146 Biofechnol. Frog. 1995, 11, 146-152

Batch and Semicontinuous Aggregation and Sedimentation of Hybridoma Cells by Acoustic Resonance Fields

Phylis W. S. Pui? Felix Trampler$,* Stefan A. SonderhoffJVg Martin Groeschl,8 Douglas G. Kilburn? and James M. Piret*J' Biotechnology Laboratory & Department of Chemical Engineering and Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 123, Canada, SonoSep Biotech Inc., Richmond, British Columbia V6V 2L1, Canada, and Institute for General Physics, Technical University of Vienna, A-1040, Vienna, Austria

Ultrasound was used to enhance the sedimentation of hybridoma cells from medium in a 75 mL resonator chamber. Forces in the acoustic standing waves aggregated the cells, and the aggregates were then rapidly sedimented by gravity. Cell separation increased with acoustic treatment time and cell concentration. The separation efficiency was over 97% for cell concentrations between lo6 and lo7 cells/mL. During acoustic treatment a t 180 W/L, the medium temperature increased at a rate of 1.3 "C/min. Ultrasonic exposures up to 220 W/L did not influence the viability or subsequent growth and antibody production of the cells. A decrease in cell viability was observed a t a power level of 260 W/L. Batch separation efficiencies were as high as 98%. Acoustic separation was tested under semicontinuous operation, and above 90% separation efficiency was achieved at a flow rate of 0.7 L/h.

Introduction Mammalian cell culture is used to produce many

secreted proteins with valuable therapeutic and diag- nostic applications. After bioreactor production, continu- ous filtration or centrifugation is commonly used to clarify the medium of suspended cells. Stirred suspen- sion bioreactors are often operated with continuous or semicontinuous addition of fresh medium and removal of spent medium containing the product. In perfusion bioreactors, cells are retained within the bioreactor or recycled from the spent medium stream to increase bioreactor productivity. Continuous filtration (Broise et al., 1992; Yabannavar et al., 19921, centrifugation (Jager, 1987; Tokashiki et al., 19901, and inclined sedimentation (Hansen et al., 1993, Searles et al., 1994) have been used to aseptically separate suspension-cultivated mammalian cells from the medium. One advantage of perfusion bioreactors is that downstream cell clarification process- ing requirements are greatly reduced. Antibody concen- trations have been recovered at up to 5 times batch levels (Hansen et al., 1993). This represents a 5-fold increased yield on medium and reduced downstream protein puri- fication requirements. However, cell separation devices are difficult to scale up and operate for extended periods of time. The usefulness of existing cell recycle systems is limited by progressive protein and cellular fouling of the devices. In addition, it is often necessary to continu- ously bleed cells from the bioreactor to limit the ac- cumulation of nonviable cells.

Acoustic particle separation technology can separate particles from fluid using resonant ultrasonic standing waves. The ultrasonic field forces aggregate particles,

Author to whom all correspondence should be addressed.

Technical University of Vienna. + SonoSep Biotech Inc.

8 Department of Microbiology and Immunology, University of

Biotechnology Laboratory & Department of Chemical Engi- British Columbia.

neering, University of British Columbia.

and then larger aggregates rapidly sediment from the fluid. Acoustic aggregation (aggregation of particles by acoustic forces in a standing wave field) has been used to enhance the sedimentation of mammalian cells (Kil- burn et al., 1989; Trampler et al., 1994; Doblhoff-Dier et al., 1994). We have used acoustic cell separation to continuously perfuse a 1000 h hybridoma culture at cell concentrations exceeding lo7 cells/mL (Trampler et al., 1994). Acoustic particle separation technology uses lower power and higher frequency than are required to cause cavitation and cell disruption. Figure 1 shows the forces responsible for the aggregation of cells in the resonance field (Coakley et al., 1994). Of the three forces, the primary radiation force generally has the greatest mag- nitude. Almost immediately following the application of an ultrasonic resonance field, the primary radiation force drives dispersed cells toward the velocity antinodes of the resonance field, so that the average distance between the cells is considerably decreased. The magnitude of the force depends on the difference in compressibility and density between the cells and the medium. The second- ary radiation force results from the interaction between the cells and becomes relevant only at very short dis- tances. Since both primary and secondary radiation forces are proportional to the cell volume, their magni- tudes increase as the aggregates become larger. A third force, called the Bernoulli force, is a result of the nonuniform acoustic field. This force drives the cells within the velocity antinode planes to the local maxima of the acoustic velocity amplitude, resulting in the formation of striated columns oriented perpendicular to the antinode planes.

This paper reports greatly enhanced batch aggregation and separation of hybridoma cells from growth medium using acoustic fields. The dependence of the separation efficiency on the ultrasound exposure time, the power level, and the cell concentration was determined. The effects of ultrasonic resonance fields on cell viability, growth rate, glucose consumption, and antibody produc- tion also were investigated.

8756-7938/95/30ii-Oi46$09.00/0 0 1995 American Chemical Society and American Institute of Chemical Engineers

Biotechnol. Prog., 1995, Vol. 11, No. 2 147

a

b

Velocity Velocity Velocity Node Antinode Node

I - I F P I 1q-E - I I -

I - I -

I 1 I

C I I

1 I

1

I - c - - t - - - - I

I - - I 1 - I - --t. - I

M M

1 - = - -

, l o ” - 1 - e

Figure 1. Migration of mammalian cells caused by the primary radiation force, Fp, and the Bernoulli force, FB, in an ultrasonic resonance field at 0 s (a), 1 s (b), and 10 s (c) of acoustic treatment at 100 W L . The lengths of the two-sided arrows indicate the acoustic velocity amplitudes.

Materials and Methods Cell Line and Growth Conditions. The hybridoma

2 E l l cell line (Ziltner et al., 19881, which produces a murine IgG monoclonal antibody against interleukin-3 (IL-31, was used in these experiments. The cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY) containing 4.5 g/L glucose supplemented with 5% (vh) newborn calf serum (Gibco).

Acoustically treated cells were grown in 80 cm2 tissue culture flasks (Nunclon, Roskilde, Denmark). Cells for all other experiments were maintained in 850 cm2 roller bottles (Falcon, Lincoln Park, NJ) rotated at 0.5 rpm (Bellco, Vineland, NJ). The tissue culture flasks were incubated a t 37 “C in a 5% COz incubator (Forma Scientific, Marietta, OH). Roller bottles were aerated with 5% Codbalance air every 2 or 3 days and incubated at 37 “C.

Cell Counting and Viability Measurements. For the cell culture experiments, cell numbers were measured by an Elzone 280PC electronic particle counter (Particle Data Inc., Elmhurst, IL) (Kachel, 1990) with an orifice diameter of 76 pm. Calibration was performed with 10.2 and 20 pm diameter latex beads (Particle Data). Cell samples were pipeted several times with a 200 pL Pipetteman (Gilson) to break up aggregates prior to analysis. Samples were diluted to less than lo4 cells/ mL in 0.2 pm filtered phosphate-buffered saline (PBS). Triplicate counts, each of >2 x lo3 cells, were averaged

to estimate the total cell number. The percentage of viable cells was determined using hemocytometer counts with trypan blue staining (Sigma, St. Louis, MO). About 100 cells were counted to obtain the viability estimates. The viable cell concentrations were calculated from the product of the total electronic particle count and the fraction of viable cells. In the ultrasonic separation experiments, 200-600 cells were counted by hemocytom- eter to obtain cell counts and viability.

Glucose and Antibody Analysis. Medium glucose concentration was monitored using a Glucose Analyzer 2 (Beckman, Fullerton, CAI. IgG antibody concentration was determined using a fluorescent concentration ana- lyzer (Idexx Ltd., Westbrook, ME) by the method of Jervis and Kilburn (1991). Carboxylpolystyrene particles (0.8 pm, 0.25% (v/v), Idexx) coated with anti-mouse IgG (Sigma) were used to capture the antibody. A goat anti- mouse IgG (H+L)-fluorescein isothiocyanate conjugate (Gibco) was bound to the particle analyte complex, and the fluorescence was measured to determine the sample IgG concentrations.

Acoustic Resonator System. Figure 2 shows a schematic diagram of the 75 mL ultrasonic resonator used in the investigations. Since the resonator was not designed to be autoclaved, it was sterilized with 70% ethanol and then rinsed with sterile PBS before use. The sterilization and acoustic treatment were performed in a laminar flow hood to avoid contamination. The resona- tor consisted of a composite transducer and a Pyrex glass reflector making up two opposite walls of the chamber. The composite transducer comprised of four PZT piezo- electric ceramics (Vibrit M202, Siemens, Munich, Ger- many), with a fundamental resonance frequency of 2 MHz, glued onto a borosilicate glass carrier (Schott, Hofheim, Germany). The piezoelectric ceramics were arranged as shown in Figure 2 with a 1 mm gap between them and electrically connected in series. The composite transducer was powered by an electronic unit consisting of a power amplifier, a voltage-controlled oscillator, and an automatic frequency control device (UCCS 03, SonoSep Biotech, Richmond, BC). The automatic frequency con- trol device utilized the true-phase signals of current and voltage consumed by the resonator to determine the active (root mean square) electrical power consumption of the resonator. Local maxima of active power consump- tion indicated resonance frequencies of the system (Schmid et al., 1990). The fraction of electrical power utilized to compensate for acoustic attenuation within the suspen- sion was estimated to be not higher than 50% (Nowotny and Benes, 1987). Other losses are due to acoustic attenuation within the transducer and the reflector, dielectric loss of the transducer, and electromagnetic transmission. The voltage-controlled oscillator was tuned by the voltage signal output of the automatic frequency controller toward preferred resonance frequencies of the resonator. To achieve a high-quality resonance field and to prevent acoustically induced streaming (Hager, 1991), the dimensions of the resonance chamber were made very precise. The transducer-reflector distance tolerance was f0.05 mm. The frequency range used was 2.45 and 2.5 MHz. To maximize the fraction of accumulated acoustic energy of the resonance field within the medium, the preferred high overtone resonance frequency of the resonator system was selected such that the composite transducer was not in resonance (Trampler et al., 1993). Table 1 summarizes the dimensions, materials, and frequency range of the acoustic chamber.

Batch Acoustic Separation Experiments. For each experiment 75 mL of cells were used. Initial samples were taken before acoustic treatment. After

148 Biotechnol. Prog., 1995, Vol. 11, No. 2

Composite Transducer

a

/ Composite Transducer

75 mL Resonatoi Volume

1--

b

Reflector

/

Figure 2. Schematic diagram of the batch acoustic resonator, front (a) and side views (b).

Table 1. Description of Dimensions, Materials, and Frequency Range of the Batch Resonator

component descrbtion transducer surface 55 x 55" transducer reflector

resonator volume 75 mL

piezoceramics

transducer glass

25.3 f 0.05 mm distance

frequency range 2.45-2.50 MHz 4 x Vibrit M 202,25 x 25 x 1 mm

Tempax 3.3 mm (borosilicate glass) (Siemens)

acoustic treatment was stopped, the aggregates were allowed to settle for 5 min and then the supernatant was sampled. The cells were then resuspended and total cell samples were taken. All samples were taken from the middle point of the chamber.

To obtain concentrations of up to lo7 cells/mL, cells from roller bottle cultures were concentrated by centrih- gation at 200g for 5 min and then diluted with super- natant to obtain the desired concentrations. All of the acoustic separation experiments were initially at room temperature.

To investigate the effect of acoustic treatment on cell growth and antibody production, cells were acoustically treated and then cultured at an inoculum level of 2 x lo5 cells/mL in 80 cm2 tissue culture flasks (Nunclon). Samples were taken daily for cell count and viability measurements. The samples were then stored frozen for antibody and glucose analyses.

Semicontinuous Flow-Through Separation. Fig- ure 3 is a schematic diagram of the flow-through con- figuration. The resonator was filled with medium con- taining 9.1 x lo5 cells/mL and tilted 15" from horizontal. After 2 min of static acoustic treatment, a cell suspension containing 9.1 x lo5 cells/mL was pumped at 0.7 L/h to the bottom of the chamber through 0.6 cm 0.d. polyethyl- ene tubing (Imperial Eastman, Manitowoc, WI). The tube was passed through the edge of the ultrasonic resonance field to minimize field disruption. The clarified medium overflowed from the chamber, and aggregated cells settled to the bottom of the chamber. Samples were taken from the overflow stream. During the first 24 min, a power level of 44-88 W/L at a resonance frequency of 2.435 MHz was used. From 24 to 75 min, a power level of 180 W/L at a frequency of 2.415 MHz was used. At the end of the experiment, the sedimented cells in the chamber were resuspended and sampled.

Results and Discussion Effect of Acoustic Treatment Time on Separation

Efficiency and Viability. A suspension of 1.3 x lo6

Resonator Chamber Agitated Cell Suspension

Reservoir Figure 3. Schematic diagram of the semicontinuous flow- through separation. The cell suspension was delivered a t 0.7 L/h to the bottom of the resonator through a polyethylene tube.

cells/mL was acoustically treated at 180 W/L applied power in the 75 mL static chamber. Suspended mam- malian cells sediment at about 1 cm/h (Batt et al., 1990). There was no discernible sedimentation of cells in the absence of acoustic treatment on the time scale of these experiments. Within a second of the initiation of acoustic treatment, the primary radiation force concentrated the cells into visible planes parallel to the transducer (Figure 4a). These planes were at the acoustic velocity antinodes spaced 310 pm apart (a half-wavelength). The cells then migrated within these planes into striated columns perpendicular to the transducer surface as a result of the Bernoulli forces, which act normal to the direction of sound propagation within the planes (Figure 4b). The Bernoulli forces and the formation of columns were a result of the nonhomogeneity of the acoustic field and have been reported by a number of other workers (Kilburn et al., 1989; Hager and Benes, 1991; Whitworth and Coakley, 1992). As more and more cells were concentrated, the columns became wider. Within 1-3 min, most of the cells were concentrated in 5-10 col- umns, and the rest of the chamber volume was visibly clarified.

The temperature in the chamber increased as a func- tion of acoustic treatment time (Figure 5). The average rate of temperature increase inside the chamber was 1.3 "C/min. This heating resulted from attenuation of the acoustic energy in the transducer, reflector, and cell suspension. Since mammalian cells cannot tolerate temperatures significantly greater than 37 "C, the heat- ing of the medium must be limited either by restricting the treatment time and/or by cooling the resonator chamber or the cell suspension before acoustic treatment.

Biotechno/. Pro& 1995, Vol. 11, No. 2 149

a

I b 1

I

Figure 4. Cell suspension inside the batch acoustic resonator a t 1 s (a) and 10 s (b) of acoustic treatment. The vertical planes of concentrated cells were a t 310 pm (half-wavelength) intervals parallel to the transducer and reflector.

m

/’”

a, Q E 101 m

‘ l . m m , , , , , ,

C 2 4 6 8

Acoustic Treatment Time (min)

25

Figure 5. Average temperature in a 1.3 x lo6 celldml suspension at a power level of 180 W5.

Temperature differences in the chamber also induced convection currents, which disrupted cell aggregation. Free convection currents moved some columns of cells to join with other columns. At cell concentrations above lo6 cells/mL, and for acoustic treatment times greater than 1 min, some aggregates became large enough to sediment before the acoustic field was turned off.

The impact of acoustic treatment time on cell sedi- mentation was investigated at a power level of 180 W/L. The separation efficiency, E, was the percentage of cells that sedimented within 5 min after the end of acoustic treatment:

where ci is the initial concentration prior to acoustic treatment and cs is the supernatant concentration after sedimentation at the midpoint of the static chamber. Separation efficiency increased sharply during the first

a )loo Separation Efficiencv J

v L

3 120 -3 \., Supernatant Concentration - Q) Q

tn 1

0 ‘0--0 0.01. # . I . I . 1 ’ I . I . I . , . I 4 0 1 2 3 4 5 6 7 8 9 10

Acoustic Treatment Time (min)

Figure 6. Supernatant cell concentration and separation efficiency after acoustic treatment at an average power level of 180 W 5 and 5 min of sedimentation. The initial cell concentra- tion was 1.3 x lo6 celldml a t a viability of 92%.

3 min (Figure 6). A separation efficiency of 92% was achieved within an acoustic treatment time of 3 min. An additional 2 min of acoustic treatment increased the efficiency to 97%.

The viability of the cells remaining in the supernatant was up to 20% lower than the initial viability. However, the viability of the total resuspended cell population after acoustic treatment was the same as the initial viability. Thus, exposure of cells to resonance fields over the range of treatment times tested had no discernible effect on cell viability. Instead, acoustic separation tended to separate viable cells preferentially. This selective separation was reproducible and likely due to the greater acoustic forces on the larger and less compressible viable cells.

Effect of Treatment Power on Separation Ef- ficiency and Viability. Cells were acoustically treated for 3 min at power levels of up to 260 W/L and then allowed to sediment for 5 min before samples were taken. Figure 7 shows that the separation efficiency was be- tween 88% and 96% at power levels between 88 and 220 W/L. At 260 W/L, increased acoustic streaming and convective flows due to heating of the transducer were observed. Acoustic streaming is liquid flow induced by the sound pressure of running waves (Rooney, 1988; Schram, 1991). A vertical biocompatible polypropylene sheet was placed normal to the antinode planes at the midpoint of the chamber to reduce acoustic streaming at 260 W/L. The polypropylene sheet was chosen as it had acoustic properties similar to those of the medium. However, the electronics did not maintain resonance and poor separation resulted. Because resonance was not maintained, the proportion of running waves was in- creased and the resulting shear forces might have disrupted cells at the 260 W/L power level. The operating range of power levels may be extended with more precisely built acoustic chambers, which reduce the running waves in the acoustic field. The power levels used in this experiment were lower than required for cavitation. Gas bubbles were not observed in the acoustic chamber.

Effect of Cell Concentration on Separation Ef- ficiency and Viability. Cells were acoustically treated at an average power level of 180 W/L for 3 min over a range of cell concentrations (Figure 8). The supernatant concentration after acoustic treatment and 5 min of sedimentation was always less than 1.5 x lo5 cells/mL. As in previous experiments, acoustic treatment did not affect viability. Increasing separation efficiency was obtained at higher cell concentrations. The separation efficiency was greater than 97% at lo6 cells/mL and

150

-a ' ,-us' -

Biotechnol. Prog., 1995, Vol. 11, No. 2

- 6 0 $' 50 m

- 3

\ 1'"

- -

0 0 40 80 120 160 200 240 280

Power (WIL) Figure 7. Viability and separation efficiency after 3 min of acoustic treatment over a range of power levels and 5 min of sedimentation. The initial cell concentration was 1.3 x lo6 cells/ mL and the viability was 92%.

. s 10 - m

5 30- 1 0 4 4 0

1 o5 1 o6 10' Initial Cell Concentration (Cel lshL)

Figure 8. Supernatant cell concentration and separation efficiency at various cell concentrations after acoustic treatment at an average power level of 180 W/L for 3 and 5 min of sedimentation. The viability of the cells was 92%.

above. Below lo6 cells/mL, the low concentration of cells presumably limits the size of the aggregates and thus the overall sedimentation performance of the system. Since batch cultures of mammalian cells routinely attain concentrations exceeding lo6 cells/mL without perfusion, acoustic separation will be required in the concentration range where the separation efficiency is high (Trampler et al., 1994). For cell concentrations greater than lo6 celldmL, sedimentation of aggregates began before acous- tic treatment was terminated. Some remixing of the cells occurred as these aggregates settled. The extent of remixing increased with the cell concentration.

Cultivation and Productivity of Acoustically Treated Cells. To investigate the long-term effect of ultrasonic treatment, cells were subjected to a single 3 min ultrasonic exposure at power levels from 0-220 W/L and then cultured in triplicate T-flasks. Figures 9-11 show that the viable cell concentrations, viability, and glucose utilization were not measurably influenced by the acoustic treatment. Table 2 compares the maximum antibody productions at the end of 6 days. Acoustic treatment had no adverse effect on antibody production within the range of power levels tested.

Other experiments have shown that, for acoustic treatment times up to 7 min with cell concentrations from 2.5 x lo5 to 2.5 x lo7 cells/mL, acoustic exposure had no discernible effect on cell viability, growth, or productivity.

Semicontinuous Flow-Through Separation. The batch sedimentation results suggested that it might be feasible to separate cells in a flow-through acoustic chamber. To test this concept, a simple experiment was performed using the resonator set-up shown in Figure

c 0 z

c

C 0 0 0, 0 Q) E m 5

e 8 c

- -

7

6

5

4

3

2 -0- 44 WIL -*- 130 WIL 1 -v- 220 WIL I '1

O ! . , ' , ~ , ~ , ~ , ' I ' l

0 20 40 60 80 100 120 140

Time (h) Figure 9. Viable cell concentration over the course of batch hybridoma 2 E l l cultures. Before inoculation the cells were acoustically treated at 8.6 x lo5 celldml for 3 min a t power levels from 0 to 220 W/L. The error bars represent standard deviations from triplicate T-flask cultures.

8ol 70

4- 44 WIL -*- 130 W/L

::I , , , , , , , , , , , , , , 0

0 20 40 60 80 100 120 140

Time (h) Figure 10. Cell viability over the course of acoustically treated hybridoma 2 E l l cultures. The error bars represent standard deviations from triplicate T-flask cultures.

~

4 5

4.0

P) 3.5

.- 3.0

2 2.5

4- 44 WIL -*- 130 WIL

i v

- c g 2.0

8 1 5 d)

I3 1.0

o . o I , I , , , , , , , I , , , I

0 20 40 60 80 100 120 140

Time (h) Figure 11. Glucose concentration over the course of the acoustically treated hybridoma 2 E l l cultures. The error bars represent standard deviations between triplicate T-flask cul- tures.

3. Semicontinuous ultrasonic separation was tested with an initial concentration of 9.1 x I O 5 cells/mL at a continuous 0.7 L/h in flow. The average residence time of the fluid in the resonance chamber was 7 min. The sedimented cells accumulated within the resonator.

The separation efficiency shown in Figure 12 was calculated using eq 1, on the basis of the concentration of cells flowing into the chamber. This high level of separation was achieved despite disruption of the acous-

Biotechnol. Prog., 1995, Vol. 11, No. 2

Table 2. Final Antibody Concentration in Batch Cultures of 2 E l l Hybridoma Cells That Had Been Acoustically Treated at a Range of Power Levelsa

power level (W/L) IgG concentration @g/mL)

-

151

. $ U

- 6 0 $ m 3

40 Q. a

a

0 17.3 & 2.2 44 17.7 * 2.4

130 17.7 f 2.8 220 17.9 f 3.3

a The errors represent 90% confidence intervals.

100 Separation Eficiency

*\@/@-*.*-e \*-*, 6 0.8 3 80 E .- s - +.

Supernatant Concentration o l o - ~ ~ o

0 0 4 . l o 0 20 40 60 80

Time (min)

Figure 12. Total cell concentration in supernatant and separa- tion efficiency versus time during semicontinuous flow-through (0.7 L/h) acoustic treatment.

tic field by the nonacoustically transparent inlet tube inside the chamber and by turbulence induced by the entering medium, which flowed into the bottom portion of the chamber. Both of these factors probably lowered the separation eficiency. At the end of the experiment, the total concentration in the chamber was 9.4 x lo6 cells/mL or 10-fold higher than the initial concentration.

A slight decrease in viability from 91% initially to 87% at the end of the experiment was probably due to the depletion of oxygen and nutrients and the accumulation of metabolites in the sedimented cell layer at the bottom of the chamber. The viability of the outflowing cell suspension was 80% throughout the test or 7% lower than the viability of the final resuspended sample. This again illustrated selective aggregation and retention of viable cells.

Conclusions Acoustic standing waves can be used to aggregate and

sediment mammalian cells without affecting growth rate, viability, or antibody production. Batch separation ef- ficiencies of hybridoma cells were as high as 98%. Acoustic separation increased with increasing cell con- centration. Acoustic separation also selectively aggre- gated and sedimented viable cells. Thus, a bleed stream to remove nonviable cells may not be necessary in perfusion bioreactors with acoustic separation systems.

A cell recycle system using an external cell settler a t a flow rate of 0.1 L/h has been used to maintain a 1.5 L bioreactor a t lo7 cells/mL (Batt et al., 1990). Semicon- tinuous acoustic separation achieved over 90% separation efficiency at a flow rate of 0.7 L/h. This flow rate could sustain a 10 L perfusion bioreactor. High cell separation efficiencies thus were achieved at flow rates significantly higher than those used with gravity sedimentation systems and some spin and cross-flow filters (Reuveny et al., 1986; Fenge et al., 1987; Velez et al., 1989; Batt et al., 1990; Tokashiki and Arai, 1991; Hulscher et al., 1992). Higher flow rates and separation efficiencies are possible using acoustic sedimentation devices specifically designed for flow-through applications (Trampler et al.,

1994). Autoclavable flow-through acoustic chambers are being developed and tested in long-term aseptic cultures in our laboratory.

Acknowledgment This work was funded by SonoSep Biotech Inc., the

National Research Council (Industrial Research As- sistance Program), and the Natural Sciences and Engi- neering Research Council of Canada. We express our gratitude to J. Poppleton at SonoSep Biotech and S. Woodside at UBC for their contributions to this work.

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1989,33, 938-940.

Accepted October 31, 1994.@

BP9400881

* Abstract published in Advance ACS Abstracts, January 1, 1995.