Cell suspension culture: an overview

Did you know that you can generate complete plants using single plant cells in liquid medium?

It could be a cell from a plant leaf, buds, flowers, or any other part of the plant.

This method of plant tissue culture is called "cell suspension culture".

Plant cells have an interesting ability, called totipotency. Because of this, individual plant cells have the potential to develop into complete plants. We exploit the totipotency of plant cells using different plant tissue culture techniques. Thus, it is possible for you to establish cultures not only using meristematic, embryonic, and mature tissues of a plant species, but also using single cells.

Apart from cell suspension culture, there are some more methods of tissue culture to obtain healthy in vitro plants. You can read more about them in our article on "7 methods of plant tissue culture".

But for now, let us dive deeper into the topic of cell suspension culture:

What is cell suspension culture?

Cell suspension culture is simply multiplying single cells at a higher rate in a liquid medium. The liquid medium is continuously agitated on an orbital shaker. This culture method is used by scientists for studying cell growth and development. It is also a popular tissue culture method for many industries to extract certain components from plant cells.

You must be wondering, why do you need to provide constant agitation to medium? This is necessary for cells to have an efficient gaseous exchange.

Agitation of the medium also exerts a mild pressure on tissue and breaks it into smaller cell pieces and single cells. This agitation helps to maintain the uniform distribution and movement of cells in the medium. Thus, it is an important procedure for cell suspension culture.

However, in order to establish cell suspension culture, we first need single plant cells. How do we get those? We can obtain pieces of undifferentiated, friable calli from callus cultures.

The callus is an irregular mass of plant tissues that do not have any structure, but possesses the ability to differentiate into different plant organs. We can obtain different calli from a single explant. However, these calli from the same explant might show variation in color, texture, etc. These calli may be compact or friable in texture, light or dark in color, and so on. You need well-established friable callus cultures for your cell suspension method.

One of the interesting facts about callus culture is that you can multiply them for an unlimited period of time without any specific differentiation. You can do this by periodically subculturing on a fresh medium containing auxin for constant cell division.

Types of cell suspension cultures

Broadly, there are two types of cell suspension cultures. These are:

• Batch cultures; and
• Continuous cultures.

Batch cultures

It is a closed system culture whereby we can grow cells in a fixed amount of culture medium under appropriate conditions. This kind of cell suspension is performed using flasks up to the volume of 250 mL. You can treat the first flask with cells as inoculum for further flasks in suspension. For each subsequent subculture, a small aliquot is taken from the initial suspension and then transferred to a fresh medium.

One major drawback of batch cultures is that the cells grow up to a certain point and then the cell growth becomes stationary. In this stationary phase, the number of cells and size of cells remains constant. Cells reach the stationary phase either because of exhaustion of some growth factors or accumulation of toxic metabolites in the culture medium. However, you can avoid this phase by providing fresh media at a faster rate.

Continuous cultures

In this type of cell suspension culture, you can maintain a constant phase of cell growth. Here the fresh medium is continuously added and the leftover nutrients and metabolic end products are constantly removed from the medium. Thus, by using this method you can avoid the detoxification of the media, and hence, overcome the drawback of batch cultures.

There are two types of continuous cultures:

Chemostat: In this method, you can add the fresh medium while harvesting the same amount of the culture. This maintains steady cell growth. However, you need to adjust the concentration of one of the nutrients, nitrogen, phosphorus, and glucose, so as to be the growth limiting factor.

Turbidostat: In this method, the biomass of the cells is constantly maintained at the level below the maximum yield. You can provide all nutrients in excess, unlike one limiting factor in chemostat culture. You can control the addition of the fresh medium by observing the turbidity/thickness of the culture.

How to determine growth of cells in suspension culture?

Scientists can determine the growth of cells in suspension culture using instruments like hemocytometer, hartley funnel, etc in a laboratory. In simple terms, they try to measure:

• Number of cells in culture;
• Packed cell volume;
• Fresh weight of cells in culture; and
• Dry weight of cells in culture.

Based on the values obtained from these measurements, changes in medium composition, as well as the rate of agitation, are decided.

What are the methods to assess the viability of cells in suspension culture?

Here we need a laboratory with instruments like a microscope and specific chemical compounds like tetrazolium salt, staining dyes, etc. Different methods to evaluate the viability of cells are:

Phase-contrast microscopy: Here scientists evaluate viability by observing healthy cell components and movements within a cell obtained from the cell suspension.

Tetrazolium salt reduction: With this method, scientists can measure the respiratory efficiency of the cells.

Fluorescein diacetate (FDA) method: With this method, scientists can measure the percentage viability of the cells. It works on the principle of illumination of the cell to a fluorescent green color.

Evan's blue staining: In this method, scientists treat cells with Evan’s blue stain where the dead cells take up the stain and the viable cells remain unstained.

We hope you got a glimpse of what is cell suspension culture. For more informational posts on different aspects of cell suspension culture, keep checking this space!

By Nancy Bhatia | 11-August-2021

References

• Kyte, Kleyn, et al (2013) Plants from test tubes: An introduction to micropropagation. Timber press, Inc.
• Bhojwani, S.S., & Dantu, P.K. (2013). Plant Tissue Culture: An Introductory Text. Springer India
• Sharma, V., & Alam, A. (2015). Plant Tissue Culture. I.K. International Publishing House Pvt. Ltd.
• https://www.urbanpro.com/class-xi-xii-tuition-puc/cell-suspension-culture-plant-tissue-culture
• https://www.biologydiscussion.com/plant-tissues/cell-suspension-culture/cell-suspension-culture-definition-principle-protocol-and-importance-plant-tissue.