The isolation of DNA-containing particles from lyophilized cells by a new procedure

ANALYTICAL

2, 5X3-520

BIOCHEMlSTFtY

The

isolation Lyophilized DONALD

From.

the

of

(1961)

DNA-Containing Cells by a New

F. CLAUSEN

Department

AND

Particles Procedure’

LELAND

of Physiology, University Minneapolis, Minnesota Received

January

from

JAHNKE of

Minnesota,

4, 1961

INTRODUCTION

Presently there are two methods for the separation of intracellular particulate mat,ter from cells: (a) separation in aqueous media by means of differential centrifugation and (b) separation by flotation in nonaqueous organic media. It has been reported (1,2) that these methods can result in losses of enzymes, lipids, and proteins as well as in undesirable adsorptions. The work reported herein is a new approach in that lyophilized cells were ground in a ball mill or in a fluid mill and used as such or made into an aerosol, from which point attempts were made to separate intracellular particulate matter by physical means without the use of organic or aqueous media. The cells used were those of the GC,HED lymphosarcoma of Gardner (3) grown in ascites form in C&H mice (4). The cells are obtainable 100% viable and over 96% pure after 150 hr of incubation within the mice.2 From a smear stained with the Feulgen reagent and photographed at 440~ with a Whipple disk in the field, size estimations were made of the nuclei and cell diameters from 42 intact whole cells. It was found that the nuclei ranged in diameter from 2.9 to 7.2 p with an average of 4.6 p. Whole cells ranged from 6.4 to 12.2 p with an average of 8.1 F. METHOD

FOR

LYOPHILIZATION

After inoculation of mice with 0.1 ml ascites fluid each and incubation for 150 hr, cells were harvested from batches of 100 mice. The cells were then washed three times in saline from which they were separated by ‘This Research Service. ‘Private

investigation was supported (in Grant C-3583 from the National correspondence

with

Dr.

part) by Cancer

G. A. LePage, 513

Public Institute, University

Health Service U. S. Public of Wisconsin

Special Health

514

CLAUSEN

AND

JAHNKE

centrifugation. The final sludge of cells, in a layer l.O-mm thick spread on 0.35-mil aluminum foil, was quick-frozen by compression between two vertically stacked 40-lb cakes of Dry Ice, each previously chilled 30 min in liquid nitrogen. The disk of frozen cells was broken up with cold spatulas, placed into the chamber of a vacuum lyophilizer, and partially covered with liquid nitrogen. The system was placed under a pressure of 1O-4 mm Hg for 48 hr t.o remove first nitrogen, then residual carbon dioxide, and finally water. The final sample averaged 4.3 gm dried weight, contained 5 X lOlo cells, and lost 1% of its weight when desiccated to constant weight over Drierite. It was ground to a coarse powder in a glass mortar for weighing purposes and stored in a desiccator, It is subject to the influence of static electricity. It is hygroscopic to the extent that, in an atmosphere which is held at 90% relative humidity at room temperature, it will absorb up to 45% of its weight of atmospheric moisture in 48 hr; absorption of atmospheric moisture under ordinary laboratory conditions is slow to the point that equilibrium may not be reached before several weeks, and absorption does not interfere with the weighing of samples for analysis. METHODS FOR GRINDING Three methods were used to grind the lyophilized cells: (a) for 52-350 hr by means of 1/4-in. glass or steel balls at -25°C in a rotating steel bomb with a grinding medium of liquid carbon dioxide, (5) for 300-330 hr in a medium of liquid nitrogen by means of a single l-in. steel ball activated by a rotating magnet, (c) in a fluid mill; the cells were fired through the deagglomerator head of the Sharples Micromerograph (5) at narrow settings of the annulus. ATTEMPTS TO SEPARATE INTRACELLULAR PARTICULATES Five methods were used in attempts to separate intact nuclei from lyophilized and ground cells, following the progress of each by means of microscopic size-frequency (6) counts and chemical analyses for deoxyribonucleic acid (DNA) by the Dische reaction: (a) centrifugation, using the Goetz Aerosol Spectrometer (7), of the aerosol produced by the firing of ground cells through the Sharples deagglomerator; (5) gravity settling of the particulates, according to the expressions of Stokes and Cunningham (S), from either stationary or moving aerosols; (c) segregation of particles in the aerosol by means of photophoresis (9), (d) gravity or centrifugal separation of particles suspended in a medium of liquid carbon dioxide at room temperature and at refrigerator temperatures; (e) separation of particles from a moving aerosol stream by means of electrostatic precipitation (10).

ISOLATION

Separations

OF

DNA-CONTAINING

515

PARTICLES

Using the Goetz Aerosol

Spectrometer

(7)

Lyophiliaed cells ground in a ball mill were made into an aerosol by firing through the Sharples deagglomerator at wide settings of the annulus and were then drawn through the Goetz aerosol spectrometer running at various speeds. The DNA content rose somewhat in the fraction representing particle sizes less than 0.6 p, indicating the friable nature of the nucleus. This is shown in Table 1. The Goetz instrument may be useful for the separation of submicron-sized particulates after TABLE SEGREGATION

1

OF DNA IN AN AEROSOL BY MEANS GOETZ AEROSOL SPECTROMETER Size (P)~

Per cent DNAa

3.5 1.5 1.0 0.8 0.6 0.5

5.6 5.3 5.5 6.2 5.9 7.7

OF THE

a Samples were taken from small areas down the channels of the rotor cone. The size reported is the average particle size for that area from the calibration tables accompanying the instrument. b The DNA content of the intact untreated lyophiliaed cells was 6.7yc. DNA determinations were reproducible to zb:S.S% of the average value. For example, the range of the intact untreated lyophilized cells was 6.3-7.1% DNA.

the removal of intact and fragmented nuclei, but slow gas-flow rates through the instrument when it is adjusted for the collection of particles the size of intact nuclei probably disqualify it for that purpose in view of the rapid reagglomeration rates of aerosols (11). Separations in Air or Other Gases under Gravity

Aerosols were made by firing the lyophilized cells at sonic velocity through the Sharples deagglomerator at narrow settings of the annulus. When used in this manner the Sharples instrument acts as a fluid mill which can be used to grind the cells to varying degrees of fineness, depending upon the annulus setting and firing pressure. The fall under gravity of the particles of such an aerosol follows the mathematical equations of Stokes and Cunningham (8). Particles smaller than 0.1 p in diameter show Brownian movement. Because of the rapid rate of reagglomeration of aerosols (ll), separations under gravity are more effective if the vertical-motion component is short and there is a long horizontal-motion component. Resolution is gained, with a horizontal

516

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component, where there is a !ayer of aerosol-free gas moving at the same rate as the aerosol stream and lying between the aerosol and the collection plate. Attempts to effect separations by allowing the aerosol to fall vertically were failures. Attempts to impose a horizontal-motion component to the system by guiding the falling aerosol into the top 0.5 cm deep layer of a 3.0-cm deep stream of gas flowing through a horizontal channel 3.0 cm deep, 12 in. wide and 8 ft long at 2.4 cm/set initially failed because of the instability of gas flow observable at such TABLE

2

SEGREGATION OF DNA BY STOKES-CUNNINGHAM FALL IN AN AEROSOL MOVING HORIZONTALLY IN AN EIGHT-FOOT CHANNEL AIicroscopic Distance from entrance orifice (in. )

10

15 20 25 30 35 40 50 60 70 80 90

PI%i?t

8.2 8.6 8.1 7.1 6.3 5.9 5.3 5.4 5.5 5.7 -

-

D

Sm;tcr;ihhan

0 5.0 6.0 2.0 0 1.0

3.0 8.0 16.0 24.0 32.0 38.0

size-frequency estimations: d(D) of particles* Sire of nucleic

“%’

0 0 2.0 15.0 36.0 52.0 62.0 74.0 68.0 67.0 65.0 56.0

4.0 20.0 53.0 53.0 46.0 38.0 30.0 19.0 13.0 8.0 6.0 3.0

Larger than eds

84.0 64.0 46.0 30.0 17.0 10.0 5.0 1.0

0 0 0 0

0 The DNA content of the intact untreated lyophilieed cells was 7%. DNA analyses were reproducible to f6.6’% of the average value. b 6(D) represents the per cent of the total number of visible particles that are of the size of the classification considered. 0 The predicated area for the accumulation of particles the size of nuclei is 20-96 in. from the entrance orifice.

low Reynolds numbers (12). At the suggestion of Dr. Kenneth T. Whitby of the Dept. of Mechanical Engineering, a density gradient was obtained in the gas by layering, in l.O-cm deep layers, nitrogen over carbon dioxide over methyl chloride. The aerosol was suspended in the top 0.5 cm of the nitrogen layer. Smoke streamers (13) indicated that a stable flow was obtained. A row of clean microscope slides was laid down the center of the bottom of the eight-foot channel and one lO.O-mg shot of lyophilized cells was fired from the Sharples, at an annulus clearance of 200 p and at a pressure of 400 Ib/sq in., and allowed to flow as an aerosol through the

ISOLATIOS

OF

DNA-COh-TAINING

.il 7

P.iRTICLES

channel at 2.4 cm/set. After the particles of the aerosol had either settled or passed from the channel as exhaust, 1000 particles on each slide were sized by means of a microscope equipped with a Whipple disk, and size-frequency curves (6) were determined for the points represented by each slide. After removal of the slides, additional shots were fired at IS-min intervals until enough material had collected on the channel bottom to permit analyses for DNA content. The data obtained are shown in Table 2. It can be seen that the DNA tends to concentrate in an area where large percentages of particles as large or larger than intact cells are found microscopically. This suggests that nucIei and nuclear fragments tend to reagglomerate more rapidly than do fragments of cytoplasm. The lowered DNA content of fractions further than 25 in. from the entrance orifice suggests friability of the nucleus, also observed from data obtained with the Goetz Aerosol Spectrometer. Separation

by Means

of Photophoresis

(8)

While photophoresis (lateral movement of falling aerosol particles away from and toward a beam of light) appeared to effect some separation according to microscopic count, there was no evident separation as determined by DNA analyses. Separations

under

Gravity or by Centrifqation Carbon Dioxide

in

Liquid

Lyophilized cells were fired through the Sharples dengglomerator used, at narrow settings of the annulus, as a fluid mill, and collected in a plastic bag. The ground cells were dispersed in liquid carbon dioxide and allowed to settle through 3-10 in. of aerosol-free liquid carbon dioxide in steel bombs under gravity or by centrifugation at room temperature and at refrigerator temperatures (to elevate the density of the liquid). No consistent concent,ration of DNA was found in any fraction. Separation by Electrostatic Means -4 series of lO.O-mg aliquots of lyophilized cells were fired through the Sharples deagglomerator at an annulus clearance of 200~ and at a pressure of 400 lb/sq in., and the resulting aerosol of fluid-milled cells was forced, by means of a stream of nit,rogen metered from a tank, through the corona discharge of an electrostatic particle sizer built after the design of Langer (10). The charged aerosol was then driven between the charged plates of the particle sizer collector, with a layer of aerosolfree gas moving between the aerosol and the collection plates in order to increase resolution. All gas flows were metered at rates compatible

518

CLAUSEN

AND

.JAHNKE

with stability of flow (12) as indicated by smoke streamers (13). Deposition was almost quantitative on the negatively charged collection plate. After the first shot the negative plate was removed and microscopic size-distribution counts (6) were made, using oblique light, at intervals along the plate. The plate was then replaced and shots were fired at 15-min intervals until enough material had collected to permit semimicro analyses of the DNA content of fractions collected at intervals along the length of the plate. The results are shown in Table 3. As with SEGREGATION OF DNA Distance from entrance orifice (in.) % (0%)

3t &s-Ji) ?4 (?&?a 94 1.0 (X-1.0) 2.0 (1.0-2.0) 6.0 (2.0-9.75)

TABLE 3 BY MEANS OF ELECTROSTATIC PRECIPITATION Microscopic sire-frequency estimrttions: e(D) at (size rrtnge in cc)

Yield (mg)

P;NcPt a

9.8 11.5 5.8 7.4 5.1 2.8

10.0 8.0 6.0 4.4 2.6 1.8

o&1.5*

4.5-5.5’

9.510.5d

0 2.0 5.0 15.0 66.0 92.0

4.0 6.0 15.0 20.0 16.0 1.0 0

49.0 8.0 3.0 0 0

Q The DNA content of the intact untreated lyophilized analyses were reproducible to &6.6% of the average value. b Smaller in size than nuclei. 5 The size of nuclei. * Larger in size than nuclei.

cells was 7%. The DNA

separations under gravity in the horizontal channel, the DNA tends to collect in an area populated by particles larger than nuclei, suggesting rapid reagglomeration of nuclei and fragments of nuclei. The lowered DNA content of fractions more distant than %-in. from the entrance orifice again suggests the considerable friability of the nucleus. The size-distribution data from small particles in oblique light is somewhat in error because of the influence of scattered light on the particle size estimation.3 CONCLUSION

It has been shown that it is possible to concentrate the DNA of certain lyophilized cells from 7% in the intact cells to as high as 10% by electrostatic means and to a lesser extent by means of gravity settling. The nuclei of these cells appear to be more friable than does the cytoplasm. If the cells, averaging 8.1 ,u in diameter, are spheres containing ‘Private Correspondence with Dr. Gerhard tion, Ill. Inst. Technol., Chicago, 111.

Langer,

Armour

Research

Pounda-

ISOLATION

OF

DNA-COILTAIKiINt;

PAUTICLES

519

spherical nuclei, averaging 4.6 p in diameter, and if the DNA content of the intact lyophilized cells is 7%, t,hen the DNA content of the intact isolated nuclei can be calculated to be 50.8%. According to this calculation, the electrostatic method has concentrated the DNA to approximately 20% of the theoretical content. This estimation would be elevated by consideration of the density differential existing between the nucleus and the cytoplasm. While actual operation of the apparatus is not difficult, poor or no control of certain variables reveals the following areas for criticism: 1. Gas flow metered by hand as indicated by flowmeter readings can be irregular. An automatic feedback control should be designed for this purpose. 2. The Sharples deagglomerator fires samples in small batches, each of which must be discharged into a container, disconnected from the rest of the system, during the discharge. As a result there is a loss of about 0.9 of each sample on the walls of the chamber. Under design is a continuous fluid milling and deagglomeration system which will feed directly into the electrical system. 3. It has not been possible to devise a fluid mill which will produce more than 1 or 2% of the theoretical number of microscopically intact nuclei. This extremely low yield requires that grams of lyophilized cells must be used to obtain milligrams of what appear to be intact nuclei. 4. There is a question as to possible damage to cell constituents from the ionized gas molecules and radiation produced by the corona discharge. To circumvent this it is planned to convert the electrical part of the collection system to a nonuniform electric field, which will eliminate the necessity of precharging the aerosol particles. 5. The lyophilization process is destructive to cells. SUMMARY

Attempts have been made to separate the nuclei from lyophilized cells by means of physical methods. After grinding the cells in a ball mill, converting them into an aerosol by blowing them at sonic velocity through a defined annulus, and effecting a particle size separation in an aerosol centrifuge, the deoxyribonucleic acid (DNA) is found to be slightly concentrated in the fraction representing particles less than 0.6 ,U in size. After blowing the cells through a defined annulus at sonic velocity to grind them (fluid mill process) and to form them into an aerosol in one operation, it was found that the DNA could be concentrated by (a) gravity from a horizontally moving stream of aerosol and (5) differential precipitation on charged metal plates after precharging in a corona discharge. In the latter two cases it was found that the

520

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AND

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DNA concentrated in the fraction representing particles larger than the nuclei, indicating rapid reagglomeration of particles containing DNA. REFERENCES V., STERN, H., .~ND MIRSKY, A. E., Nature 169, 128 (1952). V., STERN, H., MIRSKY, A. E., AND SAETREN, H., J. Gen. Physiol. 35, 529 (1952). 3. GARDNER, W. U., DOUOHERTY, T. F., AND WILLIAMS, W. L., Cancer Research 4, 73 (1944). 4. KLEIN, G., Exptl. Cell Research 2, 518 (1951). 5. P.~YNE, R. E., Third National Conference of the American Instrument Society, Philadelphia, Sept. 14, 1948 (Published as Bull. No. 1244, The Sharples Co., Philadelphia, 1948). 6. DALLAVALLE, J. M., “Micromeritics,” 2d Ed., p. 51. Pitman Publ. Co., New York, 1948. 7. GOETZ, A., Geofis. pura e appt. 36, 49 (1957). S. GREEN, H. L., AND LANE, W. R., “Particulate Clouds: Dusts, Smokes and Mists,” p. 64. Van Nostrand Co., New York, 1957. 9. WHYTLAW-GRAY, R., AND PATTERSON, H. S., ‘Smoke: A Study of Aerial Disperse Systems,” p. 120. Edward Arnold, London, 1932. 10. LAXER, G. “Particle Size Classification by Electrostatic Precipitation.” Armour Research Foundation, Ill. Inst. Technol. Final Rept. ARF 3108-9, July, 1959. Contract #AF 19 (6041-2411. Air Force Cambridge Research Center, Air Research and Development Command, U. S. Air Force, Bedford, Mass. [Also, J. AppZ. Phys. 32, 955 (196lj.l 11. FUKS, N. A., “The Mechanics of Aerosols,” p. 333. CWL Special Publ. 4-12: 1958. U. S. Dept. of Commerce, Office of Tech. Services. 12. ECKERT, E. R. G., SBHNGE, E., AND SCHNEIDER, R. J., “50 Jahre Grenzschichtforschung” (H. GGrtler and W. Tollmien, eds.), p. 407. Friedr. Vieweg und Sohn, Braunschweig, 1954. 13. CLAUSEN, D. F., J. Aerospace Sci. 28, 587 (1961). 1. ALLFREY, 2. ALLFREY,