The aim of the following experiment is to observe the effect that different types of freeze drying formulations have on cell survival. To begin, different formulations to freeze dryer cell culture are prepared. Next, each formulation is mixed with the cell culture, then freeze dried and later rehydrated and the survival rates are measured.
The results show differences in survival rates that can be linked to the characterized properties. This method can help you understand what physical and structural properties of a formulation that are importance for cell survival during dehydration. To prepare a starter culture of pseudomonas putter begin by inoculating 100 milliliters of triptych soy broth, or TSB with a colony of cells that have been cultured on aga supplemented with triptych soy broth or TSB incubate the culture at 30 degrees Celsius, shaking at 130 RPM for seven hours.
Transfer an aliquot of the culture to fresh TSB and grow for 16 hours. Harvest the cells by centrifugation at 1, 500 times G for 20 minutes of room temperature. Wash the cell pellet by Resus, suspending it in 150 millimolar sodium chloride.
Pellet the cells and resus. Suspend them in formulation medium. To prepare formulation solutions for freeze drying cells.
Weigh out the respective matrix components and dissolve them in water to achieve isotonic conditions. Adjust the solution with the excipient, sodium chloride, or other cell compatible solutes. To homogeneously resuspend cells in a low viscosity solution, vortex them the high viscosity solutions.
Add a stir bar and shake the mixture. Next, divide the formulations into pre weighed freeze dryer vials and weigh the vials. Draw 100 microliters from each sample and make tenfold cereal dilutions plate the desired dilutions and incubate them at room temperature if the vials will be sealed inside the freeze dryer, place a rubber stopper on each vial from the plated cells.
Enumerate the cells in each formulation. Using the dune, we method to measure the surface tension of the under dried bacterial mixtures. Clean the platinum ring by placing it in a colorless flame until it goes red.
Using acetone and a lint-free tissue, rinse and wipe the measuring vessel and use a flame to burn the inside. Fill the vessel with the solution and let the surface settle before measuring for solutions that take time to reach equilibrium. The setup used here will provide qualitative data for formulations with high viscosities.
Use a lower concentration solution to measure the surface tension and extrapolate the freeze drying conditions have to be adjusted to the physical properties of the formulation. The most important parameter is the glass transition temperature or TG of the formulation denoted TG prime for the freeze concentrated sample that indicates that water is still present. To determine the formula's freezing behavior, enclosed five to 10 milligrams of the sample in a differential scanning colorimetry or DSC aluminum pan with lid.
Prepare an empty pan as a reference. Set the DSC temperature scan program to mimic the freezing step in the freeze drying program. This is done because the cooling kinetics can influence the TG prime of the UN dried formulations before the heating scan begins.
Bring down the temperature to well below the expected tg. Prime of the investigated formulations choose an appropriate heating rate. 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 Prime, set the temperature range so that it covers both TG prime and the melting of the formulation. Once the freezing behavior has been determined, 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 fast sublimation of ice and residual water in the formulation. To protect the dry products after freeze drying.
Control the sample atmosphere by sealing 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.
Finally, weigh the vials to analyze the samples by x-ray. Load a small amount of the bacterial matrix onto a sample holder and mount the sample holder into the x-ray refractometer. Use the software compatible with the x-ray equipment to analyze any crystalline phase present in the sample.
To analyze the bacterial matrix by electron microscopy, mount a small amount on carbon tape fixed on a scanning em stub. Load the SEM holder into a spotter coter, and coat the sample with gold and palladium or platinum to image the sample. Load the SEM holder into the chamber and select the appropriate imaging parameters.
For example, on this instrument we use a five kilovolt accelerating voltage, a spot size of three, a working distance of five millimeters and the secondary electron detector. This table lists data for different formulations, including 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 prime of sucrose is minus 40 degrees Celsius and can be difficult to detect the sucrose concentrations below 20%weight per weight.
The thermal event at minus 35 degrees Celsius is likely related to the onset of ice dissolution shown here. Survival data for gram-negative P putter and gram-positive, a CHLOROPHYL cus formulated in different saccharide based formulations. Note that the trend in how well the formulation support cell survival is similar for both bacteria speciess.
This plot illustrates the correlation between survival after freeze drying and the surface tensions of the formulations indicating that higher surface tensions result in greater rates of survival. The SEM images shown here represent dry formulations for four different polymers whose matrices have the appearance of crisp paper or interconnected smooth sheets with corrugated patterns of embedded bacteria for fi, ccal and sucrose. The sheets are about one micron thick and 10 to 20 microns wide with fi ccal having a smoother surface HPMC and HEC for much thinner sheets.
And the bacteria are more easily visible, possibly because the polymer bacteria ratio is lower in these cellulose based formulations. 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. After watching this video, you should have a good understanding on how cell survival depends on formulation properties using the techniques shown here.
