Suspension Culture and Production of Secondary Metabolites

Introduction
Plant cell suspension cultures are mostly used for the biochemical investigation of cell physiology, growth, metabolism, and for large- or medium-scale production of secondary metabolites. For such purposes, normally suspension cultures are used which are propagated in Erlenmeyer flasks on a gyratory shaker and are maintained by regular subculturing after short intervals (usually 1 or 2 weeks). For such use, callus cultures are also maintained under a mineral oil layer that reduces the subculture frequency. However, from the callus phase, it takes additional time to establish a new suspension culture again, and this new suspension may exhibit changed biochemical traits.

For storage purposes, several strategies have been developed, all aiming at reduction of labor and costs of maintenance, while preserving all properties of the cells. Long-term conservation of suspension cultures is usually successful by cryopreservation. However, it requires special and expensive equipment and is not suitable for routine work, as reactivation of cells requires prolonged incubation and has to pass a callus phase. Therefore, for practical work there is a need for an easy and a time-saving method that could allow the storage of actually investigated suspension cultures for relatively longer terms.

A simple procedure is described here to maintain suspended plant cell cultures for medium terms. With this method suspension cultures of Agrostis tenuis, Nicotiana tabacum, Nicotiana chinensis, Oryza sativa, and Solanum marginatum, could be maintained viable under reduced temperatures for more than 4 months without transfer to fresh medium. The suspension cultures were kept without shaking at 10°C (in dark or in dim light at about 50 lux) in screw-cap plastic bottles (tissue culture flasks with membranes) that permitted sterile air to pass through easily. Some effective adsorption materials or stabilizers such as activated charcoal, gelatine, glutamic acid, starch, sugar, etc. were added to the cell suspensions. In the case of sensitive microorganisms and unicellular green algae, such substances have already shown protection during maintenance and against known freezing and drying injuries. In the case of plant cell suspensions, only with Nicotiana tabacum did the addition of 0.01% charcoal +1.0% gelatine result in slightly improved cell survival as compared to the cells grown without additives. However, the simple maintenance conditions used proved effective to prolong the storage life of suspension cultures as compared to control samples grown under normal conditions. The stability of the stored cells usually appeared unchanged, as evident from HPLC-fingerprints, and from growth characteristics after re-establishment of the cultures.

The method is convenient and the equipment described here is easily available. It is useful for routine work, as during such long-term maintenance a ready inoculum can continuously be obtained (from the same batch of cell suspension) for immediate use.

Material and Methods
Preparation of Cultures
Plant cell cultures are grown under normal conditions using routine methods. The suspension cultures should be propagated in Erlenmeyer flasks on a gyratory shaker (100 rpm) at 24°C under continuous light at 600 lux. These should be transferred to a fresh medium every 7–10 days.

Storage of Cell Suspensions
For storage, 100 mL of the cell suspension from the early exponential growth phase is transferred to a 650-mL screw-cap plastic bottle with membranes, which permits sterile air to pass through. Depending on the volume of cell suspension to be maintained, smaller 250-mL bottles with 43-mL cell suspension can also be used. However, the surface to volume ratio should remain the same as with big bottles. These bottles are supplied as tissue culture flasks with closure, secure against contamination. Alternatively, wide-mouth Erlenmeyer flasks with new cotton plugs can also be used. The volume of the added cell suspension in the vessels should lead to a high surface to volume ratio in order to assure sufficient oxygen supply for the cells. The suspension cultures are stored without shaking in the dark and at a reduced temperature of 10°C. To prolong cell survival, it is recommended to add some stabilizers, such as 0.01% activated charcoal + 1.0% gelatine, or doubled to tripled concentration of sugar

Recultivation and Viability Assay
For recultivation, an aliquot of 5 mL of the stored cell suspension is transferred with a sterile pipette from the plastic bottles to 100 mL Erlenmeyer flasks containing 25 mL of medium. The cells are cultured in these flasks for 1–2 weeks at 24°C on a gyratory shaker (100 rpm) under continuous light (600 lux). To determine regrowth on solid media 1 mL of cell suspension is placed on a Petridish (60-mm diameter).

For a periodic viability assay of the preserved cultures, the ability of regrowth is determined. After appropriate intervals of storage (every 2–3 weeks or longer, depending on the sensitivity of the cell suspension) aliquots of the stored cells and the control samples (grown under normal conditions) are recultivated in equal amount of liquid media. After a definite period of growth (about 1 week when the cells are in exponential growth phase), the cells are harvested by suction filtration. The dry weight of the regrown cells is determined, which is taken as a measure of the inoculum quantity (the cell density developed from the surviving cells during storage). The resulted cell mass from the stored cells is compared after different storage intervals and with the control samples.

Staining of a very small aliquot with fluorescence in diacetate (FDA) on a microscope slide and counting of a fraction of positive cells under the fluorescence microscope is also an easy tool to obtain a quick impression of the viability of the cells.

Stability Checking
For stability checking by HPLC, 2 g (fresh weight) of the harvested cells are extracted in boiling methanol for 1 hour. Twenty mL of the resulting extracts are analyzed by reverse phase high pressure liquid chromatography (HPLC). The result of the chromatographic procedure is a characteristic “fingerprint” of cell metabolites. Methanol is used as an organic solvent. Substances are eluted by a standard gradient system, which runs from 0% methanol to 100% methanol within 30 minutes. The majority of cell cultures show most characteristic “fingerprints” when a slightly acidic solvent system is used. Comparability of such “fingerprints” has to be assured by extracting cells from the same growth stage of the cell culture

High Pressure Liquid Chromatography (HPLC) Protocol

Column:	Nuleosil 100-7 C18, 7 mm, 4 × 100 mm
Flow rate:	1 mL/min
Solvent A:	Water + 0.1 mL H3PO4
Solvent B:	Methanol + 0.1 mL H3PO4
Gradient:	0 min: 100% Sol. A/0% Sol B
	5 min: 100% Sol. A/0% Sol B
	35 min: 0% Sol. A/100% Sol B
	40 min: 0% Sol. A/100% Sol B
	45 min: 100% Sol. A/0% Sol B
	Detector wavelength: 280 nm

Notes
Plant cell suspension cultures are normally cultivated in Erlenmeyer flasks on gyratory shakers. At normal growth temperature, when gyration of these flasks is not maintained, plant cells normally settle down very fast. Under such conditions, a lack of oxygen supply leads to cell death, or at least cell damage, within a few hours for most species. However, during a systematic study, we observed that such plant suspension cultures are able to survive at reduced temperatures and without gyration much longer (more than 16 weeks). The influence of doubling or tripling of the sugar content of the media (up to 6% sucrose or glucose) tested showed that one of the Nicotiana tabacum lines resulted in a re-established culture even after 26 maintenance-free weeks at 10°C (with 6% sucrose instead of normal 3% concentration). For Cinchona robusta, cell suspensions (recalcitrant to any tested cryopreservation protocol) after 8 weeks of standing culture at 10°C survival was still high, but no regrowth was obtained after 16 weeks. Nevertheless, after 16 weeks with FDA staining, 60% “viability” was detected. This indicates that optimized conditions may improve the regrowth.

The onset of regrowth normally occurred within a few days, and after 1 week, normal growth parameters were restored. Nevertheless, with Oryza sativa, it took about 2 weeks before rapid cell growth resumed and the re-establishment of the normal growth pattern took 1 or 2 subculture stages.

One should be prepared that during maintenance of the standing culture, much of the medium water may evaporate. Depending on the dryness of the air in the incubator or room climate, up to 1/3 of the volume may disappear within 16 weeks. The bottles or flasks should, however, not be sealed or closed air tight to minimize evaporation because of disturbances in the aeration. To avoid this, one may start with more volume if frequent samples of inoculum are required.

To optimize the maintenance conditions in one’s own laboratory, and to establish a maximum storage period for the cell suspension under study, it is recommended to conduct perodic viability assay and the stability check of the chemical traits of the preserved cultures. To check whether cell metabolism is affected by these storage conditions or the added substances, methanolic extracts should be analyzed by reverse phase HPLC. The analysis will show characteristic peak patterns (which need not be identified chemically). The storage conditions and reduced incubation temperature should not cause any changes in the characteristic of such peak patterns.

For rice culture regeneration, experiments were carried out. Dependent on the amount of extra sugar during standing culture, regeneration was higher or lower than in the controls, but high regeneration capacity appeared to be correlated with a high incidence of aberrant plants: 20% was albino. Therefore, it is recommended (as with other preservation methods) to check for the occurrence of somaclonal variations.

Although the stabilizers showed no remarkable increase in survival of few of the tested cultures, they might cause some kind of protection in other cultures and result in better stability and viability of such cell suspensions, at least over few passages.