Non-aqueous, zwitterionic solvent as an alternative for dimethyl sulfoxide in the life sciences

DMSO is the most commonly used nonaqueous solvent in life sciences, serving as a vehicle for hydrophobic compounds and as a cryoprotectant. Despite its widespread use and classification as low-toxicity by FDA/ICH, DMSO is cell-permeable and can bind intracellular proteins, altering cellular functions, epigenetic states, and potentially inducing apoptosis. Thus, DMSO has notable drawbacks. The authors previously developed a histidine-like zwitterionic liquid (ZIL) that exists as a liquid at ambient temperature and dissolves difficult biopolymers like cellulose with low bacterial toxicity. This study investigates whether ZIL can serve as a superior, biocompatible alternative to DMSO for cell and tissue applications, including as a vehicle for hydrophobic drugs (notably platinating agents) and as a cryoprotectant.

Prior literature establishes DMSO’s utility but also its cellular effects, including protein interactions, epigenetic changes, differentiation effects, and embryotoxicity. DMSO inactivates platinum anticancer drugs such as cisplatin through solvolysis, limiting preparation of concentrated stock solutions. Previous work by the authors introduced ZIL as a zwitterion-type ionic liquid capable of dissolving cellulose and compatible with E. coli fermentation, and showed that mixing with DMSO can enhance cellulose dissolution and biocompatibility. Non-cell-permeable cryoprotectants can act by dehydrating cells and stabilizing membranes; polyampholytes with strong membrane affinity have been reported as effective cryoprotectants. These findings inform the hypothesis that ZIL, with high hydrogen bond basicity and membrane affinity, may offer advantages over DMSO.

- Cells and culture: Human normal fibroblasts (hNF-1, hNF-2), mouse normal fibroblasts (mNF), human cancer cell lines (MDA-MB-231, WM266.4, PC9), MDCK cells, mouse primary astrocytes, and human iPS cells (201B7 with feeder cells) were cultured under standard conditions in DMEM-based media with FBS and antibiotics. Specific culture conditions for hNF-1 and iPS cells with feeders and growth factors are detailed. - Intracellular ZIL quantification: Cells were incubated in 5% (w/v) ZIL in DMEM for 2 h, washed, mechanically lysed in methanol, and analyzed by electrospray ionization mass spectrometry (JMS-T100TD). Cell volume was estimated microscopically to calculate intracellular concentrations. - Toxicity assays: hNF-2 and mNF in 96-well plates (4×10^3 cells/well) received solvents at indicated concentrations; viability measured after 24 h with CellTiter 96 AQueous One Solution. hNF-1 (2×10^4 cells/well) viability assessed using neutral red uptake (absorbance at 650 and 550 nm) after exposure to DMSO or ZIL. - MD simulations: A DOPE:DOPG (3:1) lipid bilayer (90:30 molecules) in TIP3P water with 5 wt% ZIL (8716 water, 32 ZIL molecules) was simulated using Amber18/AmberTools19. Systems were minimized, heated from −253 to 37 °C at 0.1 °C/ps, and run at 37 °C, 1 bar for 2.5 µs with Lipid17 for lipids and GAFF for ZIL, 2 fs timestep, SHAKE, PME, 10 Å cutoff. Radial distribution functions were computed to assess ZIL localization relative to the membrane. - Cell cycle analysis: EdU incorporation (10 µM, 1 h) with Click-iT Plus kits, DAPI staining, and flow cytometry (BD FACSAria III) to quantify G0/G1, S, and G2/M phases after 24 h treatment with 0.4–2% (v/v) DMSO or 0.4–2% (w/v) ZIL. - Rb phosphorylation: Western blot for phospho-Rb (Ser780) with standard SDS-PAGE and immunoblotting procedures; β-tubulin as loading control. - iPS cell assays: iPS and feeder cells prepared with CTK solution and TrypLE for single-cell culture; viability with Cell Count Reagent SF; expression of Nanog and Oct3/4 quantified by qPCR (Gapdh control) after solvent exposure. - Zebrafish embryo toxicity: AB* strain embryos treated with DMSO or ZIL from 1.5 to 24 hpf in E3 medium; survival and morphology assessed at 24–48 hpf; o-dianisidine staining for hemoglobin; imaging with Axiozoom V16. - Drug solubility: 1 wt% of each test compound stirred in solvents (ZIL, ZIL aq. at 50/25/5% w/v, DMSO, water) for ≥10 h at room temperature; heated to 80 °C for 2 h if needed; solubilization checked microscopically. - Cisplatin cytotoxicity with different stock solvents: 10 mM cisplatin stocks prepared in 25% or 50% (w/v) ZIL aq. or 25–100% (v/v) DMSO aq., equilibrated ≥24 h; MDA-MB-231 cells treated for 72 h; viability measured with CellTiter 96 AQueous One Solution. - Cryopreservation: Cells (1×10^6) resuspended in 100 µL of freezing media (DMEM/FBS with 10% DMSO v/v or 10% ZIL w/v; or simple aqueous media with 5–10% ZIL w/v or 5% DMSO v/v), cooled at ~−1 °C/min to −85 °C using Mr. Frosty; thawed in 37 °C medium; viable cells counted by trypan blue exclusion (Countess II). Post-thaw plating and 24 h recovery counts performed. Relative number of living cells defined versus CultureSure freezing medium (CS-FM) control. - Thermal analysis: Differential scanning calorimetry (DSC-60A plus) to determine melting and glass transitions of freezing media and ZIL aq. solutions. - Statistics: One-way ANOVA with Dunnett’s post hoc test or two-tailed unpaired Student’s t-test for two-group comparisons.

- Biocompatibility and permeability: ZIL exhibited lower cytotoxicity than DMSO to human fibroblasts (hNF-1, hNF-2) and mouse fibroblasts (mNF), especially at high concentrations (e.g., 10%). ZIL analogs lacking zwitterionic integrity were more toxic, implicating the zwitterionic structure as critical. Intracellular ZIL levels after 2 h in 5% (w/v) ZIL were 10^−4–10^−3% (w/v), whereas intracellular DMSO reached ~1.6% (w/v) after 2 h in 7.8% (w/v) DMSO, indicating ZIL is effectively non-cell-permeable. MD simulations over 2.5 µs showed no ZIL crossing the membrane and revealed ~3× enrichment of ZIL near the polar headgroups. - Cell cycle and stemness: At 1–2% (v/v) DMSO, cell cycle progression was suppressed and Rb was dephosphorylated across tested lines; ZIL at 1–2% (w/v) had little or no effect on cell cycle distribution or Rb phosphorylation. In human iPS cells, DMSO downregulated Oct3/4 and Nanog in a dose-dependent manner, whereas ZIL did not alter their expression at comparable doses. - Embryo toxicity: Zebrafish embryos treated from 1.5 to 24 hpf with 5% (v/v) DMSO showed high lethality with only 4/27 surviving to 24 hpf and severe malformations; 5% (w/v) ZIL treatment resulted in 27/27 survival without malformation and normal erythropoiesis by o-dianisidine staining. - Solubilization of hydrophobic compounds: Of 12 hydrophobic compounds tested, 8 were solubilized in ZIL and/or ZIL aq. (50, 25, or 5% w/v). Notably, zoledronic acid monohydrate (insoluble in water and DMSO) dissolved in ZIL, and adenosine 3'-phosphate and insulin (insoluble in 100% ZIL or DMSO) dissolved in ZIL aq., consistent with ZIL’s high hydrogen bond basicity. - Platinating agents: 25–50% (w/v) ZIL aq. enabled preparation of 10 mM cisplatin stock solutions that retained cytotoxicity upon dilution and treatment (72 h) in MDA-MB-231 cells, while stocks prepared in 25–100% (v/v) DMSO aq. lost cytotoxicity, consistent with DMSO-induced solvolysis. ZIL aq. is proposed as the first practical solvent for platinating agent stocks. - Cryoprotection: Replacing DMSO with ZIL (10% w/v) in standard DMEM/FBS freezing medium afforded cryoprotection comparable to CS-FM and DMEM/FBS/10% DMSO. DSC showed predominant ice formation near 0 °C with a minor glass transition, indicating an unfrozen fraction. In simple aqueous media, 5% (w/v) ZIL aq. successfully cryopreserved hNF-2 (significantly higher viability vs 5% v/v DMSO aq.; e.g., p=0.0005 in direct comparisons; post-recovery p<0.0001), and supported cryopreservation across various cell types. Long-term storage (1 year) in 5% ZIL aq. yielded recovery similar to CS-FM. MD and DSC suggest ZIL concentrates at membranes and dehydrates cells adequately during freezing. Cost estimates indicate ~2 USD/100 mL for 5% ZIL aq. versus ~100 USD/100 mL for commercial cryoprotectants.

The study demonstrates that ZIL, unlike DMSO, is largely excluded from cells while displaying strong affinity for plasma membranes, reducing direct intracellular perturbations that underlie many of DMSO’s adverse effects. Consequently, ZIL preserves normal cell cycle dynamics and stemness marker expression at low working concentrations where DMSO exerts measurable effects. In vivo embryogenesis assays further support ZIL’s superior biocompatibility. Functionally, ZIL and its aqueous solutions dissolve a complementary set of compounds relative to water and DMSO, including certain polar and otherwise intractable drugs. Critically, ZIL aq. enables stable, concentrated cisplatin stocks without loss of activity, overcoming a major limitation of DMSO that inactivates platinum agents through solvolysis. As a cryoprotectant, ZIL acts extracellularly, concentrating at the membrane and promoting adequate dehydration during ice formation, delivering cryopreservation performance comparable to DMSO-containing media and even as a minimal 5% aqueous solution across multiple cell types. These findings support ZIL as a multifunctional, biocompatible solvent and cryoprotectant that addresses several shortcomings of DMSO while opening new experimental and clinical possibilities.

ZIL is proposed as a potent, multifunctional, and biocompatible alternative to DMSO in life sciences. It is non-cell-permeable, less toxic to cells and tissues, serves as an effective vehicle for various hydrophobic and polar compounds, uniquely enables active stock solutions of platinating agents such as cisplatin, and functions as an effective cryoprotectant in both complex and simple formulations. While DMSO’s entrenched use will persist, ZIL is poised to replace DMSO in contexts where DMSO’s adverse effects are limiting, including metabolomics, epigenetics, embryology, and oncology drug handling. ZIL-based freezing media offer chemically defined, xeno-free, scalable, and cost-effective alternatives to current cryoprotectants, with potential for GMP-grade production. Future work should optimize ZIL-based cryomedia formulations, further elucidate mechanisms of cryoprotection and membrane interactions, and expand validation across additional cell types and therapeutic contexts.

- ZIL is non-cell-permeable and thus may not fully replicate intracellular cryoprotective mechanisms of DMSO; complete replacement in all cryopreservation scenarios may not be feasible. - The exact mechanism of ZIL’s cryoprotective action, including the role of glass transition and membrane stabilization, remains to be fully elucidated; further studies are needed. - Solubilization is not universal; only 8/12 tested compounds dissolved in ZIL/ZIL aq., indicating scope limitations. - Cryopreservation efficacy varies by cell type; optimization of ZIL-based media (e.g., osmolarity, protein additives, serum-free conditions) may be required. - At high concentrations, ZIL showed toxicity to some cell types comparable to DMSO (e.g., iPS cells), implying concentration-dependent constraints. - In vivo safety beyond zebrafish embryogenesis and long-term functional outcomes after ZIL exposure were not comprehensively assessed in this study.