Cell Culture

 

The culture of tissue and cells cultured in liquid nutrient medium, producing a suspension of single cells and cell clumps.”  Cell suspension culture is the primary route for studying plant cell secondary metabolismThe first attempt to culture single cells was made by Haberlandt in 1902.

Cell Culture Techniques:

The invitro cell culture technique broadly involves, the following:

1)    Isolation of single cells

2)    Suspension culture growth and sub culturing

3)    Types of Suspension culture

4)    Synchronization of suspension culture

5)    Measurement of growth

6)    Measurement of viability of cultured cells

Isolation of single cells:

Single cells may be isolated from plant organs and from cultured tissues.

A.        From plant organs

        Plant leaves with homogenous population of cells are the ideal sources for cell culture.         Cells are isolated through mechanical method or enzymatic method.

 1.     Mechanical method

Surface sterilized leaves are cut into small pieces of less than 1cm² suspended in a medium and subjected to grinding in a glass homgenizer tube. The homogenate is filtered through filters. Supernatant is removed and diluted to achieve the required cell density.

2.     Enzymatic method

The enzyme macroenzyme can release the individual cells from the leaf tissue. Macroenzyme degrades middle lamellae and cell walls of parenchymatous tissues.

B.        From Culture Tissue

Single cells can be isolated from callus cultures. Repeated sub-culturing of callus on agar medium improves the variability of callus so that free cell suspensions are obtained.

3. Suspension Cultures

The isolated cells are grown in suspension cultures. Cell suspensions are maintained by routine sub culturing in fresh medium.

Suspension culture is a type of culture in which single cells or small aggregates of cells multiply while suspended in agitated liquid medium. It is also referred to as cell culture or cell suspension culture. Establishment of single cell cultures provides an excellent opportunity to investigate the properties and potentialities of plant cells.

Types of Suspension Cultures:

There are mainly two types of suspension cultures – 1. Batch Culture and 2. Continuous culture.

Batch culture

•       A batch culture is a cell suspension culture grown in a fixed volume of nutrient culture medium For instance, cell material in 20 ml or 40 ml or 60 ml liquid medium in each passage constitute a batch culture.

•       In batch culture, cell division and cell growth coupled with increase in biomass occur one of the factors in the culture environment becomes limiting

•       The cell exhibit the following five phases of growth when the cell number in suspension culture is plotted against Y-axis and X-axis the time of incubation

1. Lag phase- Characterized by preparation of cell to divide
2. Log phase-where the rate of cell multiplication is highest
3. Linear phase-It is represented by slowness in cell division and increase in cell size expansion
4. Deceleration phase-decrease in cell division and expansion
5. Stationary phase-constant number of cells and their size

In batch culture cells can be maintained continuously by transferring small amounts of the suspension medium to fresh medium at regular intervals (2-3 days)

Continuous Cultures:

There is continuous addition of fresh nutrient medium and draining out of the used medium so that the volume is normal constant to stabilize physiological of the growing medium.

In this system, nutrient depletion does not occur due to the continuous flow of nutrients and the cells always remain in the steady growth phase. Continuous cultures are of two types – Open and Closed.

Closed continuous culture:

The used culture medium is replaced with the fresh medium, i.e., inflow of fresh medium is balanced with the outflow of corresponding spent medium. The cells present in the outflowing medium are separated and added back to the culture system. As a result, there is a continuous increase in the biomass in closed continuous culture.

Open continuous culture:

The inflow of fresh medium is balanced with the outflow of the spent medium along with the cells, i.e., both the cells and used or spent medium are replaced with fresh medium thus maintaining culture at constant growth rate.

Open continuous culture is of two types:

I.   Chemostat: in this system culture vessels are usually cylindrical or circular in shape and possess inlet and outlet porest for aeration and the introduction and removal of cells and medium.

Such a system are maintained in a steady state. The density, growth rate, chemical composition and metabolic activity of the cells remain constant. These cultures are ideal for studying growth, kinetics and the regulation of metabolic activity in higher plants.

           II.  Turbidostat Continuous culture: addition of fresh medium is done                                       whenever there is an increase in turbidity. In this system, the cells are allowed to                   grow up to a certain turbidity, when the predetermined volume of culture is                             replaced by fresh culture. The turbidity is measured by changes of optical density                  of medium.

               Importance of Cell Culture:

               Information about cell physiology, biochemistry, metabolic events.

              Understanding of an organ/embryoid formation starting from a single cell

              Mutagenesis studies may be facilitated to produce mutant cell clone form which                    mutant plants can be raised.