Cell growth on immobilized cell growth factor: 5. Interaction of immobilized transferrin with fibroblast cells

Cell growth on immobilized cell growth factor: 5. Interaction of immobilized transferrin with fibroblast cells Shu Qin Liu, Yoshihiro lto and Yukio Imanishi* Department of Polymer Chemistry, Faculty of Engineering, Kyoto University, Yoshida Houmachi, Sakyo-ku, Kyoto 606-01, Japan (Received 31 December 1992; revised 22 February 1993) Transferrin was immobilized on poly(methyl methacrylate) membrane and the interaction of the immobilized transferrin with mouse fibroblast cells STO was investigated. The immobilized transferrin was stable without decomposition over 48 h and showed higher activities of cell growth acceleration than free transferrin and transported ferric ion as effectively as free transferrin. The specific interaction of the immobilized transferrin with fibroblast cells was confirmed by experiment using the antibody. It was considered that cell 9rowth acceleration and ferric ion transportation occur by different mechanisms. Keywords." Immobilizedtransferrin;cell growth;ferricion transportation;serum-freecellculture

Introduction Serum has been used most commonly to stimulate the growth of mammalian cells ever since cell culture systems were established in vitro. However, the serum, which can support the survival, and growth of a wide variety of primary and established cells, is a complex mixture whose components are poorly characterized. In adddition, as a result of recent progress in biotechnology, the need to cultivate mammalian cells on a large scale has been increased to produce medically important substances. Therefore, a medium containing serum has several disadvantages, such as expense, inhomogeneity from lot to lot and complexity. To overcome these difficulties some chemically defined serum-free media have been developed 1. However, we found that the covalently immobilized insulin enhanced cell growth more effectively than free or physically adsorbed insulin2-8. The immobilized insulin was used repeatedly in the same way as immobilized enzymes in bioreactors are used. Therefore, this new cell culture technique using immobilized biosignal molecules is considered to be very useful. In the present paper, instead of insulin, transferrin, which is used as a supplement of serum-free cell culture as insulin is, was immobilized on a polymeric film and the effect was investigated.

Experimental Materials Human holotransferrin and apotransferrin, bovine insulin (No. 1-5500) and rabbit antihuman-transferrin antibody lgG fraction were purchased from Blamed. Technol. Inc., Sigma Co., and Cosmo. Co., respectively,

*To whomcorrespondenceshould be addressed. 0141-8130/93/040221-06 © 1993 Butterworth-HeinemannLimited

and used without further purification. Transferrin was labelled with 12sI by the chloramine-T method 3. PMMA (poly(methyl methacrylate)) was purchased from Wako Pure Chem. Ind. Co. and cast into a membrane by eluting a dimethylformamide solution (10 wt%) on a glass plate and irradiating with an infrared lamp. Surface hydrolysis of P M M A membrane and immobilization o f transferrin P M M A membrane was treated with 4 N NaOH in a water/methanol (4/1 v/v) mixture at 50°C for 90 min to introduce carboxyl groups on the membrane surface 4. The number of carboxyl groups produced using Rhodamine 6G was determined to be 1.6 x 10 -6 mol/cm 2. The surface hydrolysed PMMA membrane was placed in an aqueous solution of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (water-soluble carbodiimide, WSC) (1 mg/ml) buffered with 2-(N-morpholino)ethanesukfonic acid (MES, 0.02 M, pH 7.0), and transferrin (50-500pg/ml) was added. After the reaction had continued for i h at 30°C, the PMMA membrane was washed with phosphate-buffered saline (PBS) repeatedly until the absence of transferrin in the washing liquid was confirmed using Coomassie brilliant blue (CBB) G2503'4 and 125I-labelled transferrin. 1251Transferrin (specific activity, 2 × 105-3 x 105 cpm/g; 55% count ratio) was immobilized on to PMMA membrane by the same procedures, and the amount of 125I-transferrin before and after washing and cell culture was determined by the gamma-counting method. Transferrin-adsorbed PMMA membrane was prepared by the same procedure as for the immobilization without the addition of a coupling reagent, WSC. The amount of immobilized (covalently bonded) transferrin was determined using CBB G250 as

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Cell growth on immobilized cell growth factor. 5." S. Q. Liu et al. reported previously 3'4. A PMMA membrane immobilized with transferrin (1 c m x 4 c m ) was stained with CBB G250 for 5 min and held in a u.v. cell perpendicularly to an incident light. The amount of immobilization was determined from the ratio of u.v. absorbance at 620 nm and 465 nm by using a calibration curve which was drawn from the ratio of u.v. absorbances at 620 nm and at 465 nm of various concentrations of transferrin stained with CBB G250. The transferrin-immobilized PMMA membrane was disinfected by immersion into 75% ethanol for 10 min and used for cell culture. The amount of adsorbed (non-covalently attached) transferrin was determined by the same procedure.

Cell culture Mouse fibroblast cells STO, which were subcultured in Dulbecco's modified Eagle (DME) minimum essential medium containing 10% fetal calf serum, were washed twice with PBS containing 0.02% ethylenediamine tetra-acetate (EDTA) and suspended in a serum-free culture medium, i.e. DME containing SeO 2 (0.14 #g/ml), Gly-His-Lys (20 #g/ml), and ethanolamine (20/~g/ml). The cell suspension was used for the subsequent experiments.

Measurement of cell growth rate The cell suspension (1 x 104 cell/ml, 1 ml) was added to each well of a 24-weU tissue culture dish in which a sheet of transferrin-immobilized PMMA membrane was placed, and incubated under 5% CO2 atmosphere at 37°C for 2 days. The number of cells was determined by measuring the activity of lactate dehydrogenase (LDH) as reported previously3'4.

Measurement of cell adhesion Mouse fibroblast cells STO, which were washed twice with PBS containing 0.02% EDTA, were suspended in the serum-free culture medium and adjusted to 4 x l0 s cells/ml. The cell suspension (1 ml) was added to each well of a 24-well dish in which a sheet of transferrin-immobilized PMMA membrane was placed, and incubated under 5% CO2 atmosphere at 37°C for a definite time until cells adhered. After removing the culture medium, the PMMA membrane was washed three times with PBS containing 0.02% EDTA. The number of cells adhering to the PMMA membrane was counted by optical microscope.

Measurement of 59FeCl3 uptake Mouse fibroblast cells STO were incubated for 12 h in the presence of immobilized or free transferrin, and 59FeCI3 (0.15 pM) was added. After culturing for a further 4 h, cells were separated with 0.02% EDTA, centrifuged, and wash three times with PBS (pH 7.4). The amount of 59Fe ions taken up by the cells was determined by the gamma.-counting method 9'1°.

Statistical analysis of experimental data The experimental data were subjected to statistical analysis by Student's t-test, n = 10.

Results Immobilization of transferrin The experimental results of the immobilization of holotransferrin and apotransferrin are shown in Figure 1. With increasing transferrin concentration in the feed, the amount of immobilized transferrin increased. Under the reaction conditions employed in the present investigation, the amount of immobilized transferrin did not show a plateau value. The absence of a saturation phenomenon is explained in terms of the occurrence of unexpected intermolecular reactions of transferrin as well as desired reactions of transferrin with carboxyl groups on the surface hydrolysed PMMA membrane under the present reaction conditions, where transferrin and the coupling reagent are simultaneously added. On the other hand, the amount of adsorbed transferrin was very low and independent of the feed concentration. The amount of immobilized holotransferrin was nearly the same as that of immobilized apotransferrin under the same conditions. The similar yield of immobilized transferrins is explained by taking into consideration that both transferrins have a similar conformation 1

Stability of immobilized transferr& Table 1 summarizes the experimental results on degradation and dissociation of covalently immobilized or physically adsorbed transferrin during the cell culture procedure. The amount of adsorbed transferrin decreased a little by the addition of culture medium, and strongly decreased to nearly 60% of the original value after culturing fibroblast cells, accompanying the release of transferrin (ca 40%) into the culture medium. On the other hand, the amount of immobilized transferrin did not change with these treatments and transferrin release into the culture medium was not detected, indicating that

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Cell growth on immobilized cell growth factor. 5." S. Q. Liu et al. Table 1 Degradation and dissociation of the immobilized or adsorbed transferrin during culture

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the immobilized transferrin is neither degraded nor dissociated under conditions for cell culture. In the present experiments, transferrin is connected to a P M M A membrane through covalent linkages (amide bonds).

Cell adhesion and spreading on immobilized transferrin Figure 2 shows the experimental results of adhesion of fibroblast cells on transferrin-immobilized P M M A membrane. The presence of immobilized transferrin did not affect cell adhesion. However, after 8-h culturing, the number of cells adhering on transferrin-immobilized membrane increased, and cells were more extended on transferrin-immobilized P M M A membrane than on untreated P M M A membrane or in the presence of free transferrin, as shown in Figure 3. No significant difference was observed between apotransferrin and holotransferrin.

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Cell growth in the presence of immobilized transferrin The growth rates of mouse fibroblast cells STO in the presence of free or immobilized transferrin (apo or holo) are shown in Figure 4. Although free transferrin did not accelerate cell growth (NS at P < 0.05), the immobilized transferrin showed an approximate 1.5-fold acceleration of cell growth (P < 0.001). No significant difference was observed between apotransferrin and holotransferrin. Addition of free insulin to free, adsorbed or immobilized transferrin increased the cell growth rate, indicating the presence of synergetic interactions between insulin and transferrin.

Specific interaction of fibroblast cells with immobilized transferrin The results of fibroblast cell culture in the presence of free or immobilized transferrin, which was treated with antibody before the cell culture, are shown in Figure 5. The antibody-bound transferrin did not show any effect on the cell growth (NS at P < 0.05), when a large amount of antibody was added to free transferrin. The transferrin-immobilized P M M A membrane lost the activity of cell growth acceleration(P < 0.001), when the anti-transferrin antibody was added. A non-specific antibody did not cancel the activity of immobilized transferrin, but decreased the activity of cell growth acceleration from 2.58-fold to 2.22-fold. The slight decrease caused by the non-specific antibody could be explained by a reduced activity of immobilized transferrin due to adsorption of non-specific antibody on the membrane surface. These experimental observations imply that the immobilized transferrin is specifically blocked by the

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antibody to lose the activity of cell growth acceleration, and that non-specific antibody affects the activity of immobilized transferrin due probably to a non-specific binding to the transferrin-immobilized P M M A membrane. These investigations using antibody revealed that the biosignal is transmitted to the cell through specific interactions between immobilized transferrin and the cell. We have found similar phenomena with immobilized insulin 7.

Transportation of ferric ion Figure 6 shows that the immobilized transferrin transported ferric ions to a similar extent as free transferrin (P < 0.001). In these experiments, no difference was observed between holotransferrin and apotransferrin. Discussion

The enhanced acceleration of cell growth by transferrin might be ascribable mainly to ferric ion transportation. Different conclusions have been reported on ferric ion transportation by transferrin. Some papers aa2-14

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Cell growth on immobilized cell growth factor. 5: S. Q. Liu et al.

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Figure 4 Promotion of fibroblast cell growth by free or immobilized transferrin: (a) /X, free transferrin (holo); O, immobilized transferrin (holo); &, free transferrin (holo) + insulin (10/zg/ml); O, immobilized transferrin (holo) + insulin (10/zg/ml); E], insulin (/~g/ml); (b) A, free transferrin (apo); O, immobilized transferrin (apo); &, free transferrin (apo) + insulin (10 #g/ml); O, immobilized transferrin (apo) + insulin (10 #g/ml); [:], insulin (10 #g/ml)

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have reported that transferrin forms complex with receptor and ferric ions are released into cytoplasm before the transferrin/receptor complex enters into lysosome. Other papers 1~.~5-~7 have reported that the transferrin/ receptor complex is internalized and releases ferric ion after entering into the lysosome. It has been reported that cell growth is inhibited by removal of transferrin from culture medium or by inactivation of transferrin receptor with antitransferrin receptor antibody, but that it is restored by addition of soluble ferric ion 1°as-2°. On this basis, it has been considered that ferric ion transportation by transferrin brings about cell growth. On the other hand, investigation using melanoma cells revealed that transferrin transports ferric ion and independently

stimulates cells toward proliferation2°. May and Cuatrecasas 16 indicated the possibility of transmembrane signalling. In the present investigation, although the immobilization transferrin did not particularly enhance adhesion of cells, it induced cell spreading in the initial stage (within 8 h) of cell culture more strongly and cell growth more effectively than free transferrin. These events must have resulted from the specific interaction between immobilized transferrin and the cells as indicated by the antibody experiments. The transferrin immobilized on PMMA membrane, as well as free transferrin, transported ferric ions across the cell membrane. Our results suggest that the internalization (endocytosis) of transferrin/receptor complexes is not always indispensable for ferric ion transportation. In addition, it was further shown that immobilized transferrin was similarly active in ferric ion transportation as free transferrin, but was more active in cell growth acceleration than free transferrin. These experimental facts imply that the cell growth acceleration and the ferric ion transportation by transferrin occur by different mechanisms.

Acknowledgement This research was partly supported by the Grant-in-Aid for Scientific Research on Priority Areas 'New Functionality Materials--Design, Preparation and Control' from the Ministry of Education, Science and Culture (No. 62604565).

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Cell growth on i m m o b i l i z e d cell g r o w t h f a c t o r . 5: S. Q. L i u et al.

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