Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

Summary

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.

Abstract

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.

Protocol

1. Cultivation and harvest of P. putida

1. Prepare a starter culture of Pseudomonas putida by inoculating 100 ml of tryptic soy broth (TSB) with a colony of P. putida cells cultured on agar supplemented with tryptic soy broth (TSA). Keep the culture at 30 °C on a shaking board set at 130 rpm.
2. After 7 hr transfer an aliquot from the starter culture equivalent to 1/10 of the final volume of the main culture to fresh TSB-medium. Keep the cells at 30 °C on a shaking board at 130 rpm for 16 hr.
3. After 16 hr of growth the cells are harvested by spinning the cell culture at 1,500 x g for 20 min at RT. Decant the supernatant and wash the cells by re-suspending the cell pellet is in a NaCl-solution that is isotonic to the growth medium. In this case a 150 mM NaCl solution was used. The cells are then centrifuged once more and the supernatant is decanted before resuspending the cells in the formulation medium, see step 3.2.

2. Cultivation of other species

1. To adapt the protocol to other bacterial species the cultivation and harvest procedures should be changed according to the requirements of that species. To avoid osmotic shock when handling the cells during harvest and the washing step(s), the osmolality of the solutions needs to match that of the growth medium at harvest.

3. Formulation of cells

1. Prepare the formulation solutions by weighing out the respective matrix components and dissolve them in water. To achieve isotonic conditions, either adjust the amount of excipient or add NaCl or some other cell compatible solute to reach the desired osmolality. For substances with low molecular weight such as disaccharides the amounts can easily be adjusted to reach isotonic conditions. For substances like polymers that are of high molecular weight, the tonicity has to be adjusted with cell compatible solutes, e.g. NaCl or disaccharides. Note that any addition of a substance will influence the physical properties of the formulation, which in turn may influence freeze-drying behavior and cell survival.
2. Decant the washing solution (in this case NaCl) and re-suspend the cells in the respective formulation media.
3. Make sure that the cells are homogeneously dispersed. Formulations of low viscosity are vortexed whereas formulations of higher viscosities are mixed using by adding, e.g., a stir bar and shaking the container until homogeneous mixing is achieved.
4. Divide the formulations into freeze-dryer vials. The vials should be weighed empty, with sample before and after freeze-drying to calculate the amount of water removed during freeze-drying.
5. Add rubber stoppers to the vials if the vials are to be sealed inside the freeze-dryer, see step 4.3.
6. Enumerate the cells in each formulation before freeze-drying, see step 6.

4. Freeze-drying

1. The freeze-drying conditions have to be adjusted to the physical properties of the formulation. The most important parameter is in this case the glass transition temperature, Tg, of the formulation, denoted Tg' for the freeze-concentrated sample to indicate that water is still present. The Tg' of the freeze-concentrated formulation is easily measured using differential scanning calorimetry (DSC), see step 7.
2. Adjust the parameters of the freeze-drying process so that the temperature of the sample is always below the Tg of the sample and that the chamber pressure allows for a fast sublimation of ice, i.e. primary drying, and residual water in the formulation, i.e. secondary drying. If the sample temperature is above Tg' during the drying process there is a great risk of structural collapse of the sample which may significantly decrease the cellular survival.
3. To protect the dry products after freeze-drying the sample atmosphere has to be controlled. If possible, seal the samples in the freeze-dryer before the vacuum is released at the end of the freeze-drying cycle. Alternatively the vials can be filled with a preferred atmosphere. It is important to avoid exposing the samples to the ambient conditions as any moisture or oxygen present in the storage atmosphere will influence the survival of the cells.
4. Weigh the vials.

5. Rehydration

1. Rehydrate the samples with deionized and sterile water. The amount of water to be added should be that same as the amount removed during freeze-drying and is calculated from the weight of the empty vial, the same vial with sample before and after freeze-drying. Vortex the samples now and again then until the solutions appear homogenous.

6. Enumeration

1. Draw 100 μl from each sample and make 10-fold serial dilutions. From the dilutions of interest 100 μl is plated on TSA-plates. The plates are incubated at the temperature and time required.

7. Characterization of Formulation of Freezing Behavior

1. Take a small amount, 5 - 10 mg, of the sample and enclose it in a DSC-aluminium pan with lid. Prepare an empty pan as a reference.
2. Set the DSC temperature scan program. The cooling program is set to mimic the freezing step in the freeze-drying program. This is done as the cooling kinetics can influence the Tg' of the hydrated formulations. Before the heating scan begins bring down the temperature to well below the expected Tg' of the investigated formulations, normally -100 °C.
3. Choose an appropriate heating rate, normally a heating rate of 5 - 40 °C/min is used. The recorded heat flow signal is expressed in watts and will be influenced by the heating rate. A higher heating rate will give a larger signal of Tg'.
4. Set the temperature range so that it covers both Tg' and the melting of the formulation.

8. Surface Tension Measurements of the Hydrated Formulations

1. The experimental setup used in this experiment is according to the du Noüy method to measure surface.
2. Clean the platinum ring and the measuring vessel carefully. The vessel is rinsed with acetone, wiped with a lint-free tissue, and then burned on the inside with a colorless flame. The ring glows red in a colorless flame.
3. Fill the vessel with the solution and let the surface settle before measuring. For solutions where the equilibrium state is reached only after a long time, e.g. polymers, the simple setup used here will provide qualitative data.
4. For formulations with high viscosities, e.g. solutions of 2% (w/w) HEC or HPMC, the surface tension has to be measured on solutions of lower concentrations and then extrapolated. This can be done by taking advantage of the fact that the drop in surface tension levels off at concentrations below 0.5% (w/w).

9. X-ray Analysis of the Dry Formulations

1. Take a small amount of matrix/bacteria and load it onto a sample holder.
2. Mount the sample holder into the X-ray-diffractometer.
3. To analyze any crystalline phase present in the sample the EVA program package from Bruker can be used.

10. Electron Microscopy

1. Take a small amount of matrix/bacteria out of the vials and fix it on a SEM stub.
2. Load the SEM holder in the sputter-coater (Thermo VGScientific Polaron SC7640 used in the present work) and coat the sample with a few nm of Au/Pd or Pt. In these experiments the sputtering parameters were: 1,900 V, 20 mA, and 20 sec, for a ~ 5 - 10 nm Au/Pd coating. The coating reduces electric charging during image acquisition. It is recommended to start with a thin coating and, if charging persists and causes image artifacts, the sample should be coated again.
3. Load the SEM holder in the SEM chamber. Select appropriate imaging parameters. For the instrument used in Figure 2 (FEI Strata DB235) we used 5 kV accelerating voltage, spotsize 3, a working distance of 5 mm and the secondary electron detector.
4. Optionally, it is possible to use the Focused Ion Beam to cut cross-sections of the bacteria embedded in the polymer to reveal further details.

Results

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 T_g' of sucrose has been determined to -40 °C ^{2, 3}and 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 ...

Discussion

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 ing...

Materials

Name	Company	Catalog Number	Comments
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.