Cell Suspension Culture

Introduction:

Plant Tissue Culture

Plant tissue is a collection of experimental methods of growing large number of isolated cells or tissues under sterile and controlled conditions. The cells or tissues are obtained from any part of the plant like stem, root, leaf etc. which are encouraged to produce more cells in culture and to express their totipotency. Cells or tissues are grown in different types of glass vials containing a medium with mineral nutrients, vitamins and phytohormones.

Cell Suspension Culture

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. Such systems contribute to our understanding of the interrelationships and complementary influences of cells in multicellular organisms. Many plant biotechnologists recognized the merits of applying cell cultures over an intact organ or whole plant cultures to synthesize natural products.

Brief History

• The poineering attempts made by Haberlandt failed to achieve divisons in free cells, but his detailed paper in 1902 stimulated further studies in this area.
• H. Muir (1953) – First reported that the fragments of callus of Tagetes erecta and Nicotiana tabacum could be cultured in the form of cell suspension
• Nickel (1956) – Described the continues growth of a variety of Phaseolus vulgaris
• C. steward and E. M. Shantz (1956) – Reported the suspension cultures from carrot root explants and obtained very large number of plantlets from the culture.

Principles Of Cell Suspension Culture

1. The basic principle of single cell culture is the isolation of large number of intact living cells and cultures them on a suitable nutrient medium for their requisite growth and development.
2. Callus proliferates as an unorganized mass of cells. So it is very difficult to follow many cellular events during its growth and developmental phases. To overcome such limitations, the cultivation of free cells as well as small cell aggregates in a chemically defined liquid medium as a suspension was initiated.
3. In culture, the single cells divide redivide to form a callus tissue. Such callus tissue also retains the capacity to regenerate the plantlets through organogenesis and embryogenesis.
4. To achieve an ideal cell suspension, most commonly a friable callus is transferred to agitated liquid medium where it breaks up and readily disperses.
5. After eliminating the large callus pieces, only single cells and small cell aggregates are again transferred to fresh medium and after two or three weeks a suspension of actively growing cells are produced.
6. This suspension can then be propagated by regular sub-culture of an aliquot to fresh medium.
7. Ideally suspension culture should consist of only single cells which are physiologically and biochemically uniform.
8. Movement of cells in relation to nutrient medium facilitates gaseous exchange, removes any polarity of the cells due to gravity and eliminates the nutrient gradients within the medium and at the surface of the cells.

Methodology

1. To achieve an ideal cell suspension most commonly a friable callus is transferred to agitated liquid medium where it breaks up and readily disperses.
2. After eliminating the large cellular pieces, only single cells and small cell aggregates are again transferred to fresh medium and after 2 or 3 weeks a     suspension of actively growing cells are produced.

Isolation Of Single Cells

From Plant Organs

The most suitable material for the isolation of single cells is the leaf tissue, since a more or less homogenous population of cells in the leaves offer good material for raising defined and controlled large scale cell cultures.

Two important methods to isolate single cells from leaf are:

1. Mechanical Method

2. Enzymatic Method

From Cultured Tissues

The most widely applied approach is to obtain a single cell system from cultured tissues.

From Plant Organs

1. Mechanical Method

Gnanam and Kulandaivelu (1969) developed a procedure which has since been successfully used to isolate mesophyll cells active in photosynthesis and respiration, from mature leaves of several species of dicots and monocots including the grasses.

The procedure involves:

1. Mild maceration of 10g leaves in 40ml of the grinding medium (20µ mol. Sucrose, 10µ mol MgCl₂, 20µ mol tris HCl buffer, pH 7.8) with a mortor and pestle.
2. The homogenate obtained is passed through two layers of muslin cloth and the cells thus released are washed by centrifugation at low speed using the medium.

The mechanical isolation of free parenchymatous cells can also be achieved on a large scale.

2. Enzymatic Method

In 1968 Takabe et al treated tobacco leaf tissue with the enzyme pectinase and obtained a large number of metabolically active cells. A point to note is that potassium dextran sulphate in the enzyme mixture improved the yield of free cells.

Isolation of single cells by the enzymatic method has been found convenient as it is possible to obtain high yields from preparations of spongy parenchyma with minimum damage or injury to the cells. This can be accomplished by providing osmotic protection to the cells while providing osmotic protection to the cells while the enzyme macerozyme degrades the middle lamella and cell wall of the parenchymatous tissue. Applying the enzymatic method to cereals has proven difficult since the mesophyll cells of these plants are apparently elongated with a number of interlocking constrictions, thereby preventing their isolation.

FROM CULTURED TISSUES

1. Raise sterile tissue culture plants and obtain callus from them.
2. The callus is separated from an explant and transferred to a fresh medium of the same composition to enable it to build up a mass of tissue.
3. Repeated subculture on an agar medium improves the friablity of a callus, a pre requisite for raising the fine cell suspension in a liquid medium.
4. The pieces of undifferentiated and friable callus are transferred in a continuously agitated liquid medium dispersed in autoclaved flasks or other suitable vials.
5. Agitation is done by placing the flasks on orbital platform shaker or suitable device.
6. Movement of the culture medium exerts mild pressure on small pieces of tissues breaking them into free cells and small aggregates. Further it augments the gaseous exchange between the culture medium and the culture air and also ensures uniform distribution of cells in the medium.
7. The period of incubation during which the suspension cultured is developed from callus tissue is usually called as the initiation passage.
8. The concentration of auxins and cytokinins is often critical for the growth of cell suspension and the concentration of auxin and cytokinins used for callus culture is generally reduced for suspension culture.
9. The cells in the cell suspension may vary in shapes and sizes. They may be oval, round, elongated or coiled, but thery are thin walled, even with the presence of other lignified, trachieds like elements.

Protocol

1. Take 150/250 ml conical flask containing autoclaved 40/60 ml liquid medium.
2. Transfer 3-4 pieces of pre-established callus tissue (appx. Wt. 1 gm each) from the culture tube using the spoon headed spatula to conical flasks.
3. Flame the neck of conical flask, close the mouth of the flask with a piece of aluminium foil or a cotton plug. Cover the closure with a piece of brown paper.
4. Place the flasks within the clamps of a rotary shaker moving at the 80 – 120 rpm.
5. After 7 days, pour the contents of each flask through the sterilized sieve and collect the filtrate in a big sterilized container. The filtrate contains only free cells and cell aggregate.
6. Allow the filtrate to settle for 10 – 15 min or centrifuge the filtrate at 500 – 1000 rpm and finally pour off the supernatant.
7. Resuspend the residue cells in a requisite volume of fresh liquid medium and disperse the cell suspension equally in several sterilized flasks (150/250 ml). Place the flasks on shaker and allow the free cells and cell aggregates to grow.
8. At the next subculture, repeat the previous steps but take only one-fifth of the residual cells as the inoculums and dispense equally in flasks and again place them on shaker.
9. After 3- 4 subcultures, transfer 10ml of cell suspension from each flask into new flask containing 30ml fresh liquid medium.
10. To prepare a growth curve of cells in suspension, transfer a definite number of cells measured accurately by a haemocytometer to a definite volume of liquid medium and incubate on shaker. Pipette out very little aliquot of cell suspension at short intervals of time (1 or 2 days interval) and count the cell number. Plot the cell count data of a passage on a graph and the curve will indicate the growth pattern of suspension culture.

Different Categories Of Cell Suspension Culture

Broadly speaking there are two types of suspension cultures

1. Batch Culture

2. Continuous Culture

1. Batch Culture

Here the cell material grows in a finite volume of agitate liquid medium. For instance, cell material in 20 ml or 40 ml or 60 ml liquid medium in each passage constitute a batch culture.

Batch suspension cultures are most commonly maintained in conical flasks incubated on orbital platform shakers at the speed of 80 – 120 rpm

Different types of batch culture are

1. Slowly rotating cultures
2. Shake cultures
3. Spinning cultures
4. Stirred culture

2. Continuous Culture

The large culture vessel is kept dispersed continuously by bubbling sterile air through culture medium and the old liquid medium is continuously replaced by the fresh liquid medium (on depletion of some nutrients in the medium) to stabilize the physiological states of the growing cells.

Here nutrient depletion does not occur due to continuous flow of nutrient medium and the cells always remain in the steady state of active growth phase.

There are two types of continuous culture system

1. Chemostates
2. Turbidostates 

Importance Of Cell Suspension Culture

1. To obtain single cell clones.
2. To study the morphological and biochemical changes during their growth and development phases.
3. To understand the pathways of cellular metabolism.
4. Single cell systems have a great potential for crop improvement.
5. Free cells in cultures permit quick administration and withdrawal of diverse chemicals/ substances thereby making them easy targets for mutant selection.
6. Cells which are in a population of cultured cells invariably show cytogenetical and metabolic variations depending on the stage of the growth cycle and culture (Lindsey and Yeoman (1985) Heterogeneity)
7. To produce high yielding cultures as well as plants with superior agronomic traits.
8. Single cells derived from medicinally important plants can be studied for the production of secondary metabolites like alkaloids, glycosides.
9. For mutagenesis study. The mutagens can be added directly in the liquid medium. After the muta­gen treatment, cells are plated on agar medium for the selection of mutant cell clones. The hope is that permanent changes in the DNA patterns  of some of the cells would be achieved by such treatments.