Difference Between Primary and Secondary Cell Culture

Key Difference – Primary vs Secondary Cell Culture

Before discussing the difference between Primary and Secondary Cell Culture, let us first briefly define what cell culture is. Cell culture is the process of removing cells from an animal or plant and subsequent growth in an artificially controlled environment. The cells could be directly removed from the tissue and disaggregated by enzymatic or mechanical methods or could be derived from a culture that has been already established. The key difference between primary and secondary cell culture is that cells for primary cell culture are obtained directly from an animal or plant tissue, while cells for secondary cell culture are obtained from an already established primary culture. Therefore, secondary culture is a new culture originated from the primary culture.

Let’s look further into the meaning of primary and secondary cell culture in order to differentiate them better.

What is Primary Cell Culture?

Primary cell culture is the disassociation of cells from a parental animal or plant tissue through enzymatic or mechanical measures and maintaining the growth of cells in a suitable substrate in glass or plastic containers under controlled environmental conditions. Cells in primary culture have the same karyotype (number and appearance of chromosomes in the nucleus of a eukaryotic cell) as those cells in the original tissue. Primary cell culture could be classified into two based on the kind of cells used in culture.

  • Anchorage Dependent or Adherent Cell – These cells require an attachment for growth. Adherent cells are usually derived from tissues of organs, for instance from  kidney where the cells are immobile and embedded in connective tissue.
  • Anchorage Independent or Suspension Cells – These cells do not require an attachment for growth. In other words, these cells do not attach to the surface of culture vessel. All suspension cultures are derived from cells of the blood system; for instance, white blood cell lymphocyte is suspended in plasma.

Cells derived from primary cultures have a limited life span. Cells cannot be held indefinitely due to several reasons. Increasing cell numbers in primary culture will lead to exhaustion of substrate and nutrients. Also, cellular activity will gradually increase the level of toxic metabolites in the culture inhibiting further cell growth.

At this stage, a secondary or a subculture has to be performed to ensure continuous cell growth.difference between primary and secondary cell culture

What is Secondary Cell Culture?

As described above, when cells in adherent cultures occupy all available substrate or when cells in suspension cultures surpass the capacity of the medium to support further growth, cell proliferation begins to reduce or to entirely cease. In order to maintain optimal cell density for continued growth and to stimulate further proliferation, primary culture has to be subcultured. This process is known as secondary cell culture.

During the secondary cell culture, cells from primary culture are transferred to a new vessel with fresh growth medium. The process involves removing the previous growth media and disassociating adhered cells in adherent primary cultures. Secondary cell culturing is periodically required to provide cells with growing space and fresh nutrients, thereby, prolonging the life of cells and expanding a number of cells in the culture.

Secondary culturing a certain volume of a primary culture into an equal volume of fresh growth medium allows long-term maintenance of cell lines. Secondary culturing into a larger volume of fresh growth medium is practiced to increase the number of cells, for instance in industrial processes or scientific experiments.

What is the Difference between Primary Cell Culture and Secondary Cell Culture?

As we have now understood the two terms separately, we will compare the two in order to find other differences between them.

When to Use Primary and/or Secondary Cell Culture

This depends on what you want to learn and what type of experiment you perform.

Primary Cell Culture: This is the process to be used to culture cells from the parental tissue in concern. Cells in primary culture will have a finite lifespan due to exhaustion of substrate and nutrients and build up of toxins, with population growth. Primary culture, despite the separation techniques used in the isolation process, may contain several types of cells. However, this might not be a problem in all type of experiments and in such instances primary culture alone can be used.

Secondary Cell Culture:  Usually, the number of cells obtained from primary culture is insufficient in experiments. Secondary cell culture gives the opportunity for expanding cell population and also, prolonging life span. It enables further selection of cells with the use of a selective medium and allows genotypic and phenotypic uniformity in the population. This process is used to generate replicate cultures for basic characterization, preservation, and experimentation.

Resemblance to Parental Tissue

Primary Cell Culture: Cells for primary cell culture are directly obtained from an animal or plant tissue. Hence, cells in primary culture closely resemble its parental tissue and accordingly, the biological response may be closer to an in vivo situation than that of a secondary cell culture.

Secondary Cell Culture: Secondary cell culture originates from a primary cell culture. Though sub-culturing prolongs the lifespan of cells, there is the possibility that after a few phases, cells might be transformed or might lose control of not dividing more than a certain amount of times. This could be because of mutations or genetic alterations in primary cells during sub-culturing. For instance, some microorganisms tend to adapt to culture conditions, which is mostly different from their natural environment, by altering their biology.

Process of Culturing – Obtaining Cells

Primary Cell Culture: In primary cell culture, animal or plant tissue will go through phases of rinsing, dissection, and mechanical or enzymatic disaggregation. Disaggregated tissue will contain a variety of different cell types, and this may require the adoption of a separation technique in order to isolate cells of interest.

Secondary Cell Culture: In secondary cell culture, if the primary culture is an adherent culture, the first step is to detach cells from the attachment (surface of culture vessel) by mechanical or enzymatic means. Then, the cells have to be detached from each other to form a single cell suspension.

Number of Cells in Culture

Primary Cell Culture: It is not desirable to have an absolute single cell suspension, as many primary cells survive better in small clusters.

Secondary Cell Culture: It is sufficient to generate a single cell suspension.

Life Span of Culture

Primary Cell Culture: Primary cell cultures have finite life spans. As explained above, this is because the growth of cells exhausts substrate and nutrients and leads to accumulation of toxic metabolites. As a result, gradually the growth rate of cells drops, leading to the death of cells.

Secondary Cell Culture: Secondary cell culture prolongs the lifespan of cells. Periodic sub-culturing may produce immortal cells through transformation or genetic alteration of primary cells.

Risk of Contamination

Primary Cell Culture: Primary cell cultures are more difficult to take care of. Generally, primary cell cultures need a rich mixture of amino acids, micronutrients, certain hormones and growth factors. As a result, the risk of contamination in primary cell cultures is higher than secondary cell cultures.

Secondary Cell Culture: Secondary cell cultures are comparatively easy to maintain, and the risk of contamination is lower than primary cell cultures.

In this article, we have attempted to understand the terms primary cell culture and secondary cell culture followed by a comparison to highlight key differences between them. The basic difference lies in how cells are derived from culture; cells for primary cell culture are obtained directly from an animal or plant tissue, while cells for secondary cell culture are obtained from an already established primary culture.

Cell Culture Basics – A Handbook by Invitrogen and Gibco
Freshney, R. I. (2006). Basic Principles of Cell Culture. Centre for Oncology and Applied Pharmacology.
Image Courtesy:
“Cell Culture” by Umberto Salvagnin (CC BY 2.0) via Flickr