Optimization of Permeable Cryoprotectants for Neisseria meningitidis Based on Assessment of Ice Inhibition and Cytotoxicity

Abstract

Introduction:

Neisseria meningitidis, the primary pathogen of epidemic meningococcal meningitis, is a typical fastidious bacterium with stringent nutritional requirements and high environmental sensitivity, making routine preservation challenging. Current cryopreservation methods lack quantitative efficacy evaluation and suffer from low viability.

Objective:

This study aimed to screen optimal permeable cryoprotectants (CPAs) for fastidious bacteria typified by N. meningitidis via cytotoxicity, cryopreservation viability, and ice crystal inhibition assays and elucidate the underlying protective mechanism.

Materials and Methods:

Viable cell counting with ANOVA and Tukey’s test assessed the cytotoxicity and post-cryopreservation survival of N. meningitidis treated with 10/15/20% glycerol and 5%, 10%, 15% dimethyl sulfoxide (DMSO), formamide (FMD), and propylene glycol (PG) in PBS/brain heart infusion (BHI) at 37°C for 15 min and frozen at −80°C for 48 hours. Differential scanning calorimetry (DSC) analyzed thermodynamic parameters of CPA solutions, and cryomicroscopy determined ice-inhibiting properties by observing ice crystal morphology and growth rate.

Results:

Cytotoxicity assays showed N. meningitidis survival rates of 88.14%, 83.85%, and 81.62% in BHI broth with 5% DMSO, 5% FMD, and 5% PG, respectively. After 48 hours of −80°C cryopreservation, 5% DMSO exhibited the best protective effect in both PBS and BHI (p < 0.05), with survival rates of 64.93% and 63.11%. DSC analysis revealed decreased crystallization enthalpy (ΔH_c) and melting temperature (T_m) of DMSO with increasing concentration in BHI. Cryomicroscopy confirmed 5% DMSO most effectively inhibited ice crystal growth (921.57 μm² in PBS, 814.20 μm² in BHI within 50 s).

Conclusion:

5% DMSO in BHI broth is the optimal permeable CPA for N. meningitidis and other fastidious bacteria. Its potential mechanism is that 5% DMSO balances bacterial osmotic pressure, alleviates solute and ice crystal damage, and balances ice inhibition with biocompatibility to enhance bacterial survival.