Abstracts from the 12th Annual Extreme Cryo Meeting,University of Alberta,Edmonton,Alberta,Canada,February 3–4,2012

Cryoconservation—Opportunities and Challenges in Wildlife Species

Cryobiology is an essential discipline to basic studies and the application of assisted breeding to the management of small populations of rare wildlife species. The overall goal of these managed programs is to maintain maximum gene diversity over several generations while serving as a hedge against extinction and providing animals for reintroduction efforts. The concept of systematic collection, storage and use of biomaterials (Genome Resource Banking; GRB) for wildlife species was first proposed in 1992. Since then, numerous reviews have been published and several examples are now available that highlight the value of cryobiology in species preservation. The ability to preserve male and female genomes indefinitely, use stored sperm long after a donor's death and transport and infuse genes across geographically disparate regions are reasons for incorporating cryobiology into wildlife programs. Despite these advantages, efficient implementation of cryopreservation technologies continues to be hindered by numerous challenges. The four most significant include: 1) limited fundamental knowledge about individual species, including the difficulty of collecting gametes from some species; 2) species diversity in gamete and embryo structure, function and cryosensitivity; 3) variation among donors; and 4) influence of lack of heterozygosity on cryosurvival. These particular challenges highlight the point that findings in one species rarely translate directly to another, and there is a need for continuous species-specific cryobiological research. When such an approach has been used, though, there has been a slowly growing number of conservation success stories. This extends beyond milestone births of one or two offspring from the use of frozen sperm or embryos. For example, the recovery program of the black-footed ferret has benefited from the use of artificial insemination using both fresh and frozen thawed spermatozoa. Offspring now have been produced using sperm cryopreserved for more than two decades, an important means of infusing founder gene into the contemporary population. Recently, cryo-banking efforts have focused on developing optimal methods for preservation of gonadal tissues and in vitro culture to rescue genetic material from neonate and pre-pubertal donors. Finally, it is important to realize that GRBs will never replace conventional conservation approaches, such as habitat preservation, but would assist with preserving existing genetic diversity long into the future.

Fresh Osteochondral Allograft Transplantation—High Chondrocyte Viability 16 Months Posttransplant

The Joint Transplantation Program offers biological treatment options for young patients who are not suitable for total joint replacements. Many of these patients with osteochondral defects have failed traditional surgical treatments such as microfracture for an isolated lesion or fixation for larger defects. Transplantation of articular cartilage is necessary because chondrocytes do not repopulate once they have been damaged and no host cells can migrate into the graft.^1,2 Cadaveric donor tissue is hypothermically stored for up to 30 days in a biopreservative to maintain chondrocyte viability. For the transplant to proceed, viability must be greater than 90%, all donor serology and microbiology must be negative and the donor/recipient joint size match must be within 1–2 mm.

On November 30, 2011, there was a valuable opportunity for the first time in our program history to objectively determine if the cartilage cells were still alive 16 months after the initial transplant. A patient who had received a double dowel (snowman) osteochondral allograft in June 2010 to repair a lesion on the lateral femoral condyle in his left knee, gave consent to acquire a 1.5 mm biopsy from the graft during a follow-up arthroscopic procedure. The purpose of the scope was to investigate why the patient was having some discomfort and restricted joint movement post-operatively. The patient received a spinal anesthetic and was able to view the surgery live. During the scope, it was evident that some of the issues may have been due to scar tissue from the initial procedure, however all other joint anatomy was unremarkable. It was clear the allograft remained in place and all joint surfaces were smooth. The articular cartilage appeared intact and felt firm when probed. Upon analysis of the articular cartilage biopsy, it was evident the cell viability was 99% and the cell density was within the normal range for weight-bearing cartilage. This recent result reaffirms the effectiveness of our preservation protocol and surgical technique. We are not aware of any other studies that have been able to report post-operative chondrocyte viability results from successful osteochondral allograft procedures. There have been two publications involving cartilage biopsy results once grafts have failed.^3,4

In the last year we received ethical approval to extend the program to include joints beyond the knee. Our surgeons have since performed osteochondral dowel transplants in the shoulder of two patients to repair hill-sacs lesions. These are the first procedures that we are aware of in the shoulder joint using osteochondral allografts that were preserved beyond 72 hours of storage. In addition, the same preservation protocol was utilized for a patellar resurfacing allograft procedure in September 2011. All patients continue to report improvements in function and decreases in pain post-operatively as indicated in their WOMAC, KOOS, SF36, EuroQol and Knee Society scores. The Joint Transplantation Program will seek to publish this work once the number of patients who have received these innovative transplants has increased further for statistical significance. The program is funded by a philanthropic donation and support from the Calgary Health Trust.

1. Schachar NS, Novak K, Hurtig M et al. “Transplantation of cryopreserved osteochondral dowel allografts for repair of focal articular defects in an ovine model.” J Orthop Res 1999; 17:909–919.

2. Muldrew K, Chung M, Novak K et al. “Evidence of chondrocyte repopulation in adult ovine articular cartilage following cryoinjury and long-term transplantation.” Osteoarthritis Cartilage 2001; 9:432–439.

3. Czitrom AA, Keating S, Gross AE. “The viability of articular cartilage in fresh osteochondral allografts after clinical transplantation.” Journal of Bone and Joint Surgery, American Volume. 1990, 72(4):574–581.

4. Maury AC, Safir O, Las Heras F, Pritzker KPH, Gross AE. “Twenty-five year chondrocyte viability in fresh osteochondral allograft: A Case Report.” JBJS Case Connector, Volume 89, Issue 1. Case Reports January 1, 2007.

Cryoprotectant Agent Efflux from Vitrified Human Articular Cartilage

We have recently documented that vitrification/cryopreservation of intact human articular cartilage can preserve such tissue for long periods of time while maintaining high chondrocyte viability. This requires the use of high concentrations of cryoprotective agents (CPAs). Prior to transplantation, these CPAs must be removed because they are toxic at high doses. We hypothesized that a relationship between removal-solution volume and time required for CPA removal could be identified to aid in the clinical application of this technique.

Osteochondral dowels of 10 mm diameter from three patients undergoing total knee arthroplasty were vitrified using our recently developed protocol resulting in 6.5M CPA within the matrix. The warmed dowels were immersed in a 10 mL volume of X-VIVO for 30 minutes and this was repeated 5 times (the last wash being 5 minutes only). Removal-solution osmolality was recorded at various times and compared with pure X-VIVO. Changes in removal-solution osmolality over time were plotted normalized to osteochondral dowel volume. This experiment was repeated with 20 mL X-VIVO volume.

There was a rapid efflux of CPA from the cartilage and that rate tapered with time and repeat immersions. The osmolality stabilized during the fourth immersion indicating complete removal of the CPA. Subsequent experimentation using twice as much removal-solution demonstrated equilibration by the third immersion.

Removal of CPA from vitrified articular cartilage is essential prior to transplantation. The results of this study suggest that a mathematical relationship exists for the time of removal and amount of removal solution required to facilitate complete removal of the CPA.

Funding Canadian Institutes for Health Research (MOP 93805). JAWE holds a Canada Research Chair in Thermodynamics. * denotes co-first authors.

Cryopreservation of Insulin-Secreting INS832/13 Cells Using Plant Protein Extracts

We recently developed a cryopreservation technology based on plant proteins and recombinant wheat freezing tolerance-associated proteins as a novel alternative to dimethyl sulfoxide (DMSO). Plant proteins constitute a natural product with the important technical advantage of being nontoxic. We found that proteins isolated from cold hardy wheat (WPE, wheat protein extract) are able to cryopreserve hepatocytes with no side effects on cell metabolism. In view of these promising results, the ultimate goal of our research is to design strategies using proteins that accumulate naturally in freezing tolerant plants as universal cryoprotectants for other mammalian cell types. However, distinct cryopreservation strategies need to be designed for each cell type. We evaluated the efficacy of plant proteins as a cryoprotectant for an insulin-secreting cell line derived from an X-ray-induced rat transplantable insulinoma (INS832/13). However, the WPE that successfully cryopreserved rat hepatocytes required modification for cryopreservation of INS832/13 cells to prevent interference with insulin secretion. Various pre-incubation media were tested and the addition of lecithin, glycerol and N-methylpyrrolidone to cells for 1 hour prior to the plant proteins allowed the cryopreservation of INS832/13 cells with high viability. For cryopreservation, 0.75×10⁶ cells in 0.5 mL of RPMI medium were aliquoted into cryovials and supplemented with WPEs. Cryovials were frozen at a cooling rate of 1°C/min in a freezing container (“Mr. Frosty”; Nalgene, Rochester NY, USA) in a −80°C freezer for 24 hours, and then transferred to liquid nitrogen for at least 7 days prior to thawing. The pancreas-specific metabolic and molecular functions were well maintained in INS832/13 cells after cryopreservation. Secretion of insulin or C-peptide, glucose uptake, and the expression of pancreas-specific genes in cells cryopreserved with the plant protein formulation were similar to those of fresh cells or those cryopreserved with 10% DMSO. Oxidative stress is among the factors that have been implicated in cryopreservation-induced cellular damage. Indeed, oxidation of fluorescent probes that detect oxidative stress occurred in cells that were cryopreserved with 10% DMSO, compared to fresh control cells. When cells were preincubated with glycerol or N-methylpyrrolidone prior to cryopreservation, the DMSO-induced oxidation of the probes was decreased. We next aim to determine whether the plant protein formulations can protect against the induction of oxidative stress during cryopreservation. Our novel technology using plant proteins has promising potential for cryopreservation of sensitive eukaryotic cell types such as hepatocytes and insulin-secreting pancreatic cells.

Financial support: Natural Sciences and Engineering Research Council of Canada (NSERC) and Canadian Institutes of Health Research (CIHR) Canada.

Fitting Hydroxyethyl Starch (HES) Vapour Pressure Osmometry Data with the Osmotic Virial Equation

Long-term storage of cells and tissues can be achieved using cryopreservation. Cryopreservation of erythrocytes is an established procedure for rare and unique blood types, might be used for autologous transfusions, and offers military applications. Hydroxyethyl starch (HES) has been shown to be an effective cryoprotectant for erythrocytes, however little is known about its thermodynamic solution properties. The osmotic virial equation¹ has been shown by many researchers to apply to many types of solutes in aqueous systems including sugars, electrolytes, cryoprotectants, macromolecules, proteins, alcohols, starches, etc. Recently, the osmotic virial equation has been used to fit HES and electrolyte data.² The current study investigates fitting HES vapor pressure osmometry data with the osmotic virial equation in the following form: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*} \pi & = k_{diss}m_{NaCl} + m_{HES} + B_{NaCl} (k_{diss}m_{NaCl})^2 + B_{HES}m^2_{HES} \\ & \quad \, + (B_{NaCl} + B_{HES}) k_{diss}m_{salt}m_{HES} + C_{HES}m^3_{HES} \end{align*} \end{document}

where π is the osmolality of the solution, k_diss is the dissociation constant for NaCl, m is the molal concentration of the solute, and B and C are the second and third osmotic virial coefficients for use with molality, respectively.

HES modifications were measured after dialysis (membrane cut off: 10,000 g/mol) and freeze-drying using vapor pressure osmometry at different mass ratios of HES (up to 50%) and NaCl (up to 25%) with 3 different HES modifications (weight average molecular weights [g/mol]/degrees of substitution: 40,000/0.5, 200,000/0.5, 450,000/0.7). The data was then fit to nine different models for virial coefficient dependence on mass ratio of HES to NaCl (proposed based on the appearance of the osmotic virial coefficients) and the sum of square error and standard error were minimized.

This approach was shown to be accurate at predicting HES data of the three HES modifications for a broad range of mass ratios of HES to NaCl.

This research was funded by a Dean's Research Award for Jingjiang Cheng from the Faculty of Engineering at the University of Alberta, and by the Canadian Institutes of Health Research (CIHR) (MOP 86492). J.A.W.E. holds a Canada Research Chair in Thermodynamics.

1. J. A. W. Elliott, R. C. Prickett, H. Y. Elmoazzen, K. R. Porter and L. E. McGann, “A multi-solute osmotic virial equation for solutions of interest in biology,” Journal of Physical Chemistry B 111:1775–1785, 2007.

2. R. C. Prickett, J. A. W. Elliott and L. E. McGann, “Application of the multisolute osmotic virial equation to solutions containing electrolytes,” Journal of Physical Chemistry B 115:14531–14543, 2011.

The Role of ATP and RBC Membrane In Maintaining RBC Deformability

Red blood cells (RBCs) are hypothermically stored (1 to 6 °C) for up to 42 days prior to transfusion. Throughout this hypothermic storage (HS) period, there is an accumulation of biochemical and biomechanical changes which occur, termed “hypothermic storage lesion.” In vivo, RBCs have the unique ability to deform and pass through the restricted microvasculature of the body. This characteristic allows for the RBC to complete its necessary function of delivering oxygen to the tissues. The objective of this study is to investigate and correlate changes in RBC deformability, with changes in the ATP concentration and composition of the RBC membrane throughout HS. Leukoreduced CPD-SAGM RBC units were collected (n=30) and stored at 1 to 6 °C for 43 days. At day 2 and day 42/43, the RBC units were tested for deformability, ATP concentration, percent hemolysis, osmotic fragility, hematological indices, and morphology. At each testing point, aliquots of the RBC units (n=3) were also extracted using the Folch procedure and analyzed using HPLC and MS, to quantitate the cholesterol and phospholipids, respectively, within the RBC membrane. Throughout HS, significant changes in the RBC membrane were identified; at day 42 the cholesterol/phospholipid (C/PL) ratio of the RBC membrane had increased from 0.53±0.09 to 0.72±0.09; this occurred because of significant phospholipid, but not cholesterol, loss from the RBC membrane (p<0.001). More specifically, significant losses of phosphatidylethanolamine (p=0.002), phosphatidylserine (p=0.01), phosphatidylcholine (p=0.009), lysophosphatidylcholine (p=0.02), and sphingomyelin (p=0.02) were identified in the RBC membrane. Throughout HS, significant changes were also identified in the ATP concentration (p<0.001), and the deformability parameter, EI_max (p<0.001), which is a measure of the RBCs ability to maximally elongate. No significant changes were identified in the deformability parameter, K_EI (p=0.650), which is a measure of RBC rigidity. The relationships between the ATP, EI_max and membrane changes were investigated further, and strong correlations were identified between ATP and EI_max (R²=0.57), ATP and C/PL ratio (R²=0.57), and EI_max and C/PL ratio (R²=0.87) throughout the HS period. The strong correlation identified between the change in the C/PL ratio and the change in EI_max throughout HS, provides evidence to support the hypothesis that the composition of the membrane plays an important role in maintaining RBC deformability; while the correlations with the changes in ATP concentration suggest its role in maintaining not only maximal RBC deformability but also preservation of the phospholipids within the RBC membrane. These results suggest that throughout HS a myriad of changes occur, which are not mutually exclusive. Identifying relationships between variables is critical in identifying potential targets for the development of new preservation strategies. Future strategies need to address both the deteriorating ATP concentration and phospholipid loss from the RBC membrane to ensure high-quality, functional products are provided in critical care situations.

The authors declare no conflict of interest. Project funded by Canadian Blood Services, Research and Development and Canadian Institutes for Health Research.

Phospholipid Profiles of Microparticles in Stored Red Blood Cells

Increase in circulating cellular microparticles of different phenotypes has been demonstrated in the blood of patients with different pathologies.¹ These particles are released in response to a variety of chemical or physical stimuli, and carry molecular features related to procoagulant and proinflammatory activities.² In packed red blood cells (RBC) stored under blood banking conditions, microparticles increase during storage time.³ Many clinical studies have thus raised the hypothesis that microparticles in RBC products should be investigated as potential mediators of transfusion complications.⁴

Potent mediators of the human inflammatory response result from the oxidation of arachidonic acid (AA), a ω-6 fatty acid found in nature as free acid and covalently bound to lipid molecules. In the human erythrocyte membrane, AA is linked to different phospholipids, including phosphoethanolamine and phosphatidylserine (PS).⁵ The presence of phospholipids, specifically PS, in RBC microparticles is well acknowledged, though very little is known about their fatty acid composition and how it correlates with that of the parent RBC. Finding AA-containing phospholipids in RBC microparticles is important for developing a mechanistic understanding of their proinflammatory activity.

This work evaluates the lipid profile of packed RBC supernatant microparticles using Electro Spray Ionization (ESI) tandem Fourier-Transformed Mass Spectrometry (FTMS). Within the context of high sample throughput (1 min/analyses) of shotgun lipidomics⁶ over 100 compunds in the molecular weight range of 700–900 Da have been described and used to compare the composition of packed RBCs and respective microparticles pelleted from the RBC product supernatant through a two-step centrifugation (3,200 × g, 30 min, 4°C; 50,000 × g, 60 min, 4°C). About 70% of the listed compounds were characterized as phospholipids on the basis of the elemental composition calculations provided by the analytical platform software used (Xcalibur 2.1, Thermo Scientific). Packed RBCs (n=5 units) and corresponding supernatant microparticles (MP) presented similar overall relative distributions of phosphatidylcholines (50±2.4% RBC, 47±3.2% MP), sphingomyelins (16.5±3.4% RBC, 21±4.2% MP), phosphoethanolamines (2±0.6% RBC, 1.9±0.4% MP), phosphatidylserines (0.2±0.03% RBC, 0.2±0.07% MP) and phosphatidylcholine ethers (0.5±0.1% RBC, 0.4±0.1% MP). The most abundant phospholipid species within each class were determined and the results are consistent with previous work published on RBCs.⁵ The mass-spec features attributed to AA-containing phospholipids represented 11.2±1.5% (RBC)/8.9±1.7% (MP) of phosphatidylcholines, 27.5±1.7% (RBC)/22.8±6.3% (MP) of phosphoethanolamines and 61.1±5.0% (RBC)/64.4±7.5% (MP) of phosphatidylserines.

In conclusion, the methodology adopted enabled a high throughput way of developing large databases on lipid features of red blood cell microparticles. The results show that the RBCs and supernatant microparticle preparations analyzed are similar in terms of phospholipid composition. Such a fact will be more meaningful once the supernatant microparticle population analyzed become comprehensively described. The clinical relevance of these findings warrants further investigation. Shotgun lipidomics can be feasibly applied for quality assessment of blood products and contribute with relevant information to RBC storage/preservation research.

1. Simak J. and Gelderman MP. Cell Membrane Microparticles in Blood and Blood Products: Potentially Pathogenic Agents and Diagnostic Markers. Trans Med Rev 2006 (20) 1–26.

2. Puddu P. et al. The involvement of circulating microparticles in inflammation, coagulation and cardiovascular diseases. Can J Cardiol 2010 (26) e140–e145.

3. Rubin O. et al. Microparticles in stored red blood cells: an approach using flow cytometry and proteomic tools. Vox Sanguinis 2008 (95) 288–297.

4. Jy W. et al. Microparticles in stored red blood cells as potential mediators of transfusion complications. Transfusion 2011 (51) 886–893.

5. Leidl K. et al. Mass spectrometric analysis of lipid species of human circulating blood cells. Biochim. Biophys. Acta 2008 (1781) 655–664.

6. Han X and Gross RW. Shotgun lipidomics: electrospray ionization mass spectrometric analysis and quantitation of cellular lipidomes directly from crude extracts of biological samples. Mass Spec Rev 2005 (24) 367–374.

Design of Experiments for Investigating Complex Systems

Complexity presents challenges to the mathematical modeller and the experimentalist. Complexity is manifested in the difficult–to-quantify (or even ascertain) nature of phenomena/interactions, their multiple scales of action, and the sheer number of variables (phenomena/interactions) whose effects need to be captured.

The two basic approaches to understand systems based on fundamental modelling and experimental principles both lead to issues related to fitting/regression. With first-principles mathematical models, the model structure is specified, but parameters must be identified from data. Experiments can also be used to derive statistics-based regression models, in which case both the model structure and values of parameters must be determined. In each case, a careful choice of experiments is required to ensure that data is gathered in relevant operating regimes, and that the minimal number of experiments is performed to identify the effects of all the main factors and their interactions. Once the data is gathered and parameters are fitted, analyses related to confidence intervals provide a measure of the reliability of the parameter estimates.

The theory of optimal experimental design provides a framework for deciding on the experiments that need to be performed. Of particular relevance are factorial design methods, space-filling designs, and response surface methods. The choice of experimental design depends on the type of model (first-principles or statistical) that is being constructed. For complex systems with a large number of factors and interactions, the optimal experiment designs must be modified and approximated in order to keep the number of experiments from being excessively large; however, this leads to the loss of detail on some interactions in the models being developed.

This presentation will provide information on the proper choice of experimental designs for fundamental and statistical models, and on the approximations necessary for large-scale systems. Examples will be presented from different cryopreservation problems (cryoprotective agent toxicity interactions in human articular chondrocytes, fitting of HES-NaCl osmolality, design of experiments for vitrification optimization) to illustrate these concepts.

The Effects of Cryopreservation on Erythrocyte Adherence and Deformability

Cryopreservation in transfusion medicine serves as an invaluable tool for long-term storage of red blood cells (RBCs) especially with phenotypically rare blood groups and in military deployment. However conventional in vitro biochemical assays do not address the subtle membrane changes due to cryo-injury that may affect the in vivo quality of cryopreserved RBCs. Subtle membrane changes may arise from the osmotic and mechanical stresses associated with the cryopreservation, thawing and deglycerolization process which may result in altered rheological properties, specifically adhesion and deformability in transfused RBCs.

The purpose of this study is to determine RBC-endothelial cell adhesion and deformability changes as a result of RBC cryopreservation. Leukoreduced WB filtered and buffy coat produced packed RBC units were hypothermically stored in CPD/SAGM for 14 days, cryopreserved in 40% (w/v) glycerol and deglycerolized (COBE 2991). In vitro RBC quality evaluations were performed at pre-freeze, post-thaw and 24 hours post-thaw time points for percent recovery, percent hemolysis, hematocrit, extracellular potassium concentration, RBC indices, ATP concentration and haemoglobin content. In addition in vitro analyses of RBC pre-freeze and post thaw adherence and deformability changes were conducted using an adhesion flow chamber and laser ektacytometry analyses, respectively. Non parametric analysis was used to examine statistical differences in RBC adherence, adherence strength and deformability parameters following deglycerolization. Wilcoxon statistical analysis was conducted for pre-freeze and post thaw RBC quality parameters. Conventional in vitro RBC quality parameters such as hemoglobin content (g/unit) 49.2±1.4 (g/unit), hematocrit, 0.65±0.01 to 0.62±0.01, % hemolysis 0.46±0.02 %, % recovery (92±1.6%) and ATP (3.82±0.14 μgmol/hglb) lay within acceptable AABB and CSA quality ranges showing no statistical changes. No statistically significant change to the number of RBCs adhering to endothelial layer was determined, however there was a statistically significant increase in RBC adherence strength (4.4 to 4.6, p=0.005). RBC deformability remained statistically unchanged by cryopreservation as neither the EI_max or K_EI displayed statistical significance (p=0.735 and p=0.310) pre- or post-cryopreservation, respectively. Altered RBC rheological properties in low flow regions such as capillaries and post capillary venules have the potential to cause microvascular blockage and reduce oxygen delivery to tissues. Elevated RBC-EC interaction and deformability in the microvasculature has been correlated to many hemoglobinopathologies. Determining the subtle membrane changes associated with RBC cryopreservation is important to improving the quality of cryopreserved RBCs to ensure a safer and better quality product is available for transfusion purposes. This study demonstrated that high glycerol cryopreservation alone does not induce significant changes to deformability. However statistically significant changes to RBC adhesion strength to the endothelium layer warrants further investigation.

Use of Hemoglobin Autofluorescence for Determination of Red Blood Cell Osmotic Parameters

Knowledge of cell membrane permeability to water and solutes is critical for the design of effective cryopreservation procedures. Cell membrane permeability is determined based on the rate of cell volume changes under anisotonic conditions. For red blood cells (RBCs), osmotic permeability is most commonly determined by a stopped-flow technique based on changes in light scattering¹ or self-quenching of entrapped fluorescent dye.² However, light scattering technique can be flawed by the effects of solution refractive index, cell motion, or membrane aggregation on the intensity of scattered light.² Fluorescent quenching technique can be complicated by the absorbance of a significant portion of emitted fluorescent light by the broad absorbance spectrum of hemoglobin. It is known from the literature that RBC hemoglobin has autofluorescent properties.³

The objective of this study was to test the feasibility of using intrinsic fluorescence of hemoglobin for measuring RBC permeability to water and solutes. Packed adult RBCs for experiments were received from the Network Center for Applied Development, Vancouver. Fluorescence of RBCs equilibrated in different concentrations of aqueous sodium chloride (NaCl) solutions was measured on a SpectraMax GEMINI EM dual-scanning microplate spectrofluorometer (Molecular Devices). The kinetic changes in fluorescence of RBCs upon mixing with different concentrations of aqueous NaCl solutions were measured on a SX20 stopped-flow reaction analyser (Applied Photophysics). Hemoglobin solutions (lysed RBCs) were used instead of intact RBCs as a negative control to verify the dependance of fluorescence on cell volume changes. Spectral scan was performed on both spectrofluorometer and stopped-flow system to optimize the emission wavelength for RBC autofluorescence.

When RBCs were excited at 280 nm, maximum emission was reached at 340 nm (on spectrofluorometer) and at 320 nm (on stopped-flow system). Spectrofluorometer data demonstrated a positive correlation between autofluorescence intensity and RBC volume, which was confirmed by photomicrographs. Stopped-flow data showed a gradual increase or decrease in autofluorescence upon mixing of RBCs with hypotonic or hypertonic solutions, respectively. Tonicity of the experimental solution had no effect on autofluorescence of hemoglobin solutions.

Combined, these data showed that RBC autofluorescence is positively correlated with RBC volume and may be used to monitor rapid kinetics of RBC volume changes for the determination of RBC permeability to water and solutes.

This work was funded by Canadian Blood Services Graduate Fellowship and Canadian Institutes of Health Research Operating Grant “Preservation of red cells from cord blood as a new blood product for intrauterine and neonatal transfusions.” Special thanks to Drs. Andrew Holt and Aldo Olivieri for technical assistance. The authors declare no conflict of interest.

1. V. W. Sidel and A. K. Solomon, “Entrance of water into human red cells under an osmotic pressure gradient,” Journal of General Physiology, 41:243–257, 1957.

2. P. Y. Chen, D. Pearce, and A. S. Verkman, “Membrane water and solute permeability determined quantitatively by self-quenching of an entrapped fluorophore,” Biochemistry, 27:5713–5718, 1988.

3. Hirstch, R.E., “Hemoglobin Fluorescence,” Methods in Molecular Medicine, 82:133–154, 2003.

Mitochondria and the Membrane: A Comparative Study of Cryoinjury Using Graded Freezing Protocols

Cryopreservation protocols are routinely utilized for the long-term storage of cells and tissues. The success of these protocols is traditionally based on membrane integrity assessments after thawing. However, the exposure of cells to freezing conditions during these procedures potentially induces a variety of changes to cell structure and function that may not be readily identified with membrane integrity assays that do not assess the functional capability of the cell. There is evidence to suggest that functional assays are important factors in determining the nature of cryoinjury. It has been shown that the metabolic activity of fibroblasts, lymphocytes and granulocytes are adversely affected, in addition to membrane damage at slower cooling rates.¹ Similarly, decreases in aerobic metabolism have also been found in slowly cooled isolated keratinocytes and split-thickness skin.²The objective of this study was to investigate the progression of damage to cells during a slow cooling cryopreservation protocol, comparing membrane integrity assessments with a mitochondrial polarization assay that reflects a different aspect of cellular function. Human umbilical vein endothelial cells were subjected to freeze-thaw stresses in the absence of cryoprotectant by cooling at 0.2°C/min to temperatures between −3° C and −40° C, then either thawed directly in a 37° C water bath or plunged to −196° C in liquid nitrogen before rapid warming. The membrane integrity of cells was determined using a combination of the nucleic fluorescent dyes Syto13 and ethidium bromide assessed with fluorescence microscopy. The mitochondrial membrane potential in cells was indicated with the cationic carbocyanine dye JC-1, assessed with flow cytometry.

Cells directly thawed from experimental temperatures showed decreased membrane integrity and mitochondrial polarization corresponding with decreased temperature, though the incidence of depolarized mitochondria occurred at higher subzero temperatures. The number of membrane intact plunged cells increased with decreasing temperature to a maximum of 40.6±5.3% at −20° C, while <8% of these cells had polarized mitochondria. These results indicate that mitochondrial depolarization is evident in cells that are considered viable based on membrane integrity assessment immediately post-thaw, particularly when exposed to liquid nitrogen temperatures. Mitochondrial depolarization also occurred prior to observable membrane damage at higher subzero temperatures, indicating that sub-lethal changes and changes to functional capability of cells occurs before the structural integrity of the membrane is compromised. This demonstrates the need for a more detailed model of the progression of cell damage, as the degree and sites of damage to cells may occur at different stages in the preservation process.

This research was funded by the Canadian Institutes of Health Research (CIHR) MOP 86492, and the University of Alberta, J.A.W. Elliott holds a Canada Research Chair in Thermodynamics.

1. L.E. McGann, H. Yang, M. Walterson, Manifestations of cell damage after freezing and thawing, Cryobiology 25:178–185, 1988.

2. M.A. Zieger, E.E. Tredget, L.E. McGann, Mechanisms of cryoinjury and cryoprotection in split-thickness skin, Cryobiology 33:376–389, 1996.