Dayong Gao

Fundamental Cryobiology of Mammalian Spermatozoa

Publisher Summary This chapter discusses the fundamental cryobiology of mammalian spermatozoa. The low motility of cryopreserved mammalian spermatozoa and the often lower conception rates may be due to the fact that procedures for cryopreservation of many mammalian cell types, including sperm, have evolved empirically. The primary assay of sperm function is the use of insemination and measurement of pregnancy initiation. The approaches to measuring sperm plasma membrane integrity include supervital staining and hyposmotic swelling. The other general approach to evaluating plasma membrane integrity is to assay the maintenance of membrane semipermeability by testing the cells ability to change its volume when exposed to anisomotic conditions. Survival of cells subjected to cryopreservation depends not only on the presence of a permeating cryoprotective agent (CPA) but also on the concentration of the CPA. Viability of mammalian sperm is very sensitive to osmotic stress and the associated cell volume excursion. The optimization of CPA addition and removal procedures is also elaborated.

Cryopreservation of human spermatozoa. IV. The effects of cooling rate and warming rate on the maintenance of motility, plasma membrane integrity, and mitochondrial function

OBJECTIVE To test the hypotheses that there is a two-factor aspect of cellular damage during cryopreservation that occurs in human sperm (osmotic effects versus intracellular ice formation) and that there is a cooling rate by warming rate interaction related to this damage. DESIGN Ejaculates from healthy men were cooled at 0.1, 1.0, 10, 175, or 800 degrees C/min to -80 degrees C in a solution of 0.85 M glycerol and plunged into liquid nitrogen. Samples were warmed at 400 degrees C/min (experiment 1) or either 1 degrees C or 400 degrees C/min (experiment 2). After warming, sperm were assessed for survival using motility as the endpoint in experiment 1 and motility, plasma membrane integrity, and mitochondrial function in experiment 2. RESULTS In experiment 1, over the various cooling rates with a standard 400 degrees C/min warming rate, a plot of motility versus cooling rate produced a classical inverted U-shaped curve (n = 6) with maximum motility at the 10 degrees C/min cooling rate. In experiment 2, over the various cooling rates, both 1 and 400 degrees C/min warming rates produced similar but shifted plots of motility, plasma membrane integrity, and mitochondrial function versus cooling rate, which also produced inverted U-shaped patterns (n = 11). Maximal survival for each of the three endpoints occurred at 10 degrees C/min cooling rate for the rapidly warmed sperm and at 1 degree C/min for the slowly warmed sperm. CONCLUSIONS These data support the hypotheses that a two-factor hypothesis of cryodamage applies to human spermatozoa and that an interaction exists between cooling rate and warming rate. These data also suggest that motility, plasma membrane integrity, and mitochondrial function are not differently affected by cooling and warming during cryopreservation.

Cryopreservation An emerging paradigm change

In 1949 Polge, Parks and Smith reported on the “chance” discovery of glycerol’s cryoprotective function during their efforts to preserve avian spermatozoa in the frozen state. In the following year, Smith extended these observations by successfully cryopreserving human red blood cells (RBCs) in glycerol. These two reports identified key elements that would play a crucial role in the evolution of the field of biopreservation including the need for a cryoprotective agent (CPA), the process by which cells could successfully be exposed to penetrating CPA and the manner of freezing and thawing. In 1959 Lovelock and Bishop first described the use of dimethyl sulfoxide as a CPA with its advantage of enhanced permeability versus glycerol for many cell types. In the following decades incremental advances were made focusing on changes in and study of the carrier media containing the CPAs as well as the mechanisms of cell cryoinjury and cryopreservation. Most notable was the Mazur et al. report in 1972 which put forth the “two-factor hypothesis” to describe the interrelationships between cooling rates and survival as influenced by either toxic “solution effects” experienced at sub-optimal slow cooling rates or lethal intracellular ice present at high cooling rates. In effect, these studies established a biophysical foundation upon which cryopreservation experimentation rested for nearly four decades. Other notable developments were discoveries by Fahy et al. and Rall and Fahy in the mid-1980s. This group reported on the novel vitrification strategy of cell preservation in which high concentrations (approaching 8 molar) of a cryoprotectant mixture could be titrated over a concentration gradient to create a medium that when “frozen” was devoid of ice even at liquid nitrogen storage temperatures. Beginning in 1998 a series of studies revealed that perturbations in the cell’s proteome and genome during and following the cryopreservation process would significantly impact survival. This effect was observed regardless of the cryopreservation protocol utilized, “optimized” or other.

Development of a novel microperfusion chamber for determination of cell membrane transport properties

A novel microperfusion chamber was developed to measure kinetic cell volume changes under various extracellular conditions and to quantitatively determine cell membrane transport properties. This device eliminates modeling ambiguities and limitations inherent in the use of the microdiffusion chamber and the micropipette perfusion technique, both of which have been previously validated and are closely related optical technologies using light microscopy and image analysis. The resultant simplicity should prove to be especially valuable for study of the coupled transport of water and permeating solutes through cell membranes. Using the microperfusion chamber, water and dimethylsulfoxide (DMSO) permeability coefficients of mouse oocytes as well as the water permeability coefficient of golden hamster pancreatic islet cells were determined. In these experiments, the individual cells were held in the chamber and perfused at 22 degrees C with hyperosmotic media, with or without DMSO (1.5 M). The cell volume change was videotaped and quantified by image analysis. Based on the experimental data and irreversible thermodynamics theory for the coupled mass transfer across the cell membrane, the water permeability coefficient of the oocytes was determined to be 0.47 micron. min-1. atm-1 in the absence of DMSO and 0.65 microns. min-1. atm-1 in the presence of DMSO. The DMSO permeability coefficient of the oocyte membrane and associated membrane reflection coefficient to DMSO were determined to be 0.23 and 0.85 micron/s, respectively. These values are consistent with those determined using the micropipette perfusion and microdiffusion chamber techniques. The water permeability coefficient of the golden hamster pancreatic islet cells was determined to be 0.27 microns. min-1. atm-1, which agrees well with a value previously determined using an electronic sizing (Coulter counter) technique. The use of the microperfusion chamber has the following major advantages: 1) This method allows the extracellular condition(s) to be readily changed by perfusing a single cell or group of cells with a prepared medium (cells can be reperfused with a different medium to study the response of the same cell to different osmotic conditions). 2) The short mixing time of cells and perfusion medium allows for accurate control of the extracellular osmolality and ensures accuracy of the corresponding mathematical formulation (modeling). 3) This technique has wide applicability in studying the cell osmotic response and in determining cell membrane transport properties.

Hematopoietic SCT with cryopreserved grafts: adverse reactions after transplantation and cryoprotectant removal before infusion

Transplantation of hematopoietic stem cells (HSCs) has been successfully developed as a part of treatment protocols for a large number of clinical indications, and cryopreservation of both autologous and allogeneic sources of HSC grafts is increasingly being used to facilitate logistical challenges in coordinating the collection, processing, preparation, quality control testing and release of the final HSC product with delivery to the patient. Direct infusion of cryopreserved cell products into patients has been associated with the development of adverse reactions, ranging from relatively mild symptoms to much more serious, life-threatening complications, including allergic/gastrointestinal/cardiovascular/neurological complications, renal/hepatic dysfunctions, and so on. In many cases, the cryoprotective agent (CPA) used—which is typically dimethyl sulfoxide (DMSO)—is believed to be the main causal agent of these adverse reactions and thus many studies recommend depletion of DMSO before cell infusion. In this paper, we will briefly review the history of HSC cryopreservation, the side effects reported after transplantation, along with advances in strategies for reducing the adverse reactions, including methods and devices for removal of DMSO. Strategies to minimize adverse effects include medication before and after transplantation, optimizing the infusion procedure, reducing the DMSO concentration or using alternative CPAs for cryopreservation and removing DMSO before infusion. For DMSO removal, besides the traditional and widely applied method of centrifugation, new approaches have been explored in the past decade, such as filtration by spinning membrane, stepwise dilution-centrifugation using rotating syringe, diffusion-based DMSO extraction in microfluidic channels, dialysis and dilution-filtration through hollow-fiber dialyzers and some instruments (CytoMate, Sepax S-100, Cobe 2991, microfluidic channels, dilution-filtration system, etc.) as well. However, challenges still remain: development of the optimal (fast, safe, simple, automated, controllable, effective and low cost) methods and devices for CPA removal with minimum cell loss and damage remains an unfilled need.

Glycerol permeability of human spermatozoa and its activation energy.

Glycerol has commonly been employed as a cryoprotectant in cryopreservation of human spermatozoa. However, the addition of glycerol into the sperm before freezing and the removal of glycerol from the sperm after freezing and thawing result in anisotonic environments to the cells, which can cause cell injury. To define optimal procedures for the addition/removal of glycerol and to minimize the cell injury, one needs to know the kinetics of glycerol permeation across the sperm plasma membrane at different temperatures. For this, one has to determine the permeability coefficient of glycerol (Pg) and its activation energy (Ea). Values of Pg at different temperatures and at different glycerol concentrations were determined by measuring the time required for 50% spermolysis in hyperosmotic glycerol solutions which were hypotonic with respect to electrolytes. Value of the Ea was determined assuming an Arrhenius type temperature dependence of Pg. A dual fluorescent staining technique (propidium iodide and 6-carboxyfluoroscein diacetate) and flow cytometry were used to measure the spermolysis. The values of Pg in 0.5, 1.0, 1.5, and 2.0 M glycerol at 22 degrees C are 1.62, 1.88, 1.68, and 1.54 x 10(-3) cm/min, respectively. The values of Pg in 1 M glycerol at 0, 8, 22, and 30 degrees C are 0.33, 0.54, 1.88, and 2.60 x 10(-3) cm/min, respectively. The value of Ea is 11.76 kcal/mol.

Immunosensor towards low-cost, rapid diagnosis of tuberculosis

A rapid, accurate tuberculosis diagnostic tool that is compatible with the needs of tuberculosis-endemic settings is a long-sought goal. An immunofluorescence microtip sensor is described that detects Mycobacterium tuberculosis complex cells in sputum in 25 minutes. Concentration mechanisms based on flow circulation and electric field are combined at different scales to concentrate target bacteria in 1 mL samples onto the surfaces of microscale tips. Specificity is conferred by genus-specific antibodies on the microtip surface. Immunofluorescence is then used to detect the captured cells on the microtip. The detection limit in sputum is 200 CFU mL(-1) with a success rate of 96%, which is comparable to PCR.

The promise of organ and tissue preservation to transform medicine

The ability to replace organs and tissues on demand could save or improve millions of lives each year globally and create public health benefits on par with curing cancer. Unmet needs for organ and tissue preservation place enormous logistical limitations on transplantation, regenerative medicine, drug discovery, and a variety of rapidly advancing areas spanning biomedicine. A growing coalition of researchers, clinicians, advocacy organizations, academic institutions, and other stakeholders has assembled to address the unmet need for preservation advances, outlining remaining challenges and identifying areas of underinvestment and untapped opportunities. Meanwhile, recent discoveries provide proofs of principle for breakthroughs in a family of research areas surrounding biopreservation. These developments indicate that a new paradigm, integrating multiple existing preservation approaches and new technologies that have flourished in the past 10 years, could transform preservation research. Capitalizing on these opportunities will require engagement across many research areas and stakeholder groups. A coordinated effort is needed to expedite preservation advances that can transform several areas of medicine and medical science.

Innovation in the Treatment of Uremia: Proceedings from the Cleveland Clinic Workshop: Blood–Membrane Interactions During Dialysis

In extracorporeal renal replacement therapies, the dialyzer is not only the site at which solute removal occurs but also the extracorporeal circuit component having the largest surface area exposed to blood. Therefore, it is not surprising that interactions between blood components and the dialyzer membrane influence the dialysis procedure in several ways. Based on engineering principles, fluid flow along a surface such as membrane results in the development of a boundary layer which can influence solute removal. Furthermore, the exposure of blood to any extracorporeal artificial surface results in the activation of several pathways within the body, including those involving coagulation and complement activation. One of the byproducts of this generalized activation process is protein adsorption to the membrane surface, another phenomenon which can have a significant impact on solute removal. In this article, a detailed review of the ways in which blood–membrane interactions influence solute removal during hemodialysis and related therapies is provided. The influences of secondary membrane formation and boundary layer/concentration polarization effects on solute removal are specifically discussed. Furthermore, the importance of adsorption as a specific removal mechanism for low‐molecular weight proteins by highly permeable synthetic membranes is highlighted.

Effect of Flow Baffles on the Dialysate Flow Distribution of Hollow-Fiber Hemodialyzers: A Nonintrusive Experimental Study Using MRI

We used an innovative, nonintrusive MRI technique called the two-dimensional (2D) Phase-Contrast (2DPC) velocity-imaging technique to investigate the effect of flow baffles on the dialysate-side flow distribution in two different hollow-fiber hemodialyzers (A and B); each with flow rates between 200 and 1000 mL/min (3.33 x 10(-6) and 1.67 x 10(-5) m3/s). Our experimental results show that (1) the dialysate-side flow distribution was nonuniform with channeling flow occurred at the peripheral cross section of these hollow-fiber hemodialyzers, and (2) the existing designs of flow baffles failed to promote uniform dialysate-side flow distribution for all flow rates studies.