Selective retension of active cells employing low centrifugal force at the medium change during suspension culture of Chinese hamster ovary cells producing tPA

JOURNALOF BIOSCIENCEAND BIOENGINEERING Vol. 89, No. 4, 340-344. 2000

Selective Retension of Active Cells Employing Low Centrifugal Force at the Medium Change during Suspension Culture of Chinese Hamster Ovary Cells Producing tPA MUTSUMI TAKAGI,* MOHAMMAD ILIAS, AND TOSHIOMI YOSHIDA International Center for Biotechnology, Osaka University, 2-1 Yamada-oka, Suita-shi, Osaka 565-i-0871,Japan Received 25 October 1999IAccepted 6 January 2000

The effect of centrifugal force applied for cell separation at the medium change on the growth, metabolism and tissue plasminogen activator @PA) productivity of Chinese hamster ovary (CHO) cells suspension culture was investigated. The viability of the precipitated cells increased exponentially as the centrifugal force decreased. However, the cell recovery was lower than 91% when centrifugal forces applied for 5 min was less than 67 x g. In cultures incubated for 474 h with 7 medium changes employing centrifugal forces ranging from 67 to 364 x g, a centrifugal force lower than 119 X g resulted in higher specific rates of growth, glucose consumption, and lactate and tPA production during the whole culture period. On the other hand, daily centrifugation at 67 to 537 X g without discarding the supematant had no effect on the specific rates. The cultures inoculated with cells precipitated at a centrifugal force of 67 Xg showed apparently higher specific rates of metabolism compared to those inoculated with cells in the supernatant. The cells in the supernatant and the precipitate obtained following centrifugation at 67 X g have average diameters of 15.5 and 17.4 ,qm, respectively. The intracellular contents of amino acids, especially nonessential amino acids, of the precipitated cells were markedly higher than those of the cells in the supematant. These results indicate that large cells with high amino acid content and metabolic activity were selectively retained in the culture by means of centrifugation at low forces such as 67 Xg. Consequently, application of a low centrifugal force is recommended for medium change in order to maintain higher specific productivity of suspended mammalian cells in perfusion culture. [Key words: centrifugation, CHO, cell size, amino acids, cell separation] Various kinds of perfusion systemsfor suspensioncultures of mammalian cells, in which the cells are retained or recycled to the submergedfermentor while spent medium is replacedwith fresh medium, have been developed to increasethe cell density and reactor productivity, and to hencereduce the production costs of various therapeutic proteins such as interleukins and interferons. These practical systemsare usually based on sedimentation (l5), filtration (6-11) and centrifugation (12-17), while some novel systems employing ultrasonic resonance fields (18) and dielectrophoretic forces (19) have recently been reported. Although the sedimentation-basedsystem is simple, it suffers from the drawbacks of low sedimentation velocity and low recovery of cells due to the absence of any significant difference in specific gravity between the cells and the medium, therefore allowing only lim ited scale-up capacity. Separation filters, such as hollow fiber membranes and spin filters, may become clogged during long-term culture, and dead cells and cellular debris may accumulate in the fermentor equipped with such a filtration system. A centrifugation-based system yields a higher separation efficiency and can be used at industrial scale (15). However, few reports on the influence of centrifugal force on cell separation, growth rate and activity have been published, even though the sedimentation and retention of not only active viable cells but also dead cells and cell debris in a fermentor equipped with a centrifugation system may be a major problem. Therefore, the effects of centrifugal force on the separation and specific rates of growth, energy metabolism and tPA production of precipitated CHO * Corresponding author. 340

cells were investigatedin this study. MATERIALS

AND METHODS

Cells and medium CHO l-15500 cells (ATCC CRL9609) that produce tissue plasminogen activator (tPA) were cultivated in Ham’s F-12K medium (Gibco, NY, USA) supplemented with newborn-calf serum (lo%), streptomycin (0.1 mg/ml), penicillin (100U/ml) and methotrexate (500nM) (20). Suspension culture Thirty m illiliters of the medium in a 100ml spinner bottle (Shibata Hairo Co., Tokyo) was inoculated with approximately 3 x 106cells harvested from a culture dish (55 cm2, Sumilon, Tokyo) by trypsinization, and incubated at 37°C with agitation at 70rpm in an atmosphere containing 5% COZ. The cells were allowed to adapt to the suspension culture and grown up to a concentration of 5 x lo5 cells/ml. Then, after centrifugation of the suspensioncultures at IOOOrpm, the precipitated cells were again inoculated into fresh medium and incubated in the same manner as mentioned above. The culture medium was replacedwith fresh medium several times during cultivation after centrifugation for 5 m in (1000rpm, 186x g) to avoid depletion of glucose and glutamine. All cultures were performed twice and the reproducibility was confirmed for each duplicate. Analysis Viable cell concentrations were determ ined by the trypan-blue dye exclusion method. Mean cell diameters were determined using the EPICS Elite flowcytometer and standard beads having average diameters of 10 and 20pm (Beckman Coulter, Fullerton, USA). The glucose and lactate concentrations in the

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culture medium were determined by the glucose oxidaseperoxidase and lactic acid oxidase-peroxidase methods (Biochemistry Analyzer 2700; YSI Inc., Ohio, USA), respectively. The assay of glutamine was performed using an enzymatic analyzer (Biosensor BF-4; Able, Tokyo). Ammonia levels were measured by the indophenol colorimetric method (Wako, Tokyo). The concentration of tPA was determined by enzyme-linked immunosorbent assay (Imulyse tPA, Biopool AB, Umea, Sweden). The time courses of the changes in the cell (viable cell), glucose, lactate, glutamine, ammonia and tPA concentrations during culturing were computed to calculate the following specific rates: cell growth (p), consumption of glucose (IJ~& and glutamine (1~~~13, and production of lactate (q&, ammonia (qNH3) and tPA (qtPA). The specific rates were calculated for each duration between neighbor sampling times and the average values are shown in the figures. Determination of intracellular amino acid content After washing approximately 5 X lo6 cells two times with cold phosphate-buffered saline solution, the cell number was redetermined. Intracellular compounds were extracted by resuspending the cells in 0.5 ml deionized water and boiling in a water bath for 10min. The samples were then kept at 4°C for 1 h, centrifuged at 8000 xg for 20 min at 4°C and the cell pellet was discarded. The supernatant was passed through a filter of 0.45 pm pore size and the filtrate was stored at -20°C prior to analysis. The intracellular amino acid contents were determined using the Pica-Tag system developed by Waters (Millipore Co., Mass., USA). To identify and quantify the amino acid peaks on the chromatogram, the H-type amino acid standard (2.5 pmol.ml-‘, Wako, Tokyo) was used. The results obtained were analyzed using the software “805 data Workstation” provided by Waters (21).

l-1

0

1

100

11

200

Culture

1

300

time

341

I

400

500

(h)

FIG. 2. Cell growth of cultures subjected to various centrifugal forces for medium change, Arrows in the figure show the time of medium change. Symbols: 0, 67 xg; 0, 119Xg; A, 186xg; A, 267xg; q ,364xg.

edly as the centrifugal force decreased and became maximum (9.5%) at 17 xg. It was assumed that the dead cells were removed in the supernatant at lower centrifugal forces (Fig. 1). However, the cell recovery decreased with a decrease in centrifugal force and was only 80% at 17 x g. The cell recovery was consistently high for centrifugal forces in the range of 67-186 xg. Consequently, the low centrifugal force of 17 xg was considered to be inapplicable for medium change despite

RESULTS AND DISCUSSION Effect of centrifugal force on cell recovery and viability In order to study the influence of centrifugal force, applied for medium change, on the cell recovery and increment of cell viability of CHO cells in suspension culture, approximately 5 X lo6 cells having a viability of as low as 81% were centrifuged at different forces (17-186 x g) for 5 min. The cell viability increased mark100 $

80

f 6o 8 2 40 r=4 u” 20 0

L-

0

50 Centrifugal

100 force

150

Centrifugal

200

(xg)

FIG. 1. Effect of centrifugal force on the recovery and increment of viability of cells. Viability before centrifugation was 81%. Symbols: o , recovery; A, viability increment.

FIG. on cell culture trifugal UGluc;

force

(xg)

3. Effect of centrifugal force, for a seriesof medium change, activities. The average values of each specific rate during the shown in Fig. 2 were calculated and plotted against the cenforce applied for medium change. Symbols: 0, (1; n , qtPA; 0,

q 1 4L.c;

4

UGlo; A, Qm3.

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the increased cell viability, due to the lower cell recovery. A centrifugal force higher than 67 X g was therefore employed in the following experiments. Effect of centrifugal force for the medium change on cell activities in suspension culture Suspension cul-

6 CO" 0

tures of CHO cells were carried out for 474 h with 7 medium changes, for which various centrifugal forces (67-364xg) were applied to the cells. Some loss of cell mass at each medium change, observed in the time course of cell concentration (Fig. 2), was found to be independent of the centrifugal force, and was attributed to fluctuation in handling. The specific growth rate was markedly higher in the culture centrifuged at 67 x g compared with that in cultures subjected to higher centrifugal forces (Fig. 3). The culture centrifuged at 67 X g for the medium change also exhibited higher specific rates of glucose and glutamine consumption and of lactate, ammonia and tPA production compared with other cultures. Two reasons for this difference in the specific activities between cultures centrifuged at 67 xg and at higher centrifugal forces were speculated, namely, that cell damage may have occurred at higher centrifugal forces and that metabolically active cells may have been selectivelyretained at the lower centrifugal force.

4= M 2a ';E

Effect of centrifugal force on the activities of cells in suspension culture Cultures for 288 h were centri-

fuged daily for 10min at various centrifugal forces (O537xg), after which the precipitated cells were resuspended in the supernatant, in addition to the two centrifugations for medium change at 186xg. The specific rates of growth, metabolic activities and tPA production of the cultures centrifuged daily were almost identical to those of the control (uncentrifuged cultures) (Fig. 4). This indicates that centrifugation at force up to 537x g had no detrimental effect on the specific rates of CHO cells. These results are in agreementwith those obtamed by Tokashiki et al. (13), who observed no effect of centrifugation at force up to 500 X g on cell prolifera-

5 o-0 100 200 300 400 600 600 Centrifugal

force

(xg)

FIG. 4. Influence of centrifugal force on cell activities. Centrifugation at various forces without cell separation was performed daily during the suspension culture and the average values of each specific rate are plotted against the centrifugal force. Cells in the supernatant were returned to the culture after centrifugation together with precipitated cells. Symbols are the same as those in Fig. 3.

zm 1 : w 0.8 0

:

0.6

i 0.4 !l Em 0.2 2

SUP

PPt

auP

PPt

0 SUP

PPt

FIG. 5. Comparison of the specific activities of cells from the supernatant and precipitate after centrifugation at 67 X g. Cells in the precipitate and supematant after centrifugation at 67 X g were inoculated into other cultures in duplicate, and the average values of each specific rate during the cultures are shown. Bars indicate the standard deviation between the duplicates.

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tion and specificIgG productivity of mouse-humanhybridoma X87 cells. Comparison of metabolic activities between cells in suCells from the supernatant pernatant and precipitate

and precipitate following centrifugation at 67 X g of a suspensionculture were employed as the inoculum for second-stepcultures. Cells in the supernatant were collected by means of another centrifugation at 186x g and the inoculum concentrations were adjusted to get the same viable cell concentration in both cultures. The averagecell-specific rates during incubation for 280 h in the second-stepculture are shown in Fig. 5. The specific growth rates of the cells in the cultures of the precipitate-derived cells were slightly higher than those in the cultures of the supernatant-derivedcells. All other specific metabolic rates, including that of tPA production, were markedly higher in the former than in the latter. This might not be due to the differencein cell viability between precipitated and supernatant cells because they were almost the same (98.4 and 98.9x, respectively). This indicates that centrifugation at 67 x g removed metabolically active cells from the supernatant and consequently, the average specific growth and metabolic rates of the precipitated cells were higher than those of the cells in the supernatant. Comparison of diameter and amino acid contents between cells from the supematant and precipitate The

diameters and intracellular amino acid contents of the cells from the supernatant and precipitate, obtained following centrifugation at 67 xg, were determined. The mean diameter of the precipitated cells (17.4pm) was greater than that of the cells from the supernatant (15.7 pm) (Fig. 6). Figure 7 shows that the intracellular content of nonessentialamino acids, except proline, was higher in the precipitated cells than in the cells from the supernatant. However, there was no consistent or marked difference in the essential amino acid contents. Since nonessential amino acids are synthesizedby cells and amino acids are the building blocks of enzymesand other proteins involved in cellular metabolism, the higher amino acid content in the precipitated cells might reflect their superior metabolic state over the cells from the supernatant. The higher content of amino acids may also mean a higher specific gravity of the precipitated cells than of the cells from the supernatant. Although it is assumedthat a subpopulation having lower metabolic activity and specific gravity appear during culturing, the mechanismis not known. These results reveal that cells with higher amino acid contents and higher metabolic activities are selectively

Amp

Glu

Ser

Gly

Ala

Pro

Tyr

Total

PPt FIG. 6. Mean diameter of cells in the supernatant and precipitate after centrifugation at 67 x g. The mean diameters of the cells in the supernatant and the precipitate after centrifugation at 67 X g were estimated from 20,000 cells employing the flowcytometer and standard beads (10, 20 pm), in duplicate. Bars indicate the standard deviation between the duplicate samples.

precipitated following centrifugation at 67 x g, due to their larger diameter and higher specific gravity. In this study, the optimum centrifugation force was found to be around 67 x g when the centrifugation time was fixed at 5 min. This optimum force might depend on the centrifugation time. For example, it is assumedthat the optimum force decreases with time. Consequently, the higher activities observed in the culture after several medium changes employing lower centrifugal forces might be due to the lower rates of sedimentation of cells with low amino acid contents and low metabolic activities following centrifugation at lower forces. An optimum centrifugation condition for obtaining higher specific activity was 67 x g for 5 min in the culture system used in this study. However, the centrifugation condition for commercial production should be designed with consideration also of cell recovery. Moreover, other culture conditions, such as cell line and kind of medium, may affect cell sedimentation during centrifugation. Thus, an optimum centrifugation condition should depend on each cultivation process. In conclusion, a low centrifugal force, such as 67 x g, for 5 min is recommendedfor medium changeduring suspension cultures of mammalian cells, in order to maintain high specific productivity becausecells with higher metabolic activities and amino acid contents are selectively sedimentedmore easily at such a low centrifugal force due to their larger diameters and higher specific gravities.

IGO kg

Thr vaI Met

Ib

Leu Phe LyeTotal

FIG. 7. Intracellular ammo acid contents of cells in the supernatant and precipitate after centrifugation at 67 X g. Open bars: cells in the supernatant; closed bars: cells in the precipitate. (A) Nonessential amino acids; (B) essential amino acids.

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J. BIOSCI.BIOENG., REFERENCES

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