Biopreservation and Biobanking

Currently, vaccines are developed as either lyophilized or aqueous formulations; however, the inherent lack of vaccine thermostability requires storage at subambient temperatures. In the developing world, meeting these storage requirements can be difficult, and, as a result, vaccines are often exposed to temperatures that cause losses in vaccine efficacy. The current lack of vaccines that possess sufficient thermostability is therefore a significant barrier to global vaccination efforts. Although lyophilization and stabilizing excipients have conferred marginal stability on many vaccines, the development of adequately thermostable vaccines has historically been hindered by the inherent lability and complex nature of vaccine entities. Thus, with few exceptions, reasons for vaccine activity loss are not well understood. This review describes common mechanisms of vaccine component degradation, as well as methods used to stabilize vaccines with particular emphasis on lyophilization. The application of biophysical techniques to characterize complex vaccine components and their potential role in the future development of more thermostable vaccines is also discussed.

A technique is described to perform magnetic resonance spectroscopy (MRS) studies at hypothermia on human donor livers in a prospective fashion in the time interval between organ flushing and subsequent transplantation. The method allowed MRS to be performed without manipulating or removing the livers from the ice transport boxes. Both phosphorus (³¹P) and proton (¹H) MRS were investigated in a pilot study of six livers. The ³¹P spectra obtained demonstrated the presence of a variety of phosphorylated intermediates, including residual adenosine triphosphate in some organs. The ¹H spectra demonstrated that signals from lipid side chains could be observed only in one organ, which displayed the gross morphological signs of fatty infiltration. This pilot study is the first to report on both ³¹P and ¹H spectra in the same donor livers at hypothermia. The technique shows promise as a method for assessing donor liver status prior to transplantation.

Cryopreservation of intact articular cartilage (AC) may provide a source of tissues that will enhance long-term results of osteochondral transplantation for large, unconfined joint defects. This study investigated the effects of increasing dimethyl sulfoxide (Me₂SO) concentrations on chondrocyte recovery in intact porcine AC after a rapidcooling cryopreservation technique. Osteochondral dowels 10 mm in diameter were harvested, immersed in various concentrations of Me₂SO (1 M, 3 M, 5 M, 6 M, and 7 M), and cryopreserved using a rapid-cooling technique by plunging into liquid nitrogen after initial cooling in an ice-water bath. Fresh controls, toxicity controls, and an experimental group were compared. Slices 70 μ m thick (divided into three layers - superficial, middle, and deep) were evaluated after membrane integrity dye staining by fluorescence microscopy. High concentrations of Me₂SO (6 M and 7 M) demonstrated significant protection from cryoinjury, likely due to vitrification, but this was compromised by significant cellular toxicity. Low concentrations of Me₂SO (1 M and 3 M) exhibited significantly less cellular toxicity but did not protect against cryoinjury. The superficial layer demonstrated significantly increased cellular toxicity compared with the deeper layers at all Me₂SO concentrations. Me₂SO at 6 M demonstrated the highest recovery of chondrocytes after the cryopreservation procedure, with 42% recovery compared with fresh controls (59% recovery compared with toxicity controls), with the middle layer of the sample demonstrating the optimal balance between cellular toxicity and cryoprotection with an intact cell recovery of 56% (compared with fresh controls). This study demonstrated that concentrations of Me₂SO sufficient to result in vitrification have significant potential to successfully cryopreserve intact AC. Further investigation is required to limit potential causes cell loss, including cryoprotectant toxicity, devitrification, and heat transfer.

Use of cryopreserved small-diameter elastic arterial allografts for an arterial bypass procedure has been suggested. But, the long-term patency of the cryopreserved arteries in vivo has not been 100%. Thirty New Zealand White rabbits were used in this study. The experimental arterial segments were cooled and cryopreserved in liquid nitrogen, and warmed with a precooled water jacket apparatus. The frozen/thawed autologous carotid artery was attached end to end by anastomosis with microsurgery. Patency and structural characteristics of implanted arteries were periodically assessed for 12 months using duplex/Doppler ultrasound. Histopathological analysis was performed after 1 year of observation. The results showed that (1) the thawing device we designed was useful in preventing fractures during warming processes; (2) the immediate patency was 100% following autograft implantation; (3) after 4 months, blood flow was not observed via ultrasonography in two cryopreserved arteries and in one control group artery; (4) there were no significant differences in patency between experimental and control animals (p > 0.05); and (5) histopathologically, results showed that endothelial cells in most autografts were integrated and similar in both groups. These results suggested that the cryopreservation techniques applied in this study preserved the structures and functions of carotid arteries. Cryopreserved arteries retained a relatively high patency rate after 1 year. Further studies are needed for preserving human allografts.

Efforts to improve the tolerance of mammalian cells to desiccation have focused on the role that sugars have in protecting cells from lethal injury. The objective of this study was to examine the effect that the composition of intra- and extracellular trehalose solutions has on the survival of dried 3T3 fibroblasts. Trehalose was introduced into 3T3 fibroblasts using a genetically engineered mutant of the pore-forming α-hemolysin from Staphylococcus aureus. Cells were dried using natural convection at ambient temperatures. Plasma membrane integrity and the ability to grow and divide in culture were used to assess cell survival. We found that the post-rehydration membrane integrity and percent cell growth were a function of the residual moisture content of the cells following drying. Adjusting the initial osmolality of the intra- and extracellular sugar solutions to isotonic levels by reducing the concentration of buffer resulted in a significant improvement in the membrane integrity and growth of dried cells. 3T3 fibroblasts dried to 10% residual moisture content in an isotonic solution of 0.2 M intra- and extracellular trehalose exhibited greater than 90% membrane integrity and approximately 70% growth. Understanding the biological response to desiccation will be critical in developing effective strategies for the drying of mammalian cells.

A new technique has been developed using a tiny thermistor 0.3–0.5 mm in diameter to determine thermal conductivity of biomaterials over a wide temperature range. Based on steady-state spherical heat transfer in an infinite homogeneous medium, thermal conductivity of the measured medium can be determined from the power applied and the temperature rise of the self-heated thermistor. Compared with accepted literature, relative measurement errors of thermal conductivity of standard samples (water and ice, as well as aqueous glycol and CaCl₂ solutions) are less than 5% in the temperature range 233–313K. Using the developed technique, thermal conductivities of rabbit liver, kidney, heart, and carotid artery were determined in the temperature range 233–293K.