Oxygen Uptake by Entrapped Hybridoma Cells

Dave Wohlpart? John Gainer, and Donald Kirwant Center for Bioprocess Development, Department of Chemical Engineering, University of Virginia, Charlot tesville, Virginia 22903-2442

Received July 6, 1990Accepted December 9, 1990

Oxygen consumption by hybridoma cells immobilized in 1- and 3.9-mm-diameter calcium alginate beads was mea- sured. The entrapped cells consumed oxygen at about 10 pmol/min per lo9 cells, regardless of the bead size and cell loading. In contrast, the same cells in suspension cul- ture respire at specific rates of 3-8 pmol/min per lo9 cells (depending on the cell density). The growth rate of the im- mobilized cells was significantly reduced, while specific an- tibody production was comparable to that of free cells. Key words: hybridomas - immobilization oxygen respiration

INTRODUCTION

The physical confinement of animal cells in beads within a bioreactor offers a promising method for growth and production in a large-scale system. Immobi- lization may increase reactor cell densities, alter the physiology of the cells, and support the operation of the bioreactor in a continuous mode. Entrapment of mam- malian cells in calcium alginate beads is a popular and well-described method of immobilization capable of supporting growth and antibody production but has the potential drawback of introducing diffusional resis- tances which could offer limitations to useful bead sizes. Oxygen, in particular, might not be available in the center of a larger bead, leading to a necrotic core.

Oxygen starvation of immobilized cells has been re- ported, even for animal cells with low respiration rate^.""^ There have also been reports of growth or maintenance of hybridoma cells and antibody produc- tion in suspension culture at very low oxygen concen- t r a t i o n ~ . ~ ' ~ " ~ Except for the study of Shirai et al.,14 however, there is a paucity of data concerning the oxy- gen demand of immobilized cells. Thus, we investigated the effect that immobilization of hybridoma cells in 1- and 3.9-mm-diameter alginate beads has upon the specific oxygen uptake rate as well as growth and anti- body production.

MATERIALS AND METHODS

Hybridoma Cells

Hybridoma cells designated 63D3 were obtained from the American Type Culture Collection (catalog number

* Present address: Merck & Co., Inc., West Point, PA 19486. To whom all correspondence should be addressed.

Biotechnology and Bioengineering, Vol. 37, Pp. 1050-1053 (1991) 0 1991 John Wiley & Sons, Inc.

ATCC HB44). This hybridoma line produces a mono- clonal antibody to a 200,000-MW protein molecule on human monocytes. Cells were routinely cultured in T flasks (5% C 0 2 in air) with RPMI 1640 (HybriMax, from Sigma) containing 60 p M MEM nonessential amino acids, 1 mM sodium pyruvate, 50 p M 2-mercap- toethanol (Sigma), and 10% horse serum (Hyclone). The cells were routinely tested for mycoplasma contami- nation.

Entrapment

Hybridoma cells were centrifuged and resuspended to a density of about lo6 cells/mL in 2% sodium alginate (Kelco Gel LV) with 0.85% NaCl and 1 g/L glucose. This suspension was then pumped through a 21-gauge syringe needle at a flow rate of 1.7 mL/min. Alginate beads were sheared off the needle tip by a coaxial air stream and fell into a chilled 0.1M CaC12 solution. Beads with a diameter of 1 mm were made by adjusting the air flow rate, while 3.9-mm beads were produced by pumping the alginate-cell mixture through a glass tube (2.5 mm ID) with no external air stream. The beads were cured for 30 min before washing and subsequent resuspension in culture medium. Measurement of the viable cell population was accomplished by first dissolv- ing the calcium alginate matrix with sodium citrate 22 g/L, trisodium salt dihydrate (Sigma), pH 7.4. Vi- able cell counts were made in a hemacytometer after staining with trypan blue as described previo~sly.'~ Control experiments showed that cell viability and anti- body stability were not compromised by exposure to the citrate solution.

Culture

Hybridoma cells within alginate beads were maintained in a 250-mL spinner flask (Wheaton) with 50 mL DMEM culture medium (containing the same concen- trations of supplements as used in the RPMI medium) and stirred at 70 rpm (l-mm beads) or 50 rpm (3.9-mm beads). The higher agitation speed was found to break up the larger beads. Alginate bead destruction with RPMI (possibly through phosphate-induced leaching of calcium) was not observed with DMEM. The culture fluid was replaced once (l-mm beads) or twice (3.9-mm

CCC 0006-3592/91/01101050-04$0400

beads) daily to maintain reasonably constant environ- mental conditions in the spinner flask culture. After removal of old medium, the beads were washed twice with 25 mL DMEM and then resuspended with 50 mL medium. As described below, growth rate and specific oxygen uptake and antibody production were deter- mined in short-term experiments in which a few beads were removed from the spinner flask, washed with media, and incubated for 1 h in fresh media prior to measurement.

’ 10 Viable Cells 1’

Measurement of Oxygen Uptake

Oxygen uptake was determined by following the deple- tion of dissolved oxygen using a YSI microelectrode ap- paratus as described previously for suspension cell measurements. l9 Enough cells were removed from the spinner flask culture to provide a cell concentration of about 2 x 16 cells/mL of chamber fluid for each ex- periment. In this way oxygen depletion could be fol- lowed for 10-30 min. Typically, about 100-300 1-mm beads and 5-20 3.9-mm beads were used. At the con- clusion of each experiment total and viable cell num- bers per bead were determined by dissolution of the beads in the chamber. The oxygen concentration in air- saturated medium was found to be 0.2 mM by use of the chemical method of Slinger et a1.I6

i

RESULTS

Growth of Cells

The growth, oxygen uptake, and antibody synthesis characteristics of the 63D3 cell line in suspension (non- immobilized) T-flask batch cultures were previously measured.” The cells exhibited a maximum specific growth rate of about 0.06 h-’ and maximum antibody productivity of 8 pg/min per lo9 cells. The specific oxy- gen uptake rate in batch culture ranged from 5 pmol/ min per lo9 cells at a lower cell density to about 3 pmol/min per lo9 cells at the maximum cell density of 1.2 x lo6 cells/mL. Separate specific respiration rate measurements of these suspension-cultivated hybri- doma cells under various environmental conditions using the YSI apparatus were also previously re- ported.” An inverse relationship of specific respiration rate and cell density was observed: 10 pmol/min per lo9 cells at lo5 cells/mL to 2 pmol/min per lo9 cells at lo7 cells/mL.

The growth characteristics of the immobilized cul- tures are presented in Figures l and 2. The viable cell loading per bead initially declined in all beads from ini- tial values of 1 x lo3 and 1.3 x lo5 cells per bead (2 x lo6 and 3.8 x lo6 cells/mL) for the 1- and 3.9 mm beads, respectively, and the nonviable population in- creased. Immediately after entrapment, the cells in the 1-mm beads appeared in excellent condition, but be- tween 25 and 100 h the cells appeared bloated and ir-

0 50 100 150 200 250 300 350 4CO 450

Time (hours)

Figure 1. ginate beads.

Hybridoma growth and oxygen uptake rate in 1-mm al-

regularly shaped. Thereafter, the cultures were domi- nated by cells that appeared healthy. However, the cell population in the 3.9-mm beads looked healthy throughout the life of the culture. Rapid growth of cells within the beads occurred during the 100-200-h period, with apparent doubling times of about 25 h in the 1-mm beads and about 50 h in the 3.9-mm beads. Both values are significantly greater than the free cell values. Since a significant amount of cells leaked from the beads, these values are upper limits to the doubling times of the entrapped cells.

The cell population reached a “steady state” in the 1- and 3.9-mm beads, with lo4 and 2.8 x lo5 cells per bead, respectively. (Note that the bead diameters or vol- umes did not appear to change during the course of the experiments.) A 1-mm-diameter bead with a cell popu- lation of lo4 cells has a cell density of 2 x lo7 cells/mL, but since the entire bead volume is not occupied (see below), the actual density within colonies probably ex- ceeds lo8 cells/mL. Although the 3.9-mm beads have a total volume about 60 times greater than the 1-mm beads, the total cell number was just 28 times greater (0.9 x lo7 cells/mL). This reduced cell density in the larger beads along with the increased doubling time suggests some transport limitations (see Discussion section).

The fluorescence staining technique described by WidholmI8 provided an excellent method for observing

WOHLPART ET AL.: OXYGEN UPTAKE BY ENTRAPPED HYBRIDOMA CELLS 1051

the location of the viable cell population within an algi- nate bead. A scalpel was used to slice a thin piece from the middle of the bead, which was then treated with fluorescein diacetate in phosphate-buffered saline. Ex- amination under UV light caused live cells to fluoresce. Initially, live cells were present throughout the bead in a uniform density. Subsequent growth produced several large colonies throughout the 1-mm beads, but they were generally located near the periphery of the 3.9- mm beads. Although there was a tendency of cells to grow in the peripheral volume of the larger beads, dense colonies of cells were also found in the center of beads. In addition, the cell population growing near the outer surface was not a continuous, dense colony. Con- sequently, it is possible that the supply of essential nutrients (glucose, oxygen, etc.) is not depleted so as to restrict the growth of cells in the inner portion of the bead.

Oxygen Uptake Rate

The specific uptake rates are also shown in Figures 1 and 2. The cells entrapped in the 1-mm beads had ini- tial respiration rates of about 3 pmol/min per lo9 cells, a value typical of a high-density suspension culture.” The observed respiration rate was elevated during the “lag” phase (to 15 pmollmin per lo9 cells) when a dis- tressed cell population predominated. The specific con- sumption rate then declined to a fairly constant rate of 10 pmol/min per lo9 cells during the active growth and steady-state phases. The specific respiration rate in the 3.9-mm beads remained at approximately the same value of 10 pmol/min per lo9 cells throughout the course of the culture. Recall that the cells in these beads always appeared healthy, in contrast to the 1-mm beads. The specific consumption rates of the immobi- lized cells are, at least in the steady-state region with a cell density of the order of 107/mL bead volume, about three times the rate seen previously for suspension cells at this cell density.

Antibody Production

The specific antibody production rate of the immobi- lized cultures was measured by incubating washed beads (from the spinner flask) in fresh medium in a sterile culture tube. The beads were dissolved with ci- trate after 4 h incubation to determine the cell density and then frozen for later ELISA assay19 to determine the antibody concentration. The initial antibody level present in the alginate beads was also measured; thus the net antibody production could be calculated. The exposure of antibody to the citrate concentration present during bead dissolution was shown not to result in destruction of MAb.

In early culture times the measured specific antibody production of the cells entrapped in 1- and 3.9-mm algi-

nate beads was elevated (-30 pg/min per lo9 cells) compared to free cells. During the steady-state period production was comparable (7-8 pg/min per lo9 cells). However, in the early phase the antibody concentra- tions and cell numbers were lower so that the results are less reliable. Thus, it appears that specific antibody production was little affected by gel entrapment.

DISCUSSION

Both internal and external mass transfer resistances could be important in the delivery of nutrients and, es- pecially, oxygen from the bulk fluid to the gel- entrapped cells. However, estimates of an external mass transfer coefficient by the method detailed by Harriott7 shows that the observed oxygen consumption rates are about two orders of magnitude less than the possible external mass transfer rate. Thus, external transport resistance is not influencing the observed oxygen consumption kinetics. This is further supported by the fact that the experimental oxygen depletion rate was always observed to be zero order in oxygen.

The uptake of oxygen by cells immobilized in cal- cium alginate is often claimed to be under the control of internal diffusional mass transfer, at least within beads with diameters greater than 1 mm.’ The growth of cells within the pore infrastructure observed with the prepa- rations here could increase the resistance to oxygen diffusion, with the diffusion of oxygen hindered in pro-

If internal mass transfer indeed governs the delivery of oxygen to cells in a microbead, then the specific oxygen consumption rate should decline as the cell loading in- creases with growth. Additionally, the bead size should affect the distribution of cells within the bead if severe limitations exist.

The possible role of oxygen diffusion limitations within the bead can be approximately assessed using the well-known Weisz criterion in terms of the ob- served rate: This criterion states that if

portion to the amount of cells in the

diffusional limitations are likely. The observed con- sumption rate, Q, is equal to the cell density times the specific rate; L, a characteristic dimension, is R/3 for a sphere; D is the diffusivity within the matrix; and c b is the oxygen concentration at the bead surface. For the steady-state region in the 1- and 3.9-mm-diameter beads with D = 2 x cm2/s, we find 4’ is 0.2 and 1.4, respectively, suggesting some diffusional limitation in the larger beads. Since the volume occupied by cells was less than lo%, no reduction of the diffusivity from the solution value was made.

A more detailed analysis of diffusional effects with the bead for zero-order kinetics must consider two

1052 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 37, MAY 1991

cases8:

I. If the oxygen concentration at the center of the particle is greater than zero, the concentration at the center is

provided the second term on the right is less than unity. For the 1-mm-diameter beads this condition is fulfilled and the oxygen concentra- tion at the center is about 66% of that in the fluid. For the larger beads this condition is not fulfilled.

11. If oxygen concentration goes to zero at some ra- dius, R, > 0, then the concentration profile can be obtained by equating the diffusive flux times the external area to the total consumption in the region, R, I r I R . The critical radius R , is then given by

1 - 6Dcb/QR2 = (R,/R)2(3 - 2R,/R) (3) For the 3.9-mm beads, R, = 0.55R so that only about 17% of the bead volume is hypoxic.

The above analyses assume uniform distribution of cells within the bead volume, which is not true in the later stages of the culture where large colonies have de- veloped in particular regions within the beads. A more exact analysis would require taking into account the heterogeneous nature of the cell distribution and more detailed experimental information on the actual cell distribution. In the absence of such information, these analyses suggest that the observed specific respiration rates for the cells in the smaller diameter beads are not influenced by diffusion while those of the cells in the larger beads may be somewhat reduced due to diffusion limitations.

The most striking observation is the high specific res- piration rates of the entrapped cells. If there were any diffusional limitations, then the true kinetics of the cells would be even higher than the observed 10 pmol/ min per lo9 cells. This rate is some factor of 3 larger than that observed for freely suspended cells at densi- ties greater than lo7 cells/mL. The most likely hypothesis is that entrapped cells have greater mainte- nance energy requirements because of their' environ- ment and therefore must respire at higher specific rates. Changes in the metabolic activity of yeast cells after immobilization have been observed previously by Doran and Bailey! Galazzo and Bailey' reported that the immobilization of Saccharomyces cerevisiae in algi- nate accelerated the rate of glucose uptake or of glu- cose phosphorylation. Reardon and Baileyl3 proposed that the enhanced metabolic activity of Clostridium acetobutylicum immobilized in calcium alginate was due to the greater availability of cell lysis products, such as nucleic acids and important proteins. With mam- malian cells, altered concentrations of important factors might be found in the alginate beads, which could in-

fluence growth and respiration in the cells. Addition- ally, these cells grew in colonies, which may induce metabolic changes in the cells. Indeed, Shirai et al.I4 cor- related an increased specific oxygen utilization rate to the increase in the number of cells contained in a colony.

The maximum specific growth rates of the immobi- lized cells are significantly lower than those of free cells under similar medium conditions with the growth rate lower in the larger beads. Although diffusional limitation of oxygen and possibly other nutrients or toxic metabolites may be partly responsible for the ob- served growth reductions, we suspect that the altered environment within the gel causes reduced growth and higher maintenance rates. It should be noted that a stationary viable cell population can be maintained in the beads for extended periods of time with antibody production.

The altered metabolism implied by the elevated res- piration rates did not translate into altered antibody synthesis as compared to free cells. This is probably not surprising in that antibody synthesis is not strongly tied to growth or oxygen availability. Indeed, antibody pro- duction occurred during the stationary period of these experiments. There have also been recent reports by our lab2 and others" on antibody production under hy- poxic conditions.

The support of Virginia's Center for Innovative Technology is appreciated.

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