Cell Suspension Cultures

Abstract

Most cell suspension cultures originate from callus cultures due mainly to mechanical impact in agitated liquid media. In stationary cultures on agar, a suspension can be produced commonly by use of a sterile glass rod, or squeezing with a scalpel. In particular with 2.4D in an agar medium, a loosely connected cell population develops on the opposite side of the agar, which can be easily scraped off with a scalpel. An improvement can often be obtained by using ammonia as nitrogen source, probably due to the excretion of protons as exchange for its uptake by the cells.

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Most cell suspension cultures originate from callus cultures due mainly to mechanical impact in agitated liquid media. In stationary cultures on agar, a suspension can be produced commonly by use of a sterile glass rod, or squeezing with a scalpel. In particular with 2.4D in an agar medium, a loosely connected cell population develops on the opposite side of the agar, which can be easily scraped off with a scalpel. An improvement can often be obtained by using ammonia as nitrogen source, probably due to the excretion of protons as exchange for its uptake by the cells.

Callus cultures in a liquid nutrient medium are usually agitated, and after 10–14 days, this mechanical impact results in the development of cell suspensions consisting of cells from the periphery of the explants (Fig. 4.1). Beside healthy cells that continue to grow, such a suspension contains also dead or decaying cell material. If the methods described above fail to succeed, then an enzymatic maceration of callus material should be attempted (0.05% crude macerozym, 0.05% crude cellulose Onozuka P-1 500, and 8% sorbitol; King et al. 1973). Another possibility to produce a cell suspension is to first obtain protoplasts, as described later (Chap. 5).

Fig. 4.1

Histomorphological characterization of suspension cultures of Datura innoxia (Kibler and Neumann 1980). a Inoculum (250 µm filtrate, ca. 25×), b microcluster (ca. 125×), c histological structure of a secondary cluster cultured with kinetin (ca. 25×), d histological characterization of cell material cultured without kinetin, e cell suspensions of Datura innoxia without kinetin, and 
f with kinetin in the medium (1×)

The definition of a cell suspension still provokes controversial discussions. The original aim in the 1950s was to establish culture systems in which, similarly to algae cultures, a suspension of cells of higher plants would consist solely of single cells. In practice, this aim was reached only for a few systems, using a hanging drop method. All other attempts failed. Even in experiments starting with a population of single cells in a liquid medium, cell aggregations of various size will develop soon after initiation of growth, coexisting with some free cells (Fig. 4.1b).

Methods to Establish a Cell Suspension

As done for callus cultures, the description of how to obtain a cell suspension shall be illustrated in a practical example that can be easily adapted to many other systems. In this example, shoot explants of Datura innoxia were originally used to produce callus cultures to study the synthesis of secondary metabolites. For a better understanding, the establishment of callus cultures, now from shoot tissue, will be briefly described.

Explants of the uppermost (youngest) internode are cut using an extremely sharp scalpel. For sterilization, cut ends are briefly dipped into liquid paraffin to prevent the entrance of the agent used for surface sterilization, for 5–6 min in the hypochloride solution already described (Sect. 3.1). Using a laminar flow (aseptic working bench) for all further handling, internode segments 1–2 cm long are rinsed 4–5 times with sterilized distilled water. After this, the paraffin cover and the epidermis are removed with the help of a sterilized scalpel, and with a second sterilized ­scalpel, the tissue is cut into segments about 1 mm thick. These discs are cut into halves, which then serve to establish callus cultures. These segments are bigger than those used to establish primary carrot cultures, having a weight of about 7–8 mg, and consisting of about 30,000 cells each. If the diameter of the disc is even larger, then even more explants can be obtained. The nutrient medium is given in Table 3.3 (NL medium), and is suitable for both stationary and liquid cultures. Within 3 weeks of culture, a highly proliferate callus develops from which peripheral cells can easily be scraped off with the help of a scalpel. This cell material is transferred to the MS medium with agar (Table 3.3) supplemented with kinetin, for growth at 27°C at a 12 h light/dark rhythm. After two subcultures at an interval of 3–4 weeks, the subsequent subcultures are initiated every 2 weeks. Also for subculturing, only peripheral cell material from newly developed callus pieces is used.

After the production of sufficient cell material, the rather loosely connected clusters are transferred into a liquid medium of the same composition in Erlenmeyer flasks on a shaker. Within 1 week, a dense cell suspension develops. An inoculation of 5 g fresh weight corresponds to a cell density of 40,000 cells per ml of nutrient medium, sufficient for optimal proliferation of the cell population (Fig. 4.2).

Fig. 4.2

Growth of haploid (top) and diploid (bottom) cell suspensions of Datura innoxia (Kibler and Neumann 1980)

Cell Population Dynamics

A cell suspension usually consists roughly of three fractions, i.e., free single cells of various shapes, cell aggregates consisting of up to ten cells or more, and finally cell groups with a threadlike morphology. These fractions can be isolated by suitable sieving techniques. Investigations to characterize these three fractions indicated that cell proliferation by division occurs predominantly in cell aggregates, which are comparable to the meristematic nests of callus cultures (Chap. 3) In both, very small cells can be seen in the center, and cell size increases toward the periphery. Highest cell division activity occurs in the center of these structures.

Due to the agitation of the shaker, the outermost cells of the cell aggregates are mechanically removed (see Fig. 4.3), and then represent the fraction of free single cells. These cells should be older, and mostly quiescent in terms of cell division activity. However, some of these cells preserve the ability to divide, or this is re-induced. Such cells are possibly the origin of the third fraction, the cellular threads. A similar organization can be observed in carrot cell suspensions. As an example, the threadlike structure in Fig. 4.4 observed in a carrot suspension seems to be the result of three cell divisions. One terminal cell differentiates into a tracheid-like structure, the other accumulates anthocyanin, and the four central cells showing chlorophyll accumulation would be the youngest cells derived from the last rounds of cell division. The great differences in the structure of the two terminal cells point to an unequal first cell division, with differences in the distribution of cytoplasm. The nutrient medium can be regarded as identical for both cells.

Fig. 4.3

Loosely structured surface of a callus (top), and remains of a mechanical break-off of a cell in a suspension (bottom, see arrow; photographs by A. Kumar)

Fig. 4.4

A thread of cells in a cell suspension culture of carrot in White’s basal medium containing 10% coconut milk. Top A thread consisting of six cells, resulting from three divisions of a single cell. By the third division, the inner four cells seem to be produced. Bottom The right terminal cell contains anthocyanin, and the left terminal is a trachea. The differences of differentiation of the terminal cells would be due to an unequal first cell division of the “mother cell”. The higher degree of specialization of the terminal cells, compared to that of the four inner cells, could be due to more time elapsed since division took place relative to the last division

A determination of DNA concentration indicated a near-cytogenetic homogeneity only for cells in the aggregates (secondary calli). In the population of free single cells, a strong inhomogeneity exists, sometimes with very high DNA content per cell (Fig. 4.5). This observation is consistent with results obtained from callus material. Here, also the lowest C-values of a ploidy level can be found in the center of the meristematic nests with high cell division activity.

Fig. 4.5

DNA content of nuclei (microfluorometric determination, relative units) of haploid cell suspension cultures of Datura innoxia at inoculation (t0), and after 28 days of culture (n = 13.5; after Kibler and Neumann 1980)

In both cases, these small cells in haploid cultures were found to have a DNA content essentially identical to that of microspores of the same species (G1-phase cells), or twice that of G2-phase cells. In diploid cultures, the DNA content was either twice that of G1-phase cells of haploids, or 4 times the value of microspores in G2-phase cells. In older cells located between meristematic nests in callus material, which would be comparable to the fraction of free cells in the suspension, a broad variation in C-values was determined. Apparently, cytogenetic stability is linked to the age of the cells, i.e., the length of time elapsed since the last division. In young material with high cell division activity, a high percentage of cells contains DNA content characteristic of the ploidy level. A supplement of kinetin, which increases cell division activity, results in a higher cytogenetic stability and homogeneity of the cell population (Sect. 13.1).

In cell suspensions, many cell structures occur that are morphologically difficult to classify. However, some well-defined cell types can also be observed, e.g., tracheids, as described above (Fig. 4.4). In a cell suspension, all free single cells are bathed in the same nutrient solution, and therefore the morphological diversification of its components should be based on the origin of the individual cell. The significance of unequal cell divisions has already been mentioned above—whatever the cause of this phenomenon may be. A direct relation between cell shape and vitality has not been observed.

In cell suspensions of some species like Daucus in an IAA-supplemented medium (NL medium, Table 3.3), after some weeks of culture the formation of early stages of embryo development can be observed, and these can eventually be raised to intact plants (somatic embryogenesis; for details, see Sect. 7.3).

Using the methods described above, only limited amounts of cell material can be produced, usually not sufficient to study physiological or biochemical problems of primary or secondary metabolism or, e.g., somatic embryogenesis. If greater amounts of material are required, fermenter cultures are performed (see also Sects. 3.2, 10.9). As an example, fermenter cultures of Datura innoxia shall be described. Here, within 2 weeks it was possible to produce 1 g of dry weight per day in a liquid nutrient medium of 3.5 l originally inoculated with a cell suspension of 30 g fresh weight. The cell suspension was obtained by a method used to raise cytogenetically stable material, as described later. Pre-culture is carried out in 200 ml nutrient medium (MS+kinetin, see Table 3.3) in a 750-ml Erlenmeyer flask on a shaker (see above). For initiation of the pre-culture, the vessel is inoculated with 1–2 g fresh weight (90–250 µm fraction). The main aim of the pre-culture is to propagate the cells. After 10–14 days of pre-culture, the content of the vessel (cells and nutrient medium) is transferred to the fermenter, as described above (see Sect. 3.2). In the fermenter, cell aggregates as well as free single cells occur.

The principle to distinguish between a propagation phase and a production phase is also applied to fermenter cultures used for biotechnological purposes. Here, fermenters of much larger volume are used; to produce cell suspensions for inoculation, however, smaller laboratory fermenters are used initially, as described later. Usually, the cell suspension is transferred with some nutrient medium from the smaller to the next bigger fermenter. For a semi-continuous culture, it is common practice to remove part of the cell material in certain intervals of time for processing, and to apply fresh nutrient medium. As described later (Chap. 10), plant cell suspensions are already today cultured in fermenters with a volume of thousands of liters (Mitsu Petrochem. Ind. Ltd), e.g., to produce shikonin derivatives using cultures of Lithospermum officinale. Also propagation via somatic embryogenesis has been carried out in a fermenter (e.g., Daucus; see Sect. 7.3).

To maintain cell strains in a healthy condition for prolonged periods, subcultures have to be made frequently, usually at 1- or 2-week intervals, and with a dilution of 1:5 after 1 week, and 1:10 after 2 weeks with the fresh nutrient medium. The optimal dilution, and the subculture frequency have to be determined for each individual strain. As described above, also cryopreservation is often used to maintain cell suspensions (Sect. 3.6).