Mammalian cell cultures are essential for clinical and pharmaceutical research and applications. They allow flexibility in model systems used for health and disease, e.g., functional studies, help support in-silico analysis, and can be an alternative to animal models.
Cell proliferation is dependent on the cell type, whether they are primary cells (isolated from animal tissues), embryonic stem cells, self-renewing cells, immortalized stable cell lines, or transformed cells, and on the culture conditions (media, 2D/3D extracellular matrix, temperature, PH, CO2/O2 levels). Once in optimal culture conditions, cells can be grown for different experimental analysis. With advancements in animal-cell-culture technology, cell lines have evolved and are also used for vaccine production, therapeutic proteins, pharmaceutical agents, and anti-cancerous agents.
For a successful maintenance of mammalian cell lines, cells must be grown under controlled conditions and require their own specific growth medium. In addition, to guarantee physiological and phenotypical stability, their growth must be monitored at regular intervals. In general, when a cell line reaches about 80% confluency, the cells must be subcultured to ensure proper growth and health. A confluency of 80% means that 80% of the culture vessel surface is covered with cells. Note that the ideal confluency for subculturing cells depends on the cell type and might have to be optimized.
This article describes a typical workflow for subculturing an adherent cell line with a step-by-step description and detailed illustrations (Figure 2).
Why do I need to split my cells?
Once a cell culture is initiated, it cannot be grown indefinitely, due to the ever-increasing number of cells in a confined area, consumption of nutrients, and increase in toxic metabolites which eventually results in cell death. Moreover, researchers usually need to replicate experiments and, therefore, rationalize or expand the cells in culture. Subculturing or splitting the cells produces new cultures with lower cell density than the original culture. By removing the medium and transferring the cells into fresh growth medium and matrix, the cells are given fresh nutrients and toxic metabolites are removed, allowing long-term maintenance of the culture. After initially seeding the cells, growth starts with lag phase and proceeds to a log phase, where the cells proliferate exponentially followed by a stationary phase where growth rate and death rate are equal. (Fig. 1). In the death phase, cells die due to lack of nutrients or inadequate culture conditions, such as when cells start competing for space due to high or overconfluency.
To keep cells in optimal culture conditions and actively growing, it is necessary to renew the growth medium and to subculture them at regular intervals. Change of culture medium can take place several times in the log phase dependent on the cell type. The best time to subculture cells is between the log phase and the stationary phase, before the cells reach confluence.
Why do I need to examine my cells?
It is important to examine the cell culture every day, and immediately prior to subculturing, as a means to monitor cell health, check for contamination, and determine when to split the cells. A first examination of the culture for fungal contamination, turbidity, and particles in the medium as well as unexpected pH shifts, indicated by color change of the medium, can be done at the macroscopic level by eye. However, the absence of bacterial contamination should be confirmed at high magnifications. After this, a closer check of the general cellular morphology and growth patterns should be examined using an inverted microscope. The optics of an inverted microscope are located below the specimen. Because the cells are attached to the bottom of the dish, they can be viewed easily from this perspective. Observation should take place with a total magnification of 100 – 200x and phase contrast , because most cells are difficult to observe in normal brightfield illumination.
There are many types of mammalian cell morphology, but most mammalian cells in culture can be divided into 3 categories: fibroblast cells (Chinese hamster ovary (CHO) cells), epithelial-like cells (human cervix (HeLa) cells), and lymphoblast-like cells (human leukemia (HL60) cells). In addition, certain cell lines can have specific morphological characteristics, e.g., neurons (SH-SY5Y) which have long dendritic elongations. Cell morphology is also affected by events in the cell lifecycle. During mitosis many cells become more round, forming very refractile bright spheres that may float around in the medium. Dead cells show changes in their membrane integrity, become more round, and, in the case of adherent cells, detach from the growth surface. Under a microscope, dead cells are usually not bright and refractile and may show changes in their typical membrane morphology.
Various cell lines not only differ in size and shape, but also in their growth behavior. They either grow as adherent cells (fibroblastic and epithelial cells) or in suspension (lymphoblast-like cells). Most adherent cell lines grow as a single cell layer (monolayer) attached to glass or treated plastic substrates (e.g., coated with poly-lysine, fibronectin, collagen, or gelatin).
How can I subculture my cells?
The most common method to prepare cells for subculture is by breaking the intercellular and cell-to-substrate bonds with proteolytic enzymes like trypsin. Trypsin in combination with Ethylenediaminetetraacetic acid (EDTA) causes cells to detach from the growth surface. Trypsin cuts away the focal adhesions that anchor the cell to the culture dish and EDTA acts as a calcium chelator.
Depending on the cell type or downstream experiments, other proteases may be used as an alternative to trypsin, such as natural collagenases or synthetic dissociation cocktail solutions.
By removing calcium, cadherins which are involved in cell-cell interactions are broken and the cells separate from one another. Once no longer bond to the growth surface and surrounding cells, they can be easily separated and grown in new cell culture dishes. Cell culture conditions and subculture methods vary for each cell type. Figure 2 describes the basic steps in the subculture workflow. During the whole subculture process, it is important to work in a contamination-free environment. Examination of the cells at the beginning, during trypsinization, cell counting, and after splitting is essential. For consistent results and tracking of experiments, maintaining good records and documentation is also important.
The following protocol describes the basic principles of the subculture routine for Madin-Darby canine kidney (MDCK) cells grown in a 90 mm petri dish. These are epithelial cells isolated from the distal tubules of a dog. In culture, they grow adherently and form a monolayer of polygonal cells after they have reached confluence.
The following material and equipment are needed for subculture:
- Medium pre-warmed to 37° C (for MDCK cells: MEM with 5%