Attachment, spreading and growth of VERO cells on ... - Springer Link

Bioprocess Engineering 20 (1999) 565±571 Ó Springer-Verlag 1999

Attachment, spreading and growth of VERO cells on microcarriers for the optimization of large scale cultures R.Z. MendoncËa, J.C.M. Prado, C.A. Pereira

Abstract The VERO cell attachment, spreading and growth were measured as a function of the substrate and temperature used for cell cultivation, the presence of fetal calf serum (FCS) in the medium and the initial cell inoculum used for cultivation on MCs. The data show that the cell attachment kinetics were comparable at RT or 37 °C, a higher rate of cell attachment occurred to MCs and the presence of FCS inhibited the cell attachment to glass or plastic but not to MCs. The cell spreading, in general higher at 37 °C, was dependent on the presence of FCS, comparable on glass or plastic substrate and lower on MCs. The spread of VERO cells over MCs was fully dependent on the presence of FCS and decreases progressively with a delayed addition of FCS into the medium. The cell detachment by trypsin was slower from MCs and the cells recovered showed lower viability and reattachment. Better results of detachment, viability and reattachment were obtained by treatment with the trypsin at pH of 8 instead of 7. The lower was the number of cells/MC for the initial inoculum, the higher was the percent of unoccupied MCs (with 1 cell/MC we had 35.6% of unoccupied MCs), which were shown to remain uncovered during the whole period of culture. With an initial inoculum of 4, 6 and 8 VERO cells/MC, respectively 46%, 76% and 83% of the MCs were totally covered by cells after 7 days, the cultures showing at this time, respectively, 5.1 ´ 105, 8.8 ´ 105 and 1.8 ´ 106 cells/ml, which represented a biomass production of respectively 8.5x, 9.7x and 15.5x. When compared to 175 cm2 T-¯asks, using the same amount of medium, a VERO cell culture on 2 mg/ml of MCs offers about 10 times more available

surface for cell growth and allowed the obtention of 7 times more cells. The optimization procedures concerning initial steps of VERO cell cultures, such as the attachment, spreading and growth as a function of parameters like initial cell inoculum and medium supplementation are of special interest mainly due to the perspective of a large use of VERO cell cultures for human viral vaccine production.

1 Introduction Basically two types of animal cells are available for cultivation and use in research and production. The cells, such as hybridomes, insect cells and some cells of mammalian origin, that do not attach to a surface and as a consequence can grow free in suspension, and the cells, such as most of mammalian origin, that can only grow when attached to a surface and are called anchorage-dependent cells. Free suspension cell cultures and anchorage-dependent cell cultures are suitable for different purposes and efforts have been done in order to optimize the cell or cell product yield and minimize the ®nal cost of derived products (1±9). The microcarrier system, where the cells can multiply attached to microspheres (microcarriers) in suspension, represents a suitable system for large scale cell culture (10±12). The VERO cell line is a continuous cell line recommended by the World Health Organization as a substrate for the production of immunobiologicals for human use (13,14) and has been employed in the preparation of human vaccines (15). By using VERO cells and the microcarrier system, we have established a methodology for the preparation of rabies virus vaccine and measles virus antigen (16,17). In view of an optimization of the cell culture procedure, we have studied some metabolic parameters, Received: 10 August 1998 such as the consumption of glucose and glutamine and R.Z. MendoncËa, J.C.M. Prado, C.A. Pereira the production of ammonia and lactic acid, showing Instituto Butantan, Laboratorio de Imunologia Viral, Av. Vital that the ®nal cell yield can be improved by the modulation Brasil 1500, 05503-900 Sao Paulo, Brasil of the supply of nutrients and control of metabolites Tel.: +55 11 8137222 ext 2233, Fax: +55 11 8151505, production (8). Besides the metabolic parameters that e-mail: [email protected] or [email protected] can undoubtedly contribute for the optimization of cell cultures, physical parameters, such as good conditions Correspondence to: C.A. Pereira for the attachment of VERO cells to microcarriers, have This work was supported in part by grants from the FundacËao de been explored in technological studies recently published Amparo aÁ Pesquisa do Estado de S. Paulo (FAPESP), FundacËao (5,18). Instituto Butantan and Conselho Nacional de Desenvolvimento In the present work, we investigated the culture conCienti®co (CNPq). C.A. Pereira is recipient of a Conselho ditions for VERO cell attachment and spreading on solid Nacional de Desenvolvimento Cienti®co (CNPq) fellowship. spherical microcarriers, the detachment, reattachment and We thank A.C. Barbosa and C. Nascimento for technical viability of the cells after trypsinization as well as the assistance.

565

Bioprocess Engineering 20 (1999)

optimal cell yield and biomass production obtained with different initial cell inocula.

2 Material and methods

566

2.1 Cell cultures VERO cells at the 142 passage were cultivated in Roux bottles in Leibowits 15 medium containing 5% fetal calf serum (FCS). A cell bank prepared with the cells at the 145th passage was used in all the experiments. Cell cultures were performed in glass bottles, plastic culture T¯asks, or spinner ¯asks with a working volume of 100 ml. Cytodex 1 (Pharmacia Fine Chemicals, Uppsala, Sweden) was used as MCs for VERO cell cultures at concentration of 2 mg/ml of ®nal culture volume. They were washed three times in phosphate buffered saline without Ca++ and Mg++, at pH 7.2 and autoclaved at 120°C for 20 min. Several independent experiments were performed and all points shown are the means from three independent cultures. 2.2 Cell attachment and spreading on glass, plastic and MCs These experiments were carried out inoculating 106 VERO cells in 25 cm2 glass culture bottles or plastic cultures T¯asks or inoculating 8 cells/MC (107 VERO cells) in a spinner ¯ask with a working volume of 100 ml containing 2 mg/ml of MCs. The spinner cultures were performed with an intermittent stirring regimen of 2 min. stirring at 30 rpm per 15 min. The cultures were performed in presence or absence of 5% FCS and incubated at room temperature or 37 °C with 5% CO2. At different times the glass bottle or plastic T-¯asks cultures were examined microscopically and the rate of attachment/spreading was recorded. For the spinner cultures, samples of 1 ml were removed into Eppendorf tubes and then examined microscopically to determine the rate of attachment/spreading. At least 100 cells were counted for each determination to allow a statistical signi®cance and the results were expressed in percent of attachment/spreading. Alternatively, experiments were carried out in order to investigate the in¯uence of FCS on the cell spreading on MCs. In these experiments 8 cells/MC (107 VERO cells) were inoculated in a spinner ¯ask with a working volume of 100 ml containing 2 mg/ml of MCs and incubated at 37 °C with 5% CO2. Cultures were performed in the presence or absence of 5% FCS or it was added to the cultures 30, 60, 90, 120 min. after the cell inoculation. Cells were examined as described above and the results expressed in percent of spreading. For the study of cell detachment, viability and reattachment, the cultures were incubated at 37 °C and for observation the monolayers were washed once with PBS and treated with trypsin. The rate of detachment was microscopically evaluated in minutes, the rate of viability was microscopically evaluated by the percent of viable cells after trypan blue staining and the rate of reattachment was microscopically evaluated by the percent of reattached

cells on the respective surface after washing the cells with medium and reintroducing a fraction of them into new bottles, ¯asks or MCs.

2.3 Cell distribution and growth on MCs For these experiments, the VERO cells cultivated in Roux bottles were resuspended in 15 ml and transferred to the spinner ¯asks at the proportion of 1, 2, 4, 6, 8 cells/MC. The cultures were incubated at 37 °C and maintained with intermittent stirring regimen of 2 min. stirring at 30 rpm per 15 min. Samples were periodically removed and the cell distribution on the MCs were evaluated by the number of attached cells per MC, the percent of MC-bearing cells were evaluated by the number of MCs totally or partially covered with cells or uncovered and the kinetics of cell growth were evaluated by the number of cells per ml in the culture. The biomass production was calculated as ®nal cell yield (cells/ml)/initial cell inoculum (cells/ml). In order to compare the VERO cell yield in plastic T-¯asks and 100 ml spinner ¯asks with 2 mg/ml of MCs, the cell yield was also calculated as the number of cells/cm2. 3 Results 3.1 Kinetics of VERO cell attachment, spreading and growth VERO cell cultures were performed on glass, plastic or microcarriers at room temperature (RT) or 37 °C in the presence or absence of FCS. The data shown in Fig. 1 indicate that a higher rate of cell attachment occurred to MCs and a lower one to plastic surface, the presence of FCS in the culture medium having an inhibitory in¯uence on the kinetics of cell attachment to glass or plastic surface but no in¯uence on the kinetics of cell attachment to MCs. These kinetics were shown to be comparable at RT or 37 °C. The kinetics of cell spreading (Fig. 1), which were shown to be highly dependent on the presence of FCS, were comparable on glass or plastic surface and slower on MCs. The rates of cell spreading were higher at 37 °C than at the RT, with exception for the plastic surface where the temperature did not in¯uence the cell spreading. The data in Fig. 2 show that the ability of VERO cells to spread over MCs, was fully dependent on the presence of FCS and decreases progressively with a delayed addition of FCS into the culture medium. When VERO cell suspensions in 5% FCS supplemented medium were put in contact to MCs which were already incubated with FCS supplemented medium, the cell spreading started after 1 hour and reached 87% at 8 hours of culture. In cultures without FCS, the cell spreading started only about 2 hours after the FCS addition and then showed a faster progression reaching 82 to 88% of the cells at 8 hours (Fig. 2). The cell replication on glass, plastic or MCs, as evaluated by the number of cells in culture, showed comparable kinetics (data not shown). In Table 1 we show the rates of VERO cell detachment, viability and reattachment. The rates of cell detachment

R.Z. MendoncËa et al.: VERO cell growth on microcarriers

MCs with 7 cells/MC to 2% of MCs with 1 cell/MC and 1% of MCs with 15 cells/MC. Initial inocula of 1 and 8 cells/ MC led to a ®nal cell yields of, respectively, 1.1 ´ 105 and 1.8 ´ 106 cells/ml and a biomass production of, respectively, 7.3 and 15.5 (Table 2). An initial cell inoculum lower than 8 cells/MC were shown to leave unoccupied MCs, and the lower was the 3.2 number of cells/MC for the initial inoculum, the higher was the percent of unoccupied MCs. With 1 and 8 cells/MC Effect of initial VERO cell inoculation on cell distribution we had, respectively, 35.6 and 0% of unoccupied MCs and growth on MCs As shown in Fig. 3, a better cell distribution on MCs was (Table 2). As indicated in the Fig. 4, these MCs were shown to remain uncovered during the whole period of ensured by an initial inoculum of 8 cells/MC, which allowed, 4 hours after the inoculum, a variation from 13% of culture. With an initial inoculum of 4, 6 and 8 VERO cells/ mediated by trypsin treatment indicate that VERO cells detached faster from glass and slower from MCs. Cells detached from MCs showed lower rates of viability and reattachment. The data indicated also that better results of detachment, viability and reattachment were obtained with the trypsin at pH 8 instead of pH 7.

Fig. 1A±F. Kinetics of VERO cell attachment (*,h) and spreading (e,n) on glass (A,D), plastic (B,E) and microcarriers (C,F) as a function of the presence (h,n) or absence (*,e) of 5% FCS in the culture medium, at room temperature (A,B,C) and at 37 °C (D,E,F). 106 VERO cells were inoculated into 25 cm2 glass culture bottles or plastic culture T-¯asks. 8 cells/MC and a concentration of 2 mg/ml of MCs were used to perform the cultures in a spinner ¯ask with a working volume of 100 ml. The cultures were incubated at the indicated temperature with 5% CO2. At different times the cells were examined microscopically and the percent of attachment/spreading was recorded. The results are the average of three independent experiments

567


568

Fig. 2. Kinetics of VERO cell spreading on MCs as a function of the presence of FCS in the culture medium. 8 cells/MC and a concentration of 2 mg/ml of MCs were used to carried out the cultures in a spinner ¯ask with a working volume of 100 ml. The cultures were incubated at 37 °C with 5% CO2 and performed with culture medium without FCS (Ñ), with 5% FCS in the culture medium (*) or with the addition of 5% FCS into the culture medium 30 (h), 60 (s), 90 (e) or 120 min. (n) after the cells were inoculated. Different times after the cells were inoculated, samples were examined microscopically and the percent of spreading recorded. The results are the average of three independent experiments Table 1. Rates of detachment, viability and reattachment of VERO cells as a function of the surface used for cell cultivation and the pH of the trypsin used for cell detachment Surface

Trypsin pH Detachment Viability min. %

Reattachment %

glass plastic MCs

7

1 (0.5) 2 (1) 10 (5)

98 (2) 98 (2) 85 (5)

95 (3) 85 (4) 74 (6)

glass plastic MCs

8

0.75 (0.25) nd 2 (1)

98 (2) nd 90 (4)

95 (3) nd 85 (5)

VERO cell cultures were carried out in 25 cm2 glass culture bottles, plastic culture T-¯asks or in spinner ¯asks with 2 mg/ml of MCs and working capacity of 100 ml of culture medium with 5% FCS. The cultures were incubated at 37 °C and when monolayers were observed the cultures were washed once with PBS and treated with trypsin. The rate of detachment was microscopically evaluated in minutes, the rate of viability was microscopically evaluated by the percent of viable cells after trypan blue staining and the rate of reattachment was microscopically evaluated the percent of reattached cells on the respective surface after washing the cells with medium and reintroducing a fraction of them into new bottles, ¯asks or MCs. The results are the average (standard deviation) of at least three independent experiments

MC, respectively, 46%, 76% and 83% of the MCs were totally covered by cells after 7 days of culture (Fig. 4), showing a cell number in culture of respectively 5.1 ´ 105,

Fig. 3. Effect of the initial VERO cell inoculum on the cell distribution on MCs. Initial inocula of 1 (n), 2 (Ñ), 4 (h), 6 (s) or 8 (e) cells/MC and a concentration of 2 mg/ml of MCs were used to carried out the cultures in a spinner ¯ask with a working volume of 100 ml of culture medium with 5% FCS. The cultures were incubated at 37 °C with 5% CO2 and 4 hours later the MCs were examined microscopically and the number of attached cells per MC was recorded. The results, expressed as the percent of MC showing a given number of cells/MC, are the average of three independent experiments

8.8 ´ 105 and 1.8 ´ 106 cells/ml, which represented a biomass production during the culture period of, respectively, 8.5x, 9.7x and 15.5x (Fig. 5 and Table 2). When compared to 175 cm2 T-¯asks (150 ml of medium), a VERO cell culture on 2 mg/ml of MCs (150 ml of medium) which offers about 10 times more available surface for cell growth, allowed the obtention of 7 times more cells (Table 3).

4 Discussion The VERO cells are among those suitable for the production of immunobiologicals for human use such as the viral vaccines, since they are recommended by the World Health Organization (13,14) and because they allow a satisfactory multiplication of different viruses. Due to inherent characteristics of these cells, showing an anchorage dependent trait, they are best cultivated using a support where they attach, spread and then replicate. Among the essential conditions to be employed in large scale production of viral vaccines or antigens, this support must provide a large area for cell growth and enable an ef®cient virus infection of the whole culture. Both conditions are ful®lled by the solid dextran-charged microcarriers (MCs) (Cytodex 1). We have shown that a good cell culture performance can be achieved by using perfusion of culture medium and inlet of air to the culture, as indicated by parameters such as the number of cells/ml of medium or cells/mg of MCs, percent of MCs totally covered by cells or showing multilayered cells, maintenance of suitable levels of glutamine and galactose and elimination of NH3 and accumulation of


Table 2. Effect of the initial VERO cell inoculum on the proportion of unoccupied MCs and on the ®nal cell yield in culture

Initial cell inoculum cells/MC

cells/ml ´ 104

1 2 4 6 8

1.5 3 6 9 12

Final cell yield cells/ml ´ 104

Biomass production Final/Initial

Unoccupied MCs %

11 25 51 88 186

7.3 8.3 8.5 9.7 15.5

35.6 14.1 9.4 2.4 0

(2.6) (3.1) (2.5) (2.4) (5)

(0.2) (0.1) (0.2) (0.3) (0.3)

(8.5) (3.9) (2.8) (0.5)

Initial VERO cell inoculum and a concentration of 2 mg/ml of MCs were used to carried out the cultures in a spinner ¯ask with a working capacity of 100 ml of culture medium with 5% FCS. The cultures were incubated at 37 °C and after 7 days the MCs were examined microscopically and the % of unoccupied MCs as well as the ®nal cell yield were evaluated. The biomass production was calculated as ®nal cell yield/initial cell inoculum. The results are the average (standard deviation) of at least three independent experiments

low levels of lactate. In these conditions and cultivating the VERO cells on 10 mg/ml of MCs in a 3.7 liters bioreactor, we were able to obtain an amount, as great as 1010 cells, in a period of 7 days (or 2.5 ´ 1010 cells after 10 days) (8). The data presented in this paper show a study of the in¯uence of very initial steps of cell culture in view of a further improvement for the optimization of VERO cell cultures. We have investigated the VERO cell attachment, spreading and growth on different supports and the effect of initial cell inoculation on cell distribution and growth on MCs. In a recent publication, Shiragami et al. (9), studying the hydrodynamic effect between CHO-suspended cells and Cytodex 1 microcarriers, in cell attachment process, have shown that best performance was obtained by continuous agitation, and Ng et al. (5), in agreement to our data, have shown that the VERO cell attachment process to

Fig. 4A±C. Effect of the initial VERO cell inoculum on the percent of MC-bearing cells. Initial inocula of 4 (A), 6 (B) or 8 (C) cells/ MC and a concentration of 2 mg/ml of MCs were used to carried out the cultures in a spinner ¯ask with a working volume of 100 ml of culture medium with 5% FCS. The cultures were incubated at 37 °C with 5% CO2 and at different times the MCs were examined microscopically and the percent of MCs totally covered (s), partially covered (n) or uncovered (h) were recorded. The results are the average of three independent experiments

MCs was more ef®cient with intermittent stirring regimen. They have studied the VERO cell growth on gelatin-based macroporous microcarrier (Cultispher-G) showing the advantages of these macroporous MCs for VERO cell cultivation, although its use for viral vaccines production remains to be established due to the virus infection step in the process. We show here that the VERO cells showed a higher rate of attachment to MCs, when compared to plastic or glass, not being in¯uenced by the presence of FCS or by the temperature (Fig. 1). This observation is of particular importance for cultures on MCs due to the fact that the cell attachment, the ®rst event occurring in the cell culture process, takes place under stirring regimen which can be continuous or intermittent. For the cell spreading, the event following the cell attachment, our data show that it was always highly dependent on the presence of FCS and

569


570

Fig. 5. Effect of the initial VERO cell inoculum on the kinetics of cell growth. Initial inocula of 2 (h), 4 (e), 6 (n) or 8 (s) cells/ MC and a concentration of 2 mg/ml of MCs were used to carried out the cultures in a spinner ¯ask with a working volume of 100 ml of culture medium with 5% FCS. The cultures were incubated at 37 °C with 5% CO2 and at different times the cells were counted and the results are expressed as the number of cells/ml in the culture. Numbers indicate the biomass production (standard deviation) calculated as ®nal cell yield/initial cell inoculum. The results are the average of three independent experiments Table 3. VERO cell yield obtained in cultures on MCs and tissue culture ¯asks Area cm2 2 mg/ml MCs 1800 plastic T-¯asks 175

Medium ml

Space cm3

Cell yield ´ 105

150 150

800 920

1.25 (0.4) 15 (5) 1.7 (0.7) 2 (0.8)

cells/cm2

cells/ml

constitute important steps that may cause a loss in productivity. Our data (Table 1) show that VERO cell detachment by trypsin from MCs, when compared to those obtained from glass or plastic substrate, took longer and induced a loss of viability and lower rates of reattachment. Although not reaching the values obtained with glass or plastic, an improvement in the ef®ciency of cell detachment and reattachment was achieved by using the trypsin at pH 8 instead of pH 7. Nevertheless, the transfer of cells from occupied beads to new ones either by trypsinization or other processes of bead to bead cell transfer, are important subjects of study and source of great improvements in the cell culture scaling-up. Another initial step that in¯uences biomass production and also de®nes procedures of scaling-up, is based on the determination of the lowest number of cells necessary to constitute a good initial cell inoculum in order to allow the obtention of the highest ®nal cell yield. Our data (Table 2, Figs. 3, 4 and 5) show that the lower the number of cells/MC for the initial inoculum, the higher was the percent of unoccupied MCs, which were shown to remain uncovered for the whole period of culture. A better cell distribution on MCs was ensured by an initial inoculum of 8 cells/MC, which led to 0% of unoccupied MCs, 83% of the MCs totally covered by cells after 7 days of culture, and a higher ®nal cell yield (1.8 ´ 106 cells/ml) and biomass production (15.5). This study represents a further improvement for the optimization of VERO cell cultures on MCs and shows that relatively simple procedures in the initial steps of cell culture may allow the obtention, with 2 mg/ml of MCs, of about 10 times more available surface for cell growth and about 7 times more cells than other cell culture systems by using the same volume of medium (Table 3). The convenience of the VERO cell line for the human viral vaccine production as well as for other products, together with the capacity of MCs to support high cell densities, make these partners strong candidates for further developments of procedures of large scale cell cultures and a useful tool for industrial application.

VERO cell cultures were carried out in plastic culture T-¯asks or in spinner ¯ask with a working capacity of 150 ml of culture medium with 5% FCS. The cultures were incubated at 37 °C and References after 7 days the ®nal cell yield was evaluated. The results of cell 1. Bernard, A.R.; Kost, T.A.; Overton, L.; Cavegn, C.; Young, J.; yield are the average (standard deviation) of at least three inBertrand, M.; Yahia-Cherif, Z.; Chabert, C.; Mills, A.: Redependent experiments. Approximately space volume of ¯asks combinant protein expression in a Drosophila cell line: 3 was calculated in cm and the area available for cell culture calcomparison with the baculovirus system. Cytotechnol. 15 2 culated in cm (1994) 139±144 2. Singh, R.P.; Al-Rubeai, M.; Gregory, C.D.; Emery, A.N.: Cell death in bioreactors: A role for apoptosis. Biotechnol. Bioeng. higher at 37 °C. For the MCs, our results demonstrated that 44 (1994) 720±726 the presence of FCS was essential for the obtention of a 3. Mendonc Ëa, R.Z.; Pereira, C.A.: High density VERO cell culsuitable kinetics of cell spreading (Figs. 1 and 2). The ture on microcarriers in a cell bioreactor. Bioprocess Engiimportance of the presence of FCS from the very beginning neering 12 (1995) 279±282 of the culture on MCs, lays on the evidence that MCs do 4. Blasey, H.D.; Aubry, J.P.; Mazzei, G.J.; Bernard, A.R.: Large not constitute a good substrate for cell spreading, when scale transient expression with COS cells. Cytotechnol. 18 (1996) 183±192 compared to glass or plastic substrates (Fig. 1). In view of scaling-up procedures, an important step in 5. Ng, Y-C.; Berry, J.M.; Butler, M.: Optimization of physical parameters for cell attachment and growth on macroporous the cell culture process which basically constitutes cell microcarriers. Biotechnol and Bioeng 50 (1996) 627±635 passage to a higher capacity environment, the current 6. Di Falco, M.R.; Bakopanos, E.; Patricelli, M.; Chan, G.; Conmethodology is based on cell trypsinization and then digote, L.F.: The in¯uence of various insect cell lines, p10 and lution into a larger bottle (for glass or plastic substrate) or polyhedrin promoters in the production of secreted insulinbioreactor (for MCs). For this purpose cell detachment like growth factor-interleukin-3 chimeras in the baculovirus expression system. J. Biotechnol. 56 (1997) 49±56 from the substrate and cell reattachment to a new one


7. McCarroll, L.; King, L.A.: Stable insect cell cultures for recombinant protein production. Current Opinion in Biotechnology 8 (1997) 590±594 8. MendoncËa, R.Z.; Pereira, C.A.: Cell metabolism and medium perfusion in VERO cell cultures on microcarriers in a bioreactor. Bioprocess Engineering 18 (1997) 213±218 9. Shiragami, N.; Hakoda, M.; Enomoto, A.; Hoshino, T.: Hydrodynamic effect on cell attachment to microcarriers at initial stage of microcarrier culture. Bioprocess Engineering 16 (1997) 399±401 10. van Wezel, A.L.: Growth of cell strains and primary cells on microcarriers in homogeneous culture. Nature 216 (1967) 64± 69 11. Pharmacia: Microcarrier cell culture: Principles and methods. Pharmacia. Uppsala, Sweden 1981 12. Butler, M.: A comparative review of microcarriers available for the growth of anchorage-dependent animal cells. In: Grif®ths, J.B. and Spier, R.E. (eds.): Animal cell biotechnology, vol. 3, pp. 284±303 Academic Press-London. 1988

13. World Health Organization: Acceptability of cells substrates for production of biologicals, WHO Technical Report Series, v. 747, pp. 5±24. Geneva: WHO 1987 14. World Health Organization: Requirements for continuous cell lines used for biologicals substances, WHO Technical Report Series, v. 745, pp. 99±115. Geneva: WHO 1987 15. Montagnon, B.; Vincent-Falquet, J.C.; Fagnet, B.: Thousand litre scale microcarrier culture of VERO cells for killed polio virus vaccine: Promising results. Devel. Biol. Stand. 55 (1984) 37±38 16. MendoncËa, R.Z.; Ioshimoto, L.M.; MendoncËa, R.M.Z.; De Franco, M.; Valentini, E.J.G.; BecËak, W.; Raw, I.; Pereira, C.A.: Preparation of human rabies vaccine in VERO cell culture using a microcarrier system. Braz. J. Med. Biol. Res. 26 (1993) 1305±1319 17. MendoncËa, R.Z.; Vaz de Lima, L.R.A.; Oliveira, M.I.; Pereira, C.A.; Hoshino-Shimizu, S.: Studies on the ef®ciency of measles virus antigen production using VERO cell culture in a microcarrier system. Braz. J. Med. Biol. Res. 27 (1994) 1575±1587.

571