Suspension culture method

Alejo, N.O. (2006) Capsaicin accumulation in Capsicum spp. suspension cultures. Methods in Molecular Biology 318, 327-334. 

The purpose of this chapter is to review relevant work in the area, including an evaluation of methods employed for shear sensitivity studies, comparison of bases for data analysis and an outline of current knowledge of the interface between the hydrodynamic environment and the plant cells themselves. Coverage is limited to cell suspension cultures and does not extend to other tissue or organ systems. 

In the dialyzed batch start-up phase and the subsequent continuous operation a substantial increase in viable cell density and monoclonal antibody (MAb) titer was observed compared to a conventional suspension culture. The raw data, profiles of the viable cell density, viability and monoclonal antibody titer during the batch start-up and the continuous operation with a dialysis flow rate of 5 L/d are shown in Figures 17.6 and 17.7. The raw data are also available in tabular form in the corresponding input file for the FORTRAN program on data smoothing for short cut methods provided with the enclosed CD. 

The ES cells were encapsulated in the PMBV/PVA hydrogel by the same method used for the L929 cells. In general, ES cells form a cell aggregate called an embryoid body in suspension culture. However, it was observed that the encapsulated ES cells in the hydrogel did not form any embryoid body for 72 h. 

Roberts SC, Naill M, Gibson DM, Shuler ML. (2003) A simple method for enhancing paclitaxel release from Taxus canadensis cell suspension cultures utilizing cell wall digesting enzymes. Plant Cell Rep 21 1217-1220. 

The most studied nucleus, particularly for in-vivo studies has been 31P. Naturally abundant 31P occurs in metabolites that move freely in the tissue. The most abundant of these are inorganic phosphate sugar phosphate nucleotide and nucleoside di- and triphosphates. The assignments of the signals have been discussed by Roberts (1987). The chemical shift of P in phosphates is sensitive to pH over the physiological range of 5.8 to 7.8. Estimations of intracellular pH can thus be made by comparison of chemical shifts in vivo with those in vitro (Roberts et at., 1981). Use of this method has shown that in oil palm cell suspension cultures, there is loss of pH control with sudden increases in the external pH (Fox and Ratcliffe, 1990). 

The choice of cell culture method for cytotoxicity studies for a certain substance depends on the target cells and assay duration. Organ, spheroids, suspension cells, flask surface, and multi-well plate monolayers, as well as agar surface cultures can be employed. 

The advantage of microcarrier culture methods can be obtained for suspension cells by their entrapment within a variety of different beads. 

Table 5. Methods for assessing shear-related effects in TTC Triphenyltetrazolium chloride plant cell suspension cultures [59,60],...

In plant cell cultures, shake flask culture is an indispensable stage of cultivation. Investigations in a shake flask are very essential and critical to bioprocess scale-up and optimization. We have developed a simple and convenient technique based on the principle of the Warburg manometric method to measure 02 uptake rate (OUR) and C02 evolution rate (CER) of suspended cells in a shake flask culture. This technique has been successfully applied to suspension cultures of Panax notoginseng cells, and some important bioprocess parameters, such as OUR, CER, respiratory quotient (RQ), SOUR and specific CER (SCER), were quantitatively obtained [99]. As long as the environment temperature is strictly controlled to within an error of 0.1 °C, the measuring system is accurate and reproducible, is easy to operate, is economical, and is also able to treat many samples simultaneously. 

The aqueous two-phase system can be applied to plant cell suspension cultures as an in situ extraction method to enhance the production of hydrophobic secondary metabolites or extracellular proteins such as enzymes. Selective removal of the product during bioconversion using plant cells may also be possible in ATPS. Prior to obtaining phy to chemicals with high yields using ATPS, however, reliable cultivation of plant cells in ATPS should be proven. 

ALS was assayed by quantifying the amount of acetoin formed from acetolactate (27). The enzyme from seedlings were assayed at pH 7.15 and that from cultures at 8.3. ALS from a number of higher plants has been readily extracted and assayed (19.22). However due to the instability of the enzyme, there is no efficient procedure for its purification. Recently, Muhitch et al (28) described a four step purification procedure for ALS from maize suspension cultures. This method yielded enzyme preparation with high specific activity, but the recovery was low. We have examined ALS from different species in order to identify the best source for purification and the results are given in Table II. 

Aggregation of cells in suspension. The method was originally set up by Orr and Roseman (1969), and next modified by Tao et al. (1983) to measure differences in homotypic adhesiveness due to cell surface carbohydrates changes. A similar assay was also set up by others in order to evaluate how different parameters could influence the aggregation ability of BHK cells in culture (Urushihara et al., 1976 Takeichi, 1977). These methods measure the propensity of a... 

Suspension culture is the preferred method for scale-up because it is easier and cheaper, it requires less space, cell growth can be monitored and environmental parameters can be controlled more easily. However, many cell lines have higher specific productivity when attached to the substrate. In the urge to move to suspension systems, the following advantages of anchorage-dependent systems should not be overlooked ... 

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