Cryomicroscopy of granulocytes to determine membrane permeability at freezing temperatures

Cryomicroscopy of granulocytes to determine membrane permeability at freezing temperatures

ABSTRACTS, 24th ANNUAL States Department of Health and Human Services, National Institute of General Medical Sciences, Grant No. 1 ROl GM3757501.) 5. ...

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ABSTRACTS, 24th ANNUAL States Department of Health and Human Services, National Institute of General Medical Sciences, Grant No. 1 ROl GM3757501.) 5. Cryomicroscopy of Granulocytes to Determine Membrane Permeability at Freezing Temperatares. DAVID YORDY, SHANTI J. AGGARWAL,

AND KENNETH R. DILLER (Biomedical Engineering Program, Department of Mechanical Engineering, The University of Texas, Austin, Texas 78712). The response of fresh human granulocytes in the presence of extracellular ice was experimentally studied using a cryomicroscope. The cells were suspended in Hank’s balanced salt solution with glycerol added as a cryoprotective additive (CPA) to produce two solutions at 1.0 and 2.0 M glycerol concentrations, respectively. The glycerol was added stepwise to the solutions to minimize osmotic shock. Freezing was achieved by slowly cooling the cells on a microprocessor controlled cryostage to a preset nucleation temperature ranging from 269.15 to 254.95”K. The solution was then nucleated and held at a constant temperature. The osmotic response of the cells was recorded on 35-mm film and videotaped with corresponding temperature recorded on chart paper. Both 35-mm prints and video frames were analyzed to determine cell volume changes over time. Intracellular ice and bubble formation were noted in some experiments. Viability was determined both before and after freezing by diacetate fluorescein (FDA) stain. The volume, time, temperature, and concentration data were analyzed by a microcomputer version of the Kedem-Katchalsky transport model to determine the optimum hydraulic permeability to fit the empirical data. The resultant permeability values generally decreased for both CPA concentrations with the decrease in nucleation temperature. The 2.0 M glycerol permeability values were found to be generally less than those for the 1.0 M glycerol concentration. Hydraulic permeability and viability results are presented in relation with all observed cell responses. Overall viability for both groups was nearly equal at just under 45%. The presence of intracellular bubbles and/or ice complicated the proper assessment of viability and appeared to often have a direct influence on the viability result. 6. A Network Thermodynamic Method of Analysis and Simulation of Membrane Transport during Cryopreservation. KENNETH R. DILLER, Jo-

SEPHJ. BEAMAN, AND J. PATRICKMONTOYA* (Biomedical Engineering Program, Department of Mechanical Engineering, The University of Texas, Austin, Texas 78712; and *EDS Corporation, Lansing, Michigan).

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A network thermodynamic model for cell membrane coupled flow of water and permeable cryoprotectant additives is presented in the form of a nonlinear bond graph which formulates into the KedemKatchalsky coupled flow equations. A block diagram model which enforces the governing K-K equations and models the cell volume is presented and simulated on the program TUTSIM. The program is executed on an IBM compatible personal computer. Both the freezing and thawing of living cells and the addition and removal of cryoprotective agents may be modeled. This method of simulation has proven to be very versatile with regard to the flexibility of changing model elements interactively on the computer and the capability of implementing the program on a microcomputer for solving the coupled nonlinear transport equations. In addition to simulation experiments the model can be used to compute the values of membrane permeability parameters from cell shrinkage and swelling data by performing optimal fitting procedures of the model to the data set. The model has been demonstrated in the calculation of membrane permeability values for a number of different cell types including granulocytes and monocytes for data obtained by cryomicroscopy and by diffusion chamber experiments. The program has been set up on a computer for testing and evaluation. (This work was sponsored by grants from the Texas Advanced Technology Research Program and from IBM Project Quest.) 7. Permeation of Corneas by Cryoprotectants termined by Changes in Immersed

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W. J. ARMITACE (Department of Ophthalmology, University of Bristol, United Kingdom). Cryopreservation by vitrification could allow storage at -196°C of organized tissues such as the cornea without the damaging effects of ice formation. Vitrification requires the presence of high concentrations of cryoprotectants in order to avoid ice crystallization at practicable cooling rates, and a balance has to be achieved between the concentration of cryoprotectant necessary for vitrification and the potential toxicity of the cryoprotectant. Information about the solute permeability of the tissue is necessary to be able to manipulate the conditions of exposure to the cryoprotectant (i.e.. temperature and duration of exposure, and rate of change in external cryoprotectant concentration) in order to minimize potentially harmful toxic and osmotic effects. The rabbit cornea is about 10 mm in diameter and about 0.4 mm thick. The stroma, which comprises 90% of the thickness of the cornea, is composed of parallel layers of collagen fibres embedded in a glycosaminoglycan matrix, and it is bounded by epithelial and endothelial cellular layers. Changes in external cryoprotectant concentration cause water and solute fluxes across the cellular