Engineering and Manufacturing for Biotechnology - Marcel Hofman & Philippe Thonart

Initiation, growth and immobilisation of cell cultures of taxus spp.

for callus initiation as it increased both the percentage of callus induction from the needles and the amount of callus produced per explant (data not shown).

3.1.2 Effect of light on callus initiation

Callus initiation was reduced when explants were kept under cycled light. Callus induced under cycled light was hard, green and grew very slowly compared to the yellow-white callus induced in the dark. Most of the calli induced under cycled light photo-regime did not proliferate when subcultured and eventually turned brown.

3.1.3 Effect of plant species and explant type on callus initiation

The effects of plant species and explant type on callus initiation were evaluated using B5 medium supplemented with 5 mg/1 NAA and 20g/l sucrose. Seedlings, stems and needles of media and T. cuspidata were used as explants.

Because mature Taxus seeds have a dormancy requirement of up to 2 years, a method for rapid germination of Taxus embryos was required. The same protocol for the rapid in vitro germination of T. baccata L. cv. Stricta embryos (Zhiri et al., 1994) was used for media and T.cuspidata seeds. A 100% germination frequency was observed after 7 days of culture of the excised zygotic embryos on modified MS medium and incubated in the dark. These embryos were obtained by dissecting the seeds after soaking in tap water for 7 days as this helped break the seed dormancy by leaching of the endogenous abscisic acid. This result is consistent with that of Zhiri et al. (1994) working with T. baccata and T. canadensis.

It was observed that 16 hours photoperiod was required for the optimal growth of the germinated embryos. However, dark incubation stimulated callus formation on the embryo axis. This result is consistent with that of Flores et al. (1993) working with T. brevifolia embryos, who found that light improved embryo germination and growth into seedlings.

The best source of explant for callus induction was the seedling since they exhibited a shorter induction time (7 days) and produced more callus. In addition, this callus had a relatively higher growth rate during the next few subcultures. As shown in Table 4, callus induction on needles started within 10 days compared 20 days on stems.

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However, smaller calli were produced on needles and their growth rate during the following subcultures was too low. No major difference in either the induction time or the rate of callus induction was observed between the two species studied.

3.1.4 Effect of coconut water on callus initiation

The effect of coconut water on callus induction from the needles and stems of media and T. cuspidata was studied using the optimum concentration of plant growth regulators (NAA 5mg/l) in the dark. Addition of coconut water (10% v/v) to the medium not only increased the percentage of induction but also shortened the induction time (Table 4).

3.2. CALLUS GROWTH AND MAINTENANCE

Although the previous B5 medium supplemented with the optimum plant growth regulator (5mg/l NAA) was found to be suitable for callus induction from different explants, the rate of callus growth on this medium was slow. In order to improve the callus growth, the calli of the different cell lines were subcultured on two different media, CWT and TM5. A substantial improvement in the callus growth was observed, however a red-brown exudate leached into the culture medium and this yielded a relatively hard, clumpy and brown callus. This browning problem is reported by many researchers working with Taxus species (e.g. Gibson et al., 1995). After modifications of TM5 medium (Zalat and Mavituna, unpublished), the production of this brown exudate by the calli was completely eliminated. Within a few subcultures (4-6), we were able to obtain soft, friable, white or pale yellow and fast growing cell lines from the seedlings of the two Taxus species.

3.2.1Effect of explant type

It was observed that the type of explant affected the appearance and the rate of growth of the newly initiated callus. The calli derived from seedlings showed a faster

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improvement in their growth during the following subcultures compared to those initiated from stems and needles. The two cell lines initiated from media and T. cuspidata seedlings were designated as TMSD and TCSD and they were used in this study in addition to another cell line (TMNO) which was already initiated in our laboratory two years earlier from media needles. It was observed that the growth of the different cell lines improved significantly with time during the subsequent subcultures. This could explain the reason for the fast growth of TMNO cell line, compared to the other two newly initiated cell lines of TMSD & TCSD (Figure 1).

3.2.2 Effect of light on callus growth

Figure 2 illustrates the growth index of TMNO callus cultured on CWT medium under either cycled light photo-regime or in the dark. It was observed that callus cultured in the dark had a better growth rate than those growing under cycled light photo-regime.

After subculturing for 10 days, the callus growth index started to show a significant difference between the two photo-regimes. At the end of 30 days, the growth index of callus grown in the dark was nearly four times that of callus under cycled light treatment. The cultures incubated in cycled light showed a long lag phase for 16 days compared with just 10 days in the dark. Callus grown in the cycled light also seemed to enter a stationary phase after 20 days.

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In general, the callus cultures grown under cycled light showed a decline in growth rate over several subcultures. By the end of the 5th subculturing, only callus induced from

WP medium could be maintained with steady growth; all other calli turned brown and their viability decreased under cycled light photo regime.

3.3. SUSPENSION CULTURES

The growth kinetics of the suspension cultures of the three cell lines from Taxus species was studied in the dark. Cultures displayed a characteristic, slightly bi-phasic pattern for the increase in fresh weight over time in all the cell lines studied.

The time course of the biomass increase (fresh and dry weight), viability of cells and medium pH of TMNO cell suspension cultured in modified B5 medium, in the dark is illustrated in Figure 3. The medium pH of the different cell lines growing in the dark dropped slightly after the first five days of culture and then increased gradually. The same pattern of the pH change with time was reported for T. cuspidata cell suspension in shake flasks (Fett-Neto et al., 1994b) and T. baccata cell suspension in bioreactor (Srinivasan, et al., 1995). They suggested that the changes in the medium pH may be related to the uptake and utilisation of nitrogen sources from the medium by the cells.

It was observed that the viability of the cells of TMNO suspension culture was higher than 95% during the first 25 days of incubation, then it began to decrease and dropped to about 75% on day 45 of culturing. Retention of the high viability of cells in suspension culture during the growth cycle is one of the most important requirements for increasing the subsequent culture productivity.

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Initiation, growth and immobilisation of cell cultures of taxus spp.

Rapid uptake of sucrose and its hydrolysis to glucose and fructose followed by preferential uptake of glucose was observed in all cell lines (Figure 4). This has also been observed in other suspension cultures of Taxus species (Fett-Neto et al., 1994b, Wickremesinhe and Arteca, 1994; and Pestchanker et al., 1996). The accumulation of biomass was closely linked to the consumption of sugars in the medium. The onset of

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reduction in dry weight concentration coincided with the exhaustion of sugars from the medium on day 12.

3.4. IMMOBILISATION

media cells were immobilised in the open (continuous) pore network of the reticulated polyurethane foam matrices initially by the process of filtration brought about by agitation in the bulk liquid (Mavituna et al., 1987). The initial entrapment through filtration led to the adhesion of cells and cell aggregates to the polyurethane fibres within the pores of the reticulated foam. As the pores of foam matrix were filled with the subsequent growth of the cell aggregates, the cells started to grow on the exterior surface of the foam matrix. Figure 5 shows a comparison of growth of suspension and immobilised media cells in 250 ml shake flasks containing 100ml CWT medium. From this figure it is clear that immobilisation promoted cell growth. This observation, which was a repeat of our previous experience with other plant cells immobilised in reticulated foam matrices (Mavituna et al., 1987), indicates that plant cells seem to benefit physiologically from being immobilised (Haldimann and

Brodelius, 1987). The biomass yield per flask was about 4 times that of the freely suspended culture, starting with the same inoculum concentration, after 30 days of cultivation.

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Initiation, growth and immobilisation of cell cultures of taxus spp.

3.5. GROWTH IN BIOREACTORS

The cells in suspension culture were small and tended to float to the top of the liquid medium. Therefore, cells aggregated easily to form a “meringue” on the liquid surface in the headspace in the reactors. There was not much indication of growth in the bubble reactor. The best growth was obtained in the airlift reactor, but it formed a “meringue” of cells too. The stirred tank reactor had two impellers and the cells settled on the top impeller and again formed a “meringue”. The immobilised cell reactor provided the best result in terms of increased growth yields and cell viability (Figure 7). Figure 6 gives the growth index after 30 days of cultivation in different bioreactors.

In terms of operation, the immobilised cell bioreactor was easier to handle. It was very easy to drain off the old medium and supply fresh medium aseptically keeping the immobilised cells in the bioreactor. This system could be run in an extended period of drain-and-fill repeated batches or in continuous mode of operation.

3.6. PACLITAXEL PRODUCTION

Suspension cultures of media yielded 159 µg/l paclitaxel in shake flasks on the 15th day of culturing in CWT medium corresponding to 12.4 µg paclitaxel per g dry weight cells. The immobilised cells under the same conditions yielded 294 µg/l paclitaxel what corresponds to 11.0 µg paclitaxel per g dry weight cells.

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4. Conclusions

Calli of media and T. cuspidata were efficiently induced from needles, stems and seedlings on Gamborg B5 medium supplemented with 20g/l sucrose, 5mg/l NAA, 10% v/v coconut water. Addition of coconut water to the culture medium was found to have an enhancing effect on callus initiation. Kinetin and cycled light photo regime had detrimental effects on callus initiation. The CWT medium formulated in this work yielded fast growing cultures.

One of the major problems in plant cell culture of Taxus species is the browning of the culture which results in very slow growth rates and often cell death. We succeeded in preventing cell browning completely in the callus by modification of TM5 medium and within 3-5 subcultures, we were able to obtain a white, soft and friable callus. media cells were immobilised successfully in the reticulated polyurethane foam matrices both in shake flasks and a 4L bioreactor. Immobilisation affected the culture performance positively. Compared to the suspension cultures, the biomass yield of the immobilised cultures were higher and the specific paclitaxel yield was almost the same. This means that our immobilised cultures lead to increased product concentration in the bulk liquid. It is also very easy to change the culture medium in our immobilised cell bioreactor allowing drain-and-fill repeated batch cycles and integration of product separation with bioreactor operation.

Strategies involving cell line selection and storage, manipulation of culture morphology and redifferentiation, control of the physico-chemical environment of the cultures through bioreactor design and mode of operation, cell immobilisation, continuous product removal, genetic manipulation of plant cells, integration of plant cell culture activity with biotransformations and combinatorial chemistry techniques should bring the potential use of plant cell, tissue and organ cultures for in vitro production of pharmaceuticals and other fine chemicals a step closer to realisation.

Advances in metabolic engineering (Stephanopoulos et al., 1998), plant genetic engineering (Collins and Shepherd, 1997; Hall, 1999), combinatorial chemistry, instrumentation and analytical methods, computers and IT should accelerate the progress in this field. There are already established techniques for the production of various pharmaceutical compounds via transgenic plants as a consequence of these developments in other fields of science and engineering (Owen and Pen, 1996; Shahidi et al., 1999). As our need for new and affordable medicine continues, so will the interest in the use plants as a valuable source of new therapeutic and chemo-preventive drugs.

Acknowledgement

We would like to thank the Egyptian Government for their financial support for E Zalat and UK CVCP, HEFCE for the ORS award and UMIST for the Graduate Research Scholarship for C W Tang during this project. We are also grateful for the taxane standards from the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment of the National Cancer Institute, USA.

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