Many Applications
The nitrogen decompression method is particularly well suited for treating mammalian and other membrane bound cells. It has also been used successfully for treating plant cells, for releasing virus from fertilized eggs and for treating fragile bacteria. It is not recommended for untreated bacterial cells, but this restriction can be eliminated by using various pretreatment procedures to weaken the cell wall. Yeast, fungus, spores and other materials with tough walls do not respond well to this method.
Applications and Techniques
Mammalian Cells
Hunter and Commerford (1) published a paper in 1961 which has become a basic “cookbook” for the disruption of mammalian tissue by the nitrogen decompression method. Although most of the work reported by these authors was done with rat tissue, they also treated spleen, white cells, lymph nodes, tumors, thymus and other tissues to establish the general applicability of the method. Their results clearly demonstrated that cells can be disrupted by this method with minimum physical and chemical damage to the components.
H & C obtained complete disruption at pressures of 1300 psi and above, while pressures below 700 psi left whole cells and clumps of cells in the homogenate. At pressures between 800 and 1000 psi, cell-free homogenates were produced with nuclei intact. A hand press was used to pre-mince tissues prior to treatment in the vessel. The condition of the nuclei was found to be dependent upon the composition of the suspending buffer solution. Good results were obtained using isotonic solutions while nuclei swelling and rupture were observed in cells suspended in very dilute solutions. This was attributed to osmotic swelling which H & C found could be controlled by adding inorganic salts such as sodium chloride or organic solutes such as sucrose or glycerol. The nuclei were extremely fragile when the suspending medium contained no calcium, but the presence of as little as 0.0002M calcium chloride was found to stabilize the nuclei. Magnesium acetate is also useful for this purpose.
To determine the extent of damage to labile cells, H & C studied Deoxyribonucleoprotein, DNP, because of its susceptibility to chemical and physical stress, obtaining recoveries of over 90% DNP from the nuclear fraction with excellent preservation of the material. They also compared the enzyme activities of mitochondrial suspensions prepared by the nitrogen decompression method with suspensions produced in a PotterElvehjem homogenizer. No differences in enzyme activities were detected.
Dowben, Gaffey and Lynch (2) used the nitrogen decompression technique to prepare polyribosomes from L Cells, fibroblasts, human fetal cells from amniotic fluid, rat livers and muscle from chick embryos. Using 600 psi pressure they obtained better than 99.9% rupture and recovered more than 95% of the nuclei intact. Polysome yield was two to three times greater than when the cells were homogenized in a Dounce tissue grinder. In addition, they had better defined and more reproducible profiles. Significantly greater activities as measured by amino acid incorporation were also reported.
Short, Maines and Davis (4) compared the nitrogen decompression method with the Potter-Elvehjem types of PTFE pestle and glass tube homogenizers for preparing microsomal fractions for drug metabolism studies. The decompression method consistently produced over twice as much microsomal protein per gram of tissue as the pestle and tube fractionation. Enzyme activities per milligram of microsomal protein was found to be essentially the same for both methods, but it must be remembered that nitrogen decompression yielded over twice as much microsomal protein per gram of starting material.
Under microscopic examination the homogenates produced by the decompression method were found to be cell-free, while numerous cell clumps were observed in the pestle and tube homogenate. Electron microscopy of the microsomal pellets showed the particles to be smaller and more uniform in size for the decompression method. In summary, these authors stated that the nitrogen decompression method was more efficient and probably less variable than the PTFE pestle and glass tube methods.
Comparison with pestle and tube methods. In a recent application at the Veterans Administration Research Hospital in Chicago, a homogenate that had required eight hours to produce with the pestle and tube was prepared in fifteen minutes with a cell disruption vessel. In another laboratory, up to 12 kilograms of brain per day are being homogenized with a cell disruption vessel.
Wallach and his associates (5) have used the nitrogen decompression method to obtain complete cell fractionation with minimum nuclear damage. Working with Ehrlich Ascites Carcinoma Cells using a 0.0002M magnesium acetate buffer, they have studied the cellular distribution of phospholipides. Wallach has published many other papers in which the decompression technique has been used to prepare cell membranes.
Vaccine Preparation
A number of commercial laboratories have found that the nitrogen decompression technique is extremely effective for releasing virus from fertilized eggs. This method can be scaled up for commercial production using larger disruption vessels that are offered for this purpose by Parr.
Bacterial Cells
Fraser (7) in 1951 published some of the earliest studies on nitrogen decompression and its effect on E Coli. Fraser’s work was limited because his vessel was restricted to 900 psi operating pressure. Nevertheless, he was able to obtain 75% rupture in one pass and over 90% rupture in two successive passes using E Coli harvested during the log growth phase. Results with other bacteria and organisms with tough cell walls have been mixed.
There are several ways in which bacterial cells with tough walls can be treated to facilitate disruption by the nitrogen decompression method. These include: (1) harvesting the cells during an early growth phase before the full wall is developed; (2) growing the cells in the presence of an agent which will inhibit the formation of the cell wall; (3) using a lysozyme to weaken the wall prior to processing, or (4) using a mechanical pretreatment to weaken the cell walls before applying the nitrogen decompression method. Although these techniques have been applied successfully to many bacteria with heavy cell walls, they are not equally effective for yeast, fungus, spores and similar cells with very heavy or hard walls. Vigorous mechanical methods are generally required to break down the cell structure in these hard-walled materials since they generally do not respond well to treatment by the nitrogen decompression method.
Plant Cells
Loewus and Loewus (10) have published a number of papers in which they describe the application of nitrogen disruption procedures to plant cells and to tissue cultured plant cells. They also report considerable success in breaking diatoms by this method.
References
| (1) | Hunter, M. J. and Commerford, S. L., 1961, “Pressure homogenization of mammalian tissues.” Biochim. Biophys. Acta, 47:580- 6. |
| (2) | Dowben, R. M., Gaffey, T. A. and Lynch, P. A., 1968. “Isolation of liver muscle polyribosomes in high yield after cell disruption by nitrogen cavitation.” FEBS Letters, Vol. 2, No. 1, pages 1-3. |
| (3) | Dowben, R. M., Lynch, P. M., Nadler, H. C. and Hsia, D. Y., 1969. “Polyribosomes from L. Cells.” Exp. Cell Research, 58:167-9. |
| (4) | Short, C. R., Maines, M. D. and Davis, L. E., 1972. “Preparation of hepatic microsomal fraction for drug metabolism studies by rapid decompression homogenization.” Proc. Soc. Exper. Biol. Med., 140:58-65. |
| (5) | Wallach, D. F. H., Soderberg, J. and Bricker, L., 1960. “The phospholipides of Ehrlich and ascites carcinoma cells composition and intracelfular distribution.” Cancer Research, 20:397-402. |
| (6) | Manson, L. A., Foshi, G. V. and Palm, J., 1963. “An association of transplantation antigens with microsomal pipoproteins of normal and malignant mouse tissues.” J. Cell and ComD. Physiol., 61:109-18. |
| (7) | Fraser, D., 1951. “Bursting bacteria by release of gas pressure.” Nature, 167:33-4. |
| (8) | Avis, P. J. G., 1967. “In subcellular components, preparation and fractionation.” (Ed. Birnie, G. D. and Fox, S. M.) Chapt. 1, Pressure homogenization of mammalian cells. Published by Plenum Press, New York. |
| (9) | Manson, L. A., 1972 “Extraction of membranous transplantation antigens by pressure homogenization.” (Ed. Kahan, B. D. and Reiifeld, R. A.) Chapt. 9,oyransplantation Antigens. Published by Academic Press, New York. |
| (10) | Loewus, M. W. and Loewus, F., 1971. “The isolation and characterization of d-glucose 6-phosphate cycloaldolase (NDAdependent) from acer pseudoplatanus L. cell cultures. Plant Physiol. (1971) 48:255-260. |