BACKGROUND: Rodent sperm cryopreservation is of critical importance for the maintenance of lines or strains of genetically engineered mice and rats. However, rodent sperm are extremely mechanically sensitive due to their unusual morphology, and are severely damaged using current methods of cryopreservation. Those methods result in poor post thaw motility (PTM) for mouse.OBJECTIVE:To investigate the mechanism of mechanical damage introduced to rodent sperm during freezing, a micro-mechanical model was established to analyze the sperm radial and axial thermal stresses generated by microscale extracellular ice formation.MATERIALS AND METHODS:PTM of mouse sperm cryopreserved in capillaries of different radii (100, 200, 344, 526, 775µm) was measured using a standard computer-assisted sperm analysis system.RESULTS:The model predicts that when one of the inner dimensions of the containers (the inner diameter of plastic straws or straw capillaries) is on the same order of magnitude of sperm length, axial stress is significantly increased. The experimental results showed that the value of PTM was decreased from 38 ± 8 % in the larger (775µm) capillaries to 0 ± 0 % in the smaller (100 µm) ones.CONCLUSION:Theoretical analysis based on the established model were experimentally validated and can be used to guide the design of novel devices to improve the efficiency of rodent sperm cryopreservation.

In Part I, we documented differences in cryopreservation success measured by membrane integrity in four mouse embryonic stem cell (mESC) lines from different genetic backgrounds (BALB/c, CBA, FVB, and 129R1), and we demonstrated a potential biophysical basis for these differences through a comparative study characterizing the membrane permeability characteristics and osmotic tolerance limits of each cell line. Here we use these values to predict optimal cryoprotectants, cooling rates, warming rates, and plunge temperatures. We subsequently verified these predictions experimentally for their effects on post-thaw recovery. From this study, we determined that a cryopreservation protocol utilizing 1M propylene glycol, a cooling rate of 1°C/minute, and plunging into liquid nitrogen at -41°C, combined with subsequent warming in a 22°C water bath with agitation, significantly improved post-thaw recovery for three of the four mESC lines, and did not diminish post-thaw recovery for our single exception. It is proposed that this protocol can be successfully applied to most mESC lines beyond those included within this study once the effect of propylene glycol on mESC gene expression, growth characteristics, and germ-line transmission has been determined. Mouse ESC lines with poor survival using current standard cryopreservation protocols or our proposed protocol can be optimized on a case-by-case basis using the method we have outlined over two papers. For our single exception, the CBA cell line, a cooling rate of 5°C/minute in the presence of 1.0M dimethyl sulfoxide or 1.0M propylene glycol, combined with plunge temperature of -80°C was optimal.

The post-thaw recovery of mouse embryonic stem cells (mESCs) is often assumed to be adequate with current methods. However as this publication will show, this recovery of viable cells actually varies significantly by genetic background. Therefore there is a need to improve the efficiency and reduce the variability of current mESC cryopreservation methods. To address this need, we employed the principles of fundamental cryobiology to improve the cryopreservation protocol of four mESC lines from different genetic backgrounds (BALB/c, CBA, FVB, and 129R1 mESCs) through a comparative study characterizing the membrane permeability characteristics and membrane integrity osmotic tolerance limits of each cell line. In the companion paper, these values were used to predict optimal cryoprotectants, cooling rates, warming rates, and plunge temperatures, and then these predicted optimal protocols were validated against standard freezing protocols.

Optimization of cryopreservation protocols for cells and tissues requires accurate models of heat and mass transport. Model selection often depends on the configuration of the tissue. Here, a mathematical and conceptual model of water and solute transport for whole hamster pancreatic islets has been developed and experimentally validated incorporating fundamental biophysical data from previous studies on individual hamster islet cells while retaining whole-islet structural information. It describes coupled transport of water and solutes through the islet by three methods: intracellularly, intercellularly, and in combination. In particular we use domain decomposition techniques to couple a transmembrane flux model with an interstitial mass transfer model. The only significant undetermined variable is the cellular surface area which is in contact with the intercellularly transported solutes, Ais. The model was validated and Ais determined using a 3×3 factorial experimental design blocked for experimental day. Whole islet physical experiments were compared with model predictions at three temperatures, three perfusing solutions, and three islet size groups. A mean of 4.4 islets were compared at each of the 27 experimental conditions and found to correlate with a coefficient of determination of 0.87±0.06 (mean ± SD). Only the treatment variable of perfusing solution was found to be significant (p<0.05). We have devised a model that retains much of the intrinsic geometric configuration of the system, and thus fewer laboratory experiments are needed to determine model parameters and thus to develop new optimized cryopreservation protocols. Additionally, extensions to ovarian follicles and other concentric tissue structures may be made.

Cryobiology is a field with enormous scientific, financial, and even cultural impact. Successful cryopreservation of cells and tissues depends on the equilibration of these materials with high concentrations of permeating chemicals (CPAs) such as glycerol or 1,2 propylene glycol. Because cells and tissues are exposed to highly anisosmotic conditions, the resulting gradients cause large volume fluctuations that have been shown to damage cells and tissues. On the other hand, there is evidence that toxicity to these high levels of chemicals is time dependent, and therefore it is ideal to minimize exposure time as well. Because solute and solvent flux is governed by a system of ordinary differential equations, CPA addition and removal from cells is an ideal context for the application of optimal control theory. Recently, we presented a mathematical synthesis of the optimal controls for the ODE system commonly used in cryobiology in the absence of state constraints and showed that controls defined by this synthesis were optimal. Here we define the appropriate model, analytically extend the previous theory to one encompassing state constraints, and as an example apply this to the critical and clinically important cell type of human oocytes, where current methodologies are either difficult to implement or have very limited success rates. We show that an enormous increase in equilibration efficiency can be achieved under the new protocols when compared to classic protocols, potentially allowing a greatly increased survival rate for human oocytes and pointing to a direction for the cryopreservation of many other cell types.

The impact of successful cryopreservation of spermatozoa can be found in many fields, including agriculture, laboratory animal medicine, and human assisted reproduction, providing a cost-effective and efficient method to preserve genetic material for decades. The success of any cryobiologic protocol depends critically on understanding the fundamentals that underlie the process. In this review, we summarize the biophysical fundamentals critical to much of the research in sperm cryobiology, provide a synopsis of the development of sperm cryobiology as a discipline, and present the current state and directions for future research in sperm cryobiology in the three major areas outlined above-agriculture, laboratory animal medicine, and human clinical assisted reproduction. There is much room for new research, both empiric and fundamental, in all areas, including refinement of mathematical models, optimization of cryoprotective agent addition and removal procedures for spermatozoa from many species, development of effective, efficient, and facile cryopreservation protocols and freezing containers for agricultural sperm cryopreservation, and tailoring cryopreservation protocols for individual human samples.

BACKGROUND:Anatomic and physiological similarities to the human make swine an excellent large animal model for human health and disease.METHODS:Cloning from a modified somatic cell, which can be determined in cells prior to making the animal, is the only method available for the production of targeted modifications in swine.RESULTS:Since some strains of swine are similar in size to humans, technologies that have been developed for swine can be readily adapted to humans and vice versa. Here the importance of swine as a biomedical model, current technologies to produce genetically enhanced swine, current biomedical models, and how the completion of the swine genome will promote swine as a biomedical model are discussed.CONCLUSIONS:The completion of the swine genome will enhance the continued use and development of swine as models of human health, syndromes and conditions.

Intracytoplasmic sperm injection (ICSI), microinjection of a single spermatozoon into an oocyte, is a routine procedure in assisted reproduction programs. This procedure uses fine control of small bore microinjection needles and precise volume control via hydraulic syringe pumps. In many early experiments mercury is placed within the injection system because its high surface tension in the system facilitates the injection procedure. However, mercury is cytotoxic and therefore alternative fluids or approaches are needed. Here we examine the main properties of mercury and their impact on the various aspects of ICSI. We conclude that the small momentum diffusivity of mercury is the most important contributing factor that facilitates the ICSI procedures.

This chapter focuses on female fertility preservation procedures because of their complexity and peculiarities. Ovarian failure leads to the impossibility of childbearing apart from other problems related to the menopause, such as vasomotor, skeletal or cardiovascular alterations. Early menopause and infertility are two of the main consequences for patients treated with gonadotoxic agents. Gonadotoxicity, a decrease in ovarian activity, depends on several factors, including the age of the patient; the initial status of the ovaries; the treatment applied and cumulative doses; and the type of agent used. Ovarian tissue freezing for later autotransplantation is alternative for fertility preservation in women with oncological or non-oncological diseases. Any patient with a high risk of premature ovarian failure is a possible candidate for fertility preservation. Oocyte and ovarian tissue cryopreservation are useful as they overcome some of the disadvantages, ethical concerns and legal restrictions related to embryo cryopreservation.

As the importance of swine models in biomedical research increases, it is essential to develop low-cost, high-throughput systems to cryopreserve swine germplasm for maintenance of these models. However, porcine embryos are exceedingly sensitive to low temperature and successful cryopreservation is generally limited to the use of vitrification in open systems that allow direct contact of the embryos with liquid nitrogen (LN2). This creates a high risk of pathogen transmission. Therefore, cryopreservation of porcine embryos in a “closed” system is of very high importance. In this study, in vitro-produced (IVP) porcine embryos were used to investigate cryosurvival and developmental potential of embryos cryopreserved in a closed system. Optimal centrifugal forces to completely disassociate intracellular lipids from blastomeres were investigated using Day-4 embryos. Cryosurvival of delipidated embryos was investigated by vitrifying the embryos immediately after centrifugation, or after development to blastocysts. In this study, centrifugation for 30 min at 13,000 g was adequate to completely delipidate the embryos; furthermore, these embryos were able to survive cryopreservation at a rate comparable to those centrifuged for only 12 min. When delipidated embryos were vitrified at the blastocyst stage, there was no difference in survival between embryos vitrified using OPS and 0.25 mL straws. Some embryos vitrified by each method developed to term. These experiments demonstrated that porcine embryos can be cryopreserved in a closed system after externalizing their intracellular lipids. This has important implications for banking swine models of human health and disease.

Cell volume and concentration regulation in the presence of changing extracellular environments has been studied for centuries, and recently a general nondimensional model was introduced that encompassed solute and solvent transmembrane flux for a wide variety of solutes and flux mechanisms. Moreover, in many biological applications it is of considerable interest to understand optimal controls for both volume and solute concentrations. Here we examine a natural extension of this general model to an arbitrary number of solutes or solute pathways, show that this system is globally asymptotically stable and controllable, define necessary conditions for time-optimal controls in the arbitrary-solute case, and using a theorem of Boltyanski prove sufficient conditions for these controls in the commonly encountered two-solute case.

A novel MEMS-based Coulter counter is designed, fabricated and tested in this paper. The Coulter counter is used for detection and monitoring the impedance changes of cells as a function of time after they are mixed with different experimental extracellular environments. The device consists of a multilayer of the SU-8 microchannel which is divided into a passive mixing region, a focusing region using negative dielectrophoretic forces and a measuring region by multiple electroplated vertical electrode pairs. The devices were tested with both fluidic and electrical functionality using dyed fluids, microbeads with different dimensions suspended in saline water and fibroblast cells in anisosmotic phosphate buffered saline. The results are presented and discussed. The device ultimately aims at testing time-sensitive cell characteristics after exposure to different extracellular media with enhanced sensitivity.

This note describes the design, fabrication, and testing of a novel microelectromechanical systems Coulter counter. The Coulter counter will be used to detect and monitor impedance changes of cells as a function of time in response to different experimental extracellular environments. The device consists of SU-8 (negative photoresist) microchannels, vertical electroplated electrodes, polydimethylsiloxane cover, and is divided into a passive mixing region, a focusing region using negative dielectrophoretic forces, and a measuring region defined by multiple electroplated electrode pairs. The devices were tested using both microbeads in saline water and fibroblast cells in phosphate buffered saline solution. The results show that the proposed microsystem is capable of monitoring impedance of cells at different positions along the Coulter microchannel.

OBJECTIVE: This study investigated a novel closed system vial (CellSeal™, Indianapolis, IN, USA) for cryopreservation of human spermatozoa. DESIGN: This study was designed to determine if the system could maintain its container closure integrity (CCI) under liquid nitrogen (LN2) submersion and still maintain post-thaw motility comparable to controls. An attached segment integral to the vial was also evaluated as a sentinel for representative testing, again evaluating post-thaw motility as compared to the control as well as the vial body. MATERIALS AND METHODS: First, vials frozen/thawed with saline or bacterial support medium were subjected to CCI testing by dye or bacterial immersion, respectively, under pressure or vacuum. Dye ingress was measured using spectrophotometry and bacterial ingress was measured via CFU assay. Next, a total of 9 semen samples from 3 different donors were processed for long term storage using either a standard 2ml Corning cryovial (control), a 2ml CellSeal cryovial and the CellSeal vial integral test segment. Each sample was then frozen for at least 24 hours at -196°C under liquid nitrogen. After freezing, samples were then evaluated for post-thaw motility using standard methods. Means (normalized to non-frozen control) and standard error of the mean were calculated and compared statistically via student's t-test. RESULTS: All vials passed the CCI testing with no dye or bacterial ingress. Post-thaw samples were comparable among the control and experimental groups and no difference in motility as per t-test comparing post-thaw control (58 ± 2%) to the CellSeal vials (57 ± 2%; P=0.70); post-thaw control to the segment (52 ± 2%; P=0.25); or segment to vial body (57 ± 2%; P=0.32) was observed. CONCLUSION: The novel closed system vials present a means of assuring sample integrity even under liquid nitrogen and allows measurement of post-thaw motility of a specimen without thawing the primary container.

Partial phase diagrams are of considerable utility in the development of optimized cryobiological procedures. Recent theoretical predictions of the melting points of ternary solutions of interest to cryobiology have caused us to re-examine measurements that our group made for the ethylene-glycol–sodium chloride–water phase diagram. Here we revisit our previous experiments by measuring melting points at five ethylene-glycol to sodium chloride ratios (R values; R = 5, 10, 15, 30, and 45) and five levels of concentration for each ratio. Melting points were averaged from three measurements and plotted as a function of total solute concentration for each R value studied. The new measurements differed from our original experimental values and agreed with predicted values from both theoretical models. Additionally, the data were fit to the polynomial described in our previous report and the resulting equation was obtained:Tm=(38.3-2.145×10-1R)w+(81.19-2.909×10-1R)w2,where w is the total solute mass fraction. This new equation provided good fits to the experimental data as well as published values and relates the determined polynomial constants to the R value of the corresponding isopleths of the three dimensional phase diagram, allowing the liquidus curve for any R value to be obtained.