Appl Microbiol Biotechnol (1990) 33:287-290

Applied Microbiology Biotechnology © Springer-Vedag 1990

Anchorage-dependent mammalian cell culture using polyurethane foam as a new substratum for cell attachment Taku Matsushita, Masayoshi Ketayama, Ken-ichi Kamihata, and Kazumori Funatsu Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812, Japan Received 10 November 1989/Accepted 19 January 1990

Summary.

Anchorage-dependent mammalian cells w e r e c u l t i v a t e d at h i g h cell d e n s i t y in a n o v e l c u l t u r e s y s t e m u s i n g p o l y u r e t h a n e f o a m ( P U F ) as a s u b s t r a t u m f o r cell a t t a c h m e n t . P U F h a s a m a c r o p o r o u s s t r u c t u r e giving a h i g h s u r f a c e a r e a to v o l u m e ratio. M o n k e y k i d n e y cells (Vero) a n d C h i n e s e h a m s t e r o v a r y cells ( C H O K1) a t t a c h e d to t h e i n t e r n a l s u r f a c e o f P U F a n d g r e w to a h i g h cell d e n s i t y ( 1 . 0 4 x 108 c e l l s / c m 3 P U F a n d 3.5 × 107 c e l l s / c m 3 P U F , r e s p e c t i v e l y ) in P U F s t a t i o n a r y cultures. I n a d d i t i o n , we h a v e d e s i g n e d a P U F - p a r t i d e p a c k e d - b e d c u l t u r e s y s t e m for h i g h d e n s i t y m a s s cell culture. A m a x i m u m cell d e n s i t y o f 2.4 x 107 c e l l s / c m 3 c u l t u r e v e s s e l v o l u m e was o b t a i n e d in a p a c k e d b e d c u l t u r e o f Vero cells.

Introduction M a n y m a m m a l i a n cells u s e d for p r o d u c t i o n o f b i o l o g i cal m a t e r i a l s a r e a n c h o r a g e - d e p e n d e n t ( G r i f f i t h s 1986). T h e r e f o r e , f o r t h e p u r p o s e o f o b t a i n i n g a large a m o u n t o f p r o d u c t f r o m a m a s s c u l t u r e o f t h e s e cells, it is imp o r t a n t to i n c r e a s e t h e s u r f a c e a r e a a v a i l a b l e f o r cell a t t a c h m e n t in r e l a t i o n to t h e c u l t u r e vessel v o l u m e . T h e c u r r e n t p r a c t i c a l t e c h n i q u e s a v a i l a b l e for s u c h a c u l t i v a t i o n a r e m i c r o c a r r i e r s u s p e n s i o n c u l t u r e s (van W e z e l 1967; N a h a p e t i a n et al. 1986) a n d h o l l o w - f i b r e cultures ( K n a z e k 1974). H o w e v e r , t h e r e a r e s e v e r a l p r o b l e m s in t h e s e c u l t u r e t e c h n i q u e s . F o r e x a m p l e , cells a t t a c h e d to m i c r o c a r r i e r s are a l w a y s e x p o s e d to s h e a r i n g stress r e s u l t i n g f r o m a g i t a t i o n o f t h e m e d i u m . This s h e a r i n g stress is k n o w n to c a u s e d a m a g e to t h e cells ( S t a t h o p o u l o s a n d H e l l u m s 1985). H o l l o w fibre is e x p e n s i v e a n d it is d i f f i c u l t to scale u p t h e c u l t u r e volu m e ( J e n s e n 1981). I n this p a p e r , w e h a v e c u l t i v a t e d m a m m a l i a n cells u s i n g p o l y u r e t h a n e f o a m ( P U F ) as a n e w s u b s t r a t e f o r cell a t t a c h m e n t . P U F is a c h e a p a n d a u t o c l a v a b l e m a t e rial, a n d has a m a c r o p o r o u s s t r u c t u r e giving a h i g h surOffprint requests to: K. Funatsu

face a r e a to v o l u m e ratio. F u r t h e r m o r e , we h a v e att e m p t e d to cultivate m a m m a l i a n cells in a n e w l y d e s i g n e d c u l t u r e s y s t e m w i t h P U F p a r t i c l e s in a p a c k e d b e d c u l t u r e vessel.

Materials and methods Cells and medium. Monkey kidney cells (Vero) and Chinese hamster ovary cells (CHO-K1) were obtained from Flow Laboratories (McLean, Va, USA). The cells were grown in DME medium (Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with 10% (v/v) foetal bovine serum (Hazleton Research Products, Lenexa, Kan, USA) containing 58.8 ~tg/ml penicillin and 100 txg/ml streptomycin. N-(2-Hydroxyethyl)piperazine-N'(2-ethanesulphonic acid) (HEPES; Dojindo Lab., Kumamoto, Japan) was added to the medium (final concentration 5 mM) to adjust the pH to 7.4 with NaHCO3. Preparation of PUF. PU1~ was kindly gifted by Inoue MTP Co. (Nagoya, Japan). PUF has a sponge-like macroporous structure with each pore made up of smooth thin films and thick skeletons, partially opened and spatially connected with each other as shown in Fig. 1A. The average pore diameter of PUF is 500 Ixm. The bulk density of PUF is 0.012 g/era 3. The block of PUF was cut into either flat plates or particles. Three different shapes of PUF flat plates, (1 × 5 x 10 mm [PUF-1], 1 x 10 × 10 mm [PUF-2] or 2 × 5 × 10 mm [PUF-3]) were prepared for stationary cultures. The particles of PUF used for the packed-bed culture were 4 mm in diameter. Before use, the PUF plates and particles were submerged in distilled water and autoclaved in order to sterilize them and remove any chemical contaminants. Then, the PUF was washed with serum-free DME medium and soaked again in serum-DME medium prior to cell inoculation. Cell inoculation. The inoculum was obtained by trypsinizing cells from seed petri dishes. The cells were centrifuged at 400 9 for 5 min and resuspended in culture medium. The viable cell concentration was determined by the trypan blue dye exclusion method (Nahapetian et al. 1986). In the case of PUF stationary cultures, the suspended cells were inoculated in a petri dish containing many fiat plates of PUF. After overnight culture, the inoculated flat plates were transferred to another petri dish containing fresh medium. In the case of PUF particles in packed-bed culture, the cell suspension was directly inoculated into the packed bed. After stationary culture for 4 h, medium circulation was started.

288 of which the bulk volume and weight were 100 ml and l a g g respectively, were packed in the lower part of the culture vessel (diameter, 50 mm; volume, 350 ml) containing 200 ml medium. Medium passed through the packed bed at a flow rate of 78 ml/min, flowed to the bubble column, and was recirculated to the culture vessel. The bubble column was built in for oxygen supply to the culture system. The gas, composed of 5% CO2/95% air, was sparged by a distributor (G-2 filter) into the medium. Foams generated by sparging were broken by silicone grease (Dow Corning, Midland, Mich, USA) precoated on the inner surface of the column. It was found experimentally that this silicone grease had no harmful effect on the growth of Vero cells (data not shown). For continuous culture, medium in the bubble column was continuously withdrawn to a waste reservoir at various perfusion rates, and simultaneously fresh medium was supplied from the medium reservoir to the bubble column at the same rate. For semi-batch culture, medium in the bubble column was withdrawn once every ca. 24 h and an equal amount of fresh medium was supplied. The dissolved oxygen concentration was monitored by an oxygen electrode in the culture vessel, and maintained within a suitable range (40-100 mm Hg) (Kilburn and Webb 1968) by controlling the flow rate of the gas in the bubble column. The cell culture was grown at 37°C in an incubation box.

Fig. 1A, B. Microphotograph of polyurethane foam (PUF) structure and Vero cells grown on the surface of PUF. A Macroporous PUF structure with skeleton and smooth thin films before cell inoculation. B Monkey kidney (Vero) cells grown on the surface of PUF-2 (1 mm × 10 mm x 10 mm) in PUF stationary culture PUF stationary culture in a petri dish. Cells were stationarily cultured in a petri dish (53 mm) containing one piece of flat-plate PUF, under 5% CO2 and 95% air at 37 ° C. Four millilitres of medium was used in this experiment. PUFparticles in packed-bed cultures. The use of PUF particles in a packed-bed culture system is illustrated in Fig. 2. PUF particles,



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Growth determination and chemical analysis. Cell growth was monitored according to the method reported by van Wezel (1967). Cultured PUF with attached cells was suspended in 0.1 M citric acid solution containing 0.1% crystal violet. After incubation for 1 h at 37°C, the stained nuclei released from the cells were counted on a haemocytometer. The ammonia and glucose contents of a sample from the culture medium were measured by commercial kits (Wako Pure Chemical Industries, Osaka, Japan).

Results W h e t h e r P U F c o u l d be u s e d as a s u b s t r a t u m for the att a c h m e n t o f m a m m a l i a n cells or n o t was e x a m i n e d b y s t a t i o n a r y culture in a petri dish. F i g u r e 3 A shows the g r o w t h curves o f Vero cells b y a P U F s t a t i o n a r y culture. M e d i u m e x c h a n g e s were p e r f o r m e d at each samp i i n g p o i n t . A l t h o u g h three d i f f e r e n t t h i c k n e s s e s a n d v o l u m e s o f P U F were u s e d , the m a x i m u m e x t e n t o f g r o w t h a t t a i n a b l e was a b o u t 8 × 109 c e l l s / g P U F i n each case. I n the case o f P U F - 2 , a v a l u e o f 8.7 × 109 c e l l s / g P U F was o b t a i n e d . F r o m this v a l u e , the cell d e n s i t y was c a l c u l a t e d to be 1.04 × 108 c e l l s / c m 3 P U F in b u l k v o l u m e . T h e d o u b l i n g t i m e o f Veto cells was 25 h d u r i n g l o g a r i t h m i c growth i n each P U F c u l t u r e a n d was the s a m e as the s t a t i o n a r y c u l t u r e i n a petri dish. F i g u r e 3B shows the c h a n g e i n a m m o n i a a n d glucose c o n c e n t r a t i o n s i n the m e d i u m o f a P U F - 2 c u l t u r e a c c o m p a n i e d b y cell growth a n d m e d i u m exchanges. T h e results i n d i c a t e that the a m m o n i a a n d glucose conc e n t r a t i o n s in the m e d i u m were w i t h i n a s u i t a b l e r a n g e for m a m m a l i a n cells to grow ( N a h a p e t i a n et al. 1986). A m i c r o p h o t o g r a p h o f c u l t u r e d P U F - 2 shows the a p p e a r a n c e o f Vero cells a t t a c h e d a n d g r o w n conf l u e n t l y o n b o t h the b o t t o m t h i n film a n d t h e s k e l e t o n o f P U F (Fig. 1B). Vero cells were f o u n d to grow i n m u l tilayers o n the i n t e r n a l surface o f P U F . We have also c u l t i v a t e d C H O - K 1 cells, u s i n g P U F

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Anchorage-dependent mammalian cell culture using polyurethane foam as a new substratum for cell attachment.

Anchorage-dependent mammalian cells were cultivated at high cell density in a novel culture system using polyurethane foam (PUF) as a substratum for c...
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