Do physical forces contribute to cryodamage?

To achieve the ultimate goal of both cryosurgery and cryopreservation, a thorough understanding of the processes responsible for cell and tissue damage is desired. The general belief is that cells are damaged primarily due to osmotic effects at slow cooling rates and intracellular ice formation at high cooling rates, together termed the “two factor theory.” The present study deals with a third, largely ignored component—mechanical damage. Using pooled bull sperm cells as a model and directional freezing in large volumes, samples were frozen in the presence or absence of glass balls of three different diameters: 70–110, 250–500, and 1,000–1,250 µm, as a means of altering the surface area with which the cells come in contact. Post‐thaw evaluation included motility at 0 h and after 3 h at 37°C, viability, acrosome integrity, and hypoosmotic swelling test. Interactions among glass balls, sperm cells, and ice crystals were observed by directional freezing cryomicroscopy. Intra‐container pressure in relation to volume was also evaluated. The series of studies presented here indicate that the higher the surface area with which the cells come in contact, the greater the damage, possibly because the cells are squeezed between the ice crystals and the surface. We further demonstrate that with a decrease in volume, and thus increase in surface area‐to‐volume ratio, the intra‐container pressure during freezing increases. It is suggested that large volume freezing, given that heat dissipation is solved, will inflict less cryodamage to the cells than the current practice of small volume freezing. Biotechnol. Bioeng. 2009; 104: 719–728 © 2009 Wiley Periodicals, Inc.     

Induction of Myocardial Infarction in Adult Zebrafish Using Cryoinjury

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction¹. By contrast, urodele amphibians and teleost fish retain a remarkable capacity for cardiac regeneration with little or no scarring throughout life^2,3. It is not known why only some non-mammalian vertebrates can recreate a complete organ from remnant tissues^4,5. To understand the molecular and cellular differences between regenerative responses in different species, we need to use similar approaches for inducing acute injuries.In mammals, the most frequently used model to study cardiac repair has been acute ischemia after a ligation of the coronary artery or tissue destruction after cryoinjury^6,7. The cardiac regeneration in newts and zebrafish has been predominantly studied after a partial resection of the ventricular apex^2,3. Recently, several groups have established the cryoinjury technique in adult zebrafish^8-10. This method has a great potential because it allows a comparative discussion of the results obtained from the mammalian and non-mammalian species.Here, we present a method to induce a reproducible disc-shaped infarct of the zebrafish ventricle by cryoinjury. This injury model is based on rapid freezing-thawing tissue, which results in massive cell death of about 20% of cardiomyocytes of the ventricular wall. First, a small incision was made through the chest with iridectomy scissors to access the heart. The ventricular wall was directly frozen by applying for 23-25 seconds a stainless steel cryoprobe precooled in liquid nitrogen. To stop the freezing of the heart, fish water at room temperature was dropped on the tip of the cryoprobe. The procedure is well tolerated by animals, with a survival rate of 95%.To characterize the regenerative process, the hearts were collected and fixed at different days after cryoinjury. Subsequently, the specimen were embedded for cryosectioning. The slides with sections were processed for histological analysis, in situ hybridization and immunofluorescence. This undertaking enhances our understanding of the factors that are required for the regenerative plasticity in the zebrafish, and provide new insights into the machinery of cardiac regeneration. A conceptual and molecular understanding of heart regeneration in zebrafish will impact both developmental biology and regenerative medicine.     

Cryopreservation of vascular tissues

Cryopreservation of human blood vessels may become an important tool in bypass surgery and peripheral vascular reconstruction. Ideally cryopreservation of a blood vessel should preserve functional characteristics comparable to those of fresh controls. The key advantage of cryopreservation is the fact that storage at deep subzero temperatures allows storage of structurally intact living vascular tissues for virtually infinite time. Originally developed for long-time storage of isolated cells, the techniques of cryopreservation of tissues are challenged by the fact that these are complex multicellular systems containing diverse types of cells with differing requirements for optimal preservation. Therefore, the post-thaw functional activity of vascular tissues is determined by the type of blood vessel and, in addition, by the cell packing effect. Moreover, evidence from pharmacological studies suggests that cryopreservation induces tissue specific changes in transmembrane signaling and the mechanisms coupling intracellular calcium release, sensitivity and calcium entry into the smooth muscle cells.