Understand Difference

Primary Versus Secondary Cell Culture: Choosing the Best Method

Introduction to Cell Culture

Cell culture is an integral aspect of modern biomedical research. In essence, it involves the growth and maintenance of cells outside their natural habitat, which generally refers to tissues, organs, or organisms.

In laboratory settings, cells are grown in a sterile environment that mimics their natural physiological conditions. Successful cell culture techniques have enabled researchers to study cellular phenomena and processes with unprecedented accuracy and detail.

Going forward, we will discuss the primary and secondary types of cell culture, and the various techniques involved. Primary vs.

Secondary Cell Culture

Before we delve into the specific techniques of cell culture, it is important to distinguish between primary and secondary types of cell culture. Primary cell culture is conducted using cells directly isolated from tissues or organs, while secondary cell culture is performed using cells that have undergone multiple rounds of culture.

Primary cell culture is a more labor-intensive and time-consuming process because it requires the extraction and isolation of cells from a specific tissue or organ.

Primary Cell Culture

Primary cell culture can be achieved through two different processes – enzymatic or mechanical. The enzymatic process involves the use of specific enzymes that break down the extracellular matrix surrounding the cells, allowing them to be liberated and cultured.

Conversely, the mechanical process involves scrapping and/or gently agitating the tissues to detach cells. The method used depends on the type of cell and its location within the tissue or organ.

Anchorage Dependent or Adherent Cell

In cell culture, cells can either be anchorage dependent or anchorage independent. Adherent cells require a substrate or surface to attach to for growth and survival.

Examples of adherent cells include fibroblasts and epithelial cells. To culture adherent cells, a substrate such as plastic or glass is coated with a layer of extracellular matrix proteins such as fibronectin or collagen.

Anchorage Independent or Suspension Cells

Suspension cells, on the other hand, can grow without any substrate or surface to attach to. Examples of suspension cells include blood cells and lymphocytes.

Suspension cells can be grown in a specialized solution that provides the necessary nutrients and growth factors.

Limited Lifespan

One important thing to note with primary cell culture is that cells have a limited lifespan in vitro. This means that after a certain number of population doublings, cells stop dividing and eventually die.

The lifespan of a cell is influenced by various factors such as the type of cell, culture conditions, and cellular senescence.


In conclusion, cell culture is an important tool for modern biomedical research. By understanding the primary and secondary types of cell culture, and the processes involved, researchers can perform targeted experiments and studies with greater precision and accuracy.

The ability to grow cells outside their natural habitat has transformed the way we study human diseases, drug development, and cellular processes. As technology continues to advance, we will likely see even more innovative uses for cell culture techniques in the future.

Secondary Cell Culture

Secondary cell culture is a process in which cells, after an initial isolation, undergo a series of subcultures to expand the population, prolong the lifespan, and maintain the genetic and phenotypic uniformity. This type of culture is frequently used since it can produce a sufficient number of cells for experiments and studies without having to obtain additional tissues repeatedly.

Definition and Process

Secondary cell culture involves taking cells from a primary culture and introducing them into a new culture vessel with fresh growth media. This process is known as subculturing.

During subculturing, the cells are typically detached from the culture vessel using enzymes, such as trypsin. Once detached, the cells are collected and transferred into a new vessel containing fresh growth media.

The cells are then incubated until they reach confluency, at which point they can be subcultured again.

Expansion of Cell Population and Prolonging Lifespan

The purpose of secondary cell culture is primarily to expand the cell population and prolong the lifespan of the cells. This is achieved by subculturing the cells repeatedly.

The more subcultures a cell undergoes, the more population doublings it undergoes, thus increasing the number of cells in culture. This expansion in the number of cells makes it possible to conduct experiments and studies that require large cell numbers.

Selection of Cells and Genotypic and Phenotypic Uniformity

During subculturing, it is important to select cells that exhibit genotypic and phenotypic uniformity, as this will ensure the reproducibility of experiments and studies. Clones of cells that exhibit the same genotype and phenotype can be developed by using techniques such as limiting dilution or fluorescent activated cell sorting (FACS).

FACS comprises a process in which cells are sorted based on specific characteristics such as size, granularity, and fluorescence.

Replicate Cultures

Due to the importance of uniformity in cell cultures, replicate cultures are created during the secondary cell culture process. Replicate cultures are created by splitting the cells into multiple flasks, each with the same number of cells.

This ensures that if any contamination or genetic drift occurs, the replicate cultures can serve as backups. Comparing Primary and

Secondary Cell Culture

When to Use Primary and/or

Secondary Cell Culture

The choice of whether to use primary or secondary cell culture depends on the specific research or study being conducted.

For studies that require cells that resemble parental tissue, primary cell culture is recommended. For example, studies that require cells for use in transplant procedures, such as skin grafts, would require the use of primary cell culture.

Conversely, studies that require large cell numbers or involve the genetic manipulation of cells, such as CRISPR/Cas9, would require the use of secondary cell culture.

Resemblance to Parental Tissue

Primary cell culture is generally known to provide cells that resemble parental tissue in terms of morphology, gene expression, and cellular function. This is because primary cultures are isolated directly from tissues and are not transformed due to long passages.

Process of Culturing Obtaining Cells

The process of obtaining cells for primary and secondary cell culture differs. Primary cell culture requires the extraction and isolation of cells from a specific tissue or organ.

This process requires significant amounts of time and effort, while secondary cell cultures require the use of previously isolated cells that undergo subculture.

Number of Cells in Culture

Primary cell culture typically results in a lower number of cells in culture, compared to secondary cell culture. This is because the isolation process in primary culture results in a limited number of cells obtained.

Conversely, secondary cell culture can generate a larger number of cells due to repeated subcultures.

Lifespan of Culture

Primary cell culture has a limited lifespan because cells have a finite number of population doublings. Conversely, secondary cell culture can be used to prolong the lifespan of cells.

With repeated subculturing, the lifespan of the cells can be prolonged.

Risk of Contamination

Both primary and secondary cell cultures carry the risk of contamination. However, the risk of contamination in primary cell culture is lower than in secondary cell culture.

This is because primary cultures are usually isolated from sterile tissues in vivo, while secondary cultures have been growing in the laboratory for an extended time. Cell culture is an essential tool for modern biomedical research.

The process of growing and maintaining cells outside their natural habitat enables researchers to study cellular phenomena and processes with greater accuracy and detail. Primary and secondary cell cultures are the two primary types that provide researchers with cells for their studies.

Secondary culture is the process of subculturing cells repeatedly to increase their population and prolong their lifespan, and it’s used in studies that require a large number of cells; on the other hand, primary cell culture is generally used in studies that require cells that resemble the parental tissue. Both primary and secondary cell cultures have their advantages and disadvantages, and it’s essential to consider specific factors before deciding which method to use.

Understanding both primary and secondary cell cultures and their differences is essential for researchers and the biomedical industry at large to make significant advancements in science and the healthcare industry.

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