1. Fresh medium vessel

2. Feeding pump

3. Effluent pump

4. Effluent vessel

5. Sensor

6. Feeding line

7. Effluent line

8. Air line

9. Sedimentation column

10. Impeller

11. Air inlet

12. Air outlet

13. Sampling system

14. Sampling line

15. Dectector

16. Recorder

17. Stirrer

Figure 14. Continuous culture system.

Figure 15. High density Namalwa cell culture in serum-free medium.
Figure 16. A 45L SUS 316L Unit.

References and Bibliography (Section 2)

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6. Katinger, H. W. D., Schrirer, W„ and Kromer, E, Ger. Chem. Eng., 2:31 (1979)

7. Hosoi, S., Mioh,H.,Anzai, C., Sato, S., andFujiyoshi.N., Cytotech. 1:151-158(1988)

8. Toth, G. M., Cell Culture and Its Application (R. T. Actone and J. D. Lynn, eds.), p. 617, Academic Press Inc., (1977)

10. Blazar, B. A., Settor, L. M. and Strome, M., Cancer Res. 43:4562 (1983)

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13. Ku, K., Kuo, M. J., Delente, J., Wildi, B. S., andFeder, J.,Biotech. Bioeng., 23:79(1981)

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15. Fujiyoshi, N„ Hakko to Kogyo, 45(3): 198 (1987)

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22. Rosenberg, S. A., J. Natl. Cancer Inst., 75:595 (1985)

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24. Havell, E. A., Vilcek, J., Antimicrob. Agents Chemother., 2:476 (1972)

25. Knight, E„ h.,Proc. Natl. Acad. Sei., U.S.A., 73:520 (1976)

26. Strander, H., Mogensen, K. E., and Cantell, K., J. Clin. Microbiol., 1:116 (1975)

28. Knazek, R. A., Gullino, P. M„ Kohler, P. O., and Dedrick, R. L., Science, 178:65 (1972)

29. Evans, V. J., Bryant, J. C„ Fioramonti, M. C., McQuilkin, W. T„ Sanford, K. K„ and Earle, W. R., Cancer Res., 16:77 (1956)

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32. Morgan, J. F., Morton, H. J., and Perker, R. C., Proc. Soc. Exp. Biol. Med., 73:1 (1950)

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37. Imamura, T., Crespi, C. L., Thilly, W. G., and Brunengraber, H., Anal. Biochem., 124:353(1982)

38. Yamane, I., Murakami, O., and Kato, M., Proc. Soc. Exp. Biol. Med., 149:439(1975)

39. Barnes, D. and Sato, G., Anal. Biochem., 102:255 (1980)

41. Chohen, S. and Taylor, J. MRecentProgr. HormoneRes., 30:535 (1974)

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44. Blundell, T. L. and Humbel, R. E„ Nature, 287:781 (1980)

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CULTURES (by Shinsaku Takayama)

3.1 Background of the Technique—Historical Overview

Haberlandt[1] first reported plant cell, tissue, and organ cultures in 1902. He separated plant tissues and attempted to grow them in a simple nutrient medium. He was able to maintain these cells in a culture medium for 20 to 27 days. Although these cells increased eleven-fold in the best case, no cell division was observed. Gautheret[2] was the first to succeed in multiplying the cells from the culture in 1934. He used the cambial tissues of Acer pseudoplatanus, Salix capraea, Sambucus nigra. After 15 to 18 months in subculture, cell activity ceased. He reasoned that this inactiveness was due to the lack of essential substances for cell division. He suspected that auxin may have been one of the deficient substances. This compound was first reported in 1928 and was isolated by Kogel in the 1930' s. Addition of auxin to the medium prompted plant cell growth. This finding was reported almost simultaneously by Gautheret[3) and White'41 in 1939. Plant cell tissue and organ culture techniques rapidly developed, and in the mid-1950's another important phytohormone, cytokinins, had been discovered (Miller, Skoog, Okumura, Von Saltza and Strong 1955).|5] By 1962MurashigeandSkoog[6] had reported a completely defined medium which allowed the culture of most plant cells. Their medium has now become the mostly widely used medium in laboratories around the world.

After these initial discoveries and some significant improvements in media, scientific research on the cultivation of plant cell, tissue, and organs shifted to the area of basic physiological research. Industrial applications were also sought in the production of secondary metabolites, clonal plants, and the improvement of various plant tissues.

Plant cell, tissue, and organ culture can be performed by either solid or liquid culture methods, however, in order to scale up the culture to the level of industrial processes, the liquid culture method must be employed.

Recently, pilot bioreactors as large as 20 kl have been constructed in the research laboratories of Japan Tobacco and Salt Co. and in those of Nitto Denko Co. Solid culture methods were used in large scale pilot experiments for the production of tobacco cells, and liquid culture methods were used in the production of Panax ginseng cells. An outstanding example of cell suspension culture in a pilot scale bioreactor (750 1) was the production of shikonins by Mitui Petrochemical Industries. In all these examples, various technologies have been used to improve the productivity of the metabolites.

The technologies include: (i) selection of a high yielding cell strain, (ii) screening of the optimum culture condition for metabolite production, (iii) addition of precursor metabolites, (iv) immobilized cell culture, and (v) differentiated tissue and/or organ culture. The productivity of various metabolites such as ginsenoside, anthraquinones, rosmalinic acid, shikonins, ubiquinones, glutathione, tripdiolide, etc., reached or exceeded the amount produced by intact plants. To date, the production costs remain very high which is why most of the metabolites are still not produced on an industrial or pilot plant scale. Development of large scale industrial culture systems and techniques for plant cell, tissue, and organs, and the selection of the target metabolites are the chief prerequisites for the establishment of the industrial production of plant metabolites.

Figure 17.

The area of plant cell, tissue and organ cultures.

Figure 17.

The area of plant cell, tissue and organ cultures.

3.2 Media Formulations

The formulation of the medium for plant cell, tissue, and organ culture depend primarily on nutritional requirements. Intact plants grow photoau-totrophically in the soil, (i.e., they use C02 as the principal carbon source and synthesize sugars by photosynthesis). In the case of aseptic cultures however, establishment of an autotrophic culture is not achieved so that heterotrophic or mixotrophic growth becomes the distinguishing characteristic. Therefore, such cultures require the addition of carbon as an energy source. Given this fact, the culture medium must be formulated as a chemically defined mixture of mineral salts (macro- and microelements) in combination with a carbon source (usually sucrose). In addition to these constituents, organic constituents such as vitamins, amino acids, sugar alcohols, and plant growth regulators are usually added to the medium. Media commonly used are listed in Table 11.

Table 11. Formulations of most frequently used plant tissue culture media

Ingredients (mg MS B5 White Heller

(NH4)2S04 134

(NH4)N03 1650

NaN03 600

KN03 1900 2500 80

Ca(N03)2 300

CaCl3-2H20 440 150 75

MgS04-7H20 370 250 720 250

Na2S04 200

KH2P04 170 125

NaH2P04H20 150 16.5

KC1 65 750

FeS04-7H20 27.8 27.8

(Cont'd next page)

Table 11. (Cont'd.) Formulations of most frequently used plant tissue culture media.

Ingredients (mg MS B5 White Heiler


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