44).Mic s pro d fo nsion culture The vield of attached to a culture surface.So suspension culture systems In general cells have the same growth kinetics on cytodex microcarriers as they do on standard glass or plastic culture surfac 5.25).Provide culture conditions 然atog are opumal (sections.5)most cells will re ow with a pr unced fibroblast like n microcarriers since the spherical growth surface cannot be completely covered by the parallel array of cells 1.4.2 Production of large numbers of cells 流暗 e m rrier system (secu igh yield om s ures ca often be 11. an stage the sua pe re volume ar there large incre he cul re cycle ore f ells from a w ide Applications for small culture volumes include situations when only few cells are ure teg.clini al di area sis.volu d at the ocarriers ible. Maintain ng high de siti of nd st th Fig.10.Chinese hamster roblasts growing on Cytodex ic culture in a Petri dish. T.Utako) ncer Inst on 17
17 • Cells growing on culture surfaces often use medium more efficiently than the same cells growing in free suspension (44). Microcarriers provide a method for reducing the medium requirements of suspension cultures. • The yield of many strains of virus is greater when the cell substrate is grown attached to a culture surface. Some viruses (e.g. Herpes) grow poorly in free suspension culture systems. In general cells have the same growth kinetics on Cytodex microcarriers as they do on standard glass or plastic culture surfaces (figs. 5,25). Provide culture conditions are optimal (sections 3.4,3.5) most cells will retain their characteristic morphology, population doubling time and saturation density when growing on Cytodex microcarriers (figs. 5,9). Cells which grow with a pronounced fibroblast like morphology may have reduced saturation density on microcarriers since the spherical growth surface cannot be completely covered by the parallel array of cells. 1.4.2 Production of large numbers of cells A major area of application for microcarrier culture is the production o large numbers of cells. The advantages of the microcarrier system (section 1.1) can be used to obtain high yields of cells from small culture volumes. Cultures can often be initiated with 105 cells/ml or less and at the plateau stage the yield is usually more than 106 cells/ml (fig.9). This high yield of cells per unit culture volume and the large increase in cell number during the culture cycle (10-fold or more) make microcarrier culture an attractive technique for production of cells from a wide range of culture volumes. Applications for small culture volumes include situations when only few cells are available to initiate a culture (e.g. clinical diagnosis, cloned material). Microcarriers can be used to increase the culture surface area in small volumes and at the same time keep the density of cells/ml as high as possible. Maintaining high densities of cells leads to conditioning of the culture medium and stimulation of cell growth. With traditional monolayer techniques for small cultures it is not possible to achieve Fig.10. Chinese hamster fibroblasts growing on Cytodex microcarriers contained as a static culture in a Petri dish. (Original photograph by T. Utakoji, Cancer Inst., Japanese Fondation for Cancer Res., Tokyo, Japan, reproduced by kind permission)
Stage Primary Secondary Tertiary Quartenary Production volume: 10L 80L 650L Perfusion Control tests: Sterility Sterility enicity Mycoplasma kidney a high culture surface area/volume ratio (a x 4 cm2/ml in Petri dishes) Miccrer cutures provide a surface area/volume ratio of approx 20 cm2/ml.The incr noans that a area of diagn yield of cells is achieved before subculturing is necessary.In the sis or production of cloned material this technique leads to a reduction in the time required to grow cells for biochemical analyses(45). ssels.Microca rier culture also apid scaling up with a minimum number ieaesahagasbgacasggo up can be through the entire range of culture volumes and can wth viruses or cell products(section 1.4.3).The yield of cells from large-scale cultures using Cydotex is usually 10 cells/litre or more. 1.4.3 Production of viruses and cell products cultur s ar viru s or cell pro and th 心o0 e produc duc d i al ca el cult A wide variety of viruses c an be produced using Cytodex.including viruses sensitive res erpes (Table 1).Th an iws c oved syste em for prd in the oduction of igures 12 and 13 nd ccines1,1040.41.46.48-50. growth c ero cells and the production of Herpes simplex virus on 18
18 a high culture surface area/volume ratio (approx. 4 cm2 /ml in Petri dishes). Microcarrier cultures provide a surface area/volume ratio of approx. 20 cm2 /ml. The increase in culture surface area means that a greater yield of cells is achieved before subculturing is necessary. In the area of clinical diagnosis or production of cloned material this technique leads to a reduction in the time required to grow cells for biochemical analyses (45). Figure 10 illustrates the high yield of cells which can be obtained from microcarrier cultures contained in traditional monolayer culture vessels. Microcarrier culture also provides a method for rapid scaling-up with a minimum number subculture steps (fig. 11). Scaling-up can be through the entire range of culture volumes and can also be achieved in the absence of subculturing steps by using a continuos propagation technique (6). This technique provides sustained periods of exponential growth. Large culture volumes of several litres or more are mainly used for production of viruses or cell products (section 1.4.3). The yield of cells from large-scale cultures using Cydotex is usually 109 cells/litre or more. 1.4.3 Production of viruses and cell products Cells cultured on microcarriers are often used as substrates for the production of viruses or cell products and the microcarrier method is compatible with standard production procedures. Cytodex can be used for the production of all substances which can be produced in animal cell culture. A wide variety of viruses can be produced using Cytodex, including viruses sensitive to growth in suspension cultures, e.g. Herpes (Table 1). The microcarrier system allows cultivation of large quantities of virus in compact culture units and provides an improved system for the production of many vaccines (1,40,46,48). Vaccines produced in the microcarrier system include polio, rubella, rabies, influenza, and foot-and-mouth disease (FMD) vaccines (1,10,40,41,46,48-50). Figures 12 and 13 illustrate the growth of Vero cells and the production of Herpes simplex virus on microcarriers. Stage Production volume: Primary Secondary Tertiary Quartenary Perfusion Control tests: Sterility Sterility Virus Mycoplasma Sterility Oncogenicity Mycoplasma Virus 10 L 80 L 650 L Fig.11. Scaling up microcarriers cultures for the production of large numbers of cells. Example illustrates the subcultivation system for production of polio vaccine from monkey (Cynomolgus) kidney cells growing on Cytodex microcarriers. (Adapted from van Wezel, A.L., van der Velden-de Groot, C.A.M., van Herwaarden, J.A.M., Develop. Biol. Standard. 46 (1981) 151)
20h 45h 96h 71h 119eh Fig12.Culture of Vero cells on Cyto photograph by B.Griffiths.CAMR.Porton Down.UK.reproduced by kind permission). owth of Verc implex 9.0 3735 8.8 pl6tocaries s of hw 0 8.6 8.4 HSV-2 8.2 20 40 6080100120 Hours Table 1.Some viruses which have been grown in cultures using Cytodex microcarriers. Polio Rous sarcoma Foot-and-mouth Herpes virus 40 eslclgrstomaits olyom abies quine rhi Endogeneous C.type The advantages of using microcarrier culture for vaccine production include increased productivity.reduced costs and reduced contar nation when compared with other cell culture methods (section 1.1).Sinskey et al(51)observed that the volumetric productivity of Sindbis virus in microcarrier cultures is in excess of 50-fold greater than that of roller bottles.Van Wezel et al(49.59)have developed a "Unit Process"for the production of polio and rabies vaccines using Cytodex aeme ethe p e pllo vaccine is shown in Figure 11.Serial cultivation on Cytodex reduces the requirement for a source of primary cells and provides a production culture of 650 litres. 19
19 Table 1. Some viruses which have been grown in cultures using Cytodex microcarriers. Polio Rous sarcoma Foot-and-mouth Rabies Herpes Vesicular stomatitis Rubella Simian virus 40 Group B arboviruses Influenza Polyoma Equine rhinopneumonitis Sindbis Pseudorabies Bovine rhinotrachteitis Sendai Vaccinia Endogeneous C-type Marek’s Adenovirus Papova virus Measles Parvovirus Respiratory syncytial virus The advantages of using microcarrier culture for vaccine production include increased productivity, reduced costs and reduced contamination when compared with other cell culture methods (section 1.1). Sinskey et al (51) observed that the volumetric productivity of Sindbis virus in microcarrier cultures is in excess of 50-fold greater than that of roller bottles. Van Wezel et al (49,59) have developed a “Unit Process” for the production of polio and rabies vaccines using Cytodex microcarriers and the productivity and efficiency of such a system is illustrated in Table 2. An example of a cell culture scheme for the production of inactivated polio vaccine is shown in Figure 11. Serial cultivation on Cytodex reduces the requirement for a source of primary cells and provides a production culture of 650 litres. Hours 37˚ 35˚ HSV-2 0 20 40 60 80 100 120 8.2 8.4 8.6 8.8 9.0 8 9 10 log total cells (- -) 10 log TCID (- -) 10 50 Fig.12. Culture of Vero cells on Cytodex microcarriers used for the production of Herpes simplex virus (HSV-2). The culture was infected with HSV-2 after approx. 50 h. CPE-cytopathic effect. (Original photograph by B. Griffiths, CAMR, Porton Down, UK, reproduced by kind permission). Fig.13. The growth of Vero cells and Herpes simplex virus (HSV-2 in stirred cultures containing Cytodex microcarriers. Fig. 12. shows photomicrographs of this culture. (Griffiths, B., Thornton, B., McEntee, I., Eur. J. Cell Biol. 2 (1980) 606, reproduced by kind permission)
Table 2.Processing of polio virus type I from microcarrier cultures using Cytodex. Step Vol ug/ml Cgr5m2imson 348 762 19 n.d n.d. 3000c 30389 on-exchange 4 0.03 <0,23 Monovalent vaccine 74 1753 <0.03 0,23 PN content after gel filtration.40mg/ml:after ion exchange.8 /ml Gel filtration vith Ser Md Hee-de Rjk.EW.Devel Biol Stand ( Table 3.The average yields of polio virus from large scale cultures using Cytodex. Vero cells:1.x10 cell/ml with 1g Cytodex 1/itre Pollo virus: Type I Type ll Type Ill PCm光pd (DL/m 93 Monkey kidney cells/ml with 1-2 g Cytodex 1/itre Polio virus Type I Type lI Type IlI 39 49 sHaChA Deve ol aard (97)15 infectivity (8.84 ml or more)when usi ing human fibrob hiMRC-5) production of a stable polio vaccine from culture volumes of 140 litres (41) 80 yto Vero cels growing onCytode have Polio virus production can so be ken asan example llustrating yield of polio vir reported th eld of polio type Ⅲvirus et a e a tha he yi polio I was greater from microcarrier cultures than from cultur ask Cytodex has be en used for the production of rabies vaccine o) e by mult /m in mice 20
20 Table 2. Processing of polio virus type I from microcarrier cultures using Cytodex. Step Vol D-antigen Recovery Albumin IgG L DU/ml % µg/ml µg/ml Virus suspension 240 76 100 n.d. n.d. Clarification 248 64,2 87 1,000 300 Concentration 1 17,530 96 >30,000 >30,000 Gel filtration 4,5 3,465 85 0,23 2,0 Ion-exchange 4,5 3,465 85 0,03 <0,23 Sterile filtration 7,5 1,964 81 <0,03 <0,23 Monovalent vaccine 7,4 1,753 71 <0,03 <0,23 PN content: after gel filtration, 40mg/ml; after ion exchange, 8 µg/ml. Gel filtration was with Sepharose® 6B in a Amersham Biosciences K 215/100 column and ion-exchange chromatography was performed with DEAE-Sephadex® A-50. (van Wezel, A.L., van Herwaarden, J.A.M., van de Heuvel-de Rijk, E.W., Devel. Biol. Stand. 42 (1979) 65, by kind permission of the authors and the publisher.). Table 3. The average yields of polio virus from large scale cultures using Cytodex. Vero cellsa : 1.2x106 cells/ml with 1 g Cytodex 1/litre. Polio virus: Type I Type II Type III D-Antigen (DU/ml) 85 20 56 Infectivity (log10 TCID50) 8,1 8,2 7,5 Monkey kidney cellsb : 106 cells/ml with 1-2 g Cytodex 1/litre. Polio virus Type I Type II Type III D-Antigen (DU/ml) 80 30 40 Infectivity (log10 TCID50) >8 >8 >8 a Montagnon, B., Fanget, B. Nicolas, A.J., Devel. Biol. Standard. 47 (1981) 55. b van Wezel, A.L., van Steenis, G., Hannik, Ch.A. et al, Devel. Biol. Standard. 44 (1978) 159. Von Seefried and Chun (46) reported high yields of polio virus having high infectivity (8.84 log 10 TCID50/ml or more) when using human fibroblasts (MRC-5) growing on Cytodex. Vero cells growing on Cytodex have been used for the production of a stable polio vaccine from culture volumes of 140 litres (41). Polio virus production can also be taken as an example illustrating yields of virus from microcarrier cultures. The yield of polio virus from cultures using Cytodex is summarized in Table 3. Giard et al (2) reported that the yield of polio type III virus from microcarrier cultures (6.5 pfu/cell) was greater than the yield from roller bottles (4.0 pfu/cell). Similarly, Mered et al (52) observed that the yield of polio virus/cell was greater from microcarrier cultures than from culture flasks. Cytodex has been used for the production of rabies vaccine by multiple harvests from primary dog kidney cell cultures (48,50). The infective titre of the harvests from these cultures was 6.0±1.0 log10 LD50/ml in mice
FMD vaccines have been produced fro cells g n Cytodex and alit sto gave ns and it was (10)S the production of FMD virus fr and Whit ide (53)ha sion Mic of FMD vir nsion with infectivity and comple than asion cultu The complement-fixing activit /of FMD virus Ty e A from microcarrier cultures was at least 5 times that obtained from suspe (53). A large-scale controlled fermenter and Cytodex have been used for the prolonged e of cells sas et al (55) -term microcarrier cultures and noted that an advantage of this technique was that addition of new microcarriers to confluent cultur s caused a new wave of cell growth an viru oduction.The production f r typesofn mcer uturesecedn references 2.51.56. Microcarrier culture rovides a potential method for the mas roduction of fish virus vaccines.Nicholson observed that the production of infectious pancreatic necrosis virus from microcarrier cultures (44.5 TCID /cell)was nearly 3-fold greater than production of virus from culture flasks(16.0 TCID./cell). Interferon has been produced in high yield from microcarrier cultures.The first report(57)described yields of 4x103 IU HulFNB/106 human fibroblasts.A more detailed study examined various parameters and yield were increased to levels comparable to those obtained from traditional monolayer systems(58).Clark and Hirtenstein(59)optimized culture procedures for cell growth and modified the induction procedure to result in vields of 3x10 iU HulFNB/10 human fibroblasts This yield correspondend to 2x10 IU HulFNB/mg of Cytodex and the technique could be used to produce 3x10s IU HulFNB/5 litre culture.A procedure for producing HulFNB is included in section 6.2.1.By using Cytodex microcarriers in roller bottles Kronenberg obtained improved yields (approx.8-fold)of mouse fibroblast interferon(L.Kronenberg.pers.comm.185)The cultures used for these experiments are illustrated in Figure 22.Cytodex has also been used for the production of immune interferon,HulFNy(G.Alm,pers.comm.,184) Microcarrier culture has enabled the growth of large numbers of human colon carcinoma cells for the production of carcinoembryonic antigen(60 and the production of plasminogen activator from transformed mouse fibroblasts (K.Dane.pers.comm.186). Further information on the production and purification of specific viruses and cell products from microcarrier cultures can be obtained from Amersham Biosciences. 21
21 FMD vaccines have been produced from pig kidney cells growing on Cytodex and the vaccines were of good quality with long storage life (10). The FMD vaccines gave good protection of animals with no abnormal local reactions and it was not necessary to concentrate the antigen (10). Spier and Whiteside (53) have compared the production of FMD virus from BHK cells grown on microcarriers and in suspension. Microcarrier culture of FMD virus Type 0 gives a virus suspension with higher infectivity and complement-fixing activity than suspension culture. The complement-fixing activity of FMD virus Type A from microcarrier cultures was at least 5 times that obtained from suspension cultures (53). A large-scale controlled fermenter and Cytodex have been used for the prolonged culture of cells persistently infected with papova virus (54). Manousos et al (55) studied the production of oncornavirus in long-term microcarrier cultures and noted that an advantage of this technique was that addition of new microcarriers to confluent cultures caused a new wave of cell growth an virus production. The production of other types of viruses in microcarrier culture is described in references 2,51,56. Microcarrier culture provides a potential method for the mass-production of fish virus vaccines. Nicholson observed that the production of infectious pancreatic necrosis virus from microcarrier cultures (44,5 TCID50/cell) was nearly 3-fold greater than production of virus from culture flasks (16.0 TCID50/cell). Interferon has been produced in high yield from microcarrier cultures. The first report (57) described yields of 4x103 IU HuIFNß/106 human fibroblasts. A more detailed study examined various parameters and yield were increased to levels comparable to those obtained from traditional monolayer systems (58). Clark and Hirtenstein (59) optimized culture procedures for cell growth and modified the induction procedure to result in yields of 3x104 IU HuIFNß/106 human fibroblasts. This yield correspondend to 2x104 IU HuIFNß/mg of Cytodex and the technique could be used to produce 3x108 IU HuIFNß/5 litre culture. A procedure for producing HuIFNß is included in section 6.2.1. By using Cytodex microcarriers in roller bottles Kronenberg obtained improved yields (approx. 8-fold) of mouse fibroblast interferon (L. Kronenberg, pers. comm., 185) The cultures used for these experiments are illustrated in Figure 22. Cytodex has also been used for the production of immune interferon, HuIFNg (G. Alm, pers. comm., 184) Microcarrier culture has enabled the growth of large numbers of human colon carcinoma cells for the production of carcinoembryonic antigen (60) and the production of plasminogen activator from transformed mouse fibroblasts (K. Danø, pers. comm., 186). Further information on the production and purification of specific viruses and cell products from microcarrier cultures can be obtained from Amersham Biosciences