Experimental Cell Research 65 (1971) 228-232

U L T R A S O N I C D I S P E R S A L OF M A M M A L I A N C E L L A G G R E G A T E S

J. L. MOORE, 1 A. R. WILLIAMS 2 and M. SANDERS 2

1Tenovus Laboratory, Velindre Hospital, Cardiff, and 2Department of Mierobiology, University College, Cardiff, Wales

SUMMARY

1. A novel ultrasonic device has been used to disperse successfully aggregates of mammalian cells into a single-cell suspension.

2. This dispersal was achieved with no significant morphological damage or loss of cell viability. 3. This apparatus has been used to determine the stress required to rupture the outer cell

membranes of HeLa Sz and Chinese hamster cells. 4. This method of dispersion could be used to eliminate some problems preventing the auto-

mation of mass cervical screening.

The production of a single-cell suspension of intact mammalian and bacterial cells is often a prerequisite for certain medical and in- dustrial tests; for example biopsy material and tissue segments from animals often have to be reduced to single cells before use. The inability to disperse aggregates of human cer- vical cells without morphological damage is the main factor preventing the automation of mass cervical screening for suspected cancer cells [1].

Partial digestion with trypsin and other enzymes has been employed to separate cells but it has been reported that mammalian cells show delay in growth accompanied by some induced synchrony [2], changes in electrophoretic mobility [3], membrane da- mage resulting in leakage of internal com- ponents [4] and if digestion is too prolonged, cells are destroyed.

A system which produces a single-cell suspension without morphological damage or loss of viability is obviously of great value. This paper describes the use of a novel

Exptl Cell Res 65

ultrasonic device which disperses mammalian cell aggregates without changes in morpho- logy or loss of viability. Low frequency ultrasound at levels above the threshold for transient or collapse type cavitation has been employed but the method is coarse and results in damaged cells [5].

Mammalian cells cultured as monolayers in bottles and as colonies of cells derived from a single cell have been used to test this new method of dispersion.

METHODS AND MATERIALS

Cell culture and assay system HeLa $ 3 0 X F ceils were grown in 4 oz glass bottles with a complete medium, i.e. Glaxo 199 supplemented with 10 % calf serum. Chinese hamster ceils were also grown in 4 oz glass bottles with F10 medium supple- mented with 10 % foetal calf serum [6]. The cultures were grown for 3-5 days until a confluent growth was obtained on glass; a single bottle yielded approx. 107 ceils. For some experiments only 200 cells were inoculated into a bottle and these were incubated for 14 days. The cells grew into small colonies which could be harvested almost intact by gentle trypsinization, or by scraping the surface of the glass with a surgical needle.

Ultrasonic dispersal of mammalian cell aggregates 229

Mammalian cells were harvested by removing the used nutrient medium and adding 1 ml of trypsin in Tris saline buffer pH 7.2 [6]. This trypsin was used to bathe the surface of the cells and was then dis- carded. A further 3 ml of trypsin was added and the bottles were incubated at 37~ for approx. 3 rain or until the cells peel off the glass when the bottle is gently rocked from side to side. A quantity of comp- lete medium was then added to stop further enzymic action by the trypsin. When aggregates of cells were not required the cells were sucked into a 5 ml pipette and then blown back into the bottle six times to obtain a single-cell suspension. The total number of cells in suspension was checked in a Coulter counter and in a haemocytometer. From the haemocytometer it is possible to determine the multiplicity of the ceil culture, i.e. the proportion of cells that exist in groups greater than one. After dispersion of the aggregates by the pipetting technique the multiplicity was found never to exceed 1.5. A predominantly single-cell sus- pension produced in this way (or cells straight from the bottle as aggregates of two to several hundred, or as a disc of cells from a single colony), were used for the disruption and breakage experiments. After ultrasonic dispersion a sample of the cells was placed into a 4 oz glass bottle containing the appropriate complete growth medium and allowed to grow for 10-14 days at 37~ The medium was then removed and any colonies in the bottle stained and counted. In this way the number of viable cells that were added to a bottle after treatment was determined.

The sonication system A resonant length (1.69 cm) of thin steel wire (radius 115/tin) was clamped at one end to the tip of a stain- less steel velocity tranSformer (probe) so that the wire axis was at right angles to the long axis of the magneto- strictive stack. The wire was therefore driven trans- versely at 20 kHz at right angles to its axis and a transverse standing wave pattern set up [7]. Time independent (d.c.) acoustic microstreaming [8] is generated around the submerged portions of the wire as it vibrates transversely in the test liquid. The hydrodynamic shear forces generated between layers of solution in laminar flow have their greatest value at a distance (d) from the wire, corresponding to the boundary layer thickness. This distance is equal to 4 /zm at 20 kHz for liquids having a viscosity and density similar to that of water.

The sample to be sonicated (0.2 ml) was placed in a small test tube 1.5 cm long by 0.4 cm in diameter and the vibrating wire inserted until it was about 1 mm from the bottom of the tube. Should an aerosol be produced when the wire is vibrating, then the vessel is raised or lowered until the liquid meniscus meets the wire at a displacement node. After treatment, the liquid in the small test tube was mixed and drawn up into a 0.2 ml pipette. Sufficient liquid to flood a baemocytometer was used for the total count and then a measured quantity, i.e. usually 0.18 ml, was added to a 4 oz bottle of complete medium for assay of viable cells. When aggregates were sonicated at displace- ment amplitudes too low to disperse them, they were pipetted six times to ensure that the aggregates were

fully dispersed. In certain instances a further dilution was made in a second 4 oz bottle if the number of viable cells was expected to be greater than 1 000.

Electron microscopy Samples were mounted by the "blot-dry" method on Formvar-coated grids after fixation in 3 % v/v glutar- aldehyde in 0.1 M phosphate buffer pH 6.8. The cells were coated with a thin film of gold/palladium and examined in a scanning electron microscope.

RESULTS

An aliquot of a single-cell suspension of each cell line was sonicated at different wire displacement amplitudes for 5 min at room temperature (23~ as described under Me- thods. The results presented in fig. 1 show that both cell lines gave similar results in that essentially no cell disruption occurred after 5 min sonication at maximum displace- ment amplitudes (measured at the free end) of up to 40 #m. However, the two cell lines did differ in their response to ultrasound at higher displacement amplitudes, and gave 50 % survival values of 62/zm and 67/~m for HeLa $3 (OXF) and Chinese hamster cells respectively.

Further experiments were made with cells harvested by mild trypsinization without sub- sequent pipetting so that cells were present as aggregates. Some aggregates were obtained by scraping cells from the bottle with a sur- gical needle. Table 1 shows the results ob- tained from typical experiments to determine the effect of sonication on cell viability. Aggregated sample of HeLa $3 and Chinese hamster cells, obtained by both mild trypsini- zation and scraping the cells from the glass were sonicated for 5 min at the maximum displace- ment amplitudes shown. Values are presented for the total number of intact cells estimated immediately after sonication using a haemo- cytometer and the number of viable cells esti- mated from growth in 4 oz bottles after an allowance was made for plating efficiency.

.Exptl Cell Res 65

230 J. L. Moore et al.

100- //'~. . . . .

,/ 50. ~

C �9 , . O 15 30 45 60 75 90 105

Fig. 1. Abscissa: ampl i tude (/~m): ordinate: % b roken cells.

Decrease in the n u m b e r of intact Chinese hams t e r cells (�9 and H e L a $3 (OXF) cells ( • ), as the dis- p lacement ampl i tude of the wire was increased.

These results show that there is little, if any, lethal damage caused by the sonication pro- cedure in intact cells that remain, i.e. cell death is accompanied by disruption of the cell membrane and dispersal of the cell con- tents.

Low magnification stereoscan photographs showed that aggregated Chinese hamster cells were dispersed to form an essentially single- cell suspension after 5 min sonication at a maximum displacement amplitude of 60/zm. Stereoscan photographs taken at higher magnification showed that the separated cells are often distorted. This distortion does not appear to affect the viability of the cell, but there did appear to be some delay in division, up to a maximum of 24 h for cells that were treated to a viability of less than 20 %. Mare-

Table 1

Cell type

M a x i m u m H a e m o - wire cyto- ampl i tude , me te r Viable /~m cells/ml cellsa/ml

Chinese h a m s t e r (mild t rypsiniza- t ion)

Chinese h a m s t e r (scraped)

H e L a $3 Oxford (mild t rypsinizat ion)

0 9.0• 8.0• 39 8.0 • 105 8.5 • 105 42 6.0 • 105 7.3 • 105 46 7.0 • 105 7.0 • 105 50 6.0 z 105 5.0 • 105

0 8.0 • 104 9.0 • 104 31 7.0 • 10 ~ 6.0 • 104 35 6.5 • 104 7.0 • 104 39 5.0 • 104 6.0 • 104 42 5 .0• 7 .0• 46 4.0 • 104 5.0 • 104 50 5 .0• 4 .0•

39 8.1 • 104 7.7 • 104 39 3.0 • 105 3.1 • 105

a This coun t is corrected for the p la t ing efficiency. Norma l ly P.E. of 70-90 % is obta ined. The viable coun t is mul t ip l ied by 100/PE % so tha t a direct com- pa r i son can be made .

malian cells irradiated with ionizing radia- tions are known to exhibit a division delay [9] which is thought to be related to the time needed for recovery of some sub-lethal da- mage. The acoustic microstreaming may bring about some disorganisation of internal components of the cell and these must be reorientated before active growth leading to division can be initiated.

A second series of experiments with discs of cells removed from glass by mild trypsini-

Table 2

Cell No. of M a x i m u m wire H a e m o c y t o m e t e r Viable Theoret ica l type discs, amp l i t ude , / zm coun t / s ample coun t / s ample n u m b e r / s a m p l e b

H e L a $3 1 0 a l . 2 - 2 x 104 1.3 x 104 1.5 x 104 2 39 1 x104 0 . 9 x 1 0 4 3 x 1 0 4 8 39 8.1 • 104 7.7 • 104 1.2 • 105

20 39 3.0 • 105 3.1 • 105 3 • 105

Chinese 15 39 2.0 • I0 s 1.8 • 105 2 • 105 h a m s t e r 40 39 8.7 • 105 6.0 • 105 6 • 105

a N u m b e r of cells in a single disc af ter dispersion by the pipet t ing technique . b N u m b e r of cells expected a s s u m i n g each disc con ta ins an average of 1.5 • 104 cells. Rela t ive un i t s

Exptl Cell Res 64

Ultrasonic dispersal of mammalian cell aggregates 231

zation are shown in table 2. Single colonies upon retrypsinization yielded between 12 000 and 20 000 cells with an average value of 15 000. The colonies were 2 or 3 cells thick in the centre and are probably a better repre- sentative of biopsy and tissue segment ma- terial. Different numbers of discs were treated to ensure that the number of cells in the me- dium did not affect the dispersal system.

DISCUSSION

These results show that the novel ultrasonic device employed in these experiments can successfully disperse aggregates of cultured mammal ian cells to form a suspension of single cells. Providing the maximum displace- ment amplitude of the wire did not exceed 40/~m, the aggregates were dispersed without disrupting the component cells. Despite the mechanical bruising shown by the separated cells in some stereoscan photographs, each intact cell is capable of giving rise to a new colony, i.e. each intact cell is still viable (table 1). This suggests that cell death in this ultrasonic system is caused by mechanical rupture of the cell membrane and dispersal of the cell contents.

The breakage curves for the two cell lines had different slopes when plotted against the maximum displacement amplitude (fig. 1). Since a steep slope covers a smaller range of displacement amplitude, and hence a smaller range of hydrodynamic stresses, it follows that the HeLa Sa cell suspension had a narrower range of cells having different mechanical strengths than did the Chinese hamster cells. I t has not yet been determined whether this spread of particle strengths was caused, in part at least, by the pretreatment the cells received (i.e. the mild digestion with trypsin to free the cells f rom the glass), or whether it represents cells having different mechanical strengths at different stages during their life

cycle, since the cell lines used were not synchronous.

From table 2 it can be seen that the number of discs and therefore the number of cells being treated did not affect the efficiency with which aggregates were dispersed. The viable count and total count are similar for cell numbers between 104 and 106 and since both are similar to the theoretically expected num- ber, little, if any, cell disruption occurs at any cell concentration.

The maximum displacement amplitudes re- quired to disrupt 50 % of the cells of each cell line in 5 min are 62 # m for the HeLa (OXF) and 67/~m for the Chinese hamster (see fig. 1). From microstreaming theory [10] these dis- placement amplitudes correspond approx- imately to 1 • 104 and 1.2 • 104 dynes/cm -2

respectively. These values are, as expected, much greater than the value of 3.5• 103 dynes/cm -2 required to disrupt 50 % of hu- man and canine erythrocytes using a similar microstreaming system [1 I].

It is the intention of the authors to use this technique to disperse biopsy material for a detailed examination of the electrophoretic mobility of tumour and normal cells.

The authors wish to thank Professor D. E. Hughes for his invaluable advice and encouragement, especially with the electron microscopy. LL.M. wishes to ac- knowledge the generous financial support of the Te- novus Organisation, Cardiff, South Wales; A.R.W. thanks the Medical Research Council for his research grant.

R E F E R E N C E S

1. Husain, O A N & Henderson, M J, Cytology automation (ed D M D Evans). Livingstone, Edinburgh (1968).

2. Berry, R J, Evans, H J & Robinson, D M, Exptl cell res 42 (1966) 515.

3. Monier, E, Zaydela, F, Choix, P & Petet, J F, Cancer res 19 (1959) 927.

4. Seaman, G V F & Cook, G M W, Cell electro- phoresis (ed E J Ambrose). Churchill, London (1965).

5. Boucher, R M G, Pisano, M A, Tortora, G & Sawicki, E, Appl microbiol 15 (1967) 1257.

Exptl Cell Res 65

232 J. L. Moore et aL

6. Paul, J, Cell and tissue culture. Livingstone, Edinburgh (1965).

7. Williams, A R & Nyborg, W L, Ultrasonics 8 (1970) 36.

8. Holtsmark, J, Johnsen, I, Sikkeland, T & Skav- lem, S, J acoust soc Am 26 (1954) 26.

9. Elkind, M M & Whitmore, G F, The radio- biology of cultured mammalian cells. Gordon & Breach, London (1967).

10. Nyborg, W L, Physical acoustics (ed W P Mason) vol. 2 (1965).

11. Williams, A R, Hughes, D E & Nyborg, W L, Ultrasonics. In press (1970).

Received September 14, 1970

Exptl Cell Res 65