Introduction to Cell Culture

This Introduction to Cell Culture provides an overview of the essential concepts, terminology, and techniques used to grow and maintain cells in vitro, including the different types of cultures researchers work with.

Culturing cells, or cell culture, refers to the removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment. The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation, or they may be derived from a cell line or cell strain that has already been established. Basic techniques of cell culture include aseptic handling to prevent contamination, preparing and maintaining culture media, subculturing (or passaging) cells to prevent overgrowth, and cryopreservation for long-term storage. Mastering these techniques is essential for ensuring the health and viability of cultured cells and obtaining reliable experimental results.

While culturing cells can be challenging due to the need for sterile conditions and precise control over environmental factors, it is a fundamental skill in many areas of biological research and biotechnology. There are three main types of cell culture:

In primary culture, cells are directly isolated from tissues and established in vitro for the first time. The cells initially sourced from a tissue sample are called primary cells. They are maintained and proliferated under the appropriate conditions until they occupy all the available substrate (i.e., reach confluence).

When primary cells reach confluence and are subcultured (passaged) for the first time, the resulting population is referred to as a secondary culture (passage 1). In the first passage, the cells are transferred to a new vessel with fresh growth medium and more room to enable continued growth. Subsequent passaging continues and, depending on the cell type, may eventually result in a finite or continuous cell line (see more in the section Finite vs continuous cell line).

Where primary cells are directly isolated from tissues or body-derived samples and cultured for the first time, cell lines are established when cells derived from a primary culture are successfully subcultured and maintained in vitro over multiple passages. In addition to primary and secondary cultures, many scientists also use pre-established cell lines for cell culture work. Using predictable cell lines is cost-effective and supports reproducible results while delivering robust growth, scalability, and ease of maintenance for efficient day-to-day laboratory work.

Cell lines are foundational in cell biology, enabling controlled, reproducible in vitro studies across a wide range of research applications. Understanding how cell lines are established, how they differ from primary cells, the distinctions between finite and continuous lines, as well as between strains and subclones, is essential for selecting the appropriate cell culture model and producing reliable results in cell culture workflows.

When primary cells are successfully subcultured and maintained over multiple passages, the resulting population is referred to as a cell line. During extended passaging, cells with the highest proliferative capacity tend to predominate, leading to increased genotypic and phenotypic uniformity within the culture. The main difference between cells and a cell line is that “cells” is a general term that refers to individual biological units that may exist in vivo or in vitro, whereas a cell line is a defined population of cells grown and proliferated repeatedly in vitro. Cell lines are therefore characterized by their ability to undergo serial passaging and maintain relatively consistent growth characteristics over time. Most cell lines derived from primary cultures have a finite lifespan unless they undergo spontaneous transformation or are intentionally immortalized.

Finite cell lines usually divide only a limited number of times before losing their ability to proliferate—this genetically determined event is known as senescence and is typical in most normal cells. A continuous cell line is a finite cell line that has undergone a process called transformation. Transformation can be spontaneously, chemically, or virally induced—producing an immortal cells; a characteristic in which the line has the ability to divide indefinitely. While choosing from finite cell lines may give you more options to express the correct functions, continuous cell lines are often easier to clone and maintain.

While choosing from finite cell lines may give you more options to express the correct functions, continuous cell lines are often easier to clone and maintain.

Read about key starting considerations for cell culture

A cell strain is created when a subpopulation of a cell line is selected through cloning or other isolation methods. This process yields a more defined subset of the original cell line, often with distinct genetic or phenotypic characteristics. Over time, cell strains may acquire additional genetic changes relative to the parent line. A subclone refers specifically to a population derived from a single isolated cell within a cell line. Because it originates from one cell, a subclone represents a genetically more uniform subset of the original culture.

Culture conditions vary widely for each cell type, but the artificial environment in which the cells are cultured invariably consists of a suitable vessel containing the following:

• A substrate or medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals)
• Growth factors
• Hormones
• Gases (O₂, CO₂)
• A regulated physico-chemical environment (pH, osmotic pressure, temperature)

Most cells are anchorage-dependent and must be cultured while attached to a solid or semi-solid substrate (adherent or monolayer culture), while others can be grown floating in the culture medium (suspension culture).

Cell lines in continuous culture are likely to suffer undesirable outcomes such as genetic drift, senescence, and microbial contamination, and even the best-run laboratories can experience equipment failure. An established cell line is a valuable resource, and its replacement is expensive and time consuming. Therefore, it is vitally important that they are frozen down and preserved for long-term storage. A properly maintained frozen cell stock is an important part of cell culture.

Read more about cell culture environment
Read more about cell freezing protocols
Read more about cell morphology and culture types

Basic cell culture involves several key steps:

Cell culture is one of the major tools used in cellular and molecular biology, providing excellent model systems for studying the normal physiology and biochemistry of cells (e.g., metabolic studies, aging), the effects of drugs and toxic compounds on the cells, and mutagenesis and carcinogenesis. It is also used in drug screening and development, and large-scale manufacturing of biological compounds (e.g., vaccines, therapeutic proteins). The major advantage of using cell culture for any of these applications is the consistency and reproducibility of results that can be obtained from using a batch of clonal cells.

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