a −85 °C freezer for 3–5 days. Thawing used prewarmed (37 °C) culture medium. Viability was assessed by trypan blue exclusion using a hemocytometer. Outcomes reported as relative number of living cells = (living cell count with sample)/(living cell count with commercial cryoprotectant). Commercial comparator: Culture Sure freezing medium (DMSO-containing).
• Toxicity assessment: Post-trypsinization floating cells (1×10^5) were incubated in cryoprotectants at room temperature (15–25 °C) or 0 °C. After 60 min, dead cell ratio was determined (dead/(live+dead)).
• Cell volume measurements: Post-trypsinization floating cells (1×10^5) were incubated in solutions for 5 min at room temperature or 0 °C. Microscopy (Olympus IX83) images analyzed in ImageJ to compute relative cell volume versus PBS (defined as 100%).
• Water content of cells: Post-trypsinization cells (1×10^7) were weighed before and after vacuum drying (1 Pa) to determine water content in original cells and after exposure to water/OE2imC3C (90/10, v/w).
• Osmotic pressure: Measured with a vapor pressure osmometer (VAPRO 5600) in a 10 mL chamber.
• Cryogenic phase behavior: DSC assessed glass transition and fraction of unfrozen water. Cooling to −100 °C at −1 °C/min, heating to 25 °C at 5 °C/min. Unfrozen water estimated from melting enthalpy relative to pure water.
• Molecular dynamics (MD) simulations: Bilayers of 200 DPPC lipids with aqueous phases either water/OE2imC3C/DMSO (75/10/15 w/w/w; 10,000 water/94 OE2imC3C/462 DMSO molecules) or water/DMSO (85/15 w/w; 12,000 water/480 DMSO). Systems minimized, equilibrated (NVT then NPT; temperature ramp from −253 to 37 °C; 1 bar), then 1 µs production at 37 °C, 1 bar, using AMBER18/Tools with PMEMD.CUDA. Force fields: Lipid17 for lipids; GAFF for DMSO and OE2imC3C; TIP3P water. Long-range electrostatics by PME; 2 fs timestep; SHAKE; Langevin thermostat (1 ps−1) and Berendsen barostat. Membrane stability assessed by electron density profiles and surface area; DMSO number density profiles across the bilayer determined.

• Zwitterion-only media performance and optimization:
  • Water/OE2imC3C binary media provided cryoprotection across several cells, with optimal concentrations at water/OE2imC3C = 90/10 and 85/15 (v/w). Too low (95/5) led to insufficient dehydration; too high (≥65/35) decreased viability, likely due to toxicity and rapid water flux.
  • Measured osmotic pressures (mOsm/kg) for water/OE2imC3C: 95/5 = 381; 90/10 = 626; 85/15 = 921; 75/25 = 1501. Cell volumes decreased linearly with the reciprocal of medium osmotic pressure, indicating controlled dehydration.
  • Across hNF, mNF, and BOSC, 90/10–75/25 (v/w) was identified as an optimal range; nevertheless, BOSC remained poorly preserved by zwitterions alone.
  • FBS addition did not improve cryoprotection for OE2imC3C- or DMSO-based media.
• Screening of 18 zwitterions:
  • Multiple zwitterions (e.g., OE2imC3C, VimC3S, C1imC3S, trimethylglycine, L-carnitine) showed cryoprotective effects at 95/5 and 90/10 (v/w) but with variability across cell lines (mNF, BOSC). Zwitterions acted as non-permeating agents promoting extracellular ice inhibition and cellular dehydration.
  • In DSC, typical permeating CPAs (DMSO, glycerol) showed ~14–15 wt% unfrozen water, while zwitterions functioned effectively as non-permeating CPAs.
• Synergy with permeating CPAs:
  • Adding DMSO or glycerol to water/OE2imC3C (90/10, v/w) increased viability; DMSO was superior to glycerol. Best-performing mixture: water/OE2imC3C/DMSO (90/10/15, v/w/w), yielding relative viability of 1.13 (mNF) and 1.14 (BOSC), surpassing the commercial medium.
  • Mixtures of DMSO with other zwitterions (OE2imC3C, C1imC3S, trimethylglycine, L-carnitine) at 90/10/15 (v/w/w) achieved viabilities comparable to or better than commercial medium across mNF and BOSC; VimC3S performed less well when combined with DMSO despite similar performance alone, indicating mixture-specific effects.
  • Universality: Eight additional lines (including Vn1919, HL-60) showed improved viability with water/zwitterion/DMSO; poor responders to zwitterions alone (B16F10, 4T1) improved with DMSO blends.
  • Freezing-vulnerable cells: K562 and OVMANA exhibited markedly higher relative viability with water/OE2imC3C/DMSO and water/C1imC3S/DMSO (90/10/15, v/w/w): K562 = 1.74 and 1.80; OVMANA also improved and showed post-thaw proliferation equivalent to commercial medium.
• Mechanistic insights:
  • DMSO alone (in water) caused cell expansion, indicating the need for a non-permeating component to control osmotic balance; OE2imC3C corrected this, maintaining dehydrated cell volume similar to OE2imC3C alone, thereby mitigating intracellular ice formation.
  • Toxicity: DMSO/water mixtures were more toxic; water/OE2imC3C/DMSO (90/10/15) showed lower toxicity than medium/DMSO (85/15), suggesting OE2imC3C reduces DMSO toxicity, potentially by suppressing DMSO influx via osmotic dehydration and molecular interactions.
  • MD simulations: In water/DMSO, DPPC bilayers partially collapsed into interdigitated phases within 250 ns; in water/OE2imC3C/DMSO, membranes remained relatively stable up to 1000 ns. DMSO number density at bilayer center decreased from 3.44×10^−7/Å^3 (water/DMSO) to 1.42×10^−7/Å^3 (with OE2imC3C), consistent with inhibited DMSO penetration. Interactions between OE2imC3C and DMSO/lipids likely underlie membrane stabilization. Mean square displacements indicated viscosity changes were modest (~80% of DMSO/water), not the dominant factor.
  • Among tested physical properties (glass transition, Tg, osmotic pressure, unfrozen water fraction), cellular dehydration emerged as the key correlate of cryoprotection, not Tg or unfrozen water fraction alone.

The study demonstrates that synthetic zwitterions function as effective non-cell-permeable cryoprotectants by dehydrating cells and inhibiting extracellular ice formation. However, for cells insufficiently dehydrated by zwitterions alone (e.g., BOSC), combining with a permeating agent (DMSO) compensates for intracellular ice inhibition needs. The optimized ternary mixtures (water/zwitterion/DMSO at 90/10/15, v/w/w) improved or matched post-thaw viability relative to a commercial medium across diverse cell types, including freezing-sensitive K562 and OVMANA. Mechanistically, OE2imC3C maintains osmotic dehydration and appears to suppress DMSO membrane permeation and toxicity, supported by MD simulations showing reduced DMSO penetration, preserved bilayer structure, and specific interactions between OE2imC3C, DMSO, and lipids. These findings clarify that the dominant factor is controlled cellular dehydration rather than bulk glass transition or unfrozen water fraction, and they support a rational design principle: pair a strong non-permeating dehydrating zwitterion with a permeating CPA to achieve broad, efficient cryopreservation with reduced toxicity.

Zwitterion aqueous solutions protect cells by promoting dehydration, inhibiting extracellular ice formation, and exhibiting low cytotoxicity. Structural tuning across 18 zwitterions showed that traditional bulk physical parameters (glass transition behavior, Tg, unfrozen water fraction) are not primary determinants of efficacy; instead, the extent of cellular dehydration is key. Since zwitterions alone could not efficiently preserve certain cells (e.g., BOSC), integrating a permeating CPA (DMSO) addressed this limitation. The formulations water/OE2imC3C/DMSO and water/C1imC3S/DMSO (90/10/15, v/w/w) provided robust, broadly applicable cryoprotection, outperforming a commercial cryoprotectant for vulnerable K562 and OVMANA cells and maintaining normal post-thaw proliferation. MD simulations suggest OE2imC3C mitigates DMSO-induced membrane disruption by limiting DMSO penetration and stabilizing bilayer structure. Future work should further elucidate molecular-level interactions and optimize formulations (e.g., by additional additives for pH/osmolarity) to extend applicability and minimize toxicity.

• Dependence on relative viability measurements: The study reports relative numbers of living cells normalized to a commercial medium because absolute counts varied biologically; this introduces variability and limits direct comparison of absolute survival.
• Counting method discrepancies: Differences between automated counters and hemocytometer readings led to notable discrepancies with prior data, indicating methodological sensitivity in viability assessment.
• Scope of storage conditions: Freezing was conducted for 3–5 days at −85 °C using a passive freezing device; long-term storage and alternative cooling profiles were not evaluated here.
• Mechanistic inference: While MD simulations and experimental correlations support proposed mechanisms, the authors note that further study is required to fully clarify cryoprotection mechanisms, including detailed interaction networks and transport kinetics.
• Cell line coverage: Although multiple lines were tested, generalizability to primary cells, stem cells, and tissues requires further validation.