To assess cryoprotection, serial dilutions of induced and uninduced RiAFP culture were plated on LB agar plates. Cultures were then frozen at -20oC overnight, diluted once more, and plated again for a cell viability count. Relative freezing tolerance was calculated by determining number of viable cells before and after freezing. Protein expression was induced by overnight exposure to 0.5mM IPTG at 20oC. Induction of RiAFP expression leads to significantly greater survivability than vector and uninduced controls. Uninduced controls also exhibit increased survivability, likely due to leaky expression. T-test analyses (P < 0.05) indicate that the differences in survival rates between transgenic strains are statistically significant.
Figure 1: E. coli expressing RiAFP show freeze resistance. The data are expressed as mean values (+/- standard deviation) of three biological replicates.
Ice Recrystallization Inhibition Assays
A modified splat assay was used to visualize ice recrystallization inhibition (Smallwood, et al 1999). 7uL of a 30% sucrose solution containing a known concentration of sample was sandwiched between two sterile glass slides. Excess liquid was removed, and the slides were sealed with epoxy. The sandwich was placed into a heptane bath cooled by liquid N¬2 for 30 seconds. The sealed samples were placed in a -10oC freezer and imaged at various time intervals. Samples were transferred to a Nikon multi-functional optical microscope on a super-cooled NaCl ice bed and imaged using a plane polarized filter and imaged at 50x unless otherwise noted. For control samples, varying the BSA concentration from 1mg/mL to 10mg/mL did not affect crystal size.
Figure 2: Splat assay shows concentration dependent inhibition of ice recrystallization. Ice recrystallization was allowed to take place for five hours at -10oC before imaging.
Figure 3: Splat assay shows concentration dependent inhibition of ice recrystallization. Ice recrystallization was allowed to take place for twelve hours at -10oC before imaging
A modified capillary assay was also used to study recrystallization inhibition. Samples were loaded into 1mm diameter glass wells using 10ul of sample. The wells were sealed with a glass cover slip and were snap frozen in liquid N2, ensuring that the sample did not contact the liquid N2. The samples were incubated at -10oC for varying time periods and visualized using a Nikon optical microscope.
Figure 4: Capillary assay indicates that RiAFP inhibits ice recrystallization formation in a concentration-dependent manner. Recrystallization took place at -10¬oC for one hour.
Before we obtained purified protein, we conducted preliminary experiments of ice recrystallization inhibition comparing cell lysate to lysis buffer controls. Although this is not an perfectly controlled experiment, as there is background from other proteins in the “juice”, we were still able to see recrystallization inhibition. Of course, the assay using purified protein is a much better experiment.
Post-freezing Survivability in C. elegans
C. elegans, a simple 1mm long worm, is a good potential model for studying the effects of RiAFP on the freezing of a multicellular organism. C. elegans stocks are routinely frozen in glycerol solutions and recovered in academic labs. The recovery of C. elegans should be in the range of 20-30% of the total number of animals frozen, according to the Caenorhabdidis Genetics Center. We aimed to count the number of nematodes before freezing, and the number of surviving nematodes after freezing. A nematode would be counted as alive if it is capable of moving.
We compared nematodes frozen in elution buffer, a glycerol freezing solution, freezing solution diluted in elution buffer, RiAFP (1mg/mL) added to freezing solution, and a solution of RiAFP (1mg/mL). Worms were frozen at -80oC overnight. This freezing treatment was completely lethal to the nematodes frozen in elution buffer, worms frozen in glycerol freezing solution, and worms frozen only in RiAFP solution. This large degree of lethality, especially for worms frozen in the typical freezing solution, is unusual. We suspect it is because the worms were not starved for an appropriate amount of time before freezing, or because the rate of cooling was too fast. However, interestingly, three nematodes survived after treatment with RiAFP + freezing solution. We are in the process of conducting further survivability experiments in C. elegans to better quantify any protective effects of RiAFP.
Cryopreservation of Rat Liver Tissue
Currently, organ cryopreservation is hindered by ice inflicted damage. Liver preservation protocols allow safe transplantation only up to 12 hours after removal from the donor, and primary nonfunction still occurs at a rate of 5-10% and remains an important cause of death and emergent retransplantation (Brockbank, 2009). Improved preservation techniques will be important to the further development of clinical transplantation.
Our team hypothesized that antifreeze proteins might have a protective effect on frozen mammalian liver tissue. Three mm cubes of rat (Sprague-Dawley, Charles River Laboratories) liver were removed from a euthanized, saline-perfused animal. Tissue samples were immediately immersed in one of four solutions: 1) 9 mg/mL RiAFP, 2) 4.5 mg/mL RiAFP, 3) 0 mg/mL RiAFP, 4) 0.9% regular saline. The immersed samples were subsequently frozen at -20oC for 12 hours. Upon thawing, the tissue was then fixed in 4% paraformaldehyde via immersion at 4oC for 24 hours. Fixed samples were paraffin blocked, sectioned, and stained with hematoxylin and eosin for further histological analysis.
The results show that RiAFP has a protective effect on the frozen liver. Upon review of H&E stained sections of the liver tissue, there is an apparent difference in histological structure. Control tissue frozen with 0.9% saline and 0 mg/mL RiAFP show large noticeable patches of perforation with numerous small shrunken nuclei indicative of the early stages of cell death. Tissue frozen with 4.5 mg/mL and 9.0 mg/mL of RiAFP showed decreased perforation on the whole as well as increased cell survival and tissue integrity. In the future, freezing the tissue at a temperature less extreme than -20oC may show even more pronounced results.
All rat tissue was generously donated from the Nihal de Lanerolle lab and handled by Alexander Li, who is trained and cleared by the Yale Animal Resources Center and Yale IACUC to handle non-primate vertebrates.
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