Cytotechnology 15: 321-328, 1994. 321 @ 1994 Kluwer Academic Publishers. Printed in the Netherlands.

Quantitative investigations of cell-bubble interactions using a foam fractionation technique

W. S. Tan, G. C. Da i & Y. L. C h e n Laboratory of Cell Culture Technology, Research Institute of Biochemical Engineering, East China University of Science & Technology, 130 Meilong Road, Shanghai 200237, P.R. China

Key words: Adsorption, cell-bubble interactions, foam fractionation, hybridoma cells, Pluronic F68, serum

Abstract

Previous work by the authors and others has shown that suspended animal cell damage in bioreactors is caused by cell-bubble interactions, regardless whether the bubbles are from bubble entrainment or direct gas sparging. As approach to measure the adsorptivity of animal cells to bubbles, a modified batch foam fractionation technique has been developed in this work and proven to be applicable. By using this technique, the number of cells adsorbed per unit bubble surface area and the adsorption coefficients have been measured to quantify hybridoma cell-bubble interactions, and the preventive effects of serum and Pluronic F68 on these interactions. It was demonstrated quantitatively that the hybridoma cells adhere to bubbles spontaneously and significant numbers exist in the foam, and that both the serum and Pluronic F68 provide strong prevention to these cell-bubble interactions. The results obtained provide criteria for bioreactor operation and medium formulation to prevent cell-bubble interactions and cell damage in the culture processes.

Abbreviations: NBCS - new born calf serum; SFM - serum-free medium.

Introduction

With the development of cell fusion and recombinant DNA technologies, large quantities of valuable biolog- icals can be produced by animal cell culture technolo- gy. A prominent example of such an animal cell is the hybridoma cell, used for in vitro, large scale production of monoclonal antibodies. In large scale suspension cultures of animal cells in bioreactors, the agitation and/or sparging are the most practical ways of sup- plying sufficient oxygen and mixing for cell growth. However, significant cell damage or injury has been observed to result from excessive agitation and gas sparging. There have been many studies on animal cell damage or injury in bioreactors (Handa-Corrigan et al., 1987, 1989; Tramper et al., 1986, 1988; Jobses etal., 1991; Murhammer and Goochee, 1988, 1990b; Bavarian et al., 1991; Chalmers and Bavarian, 1991; Kunas and Papoutsakis, 1990b; Gardner et al., 1990; Michaels etal., 1992; Oh etal., 1989, 1992; Cherry and

Hulle, 1992; Tan et al., 1993). Because the agitation intensity of most bioreactors for animal cell suspended cultures rarely exceeds 300-500 rpm, independent of impeller size and design. The Kolmogorov eddy sizes produced in such bioreactors are usually much larger than the cell size. Therefore, it can be concluded from the previous investigations that the cell damage either in sparged (bubble column, airlift) or stirred bioreac- tors is most likely the result of cell-bubble interactions.

There are three possible regions of cell-bubble interactions: the bubble formation and injection region at the sparger, the bulk medium in which the bub- ble rises, and the bubble disengagement region at the air-medium interface (Tramper et al., 1986, 1!988). Recently, the work carried out by Handa-Corrigan et al. (1989), Kunas and Papoutsakis (1990b) and Job- ses et al. (1991) demonstrated that cell damage takes place at the region of bubble disengagement at the medium-air interface. At the same time, three pos- sible mechanisms for this cell death were proposed:

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(1) oscillatory disturbances caused by rapidly burst- ing bubbles, (2) physical shearing of the cells result- ed from the liquid film draining around the bubbles, and (3) physical loss of cells in the foam. By using a microscopic, high-speed video-system, the adsorp- tion of cells to the bubbles and the transport of these cells into the foam layer have been observed (Bavarian et aI., 1991). Based on experimental results, Tan et al. (1993) inferred that only the cells adsorbed on the bub- bles can probably be damaged in a similar manner to those mentioned above. To demonstrate that the bub- ble rupturing process kills cells, Garcia-Briones and Chalmers (1992) collected a sample from the upward jet, which resulted from a bubble rupturing at an air- medium interface. This sample contained a large num- ber of cells and approximately 90% of the cells were dead. Using a more advanced microscopic system than Bavarian et al. (1991), Garcia-Briones and Chalmers (1992) were able to visualize a large area of the bub- ble film of a single bubble at the gas-liquid interface and reported that a large number of cells (>300) could be observed on the film. Recently, Trinh et al. (1994) found that approximately 1050 cells were killed per single, 3.5-ram bubble rupture and approximately the same number of dead cells were present in the upward jet. From this point of view, the adsorption of cells to bubble surfaces is the step controlling the cell damage and losses in bioreactors and plays an important role in the successful cultivation of animal cells in stirred and sparged bioreactors. To our knowledge, no general and reliable technique is yet available in the literature with respect to quantification of the adsorption of cells on bubbles and cell enrichment in the foam.

The protective effects of additives including serum, Pluronic F68, polyethylene glycol (PEG), bovine serum albumin and several protein mixtures on ani- mal cells have been examined by various investigators (Handa-Corrigan et al., 1989. Kunas and Papoutsakis, 1989, 1990a; Michaels et al., 1991a, 1991b; Murham- mer and Goochee, 1988, 1990a,. 1990b; Ozturk and Palsson, 1991; Ramirez and Mutharasan, 1990, 1992; Smith and Greenfield, 1992). It was proposed that these additives protect cells from damage by biologi- cal effects on cells to make them more shear resistant and/or by physical ones to change the shear forces pro- duced. Recently, Garcia-Briones and Chalmers (1992), Zhang et al. (1992) and Tan et al. (1993) suggested that Pluronic F68, due to its surface active proper- ties, changes the physicochemical characteristics of the foam formed and prevents cells from adhesion to bubbles.

Foam Out

f Foam

Section

O 0 U I Sparging o o Section ~176

0 0

Gas In ' F

=

L_

\ v c

Vf C f

' / F o a m Collector

Foamate

Out

Fig. I. Diagram of the modified batch foam fractionation.

In this work, a modified batch foam fractionation technique has been developed. By using this technique, the number of cells adsorbed on bubble surfaces and the adsorption coefficients have been measured to quantify the hybridoma cell-bubble interactions and the preven- tion provided by serum and Pluronic F68. Based on the results obtained, the mechanism of cell death and the protection by additives will be discussed.

Description o f foam fractionation technique

In order to quantify the hybridoma cell adsorption and enrichment in the foam layer, a modified batch foam fractionation technique has been developed, diagramed in Fig. 1. Gas is sparged into the lower end of the apparatus at a constant flow rate F (ml min-1). Ris- ing through the cell suspension, the bubbles in which the cells are trapped enter the foam. The foam rises through the foam section and is collected as it exits the top. With an attempt to find correlations between the measurable data and the number of cells adsorbed on bubble surfaces, a model is derived on the basis of the following assumptions.

(1) The foam does not rupture and drain back into the bulk suspension before exiting.

(2) Once adsorbed on bubbles, the cells do not leave the bubble surfaces as the foam rises until the foam exists.

(3) The changes of the physical properties, such as surface tension, of the cell suspension during oper- ation are negligible.

(4) No cell growth and metabolism occur over the short duration of the foam fractionation.

(5) As the bubbles just depart from the cell suspen- sion and enter the foam phase, the adsorption of cells on these bubbles reaches equilibrium with the viable cell density in the bulk solution, following the equation

Pc = / ( C ) (1)

with Pc being the number of cells adsorbed per unit bubble surface area (cell cm-2), C the viable cell density in the bulk cell suspension (cell ml-1). The number of cells adsorbed per unit surface area

Fc is considered to be proportional to C. Thus, equation (1) becomes:

Pc = aC (2)

where a is the adsorption coefficient (cm). This pro- portionality follows from the analogy of the adsorption characteristics of surface-active agents to interface in dilute solutions. The assumption of constant physical properties of the cell suspension during foam fraction- ation is implicit in equation (2).

Obviously, the batch foam fractionafion is a time- varying process. As shown in Fig. 1, the volume and viable cell density for a given cell suspension are V (ml) and C (cell m1-1) at time t, respectively. After a interval of time dt, the cell suspension volume decreas- es to V - dV, producing foam liquid with volume of dVf which equals -dV. In this foam liquid the densi- ty of cells trapped by bubbles is Cf. Meanwhile, the viable cell density in the bulk cell suspension becomes C - de. Thus, the mass balance for the viable cells over dt is

V C = dVf Cf + (V - dV)(C - dO) (3)

where dVf = --dV. With higher derivatives being neglected, the above equation becomes

dV dC - - ( 4 )

W C f - - C

Equation (4) can also be written in an integrated form as follows

ln~.~o = fc c~ dC C f - C ( 5 )

323

where the initial condition is taken as V = Vo and C = Co at t = 0. To integrate the above equation, the correlation between Cf and C should be known.

Over the short duration of time dt, the number of cells trapped by bubbles from the bulk suspension into the foam phase equals:

dN = FcdA = a C d A (6)

where dA is the surface area of bubbles produced dur- ing dt, cm 2. As defined, Cf is

dN _ a C d A a a Cf = t]Vf dVf - d_~V C = ~ C (7)

dA

where 6 = _r ' cm, is taken as the equivalent thickness of the liquid film of the foam. It should be noted that

is not the actual thickness of the liquid film, due to liquid drainage and bubble coalescence with the foam rising in the foam section. It is defined as the ratio of foam liquid volume to bubble surface area produced over dt. Obviously, (5 will be dependent on both the physical nature of the liquid and the gas flow rate. For a given cell suspension and constant gas flow rate, both a and 5 are constants, meaning that Cf is proportional to C. Thus, equation (7) can be written as

ce = 9 c (8)

where/3 is the proportionality coefficient. Substituting equation (8) into (5) and integrating

yields

lnV_~ o = 1 l l n - ~ (9) V 8 -

or

ln-? /3 = ln---~- ' + 1 (10)

It can be seen that, by only measuring the volumes of cell suspension and viable cell densities at the begin- ning and end of the foam fractionation process,/5' can be estimated easily from equation (9) or (10).

From the beginning (t=0) to the end (t=t) of the foam fractionation, the volume of foam liquid pro- duced equals to

/v Vf = - d V = Vo - V (11) o

and the surface area of bubbles sparged can be calcu- lated approximately as

324

s t F d2dt _ 6 F t A = -~lb3 -Tr db (12)

where db is the average diameter of bubbles, cm. Combining equations (11) and (12), 6 can be esti-

mated from the following equation:

_ Vf _ (V0 - V)db (13) A 6 F t

Thus, the adsorption coefficient o~ is

c~ = t 6 (14)

By measuring V, Vo, C and Co at certain experimental conditions (F, db and t) and combining equations (10), (13) and (14), c~ and I'e = c~ Co, which represent the cell adsorptivity on bubbles and enrichment in foam for a given cell suspension, can be determined.

Materials and methods

Cell culture

The cell line used in this study was 2F7 mouse-mouse hybridoma cells (Shanghai Institute of Cancer SIC, Shanghai, P.R. China) producing an IgG2a antibody against the human small cell lung cancer. The hybrido- ma cells were grown in a 1.5-liter CelliGen bioreactor system (New Brunswick Scientific Co., Edison, NJ, USA) with a working volume of 1.3 liter in semicontin- uous mode at a dilution rate of 0.55 day -1. The serum- free medium (SFM), used for the bioreactor culture and developed by SIC, was a mixture of DMEM/F12 (1:1) (D8900, Sigma Chemical Co., St. Louis, MO, USA) and some additives, and supplemented with 50 units m1-1 of each penicillin and streptomycin. The bioreactor was kept at an agitation speed of 60 rpm, temperature 36.8 ~ pH 7.25 and dissolved oxygen (DO) 60% air saturation, and employed surface aera- tion. The pH was automatically controlled at the set point by adding 5.6% sodium bicarbonate or using the CO2 content of the gas phase.

Foam fractionation experiments

The batch foam fractionation experiments were car- ried out in a 100-mL graduated measuring cylinder and performed on 100ml samples from tim bioreactor culture. Gas was fed into the bottom of the cylinder at a constant flow rate. The sparger had ten nozzles with

diameter of 0.25mm, generating bubbles ranged in size from 0.22 to 0.26cm in diameter. Foam rose through the cylinder and was collected as it exited the top. The height of the foam section inside the cylinder was 7cm at the beginning of experiments. To ensure a smooth flow of the foam out of the cylinder, the cylinder was held tipped up at a 45 ~ angle.

To examine the effects of serum and Pluronic F68 on cell-bubble interactions, new born calf serum (NBCS) (East China University of Science & Technol- ogy, Shanghai 200237, P.R. China) and Pluronic F68 (BASER Ludwigshafen, Germany) were directly added to the samples at the concentrations required just prior to fractionation.

Measurements

At the beginning and end of the experiments, sam- ples (about 1 ml) were drawn to count the viable cell densities in the cell suspension. Duplicate counts were performed on each sample by the trypan blue exclusion method in a hemacytometer.

Some considerations for experiments

The gas flow rate selected should ensure that the foam with the captured cells flows out the cylinder smoothly, not rupturing and draining back into the bulk solution. The appropriate range of the gas flow rates should be determined experimentally.

Actually, the enrichment of surface active materi- als, such as proteins, from the cell suspension to the foam is inevitable, because of hydrophobic interac- tions. Thus, as a result of this fractionation, the phys- ical properties of the bulk suspension, such as foam- forming ability, foam stability and surface tension, will change, causing the adsorption coefficient of cells on bubble surfaces to deviate from constant during foam fractionation and the true value. In order to make a applicable and reliable, the volume of the foam liquid produced should be small during the experiments. In this work, Vo - V was controlled in the range of 10 - - 20 ml.

Results and discussion

Determination of the appropriate gas flow rates

Figure 2 shows the effects of gas flow rate on the experimental results. It can be seen that the liquid film

25

20

%_

: lef luxing Flow !

Region ::

Operat ional

Region

Flooding Flow

Region

10

0

20

15

0 200 400 600 800 1000 1200 1400

10

6(3

o~ 5

F (ml mim-')

Fig. 2. Selection of the appropriate gas flow rates in foam fraction- ation experiments. Viable cell density: 13.3 • 10 5 cell m1-1.

o E 0

0 o

o

'~ f . 40

30

20

10

0 I I 0

0 5 10 15 20 Viable Cell Density (10 5cell ml")

2000

1500

100 0

500 [ - -

325

[]

t

E

v

Fig. 3. Adsorptivity of the hybridoma cells cultured in serum-free medium (SFM), Bubble size: 0.22--0.26 cm.

O

thickness of the foam 6 increases, but/3 decreases, with the gas flow rate increasing. This is attributed to a reduction of liquid drainage as the foam rises faster. This means that more liquid will be carried by the foam at higher gas flow rates. It is interesting that, although both 6 and/3 are dependent on gas flow rates, the adsorption coefficient a is almost a constant in the range of gas flow rates from 300 to 1000 ml rain -1, independent of E This constant is close to the true adsorption coefficient for a given sample. For the example shown in Figure 2, the viable cell density in this cell suspension was 13.3 x 105 cell m1-1, and the adsorption coefficient a and the number of cells adsorbed per unit bubble surface I'c were estimated to be about 11.4 x 10 -4 cm and 1516 cell cm -2, respectively.

In the region of Figure 2 corresponding to low gas flow rates, the foams produced are not able to exit completely. Some bubbles rupture and drain back into the bulk solution. Since the ceils trapped in these bub- bles remain in the foam section, the measured adsorp- tion coefficients are biased high. This region is called the Refluxing Flow Region. On the other hand, in the range of high gas flow rates, the foams carrying large amounts of liquid out of the cylinder result in a being biased low. This region is called the Flooding Flow Region. Obviously, to obtain true and reliable results,

the gas flow rates should be selected from the region between the above two. In this region, called the Oper- ational Region, the adsorption coefficients are inde- pendent of gas flow rates. For the 100-mL graduated measuring cylinder with inner diameter of 2.8-cm used in this work, the range of appropriate gas flow rates is 300-1000 ml min -1 .

Also by foam fractionation experiments, Cherry and Hulle (1992) used o~ as a characteristic paramter of the adsorption of insect cells (Sf9) to the foam layer. Their a was the same parameter as/3 used in this study. Having found an average a of 0.6, they concluded that insect cells were not enriched in the foam layer. Actually, the results in Figure 2 show that/3 is strongly affected by the gas flow rates, implying that/3 alone can not be used as a measure of cell adsorptivity and enrichment in the foam layer.

Hybridoma cell adsorptivity on bubble surfaces

The adsorption coefficients and the numbers of hybridoma cells adsorbed per unit bubble surface, cor- responding to different densities of the cells cultured in serum-free medium (SFM), are shown in Figure 3. It was found that the adsorption coefficients decrease as viable cell density increases. In contrast, the number of the cells adsorbed does not change at high cell densi-

326

ties. The number of cells adsorbed per cm 2 area of bub- ble surface is about 1500 for the viable cell densities higher than 10 x 105 cell m1-1. At these concentra- tions, saturation of cell adsorption has probably been reached. This state, in which the adsorption and des- orption of cells is in dynamic equilibrium, results from the combination of thermodynamic and hydrodynamic effects. Considering the hydrodynamics surrounding the bubbles, the bubble size is an important factor to consider, implying that the cell adsorption on bubbles will inevitably be affected by the bubble size. This phe- nomenon is currently being studied in our laboratory. So, it should be noted that the results mentioned above are valid only for the range of bubble sizes used in our experiments. This is the main difference between the adsorption of cells and that of surface-active materials at gas-liquid interfaces.

Cell-bubble attachment, cell transport into the foam layer and cells in the films of bubbles have been video- taped (Bavarian et al., 1991; Chalmers and Bavari- an, 1991; Garcia-Briones and Chalmers, 1992), and now the results obtained from the foam fractionation experiments show quantitatively that significant nume- brs of hybridoma cells do exist in the foam. Although cell damage does not take place in the process of cell adsorption, only those cells adsorbed on bubbles and existing in the foam layer can be destroyed by the shear stresses produced by bubble rupture, as Handa- Corrigan et aI. (1989), Chalmers and Bavarian (1991), and Cherry and Hulle (1992) proposed. In other words, for cell death in sparged bioreactors, the event that occurs first is the cell adsorption to bubbles, mean- ing that the cell death rate in the bioreactors will be dependent on the cell adsorptivity and the bubble sur- face area produced. From this point of view, if cell adhesion to bubbles is prevented, and/or the number of cells captured by bubbles is decreased, by either reducing the cell adsorptivity or decreasing the bubble surface area produced, cell damage in bioreactors will be suppressed in efficiency or eliminated altogether.

Effects o f serum and Pluronic F68 on cell-bubble adhe-

sion

After the cell suspensions were taken from the bioreac- tor culture, the NBCS and Pluronic F68 were added to samples just prior to sparging. The results are shown in Figure 4. It is found that both serum and Pluronic F68 provide strong preventive effects to the cell adsorption on bubbles, and their effects are strongly dependent on their concentrations in the bulk cell suspensions.

As the concentrations of serum and Pluronic F68 were increased, the adsorption coefficients decreased. Inter- estingly, great reductions of the adsorption coefficients are found at their low concentrations. As shown in Figure 4, the effect of Pluronic F68 on cell adsorp- tivity is much more significant than that of serum. At Pluronic F68 concentrations of 0.1%, the adsorption coefficient drops to zero. This indicates that no cells are adsorbed on bubble surfaces and enriched in the foam layer. These results demonstrated quantitatively that the protective effect of Pluronic F68 and serum on cells against damage is associated with the preven- tion of cell adsorption to bubbles. This suggests that the effects of protective additives on cell adsorptivity can be used as criteria for media formulations that will prevent cell damage in bioreactors.

Since both the serum and Pluronic F68 are surface- active agents, supplementations of such materials will inevitably change the physicochemical properties of the interfaces (cell-liquid, gas-liquid, and cell-gas), due to the hydrophobic interactions. Garcia-Briones and Chalmers (1992), Zhang et al. (1992) and Tan et

al. (1993) suggested that the protection of Pluronic F68 was attributed to these changes, such as decreasing the surface tension of the medium. The effects of those additives on the behavior of interfacial phenomena in animal cell culture bioreactors should be investigated further.

Conclusions

The modified batch foam fractionation technique has been developed in this work and proven to be suit- able for measuring the adsorptivity of hybridoma cells on bubbles. By measuring, it has been demonstrated quantitatively that the hybridoma cells adhere to bub- bles spontaneously and significant numbers of ceils exist in the foam. These results suggest that, by either reducing the cell adsorptivity or decreasing the bubble surface area produced, cell damage in bioreactors will be suppressed and prevented. As protective additives, the effect of serum and Pluronic F68 on cells has been found to be the result of preventing cells from adhesion to bubbles. In future, the effects of hydrodynamics sur- rounding the bubbles on cell-bubble adhesion, and the effects of protective additives on interfacial phenome- na in animal ceil bioreactors should be studied.

12

( a ) E o

,'7 l q o

~ a

o ~

o 0

t -

O 4

0 m 2

<

0 1 2 3 4 5 Serum Conc. (%)

0 0 .15

A

E o

0

o ~

.9

o 0

k . .

o "o <

12

(b)

8

6

4

2

0 . 0 5 0 . 1

Pluronie F68 Cone. (%)

327

Fig. 4. Effects of serum (a) and Pluronic F68 (b) on cell-bubble adhesion. The hybridoma cells were cultured in SFM. Viable cell density: 15.3 x i05 cell ml -~.

Notes

1. A = surface area of the bubbles, cm 2

2. C = viable cell density in bulk cell suspension, cell m l - t

3. Cf = density of cells trapped by bubbles in foam liquid, cell m - ]

4. Co = initial viable cell density in bulk cell suspen- sion, cell ml - ]

5. db = average diameter of the bubbles, cm

6. F = gas flow rate, ml min-1

7. N = number of cells captured by bubbles from bulk cell suspension to foam phase

8. t = time, rain

9. V = volume of the bulk cell suspension, ml

10. Vf = volume of the foam liquid, ml

11. V0 = initial volume of the bulk cell suspension, ml

12. c~ = adsorption coefficient, cm 13. t3 = c-z c 14.6 = ~ equivalent thickness of the liquid film of dA '

the foam, cm

15. Pc = number of cells adsorbed per unit bubble surface area, cell cm -2

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Address for correspondence: W.S. Tan, Laboratory of Cell Culture Technology, Research Institute of Biochemical Engineering, East China University of Science & Technology, 130 Meiiong Road, Shanghai 200237, P.R. China.