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1. Conservation of Endangered Species Using Sperm (Reproductive Cell) Freezing There is an urgent need to protect and conserve the endangered species of the world. The traditional programs of breeding and maintaining endangered species in captivity are not only very difficult but are also very expensive. Thus, limiting the genetic diversity of an endangered species. An alternative method to conserve endangered species is to cryopreserve (or freeze and store at very low temperatures) the reproductive tissue of an animal, as an embryo, egg or sperm. Cryopreservation is cost-effective and allows the maintenance of a very large number of animals in 'captivity' for a very long period of time, while also keeping the genetic diversity at a high level. Successful cryopreservation of any tissue or cell requires the knowledge of several cell specific parameters, including the ability of the cell to either retain or lose intracellular water during the freezing process. In this work, we propose to experimentally determine these cell membrane permeability parameters for a variety of endangered species (the species to be studied will be decided in collaboration with the Audubon center for Research of Endangered Species, New Orleans) under a variety of chemical and cooling conditions. The experimentally determined membrane permeability parameters will then be incorporated into a model of cell freezing to determine the 'optimal' cooling rates of freezing. The cryogenically preserved tissue can then be used, via in-vitro (outside the body) fertilization and related assisted breeding procedures, to establish breeding colonies and to repopulate the endangered species both in captive reservations and in their natural habitat. {Funded by the Louisiana Board of Regents}.(student: Sreedhar Thirumala, Yimeng He)
2. Rational Design of Macaca mulatta Ovarian Tissue Cryopreservation Protocols Aggressive treatments of women of child-bearing age suffering from various malignant diseases have resulted in significant increases in long-term survival of such patients. However, the consequences of treatment are often premature ovarian failure and irreversible loss of fertility. This has prompted innovative research to develop methods to cryopreserve ovarian tissue containing primordial follicles with the ultimate goal of restoring fertility of cancer survivors. The goal of the proposed research is different: to restore ovarian endocrine function so as to prevent deleterious consequences of premature menopause, especially in young women. This would obviate the need of hormone replacement therapy (HRT), eliminate issues of potential toxicity, and of patient compliance and adherence to HRT. We will use macaque ovarian tissue as a model to derive optimum methods of cryopreservation for primate ovarian tissue. The proposed research will not only enhance our fundamental understanding of the freezing process in primate ovarian tissues but has the potential to significantly improve the quality of life for female patients recovering from traditional cancer therapies. {Funded by the Whitaker Foundation}.(student: Guanglei Li )
3. Numerical investigation of a novel method to vitrify biological tissues using pulsed lasers and cryogenic temperatures. This research work was aimed to develop a two-dimensional computational model for tissues when brought in contact with cryogenic temperatures. The objective was to achieve extremely high cooling rates sufficient to vitrify the biological tissues when the tissue is perturbed by a pulsed laser and brought in contact with liquid nitrogen. This involved tracking of the phase front which is a non linear moving boundary using unstructured deforming grids and then determine the frozen and unfrozen domains to numerically investigate the propagation of thermal energy when the tissue is perturbed by a pulsed laser.(student: Deepak Kandra)
4. Dessication Protocols for Long Term Storage of Mammalian Cells Preservation by desiccation is an alternative that attempts to reproduce a naturally occurring preservative technique, namely, the phenomenon of anhydrobiosis. In anhydrobiosis, organisms survive extremes of dehydration by imbibing large intracellular concentrations of sugars. In order to optimize a desiccation protocol, it is important to understand the mechanism(s) by which cells are protected during desiccation. We hypothesize that the intracellular sugar transforms into a glass during desiccation. The formation of glass minimizes molecular mobility in the cells thereby arresting metabolic processes and other deleterious chemical reactions that would lead to cellular injury and eventual death. As a part of our study, we will first establish the conditions under which intracellular glass is formed. We will then verify that intracellular glass leads to minimized molecular mobility. After establishing the conditions under which molecular mobility is severely diminished, we shall desiccate sperm cells, store them for various time periods, and then test for structural/functional viability and phenotypic stability. These studies will form the basis of establishing a desiccation protocol that will allow for an easy and flexible solution for banking and storage of sperm cells from endangered species.(student: Dinesh Pinisetty, Surbhi Mittal)
5. Cryobiological Investigations of the Diverse Aquatic Spematozoa The brood stocks used in commercial aquaculture, have not yet been subjected to intense selection and domestication, and tremendous potential exists for genetic improvement in these animals. Thus, the importance of developing alternative stocks for meeting a wide variety of market and production conditions is clearly recognized in aquaculture. Consequently, in the future, such traits as meat quality, carcass yield, disease resistance, reproductive efficiency and adaptation to extensive environmental variations should receive increased attention. Conservation programs are therefore needed, given the risks of genetic erosion of these breeds. Genetic erosion may result from an insufficient population size, making it hardly sustainable in the long term, or from a high proportion of crossbreeding inducing genetic dilution of the breed in a foreign genome. Presently, very few breeds could indeed rely on a sound conservation program if they had to face a sudden extinction. Cryopreservation of reproductive tissue brings safety in the long term, against the risks mentioned above. The freezing or "cryopreservation" of gametes (spermatozoa) involves exchanging the water in a gamete with a cryopreservation agent (CPA). This project aims to study the membrane physiology and cryobiology of marine and freshwater gametes to understand basic membrane mechanisms, maintain genetic diversity, and provide gametes for long term storage in genome resource banks. The strategies and technologies developed here will provide new ways to conserve endangered species, improve aquaculture to feed more people and assist in human health progress.(Student: Sreedhar Thirumala , Dinesh Pinisetty)
6. Fabrication of Micro Scale Arrays of Thermoelectric Actuators for Cryobiological Applications This research work is aimed to design and fabricate actuators, based on the Peltier effect. Mathematical model is used to identify important device parameters and dimensions. The organization of the proposed device will be an array of 100 (10x10) microscale (~10 μm) thermoelectric actuators, each of which will be separated by a distance of 50 μm center-to-center and dimensioned so that each device will measure or modulate the temperature in the neighborhood of a single cell. The prototype devices are being fabricated with a modified multi-step LIGA ((Lithographie, Galvanoformung and Abformung) technique wherein a patterned photoresist, PMMA, mask defines the geometry of the device. This will enable high-density wiring required for the device. Signals from individual rows of micro-devices will be multiplexed to reduce the size of the supporting electronics. The actuators will be embedded in Polymethylmethacrylate (PMMA) matrix to improve insulation.An artificial tissue (AT) system composed of Normal Human Dermal Fibroblat (NHDF) cells from stem cells will be grown on the device for experimentation wherein a PMMA sheet will act as an interface between the cooler and the embedded cells. The thermoelectric micro device thus developed will result in the unique capability of temperature manipulation and control on cellular scales (micrometers). (student: Prabhakar, Aparna)
7. Nanoscale Measurements of Water Loss During Desiccation of Biological Cell Suspensions There is an urgent need to protect and conserve the endangered species of the world. Traditional programs of breeding and maintaining endangered species in captivity are not only very difficult but are also very expensive. Thus, limiting the genetic diversity of an endangered species. Desiccation, or the phenomenon of anhydrobiosis, offers the attractive possibility of storage at ambient temperature simplifying the logistics of storage and transportation thereby significantly reducing the cost. Cell stasis is achieved in nature by anhydrobiotic organisms through desiccation at ambient temperatures, and the pharmaceutical industry has made significant strides in storing proteinaceous drugs, membranes, and viral particles in dry state using various small sugar molecules as stabilizers. Although some success have been demonstrated in desiccation preservation of mammalian cells in the presence of sugars, the process has not been optimized due to a lack in understanding of the fundamental mechanism(s) by which sugars protect the cells. Current techniques of desiccation use a convective drying stage, an approach that has advantages both in terms of cost as well as ease of use operation. To dynamically quantify the rate of moisture loss during a prescribed desiccation protocol, we have modeled and characterized a novel micromembrane ultrasound (resonant) sensor. To model the sensor, we performed a static (stress) and dynamic (frequency change with change in imposed mass) analysis of the membrane. Rectangular membranes were selected for their structural/directional rigidity. Static analysis of the membrane was used to relate the stress induced in the membrane to the weight of the cell suspension. To prevent failure (rupture) of the membrane during the initial loading process, analytical and finite element techniques were utilized to determine the minimum dimensions of the membrane for a predetermined membrane material (silicon) and the loading weight (i.e. volume of the cell suspension). Dynamic analysis encompassed effects of the mechanical vibration i.e. the ultrasound vibrations, on the membrane with respect to time and change in loaded mass (i.e. loss of moisture content). Although the characterized device was found to have a sensitivity of ~1 nanogram/millisecond, limitations in fabrication techniques and measurement/frequency analysis suggest that the fabricated device will only be able measure weigh loss in 10’s of nanograms. Aim is to fabricate the sensor using microfabrication techniques, and to quantify the rate of water (moisture) loss during the drying protocol in the presence and absence of sugars. The results will be used to develop and perfect a computational model of water loss during drying of cells loaded with sugars. It is expected that these studies will form the basis for developing a desiccation protocol, for an easy and flexible solution for banking and storage of sperm cells from endangered species.(student: Surbhi Mittal) 8. Cryobiological Investigations of Adipose Tissue-Derived Adult Stem (ADAS) Cells Human adipose tissue provides a uniquely abundant and accessible source of adult stem cells for applications in tissue engineering and regenerative medicine. The adult stem cells are isolated by collagenase digestion, differential centrifugation and subsequent adherence to a plasticware surface. In response to chemical, hormonal or structural stimuli, the adipose-derived adult stem (ADAS) cells can differentiate along multiple lineage pathways, including adipocytes, chondrocytes, myocytes, neurons and osteoblasts. The cells can be transduced with viral vectors and have potential utility as gene delivery vehicles.A sufficient ADAS supply available on demand and on a year-round basis has the potential to revolutionize the tissue engineering and regenerative medicine industry. Successful cryopreservation of scientifically and commercially important ADAS using relatively simple methods would provide a means for meeting these needs. Therefore, one of the primary objectives of the current study will be to develop new and efficient cryobiology protocols to enhance the availability and the diversity of ADAS cells. All cell systems do share common cryobiological responses which may be exploited to better understand and alleviate the specific problems of freezing in ADAS cells. The highest rates of cellular survival are typically found for cooling rates which are fast enough to minimize dehydration solute effects injury while still slow enough to preclude large amounts of intracellular ice. Thus, to optimize and generate a firm biophysical understanding of the freezing process in any biological system, both water transport (dehydration) and intracellular ice formation (IIF) need to be experimentally determined. We are unaware of any studies that report water transport and IIF parameters in any stem cells. This study aims to rectify this lack of cryobiological knowledge in several stem cells using traditional cryomicroscopy procedures and a recently developed differential scanning calorimetry (DSC) technique.At the end of this work we should have a mechanistic understanding of how the several different ADAS cells behave during a freezing protocol. In addition, we will also know how the ADAS cells respond to a prescribed osmotic (CPA) and thermal cooling history as well obtaining for the first time the effect of freezing/long term storage techniques on the genetic integrity of ADAS cells. This information is critical to the design of optimal cryopreservation protocols for currently available ADAS cells and will suggest a general strategy towards optimization of stem cell cryopreservation. (student: Sreedhar Thirumala) 9.Molecular dynamic simulations of water permeation through phospholipids membranes in the presence of sugars DESICCATION is used as a strategy to preserve biological activity through times of extreme environmental stress. The ability to survive in a dry state for extended periods of time has been identified in variety of organisms, including plant seeds and bacteria. Extensive studies on these organisms have revealed that they generate sugars through a series of complex molecular and physiological adaptations to survive excessive water loss during desiccation process. Current efforts are now focusing on the use of desiccation as a method of long-term storage of mammalian cells. For sugars to be maximally effective at protecting against the damaging effects of dehydration, they need to be present on both sides of the membrane. Moreover, their action is believed to result from specific interactions between membrane phospholipids and the polar sugar molecules in the solvent environment. Experimental results show that the addition of small amounts of sugars leads to an increase in the interlayer distance. At small concentrations this effect is explained by moderate hydration repulsion between the layers but the detailed mechanism is still not understood. The aim of this study is to investigate using atomistic molecular dynamics simulations the detailed mechanism of sugar Bilayer membrane interactions as well as the membrane water permeation in the presence of sugar molecules. In these simulations the trajectory of all atoms in the system is tracked by integration of the Newton's equations of motion. Using these trajectories and by employing statistical mechanics we can determine various properties of interest. In this project, to validate our simulation results, we will also perform a set of experimental studies. (student: Dinesh Pinisetty)
Movie Clips
Freezing of cellular suspensions
These movie clips need Windows Media Player, click here to download |
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