Module 4 : Application of Cell Culture Systems in Metabolic Engineering

Lecture 35 : Growth And Production Kinetics Of Cell Cultures In Shake Flasks

 

1. Introduction

For the development of plant cell culture technology, accurate and rapid measurement of cell growth and assessment of growth-related bioprocess kinetics are essential to the rational development of plant cell bioprocess engineering. Although the plant cell culture system appears to be similar to a microbial cell culture system, there are important differences between the two. The major differences include cell size, aggregation of plant cells, change in plant cell physiology for its primary and secondary metabolisms, rheological properties of the medium, and requirement of plant cells for complex nutrients. Therefore, the best method of assessing the plant cell growth kinetics should be carefully examined and evaluated. The cell suspension culture is much more amenable for biochemical studies and process development than callus cultures. The success in the establishment of a cell suspension culture depends, to a great extent, on the availability of “friable” callus tissue (i.e., a tissue that, when stirred in liquid medium, rapidly disaggregate into single cells and small clusters). The cell suspension culture generally grows at a faster rate and allows cells to be in direct contact with the medium nutrients. Suspension culture could be run as batch culture or continuous culture.

In batch culture, the culture environment continuously changes and growth, product formation, substrate utilization, all terminate after a certain time interval.

But in continuous culture, fresh nutrient medium is continually supplied to a well-mixed culture, and products and cells are simultaneously withdrawn. Growth and product formation can be maintained for prolonged periods of time in continuous culture.

The reasons proposed for predominant use of batch culture is:

2. Procedure of growth measurement of plant cell suspension cultures

Growth of suspension cultures is generally assessed as the settled cell volume, the packed cell volume, fresh cell weight, dry cell weight. Medium residual conductivity and pH measurements are other indirect evaluation methods. Finally, parameters describing growth efficiency, such as specific growth rate (µ), doubling time (td), and growth index, are determined.

2.1. Growth curve

It is commonly accepted that growth of a cell suspension culture with respect to time is best described by the sigmoid curve theory. At the beginning, the cell population grows relatively very slow (lag phase). As the population size of plant cell approaches one half of the carrying capacity (defined by the nutrient status of the culture medium), the growth of culture per time unit increases. The growth rate is measured by the steepness of the curve, and it is the steepest when the population density reaches one-half of the carrying capacity (in the middle of the sigmoid). After that the steepness of the curve decreases until it reaches the carrying capacity (stationary phase). At this time the growth rate slowly decreases due to limitation of nutrients as described in Figure 35.1.

Figure 35.1: Graph showing different growth phases of a typical plant cell suspension culture.
The method needs harvesting cells at frequent intervals to determine the fresh weight and dry weight of cells per volume of cell suspension culture, thus, this is an invasive method. The method is also used to understand the nutrient uptake by the cells in suspension culture. For example, in cell suspension cultures of Lantana camara , it was observed that the cultures remained in the lag phase till the second day. Biomass increased till the 12th day following which the stationary phase started (Figure 35.2).