Genetically-modified animals: powerful tools for biomedical research

Comparative Medicine: Innovations for the Animal to Human Health Corridor PanelThe University of Missouri houses three National Institutes of Health (NIH), National Center for Research Resources (NCRR) animal resource centers. These animal resource centers are: (1) the Mutant Mouse Regional Resource Center (MMRRC); (2) the Rat Resource and Research Center (RRRC); and the National Swine Resource and Research Center (NSRRC). These animal centers provide genetically modified animal models of human health and disease for comparative medicine research investigators. Critser will describe the goals and functions of these animal resource centers and illustrate how investigators can access these animal models, as well as the ancillary services the resource centers provide

Parameter estimation and data reduction for cellular biophysical analysis

Abstract only availableAccurate estimation of cellular permeability parameters are an important part of designing and developing optimized cryopreservation protocols. Electronic Particle Counters measure cell volume by detecting changes in electrical conductivity. However, data that are obtained from these machines are noisy, making immediate application of curve fitting algorithms impossible. We attempted to reduce or eliminate noise due to both the population variance and the instrument. To eliminate the noise we grouped the original data into evenly spaced time bins, compared the point density of each bin to the average density over all the bins, and discarded those bins whose density fell outside a predetermined range that was centered around the average. Next an averaging scheme was created to remove the noise from the top and bottom. This was accomplished by grouping the remaining bins and applying a third order polynomial fit to the high and low ends of their volume histogram. Minima where found for each end in each time bin and their average was used as our high and low cut off. Any remaining noise was eliminated through the use of a Fast Fourier Transform and a high pass filter. After noise reduction a curve was then fit to the remaining data points using a least squares parameter estimation technique and a conjugate gradient method to find optimal parameters of the differential equations which model cell volume flux. These parameter values that were acquired using the best fit technique could then be used in models to more accurately represent the data that was collected.NSF-REU Program in Biosystems Modeling and Analysi

Two-parameter model of cell membrane response to cryoprotectant agents

Abstract only availableCryobiology is a widely applicable topic of biological science. From thoroughbred horse gene preservation to organ transplants, the applications of cryobiology, specifically cryopreservation, are broad. The two major problems encountered during cryopreservation are the following: intracellular ice formation and solute damage (Mazur et al, 1972, Exp Cell Res 71: 343-355). These are somewhat alleviated with cooling rate variation and the use of cryoprotectants (CPAs) such as DMSO, glycerol, and ethylene glycol. Scientists desire standardized protocols to optimize the process of adding and removing CPAs and applying optimal cooling and warming rates. Part of this requires understanding cells and/or tissues membrane permeability to water and the chosen CPA (Gao and Critser, 2004, Biomaterials and Biomedical Engineering). There are several mathematical models of the volume change process including the “two-parameter” model which is used exclusively for this project (Benson et al., 2004). The two differential equations governing the cell volume behavior are the following: To empirically find the unknown parameters Lp and Ps, water permeability and solute permeability respectively, a culture counter was used to gather cell volume change data. Cells were placed in a media then CPA was added to cells. The culture counter measures a voltage change which is directly related to the cells' volume change. The culture counter is a “dirty” way to gather data and the data sets acquired have substantial amounts of digital noise. The first step in analyzing the data was to filter the data to eliminate the majority of the noise and extract a relatively clean data set. This was done by isolating small bins of data, finding the average value, and removing the outlier datum. Next, a curve was fit to the data set to find the two unknown parameters. This was done with the Runge-Kutta numerical method for solving ordinary differential equations. The data clean up and curve fitting required a computer function and was written using the program MATLAB. The function can be applied to most sets of data to find the parameters for various cells and CPAs. These results will be used in optimizing data analysis for solute and solvent mass transport studies and will facilitate improved cryopreservation methods.NSF-REU Biosystems Modelin

Understanding a cryoprotective agent; the ternary phase diagram of proplyene glycol, sodium chloride and water [abstract]

The use of cryoprotectant agents (CPA) to aid in the preservation of cryopreserved tissues has become widely accepted as an important step in this process. Among the CPA used is proplyene glycol, known to be less toxic to some cell types than other CPA. In order to optimize the usefulness of the CPA proplyene glycol, the liquidus surface of the ternary system of sodium chloride/proplyene glycol/water must be understood. To this end, a differential scanning calorimeter was used to precisely measure melting points for varying ratios of proplyene glycol and sodium chloride, at differing concentrations of water in order to establish the isopleth and generate melting curves. (inserted only if this is indeed done) Curves were fit to a modified virial equation, and the Akaike Information Criterion (AIC) was used to determine the accuracy of fit

Bovine oocyte permeability in 1, 2-propanediol

Abstract only availableCryopreservation of oocytes is of great importance both in medical research and in the agricultural industry. Cryopreservation of oocytes allows for future breeding of selected genetic lines of animals. Effective cryopreservation of bovine oocytes has drawn considerable attention due to its application in the agricultural industry. The reproductive cells of genetic lines of cattle can now be cryopreserved and used in the future to breed superior cattle through in vitro fertilization (IVF). Currently, the preservation of bovine oocytes and other cells has had marginal success due to damage sustained to the cell during freezing and thawing due to volume fluctuations (Mazur et al; Experimental Cell Research 71(1972) 345-355). The Kedem and Katchalsky model (Biochem Biophys Acta, 1958, 27:229-246) can be utilized to model changes in cellular volume during freezing and thawing in cryoprotectant solutions. This model takes into account the specific plasma membrane permeability of the cell that is exposed to a particular solution. The purpose of this study was to determine the hydraulic conductivity, Lp, and the permeability coefficient for the cryoprotective agent, 1, 2-propandiol (PrOH), PCPA, for bovine oocytes. The activation energies of each of these parameters can be determined under the assumption that the plasma membrane permeability parameters follow an Arrhenius relationship. Experimental trials were conducted at temperatures of 30°C, 20°C, 10°C, and 4°C. In order to study the response of a single bovine oocyte to 1.5 M 1, 2-propanediol, a micro-pipette holding device (Gao et al.; Biophysics J, 71:443-450) was used to immobilize the oocyte in a small drop of TL-Hepes media. The oocyte was then abruptly exposed to 1.5 M PrOH media. The volume change of the oocyte (dv/dt) was recorded with a digital camera that was mounted to an inverted light microscope. The area of the cell in each image was calculated with a Fovea Pro Software plug-in to Adobe Photoshop. The volume of the cell was calculated from the calculated area, assuming that the cell was spherical. The constants, Lp and PCPA, were numerically approximated assuming that the cellular volume dynamics followed the Kedem and Katchalsky model. It was found that the oocytes underwent osmotically driven volume changes upon exposure to the cryoprotectant, 1, 2-propanediol. The bovine oocytes contracted more rapidly at higher temperatures. The oocytes also regained their isosmotic volume faster at higher temperatures. The permeability parameters found in this study along with there activation energies will be used in the future to develop an optimal cryopreservation protocol for bovine oocytes through computer-based modeling.NSF-REU Biosystems Modelin

Membrane permeability of Bovine Ooctyes to Propylene Glycol and the application to the improvement of Cryopreservation

Abstract only availableIn this study, the goal was to determine the permeability parameters of bovine oocytes for water (Lp) and Propylene Glycol (PPG) at temperatures of 30, 20, 10, and 5°C. By determining permeability parameters, we can model cell volume changes during addition and removal of cryoprotectants to determine a method that will prevent osmotic damage to the cells. Individual oocytes were held stationary by a holding pipette in a Petri dish on a Nikon inverted microscope. The oocytes were initially equilibrated in propylene glycol (PG) and 0.1M Sucrose for 20 minutes and then a solution of TL-Hepes with 0.1M Sucrose was added to a drop of 1.5M PG containing the oocyte. The specific initial concentration of PG and volumes of added solutions were modified for each temperature. Then digital images were captured on a regular time scale using a Spot RT Cooled CCD Digital camera in order to record shrinking and swelling. Morphometrical analysis was then performed on each image using Adobe Photoshop to measure the radius of each oocyte at the various time points during the volume excursions. Using Microsoft Excel, we were able to fit the experimental data to a best fit curve of a theoretical model for volume change, which allowed the determination of the values of Lp and PPG. These values were used to model the cell volume changes using MLAB (Civilized Software, Inc., Bethesda, MD) to developing optimized addition and removal procedures for 3.0M CPA that would minimize potential damage of the oocyte due to shrinking and swelling, and toxicity effects of the CPA due to excessive exposure. Currently, our results for the mean values of the permeability parameters Lp and PPG at 20°C are 0.3 ± 0.03 µm-min.atm and 15 ± 7.2 µm/min, respectively (mean ± SD, n=2). Further data acquisition and analysis is in progress.NSF-REU Program in Biosystems Modeling and Analysi