Vascular endothelial cells and cryoprotectant toxicity

ABSTRACTS,

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of Cryopreserved Murine Fetal Liver Cells for the Treatment of Lethally Irradiatedkjuries. Z. DING, Z. MAI, J. ZHANG, Y. XIANG, AND A. Yu (Department of Radiation

of 2”C/m from 20 to - 60°C. The adenosine triphoshate level reduced rapidly during 4°C HC-A and DMSO solution perfusion but changed slightly during freezing.

Medicine, Second Military Shanghai, China).

190. Effects

188. Transplantation

Medical

College,

We present a comparative study on the transplantation of fresh and cryopreserved fetal liver cells in lethally irradiated mice. The results showed that the 30day survival rates after transplantation were 38.8% (2%~) for recipients infused with fresh fetal liver cells and 54.5% (42/7) for those which received cells cryopreserved in liquid nitrogen for 2-103 days. The viability assay showed that the frozen-thawed cells still had an intact viability and repopulating capacity. Our investigations demonstrate that the infusion of fetal liver cells preserved in liquid nitrogen and thawed rapidly could achieve hematopoietic reconstitution in lethally irradiated mice. 189. Effects

of Cooling Rate and Helium Hypothermic Perfusion on Kidney Cryopreservation. P. GAO, Z. Q. SUN, AND H. X. HAN (Shanghai

Institute of Mechanical Engineering, Shanghai, China). Fourteen dog kidneys were tested in a liquidnitrogen cooled cryobiological freezer. After perfusion with about 200 ml HC-A solution at 4°C the isolated kidney was further perfused with 400 ml HC-A and dimethyl s&oxide (DMSO). The final concentration was 1.4 M DMSO. The two-step cooling protocol was introduced: The kidneys were cooled from 20 to - 25°C at the cooling rates 0.5, 1, and 2”C/m, respectively; then the temperature was decreased from - 2.5 to - 60°C at the same rate of 2”C/m. During the experiment the precooled gaseous helium was induced into the kidney through the artery. The temperature of the kidney surface, middle, and center were measured simultaneously by three thermocouples equipped within a tiny probe. The experimental results were judged by morphological and structural changes through microscopic and electronic microscopic observation. The adenosine triphosphate level was also measured during the experiment. Before the ice formed, the temperature gradient between the kidney surface and center with helium perfusion was up to 6°C and about 4°C less than that without helium perfusion; after freezing, the peak temperature gradient was 14°C (cooling rate 2”C/m) whether helium perfused or not. Glomerule epithelium granular degeneration was one of the most important changes during freezing of kidneys. Both light and electronic microscopic observation showed that the obvious granular degeneration took place between -20 to -40°C. The kidney with the cooling history of l”C/m from 20 to - 25°C and then 2”Clm to - 60°C was better than the kidney with the cooling rate

of 4°C Storage on Morphology and Cytochemistry of Murine Epidermal Langerhans Cells. J. YANG, D. M. CHEN, G. LIU, N. M. Qv, J. M. TANG, X. R. SHI, AND H. Y. ZHU

(Department of Histology and Embryology, Beijing Medical University, Beijing, China). Storage of allografts or heterografts becomes a popular topic of research in the sphere of plastic surgery and bums. Allogeneic skin survival has been prolonged when allografts stored at 4°C for 7 days and longer. To further investigate the role of Langerhans cells (LC) of immunological significance in the skin, the authors of this paper observed the survival duration of murine plantar skin grafts under monologous allograting at different periods of storage at 4°C. The routine H and E staining, ATPase staining, and DNA development of the stored skin grafts were performed to observe the possible changes. LC originate from the bone marrow, are dendritic in shape, ATPase positive with Fc receptor and C3 receptor. The results showed diminishing of LC dendrites after 4 days of storage at 4°C. The ATPase-positive cells were noticed to have left only a round cell body with tiny remains of dendrites after 7 days of storage. The ATPase quantitative analysis revealed marked decrease of ATPase activity after 4 days of storage. Feulgen reaction and H and E staining showed freshskin-like structure in samples stored for l-7 days, while intracellular hydrops and darker nuclear coloring were seen in the 21-day group. Allografting of grafts after 4 days of storage was conducted with marked prolongation of survival of stored grafts. The prolongation of allogeneic skin survival after 4°C storage might be related to the LC changes. 191. Vascular

Endothelial Cells and Cryoprotectant Toxicity. G. A. POLLOCK, P. A. STEWARTRICHARDSON, S. H. MAGUIRE, L. HAMLYN, AND I. R. HARDIE (Department of Surgery,

University lia).

of Queensland,

Brisbane, Austra-

There is substantial evidence that alteration of vascular integrity is a major factor in the failure to successfully cryopreserve solid organs. To avoid this damage, ice formation in the organ must be reduced to an innocuous amount or eliminated by using high cryoprotectant (CPA) concentrations. Since the use of such concentrations is limited by the osmotic stress and chemical toxicity placed on the vascular cells, these effects of CPAs were studied in cultured endothelial cells. Susceptibility to high ionic (Na+) concentrations was tested by comparing responses to NaCl

574

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and sucrose. The osmotic and toxic effects of glycerol were measured independently at 10 and 37°C for 5 mitt and 1 hr. Dirnethyl sulphoxide (Me$O), propylene glycol (PG), and ethylene glycol (EG) were then compared to glycerol at 10°C for 1 hr. Monolayers of aortic endothelial cells, grown to confluence in microwells, were incubated in CPA concentrations up to 6.OM, using vehicle solutions with intracellular (RPS-2) or extracellular (HP-5) composition, both with and without colloids. Cell damage was assessed by acid phosphatase estimation of cell number and r3Hladenine uptake (1). Findings were (a) adding a colloid improved the results, perhaps by providing osmotic support during CPA washout; (b) HP-5 (Haemaccel, low K+) was a superior CPA vehicle to RPS-2 (albumin, high K+) in every case; (c) endothelial cells were susceptible to high ionic concentrations; (d) glycerol caused some chemical toxicity and had the greatest osmotic effects; (e) Me,SO and EC were the most toxic CPAs; (f) PC could be added and removed in a single step up to 2.5 M without detrimental effects and was relatively nontoxic at this concentration with exposure times to 1 hr. The results suggest that PG is a most promising CPA for use in cryopreservation of solid organs. REFERENCE 1.

Ager, A., and Gordon, J. L. J. Exp. Med. 159, 592-603

WORKSHOP

(1984). I-IMPROVED

METHODS 192.

FOR

FREEZE-DRYING LIVING

CELLS

A Review of the Freeze-Drying Process. A. P. MACKENZIE (Center for Bioengineering, University of Washington, Seattle, Washington).

The freeze-drying process is generally known to involve: (i) the conversion of water to ice, (ii) the sublimation of that ice, (iii) the evaporation of part or all of any water that was not converted to ice. Three constituent processes are thus identified. The conversion of water to ice is accomplished by a deliberate freezing and may involve a well-defined and preferred freezing procedure. The sublimation of ice is generally accomplished at an approximately constant temperature and in such a way that the crystals do not melt but are transformed directly to a gaseous state. The sublimation of ice is very frequently termed “primary drying.” The evaporation of part or all of any water not present as ice is often begun soon after ice begins to sublime. The evaporation of water not present as ice is frequently termed “secondary drying.” Aqueous sample materials may freeze with the crystallization of one or more solutes to yield classical eutectics, or they may freeze with the continued concentration of a solute-rich phase that persists in a metastable amorphous state. Many systems freeze with the crystallization of

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one or more solutes and the persistence of a residual amorphous concentrate. Structured systems may freeze in any of a variety of ways determined by their physical structure, the continuity of the aqueous phase, the nature of the solutes and structural elements, and the water content. The nature of the frozen state helps to determine the physical course of primary and secondary drying. Primary drying may be conducted and completed at very low temperatures for certain well-demonstrated reasons, or at much higher temperatures where sample materials tolerate the treatment and retain desired characteristics. Secondary drying proceeds to limited extents at lower temperatures and is generally promoted with increase in sample temperature. Freeze-drying rates increase rapidly with increase in freeze-drying temperature. Drying rates at very low temperatures appear to be limited largely or entirely by the very low vapor pressure of ice at those temperatures and not by any barrier to heat transfer. Freeze-drying may proceed so much more readily at higher temperatures that the rate is determined partly by the way in which heat required to sublime ice is transferred to the sample material. Different sorts of samples will benefit from different freezing procedures and different temperatures during primary and secondary drying in accordance with experimental objectives and may require the use of distinctly different sorts of freeze-drying equipment. Many choices must often be made! 193. History and Development of Freeze-Drying. H. T. MERYMAN (American Red Cross Blood Services, Rockville, Maryland). Initially, freeze-drying was used primarily for the desiccation of histological sections and for the storage of bacterial cultures at a time when mechanical freezers were primative and unreliable. Freeze-drying received a major boost during the second World War when the freeze-drying of plasma required processing at a scale previously unknown. The names of Flosdorf, Mudd, Greaves, and Strumia are intimately associated with those pioneering days. Pharmaceutical applications stimulated an age of sophistication with the development of a better understanding of the requirements for sample temperature control, and the role of eutectics and of residual moisture. Rey, Hackenberg, Rieutord, and Rowe played prominent roles in the maturation of the industry. During the 1950s and 1960s the potential of freeze-drying for the preservation of foods attracted the food industry and much of the research and development during those years was stimulated by the lure of this very large-scale application. The concurrent development of the frozen food industry enabled storage economics unachievable by freezedrying and interest in the process rapidly waned, leaving it predominately in the hands of the pharmaceutical industry where the cost and quality of the products