Non-woven fabrics as new cell matrices for IMR-90 human embryonic lung diploid fibroblast cells

JOURNAL OF FERMENTATION AND BIOENGINEERING

Vol. 70, No. 4, 289-291. 1990

Non-Woven Fabrics as New Cell Matrices for IMR-90 Human Embryonic Lung Diploid Fibroblast Cells S H I N J I R O M I T S U D A , * Y O S H I A K I M A T S U D A , Y A S U H A R U I T A G A K I , A K I R A SUZUKI, E I T A R O K U M A Z A W A , K A N J I H I G A S H I O , AND GOSEI K A W A N I S H I

Research Institute of Life Science, Snow Brand Milk Products Co., Ltd., 519 Shimo-lshibashi, Ishibashi-machi, Shimotsuga-gun, Tochigi 329-05, Japan Received 9 May 1990/Accepted 6 August 1990 IMR-90 cells could proliferate on non-woven fabrics (NWF) up to a cell density of about 3 x 10S/cm z, which was about 7 times higher compared to that in monolayer culture with a T-flask. The production of tissue plasminogen activator (t-PA) by perfusion culture using a device which includes a cultivating pan fitted inside a sheet of NWF was performed. The t - P A activity at a dilution rate of 0.22, 0.33 and 0.45 d -1 was 245,220 and 160 I U / m i . Compared to plastic surfaces, NWF stably retained the cells during t - P A production using serum-free medium.

(Sigma, St. Louis, USA) was used. Two types of non-woven fabrics (NWF), which were made of polyester (PET) or nylon (NX), were supplied from the packaging group of the Snow Brand Technical Research Institute. The N W F surface was treated by irradiation (4 mHz) three times at a distance of 5 m m with a plasma discharge torch (Thomas Scientific, New York, USA). The n u m b e r of cells adhering to the N W F was determined colorimetrically by M T T assay (8). It was deduced from the results of preliminary tests that 1 mg of glucose was consumed to proliferate 1 × 105 IMR-90 cells in order to determine the transfer point from the growth to the production phase in the perfusion culture. Plasminogen activator activity was assayed by the fibrin plate method (9) using W H O t - P A as a standard. Two types of N W F were tested for the growth of I M R 90 cells. Each NWF, P E T and NX was discharged in order to examine whether further affinities for the cells were brought about. The results are summarized in Table 1. Final cell density on discharged NX reached 2.9 × 105/cm 2NWF, which was about seven times higher than that in monolayer proliferation in T-flasks. Although P E T was originally superior to NX, the discharge treatment made it slightly inhibitory for the cells to proliferate. O n the contrary, cell growth on NX was improved by the discharge treatment. Cellular t - P A productivity was not affected by

Tissue plasminogen activator (t-PA) is a serine protease which has a higher affinity for fibrin in blood clots than urokinase-type plasminogen activator (1). t - P A has been isolated from various m a m m a l i a n cells (2). A m o n g them, the production of t - P A by Bowes m e l a n o m a cells (3) and guinea pig keratocyte (GPK) cells (4) have been studied in large-scale cultivation with microcarriers. Although h u m a n diploid fibroblasts such as IMR-90 cells are k n o w n to secrete t - P A (5), no descriptions of practical production methods are available. H u m a n diploid cells are limited with respect to their life spans and are inferior to cancer cells such as Bowes m e l a n o m a cells in termes of various growth characteristics. However, cancer cells might produce cancer-causing factors such as h u m a n transforming growth factors, thereby limiting the therapeutic use of t P A (6, 7). Therefore, we have investigated perfusion culture production methods using h u m a n normal embryonic lung diploid fibroblast cells. Non-woven fabrics as new materials which would immobilize I M R - 9 0 cells were investigated. I M R - 9 0 cells were obtained from the American Type Culture Collection (ATCC, CCL186). Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS) was used for cell growth. In the production phase, D M E M fortified with 1% proteose peptone (Difco, Detroit, USA) and 4 K I U / m l of aprotinine

TABLE 1. Comparison of t-PA production with petri dishes between two kinds of non-woven fabrics (NWF) NWF

Materials

PET Polyester PET Polyester NX Nylon NX Nylon Petri dish (Control)

Discharge treatment (-) (+) ( ) (+) ( )

Cell density /cm2-NWF 2.5 × 105 2.1 × 105 1.8 × 105 2.9 × 105 4.0 x 104

Relative value

t-PA activity IU/ml

Cellular productivity IU/106/d

6.3 5.3 4.5 7.3 1.0

138 106 70 113 --

282 261 196 197 --

The cell density of IMR-90 cells proliferating on the surfaces of two kinds of NWF and their t-PA productivities are compared. After discharge treatment and autoclaving, the round-shaped NWF were set into 60 mm petri dishes. Inoculum size was 1 x 106/petridish (3.6 x 10a/cm2) and medium volume was 6 ml. The following day, each NWF was transferred to 100 mm petri dishes and medium volume was increased up to 20 ml. After 12 d of growth, each NWF was cut into two pieces, one was used for t-PA production and another was used for determination of cell numbers by the MTT method. The t-PA was produced in 50 ml of medium during 7 d. * Corresponding author. 289

290

MITSUDA ET AL.

means of irradiation with the discharge torch. Cell growth on the N W F was observed by scanning electron microscope (Hitachi $800, Fig. 1). Batch t - P A production by cells grown on N W F (PET not treated) fitted inside the roller bottles was examined. The time course of t - P A production is shown in Fig. 2. In regular roller bottles at low cell density (1.5 x 104/ml), the t - P A concentration secreted into the medium reached a plateau at about 150 I U / m l at day 7. At high cell density (1.5 x 105/ml), the t - P A rapidly increased up to 116 I U / m l but it began to decrease after day 4. The results showed that t - P A production at lower cell density was more effective. The production of t - P A by normal diploid fibroblasts is known to be regulated by negative feedback control (5). The t - P A once secreted is b o u n d to receptors on the cell membranes, internalized and then degraded (10). In production using the NWF-fitted bottles at a cell density of 1.1 × 10S/ml, t - P A continued to increase and reached 3 0 0 I U / m l by day 15. Higher t - P A activity was obtained in comparison with the production in regular roller bottles at similar cell densities (1.5 x 10S/ml). This fact implies that the cell matrice type affects t - P A production by IMR-90 cells. The initial time-lag of t - P A production in the NWF-fitted bottles might be due to c o n t a m i n a t i o n of serum components and other substances which remained in the N W F during the transfer from the growth to the production phase, and which had an inhibitory effect on t - P A production. The components were only slightly removed by washing the N W F with PBS because N W F has a complicated three-dimensional surface (Fig. 1). The cells on the smooth surfaces of plastic roller bottles were observed to be slightly detached at the end of the production. Kadouri et al. suggested that IMR-90 cells were

J. FERMENT.B1OENG., easily detached from the plastic surfaces in serum-free medium (2). Compared to the plastic bottles, the advantage of using N W F as a cell matrix is to stably retain the cells in serum-free medium. Production in the NWF-fitted bottles resulted in more cumulative t - P A , however, the productivity per unit cell was 3.6 times lower than that in the regular bottles at the same medium volume (2/). Production of t - P A by the cells immobilized to the N W F was further investigated using a perfusion system in order to reduce the influence of product inhibition. Figure 3 shows the device which was originally designed. Medium is supplied through the sintered metal filters and runs slowly down along the inclined surface of the pans. Several advantages of the reactor designed for long termed cultivation of the h u m a n fibroblasts are as follows. (i) The cells are cultivated in mild conditions without significant shear stress, such as static incubation with continuous and slow movements of medium. (ii) Depth of the liquid phase is shallow, so gas is readily transferred through. (iii) A slgiht gradient of the components in the medium occurs when the medium flows along a sheet of N W F that retains cells. C o n t i n u o u s t - P A production using a single pan was performed. After proliferation up to 2.5× l @ / c m 2, the growth medium was replaced with production medium. Cell density in the pan was estimated as 2.2 x 10S/ml. At day 3 after the start of the production, continuous production was initiated at a dilution rate of 0.22 d 1. At day 13, this was increased to 0.33 d '. At day 23, the dilution rate was increased up to 0.45 d J. t - P A production was

v

O ,< I1

±

Production in days

FIG. 1. IMR-90 cells grown on non-woven fabrics (PET).

FIG. 2. Batch production of t-PA by IMR-90 cells grown on NWF (PET, 850 cm2) fitted inside roller bottles. IMR-90 cells grown on NWF-fitted roller bottles were incubated in two liters of production medium at 0.5 rpm for 15 d. Cell densities were 1.1 x 105/rnl (2.5 x 105/cm2). As control runs, t-PA production in regular bottles was performed at two levels of medium volume, 200 ml and 2 l. Cell densities were 1.5 x 10Mml and 1.5 x 104/ml respectively. Symbols: e , with NWF, Med. vol. 2 l, Cell density 1.1 x 105/ml; O., control-l, 2 l, 1.5 x 104/ml; A,control-2, 200 ml, 1.5 × l@/ml.

VOL. 70, 1990

NOTES

250-

"100

2o0-

-8o

291

v

> 1,50-

-60

>

t3 ,< 13.

0

L IO0-

.40

o.

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FIG. 3. A cell cultivation device with NWF. The device employed for cultivation of IMR-90 cells comprises cultivating pans in a tank. The pan with a surface area of 400 cm2 contains 450 ml of medium. The fresh medium from the inlet port is supplied radially through a filter made of sintered metal and which is arranged in the central portion of the pan. Each pan has a bottom surface that inclines downwards towards the outer portion. The spent medium overflows from the edges of the pan and passes out through the lower outlet port. If necessary, the medium can be recirculated through the recirculating line. 1 Tank (~ 280 mm, H 260 mm), 2; Pans fitted with NWF on the bottom, ~:~)Sintered metal filter (pore size; 120,urn), @ Medium inlet port, ~',Medium oulet port for overflowing medium, ~'. Gas (5% CO2/ 95% Air) inlet port, 7: Gas outlet port, -S~ Fresh medium reservoir, @ Harvest reservoir, ~il~Recirculating line (Dotted line). a c h i e v e d f o r 33 d. T h e effects o f the d i l u t i o n rate o n t - P A p r o d u c t i o n are s h o w n in Fig. 4. T h e t - P A activity was 245 I U / m l at D = 0 . 2 2 d 1,220 I U / m l at D = 0 . 3 3 d 1 a n d 160 I U / m l at D = 0 . 4 5 d '. V o l u m e t r i c p r o d u c t i v i t y was d e t e r m i n e d to be 54, 73 a n d 72 I U / m l / d , respectively. In c o n t i n u o u s p r o d u c t i o n , t h e v o l u m e t r i c p r o d u c t i v i t y was s u p e r i o r to t h a t in b a t c h p r o d u c t i o n . Effects o f i n c r e a s i n g the d i l u t i o n rate o n t h e release o f n e g a t i v e f e e d b a c k c o n t r o l a n d o n t h e a c c o m p a n y i n g increase in v o l u m e t r i c p r o d u c t i v i t y w e r e n o t o b v i o u s in this e x p e r i m e n t . T h e m a x i m u m value f o r t - P A activity was o b t a i n e d at D - - 0 . 2 2 d l a m o n g t h e d i l u t i o n rates t e s t e d . T h e value, w h i c h is d e p e n d e n t o n d i l u t i o n rate, was a s s u m e d to vary w i t h cell d e n s i t y a n d the t y p e o f p r o d u c t i o n method. T h e high a n d s t a b l e p r o d u c t i o n d u r i n g a o n e m o n t h p e r i o d was a c h i e v e d using the s y s t e m with a single p a n . T h e n o n - w o v e n f a b r i c s w e r e f o u n d to be g o o d cell m a t r i c e s f o r I M R - 9 0 cells b e c a u s e n o t o n l y did t h e y e n a b l e c u l t i v a t i o n w i t h h i g h cell d e n s i t y , b u t t h e y also r e t a i n e d the cells w i t h a h i g h affinity. H o w e v e r , the cells w e r e o b s e r v e d to g r o w n o n u n i f o r m l y o n t h e N W F by s t a i n i n g viable cells a f t e r a l o n g t e r m p r o d u c t i o n . T h e p r o b l e m s o f h o w to o b t a i n a u n i f o r m d i s t r i b u t i o n o f s e e d i n g cells a n d f e e d i n g m e d i u m i n t o p l u r a l p a n s r e m a i n to be s o l v e d . We wish to thank Mr. T. Kimura and Y. Sagara of the Technical Research Institute, Snow Brand, for their electron microscopic observations of IMR-90 cells on NWF.

E o20 -~ >

50

o;1

o'.2

o~a

o'.4

o15

Dilution rate ( l / d ) FIG. 4. Influences of dilution rate on t - P A activity and volumetric productivity in perfusion culture using the cell cultivation device with NWF (PET). A single pan with 400 cm 2 of NWF proliferated at 2.5 x 105 cells/cm2 was used for the continuous t - P A production. Medium volume within the single pan was 450 ml. Dilution rate was initiated from 0.22 d ' and was increased to 0.33 d ', finally up to 0.45 d 1 every 10 d. The dilution rates were defined as output volume of medium per volume in the pan. The volumetric productivities were defined as the product of the activity in medium and the dilution rate. Symbols: 0 , t - P A activity; O, volumetric productivity.

REFERENCES

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