Freeze-drying is often an easy and convenient way to obtain dry products of viable bacterial cells. An issue of the process is cell survival. We detail here a procedure to investigate how cell survival during freeze-drying is influenced by the properties of the formulation used.
Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is considered a gentle dehydration method. To facilitate cell survival during drying, the cells are often formulated beforehand. The formulation forms a matrix that embeds the cells and protects them from various harmful stresses imposed on the cells during freezing and drying. We present here a general method to evaluate the survival rate of cells after freeze-drying and we illustrate it by comparing the results obtained with four different formulations: the disaccharide sucrose, the sucrose derived polymer Ficoll PM400, and the respective polysaccharides hydroxyethyl cellulose (HEC) and hydroxypropyl methyl cellulose (HPMC), on two strains of bacteria, P. putida KT2440 and A. chlorophenolicus A6. In this work we illustrate how to prepare formulations for freeze-drying and how to investigate the mechanisms of cell survival after rehydration by characterizing the formulation using of differential scanning calorimetry (DSC), surface tension measurements, X-ray analysis, and electron microscopy and relating those data to survival rates. The polymers were chosen to get a monomeric structure of the respective polysaccharide resembling sucrose to a varying degrees. Using this method setup we showed that polymers can support cell survival as effectively as disaccharides if certain physical properties of the formulation are controlled1.
1. Cultivation and harvest of P. putida
2. Cultivation of other species
3. Formulation of cells
4. Freeze-drying
5. Rehydration
6. Enumeration
7. Characterization of Formulation of Freezing Behavior
8. Surface Tension Measurements of the Hydrated Formulations
9. X-ray Analysis of the Dry Formulations
10. Electron Microscopy
Table 1 displays data on formulation composition, thermal events recorded by DSC during heating of the frozen formulations, structure of the dry samples and the surface tension of the formulation solutions. The Tg‘ of sucrose has been determined to -40 °C 2, 3and can be difficult to detect for sucrose concentrations below 20% w/w. The thermal event at -35 °C is probably related to the onset of ice dissolution2. The crystalline structure detected by X-ray in the HEC and HPMC samples and also seen in SEM (see Figure 2b and 2c) overlap with the normal crystal form of NaCl.
Figure 1A shows survival data for the Gram-negative P. putida and the Gram-positive A. chlorophenolicus formulated in the different saccharide based formulations. Note that the trend in how well the formulations support cell survival is the same for both bacteria species. The plot shown in Figure 1B illustrates the correlation between the freeze-drying survival and the surface tensions of the formulations.
Figure 2 shows SEM-images of the dry formulations. For the four polymers shown here, the matrix formed has the appearance of “crisp paper”: interconnected smooth sheets in which the bacteria are embedded and show up as corrugations. For Ficoll and Sucrose, the sheets are about 1 μm thick and 10 – 20 μm wide, with Ficoll having a smoother surface. HPMC and HEC form much thinner sheets, possibly due to the fact that the amount of polymer is less in these cellulose-based formulations than in the others (Table 1). Moreover, salt crystals were observed for HEC and HPMC, with the latter showing a larger amount of precipitates on the surface of the polymer sheets. Bacteria are more easily seen in the cellulose based formulations because the sheets are thinner. In Sucrose and Ficoll, they are mostly observed as corrugation of the otherwise smooth surfaces.
Formulation composition | Thermal events detected in frozen formulations | Structure of dry formulation | Surface tension of undried formulations (mN m-1) |
10% Sucrose | onset of ice dissolution, -35 °C | amorphous sucrose | 72 ± 0.1 |
10% Ficoll, 150 mM NaCl | Tg‘, -22 °C | amorphous Ficoll | 68 ± 0.3 |
2% HEC, 150 mM NaCl | eutectic melting of ice and NaCl, -28 °C | amorphous HEC, crystalline NaCl | 64 ± 0.6 |
2% HPMC, 150 mM NaCl | eutectic melting of ice and NaCl, -27 °C | amorphous HPMC, crystalline NaCl | 52 ± 0.8 |
Table 1. The composition of the different formulations and properties of the aqueous or dry formulations. The polymer concentrations of the HEC and HPMC were maximized to achieve a thick enough matrix cover of the bacteria but still have a workable solution as regards to viscosity.
Figure 1. A) Survival rates of freeze dried P. putida (white) and A. chlorophenolicus (grey) when formulated in solutions based on the disaccharide sucrose or the polymers Ficoll, HEC and HPMC, B) Correlation between cell survival after freeze drying and the surface tension of the un-dried formulations.
Figure 2. SEM images of P. putida in four different formulations: a) sucrose, b) Ficoll, c) HEC, d) HPMC. The bacteria are difficult to see in a) and b) because the polymer sheet are quite thick and can envelop the bacteria completely; in c) and d) the bacteria show up more prominently on the surface. In d), they can be distinguished from the large amount of salt precipitates because of their shape and contrast, as they appear as oblong, dark corpuscles.
The motive for this study was to investigate some formulation properties that may be of importance for cell survival during freeze-drying. Although the intrinsic drying tolerance varies between different species, as illustrated in Figure 1A, the trend on how well the different formulations support cell survival is similar. It is informative to start with a comparison of sucrose and Ficoll. It is believed that a key factor for a formulation to support cell survival is the capability of the formulation ingredient(s) to replace water during dehydration, thus maintaining the structure of the proteins and membranes of the cell also in the dry state. Disaccharides such as sucrose but also trehalose show this property, whereas polymers such as the polysaccharide starch do not5,11. Polymers are considered too large to interact with the membrane lipids in the same fashion as disaccharides. Another property important for a successful freeze-drying of microorganisms is the ability of the supporting matrix to vitrify (i.e. become amorphous solids) during freeze-drying. The amorphous structure is beneficial for shielding and keeping cells separated. Interestingly, both disaccharides and polymers readily vitrify if the freeze-drying is properly performed. Our results show that both the sucrose and Ficoll based formulations become amorphous after freeze-drying, see Table 1, and support cell survival equally well. However, there are significant differences between the two saccharides. In contrast to sucrose, the Ficoll molecule is, with a molecular weight of 400 kDa, too large to replace water in the interactions with lipid membranes during freeze-drying 4, 5. Furthermore, as the Ficoll molecules are barred from the periplasmic space of Gram negative bacteria, again owing to their size6, cellular uptake of Ficoll is unlikely. Based on this, we suggest that the protective effect provided by the sucrose formulation is not primarily due to cellular uptake of the disaccharide, important for water replacing capacity of intracellular structures 7, 8. Rather, the capability of Ficoll to support cell survival has to do with properties common to both the Ficoll and sucrose formulation, e.g. the amorphous structure.
Focusing on the polymers, it is evident from Figure 1A that the capability of the polymer formulations to support cell survival varies. The X-ray analysis showed that NaCl crystallized during freeze-drying and coexisted with the otherwise amorphous structure of the HEC and HPMC formulations. No crystalline material was detected in the Ficoll based formulation. NaCl can form complexes with the hydroxyl moieties in carbohydrates 9, 10. In Ficoll and HEC-based formulations the NaCl to hydroxyl moiety molar ratio was 1:11 and 1:1, respectively, explaining why no crystallization was detected in the Ficoll-based formulation. The phase separation between NaCl and the cellulose polymers was also observed in the DSC-investigations. A eutectic melting was recorded during heating of the frozen HEC and HPMC-formulations indicating that the phase separation takes place in the frozen state. The morphology and microstructure of the formulation is revealed by SEM imaging (Figure 2) and corroborates the DSC and X-ray data. Bacterial cells are seen protruding from the sheets with deposited NaCl crystals clearly visible on the surface of the HEC-sheets. Based on the similarity between the survival rates for bacteria formulated in the HEC-solution and Ficoll solutions as compared to HPMC we conclude that the state of the salt, whether dispersed as crystals or dissolved in the polymer, does not correlate with cell survival. Instead cell survival shows a relationship with the surface tension of the formulations, see Figure 1B. For lower surface tensions, lower survival rates are recorded for the bacteria. Note however that the measured surface tension displays the interaction between the solutes, i.e. sucrose and the polymers, and the solvent molecule. Still it allows for an indirect speculation that surface activity of polymers can be related to their tendency to interact also with the cell surfaces in a destabilizing manner. As the concentration of the solutes increases during freezing, the negative effects may be potentiated explaining why the negative effect of the surface active polymers were not seen when only storing the bacteria in the fully hydrating formulations.
The authors have nothing to disclose.
The work was supported by The Swedish Foundation for Strategic Environmental Research (MISTRA) through the DOM program and Grant 211684 (BACSIN) from the European Union Community FP7 Framework program. We thank J. Engstrand for assistance in filming the X-ray analysis and L. Tang for helping with the conceptual narrative.
Name of the reagent | Company | Catalogue number | Comments (optional) |
Ficoll PM 400 | GE-healthcare | 17-0300-10 | |
HEC (Natrosol-M Pharm Grade) | Ashland | Gift from Ashland | |
HPMC (Methocel F4M) | Dow | Gift from the Department of Pharmacy, Uppsala University | |
Sucrose | Sigma-Aldrich | S2395 | |
NaCl | Sigma-Aldrich | 71376 | |
Tryptic Soy broth | Merck | 105459 | |
Tryptic Soy Agar | Merck | 105458 | |
Lyostar II | FTS Kinetics | N.A. | |
Pyris Diamond DSC | Perkin-Elmer | N.A. | |
Bruker AXS SMART CCD 1k Diffractometer | Bruker | N.A | |
Dual-beam FEI Strata DB235 FIB/SEM | FEI | N.A | |
Krüss Educational Tensiometer | Krüss | N.A. |