In the below mentioned article, we will discuss about the growth patterns in cell suspension culture and the measurement of cell growth in cell suspension culture.
Growth Patterns in Cell Suspension Culture:
Under appropriate light, temperature, aeration and nutrient medium the growth of suspension culture follows a predictable pattern or growth curve.
The growth of suspension culture can be monitored very easily by simply counting the cell number per unit volume of culture in relation to days of culture. From such data a typical growth curve can be prepared on a graph paper.
The growth curve for a typical higher plant suspension culture consists of lag phase, logarithmic phase or exponential phase, linear phase and stationary phase (Fig 4.8). The lag phase is the period where the cells adjust themselves to the nutrient medium and undertake all the necessary synthesis prior to cell division.
This is followed by very rapid cell division causing a logarithmic increase in cell number.
This phase is called as logarithmic phase. A further period of rapid cell division results in a linear increase in number and the phase is called linear phase. As nutrients are depleted and some of the cells of the culture being to show senescent characteristics, the rate of cell division within the culture declines and it passes through the stationary phase.
At this stage the growth curve forms a plateau. If the cells are removed just before or just after the entry into stationary phase in each growth cycle and are sub-cultured to fresh medium, then identical patterns of growth of the cell line can be maintained in each culture passage.
Dry weight, total protein, DNA synthesis etc. can also be considered as other parameters for the preparation of identical growth curves. It also indicates that the chemical composition of the cell changes throughout the growth cycle and such changes are closely coupled to the cell division in most of the plant material.
However, in some material there is no corresponding increase in dry weight accumulation and consequently the divergence between the rate of cell division and rate of dry weight accumulation increases. From these studies, it has been concluded that there are independent mechanisms for controlling cell division and many biosynthetic pathways.
A synchronized cell population and the continuous changes in physiological property may also cause the divergence between the rate of cell division and the biochemical changes of the cell. It is also important to note that the degree of cellular aggregation is not constant but changes significantly during the growth cycle of the suspension culture. As the culture enters the period of most active growth the cell aggregation is maximum and during the stationary phase cell aggregation is minimal.
For experimental studies on growth of cell suspension, the inoculum or cell density is an important factor. Very low density or high density of cells in liquid medium is unable to grow. So, to induce the growth, an initial density of 2 x 106 cells/ml to 2 x 108 cells/ml is inoculated in liquid medium.
This initial density increases during growth and attains a higher density at the stationary phase. Most commonly, such high cellular density are diluted on subculture by a factor of ca. X 10. The particular initial cell density that is able to grow in liquid medium is called critical initial density (CID). The CID may vary from plant to plant.
Measurement of Cell Growth in Suspension Culture:
The cells in suspension culture grow by cell division and the number of ceils increases. Growth studies of this kind are very valuable for the characterisation of cell lines, effect of nutrient medium and hormones etc. Growth in such cultures can be monitored by determination of cell number, cell dry weight, packed cell volume, etc.
The rate of increase of cell number can be calculated simply by counting the cell number in haemocytometer under a microscope. Cell count- data obtained from haemocytometer is multiplied by a factor x 103 and the result can be expressed in terms of cell number per unit volume of culture. Therefore, by comparing the cell number at the beginning of culture and after certain days of incubation, the growth can be measured.
Again, as the cells increase in number during growth, the liquid medium will be more turbid and as a result the optical density (OD) of the suspension culture will also be altered. The changes of OD value can be detected by a calorimeter. Therefore, from OD value growth can be measured.
Definite volumes of cell suspension can be harvested from multiple replicated sets of culture. Such amount of cell suspension is transferred in a graduated conical centrifuge tube and is centrifuged at 2,000X g for 5 minutes. The cells will form a pellet after centrifugation. The volume of cell pellet then represents the packed cell volume (PCV). It is also called biomass volume.
Therefore, harvesting the cell suspension at definite periods of interval and measuring the PCV, the growth can be monitored and expressed as milliliter cell pellet per milliliter culture. From the same experiments dry weight of cell mass, can also be estimated by drying the pellet in a hot air oven (12 hours at 60°C) after replacing the supernatant and weighing the dried cell mass in a chemical or electrical balance. In this method, growth can be expressed in terms of dry weight in gram or milligram per unit volume of culture.
Cell death may occur in suspension cultures due to several factors. So, for the studies on growth the test for viability of cells is very important. Otherwise, cell count data will be erroneous without testing the viability.
The most frequently used staining method for assessing cell viability is fluorescein diacetate (FDA). FDA dissolved in 5 mg/ml of acetone is added to cell population at 0.01% final concentration. Dead cells fluoresces red. Evans blue also used at a final concentration of 0.01% is specific for dead cells. As soon as the stain is mixed with cell suspension, the in- viable cells stain blue and the viable cells remain unstained.