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Workflow for subculture of adherent cells

Many fields of biomedical research, like cancer research, drug development and tissue engineering, require the use of living cells to perform a variety of assays. Mammalian cell cultures are an essential tool in biology because they allow rapid growth and proliferation of different cell types for experimental analysis.

In order to successfully work with mammalian cell lines, they must be grown under controlled conditions and require their own specific growth medium. In addition, to guarantee consistency their growth must be monitored at regular intervals. When a cell line reaches about 80% confluence, the cells must be subcultured to ensure proper growth and health of the cells. 80% confluency means when 80% of the surface of a culture vessel is covered with cells.

This article describes a typical workflow for subculturing an adherent cell line with detailed illustrations of all of the necessary steps.

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Video: Workflow for subculture of adherent cells

Why do I need to split my cells?

Once a cell culture has been started, it cannot be grown indefinitely due to the increase in cell number, consumption of nutrients and increase in toxic metabolites which eventually results in cell death. Moreover researchers usually want to perform experiments on their cells several times, and therefore do not want to use up all of the cells at once. 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, 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 a 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 and inadequate living conditions.

In order to keep cells healthy 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 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. 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. Since 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 with phase contrast, because most cells are difficult to observe in normal bright field illumination.

There are many variations in mammalian cell morphology, but most mammalian cells in culture can be divided into three categories: fibroblastic cells (Chinese Hamster Ovary cells (CHO)), epithelial-cell-like (human cervix cells (HeLa)) and lymphoblast-like cells (human leukaemia cells (HL60)). In addition to this certain cell lines can have specific morphological characteristics, e.g. neurons (SH-SY5Y) which have very long dendritic processes. Cell morphology is also affected by events in the cell lifecycle. During mitosis many cells round up, forming very refractile bright spheres that may float around in the medium. Dead cells often round up and become detached also but are usually not bright and refractile.

Various cell lines not only differ in size and shape, they also differ in their growth behaviour. They either growing adherent (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 (coated with poly-lysine, fibronectin, collagen or gelatine).

How can I subculture my cells?

The most common method to prepare cells for subculture is by breaking the intercellular and cell-to-substrate connections 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.

By removing calcium, cadherins which are involved in cell-cell interactions, are broken and cells separate from one another. Once separated from the growth surface and the 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 trypsination, cell counting, and after splitting is essential. For consistent results, maintaining good records and documentation is also important.

The following protocol describes the basic principles of the subculture routine for Madin Darby Canine Kidney Cells (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 is needed for subculture:

Materials:

  • Pre-warmed medium to 37° C (for MDCK cells: MEM with 5% FCS , 2 mM Glutamine, 100 U/mL Penicillin, 100 mg/mL Streptomycin)
  • Pre-warmed PBS without Ca2+ /Mg2+
  • Pre-warmed 0.05% Trypsin, 0.02% EDTA in D-PBS
  • Trypan Blue (vital stain)
  • 70% ethanol or isopropanol for disinfection

Equipment:

  • Cell counting chamber
  • Pipette/ micropipette with disposable tips
  • Phase contrast capable inverted microscope (Leica DMi1, for example)
  • Personal protective equipment
  • Water bath set to appropriate temperature
  • Incubator at 37°C with 5% CO2 and high humidity
  • Centrifuge
  • Pre-labelled dishes

The splitting ratio is 1:10.

4-steps-workflow for subculture of adherent cells as conducted with the Leica DMi1

Step 1: Cell examination

Step 2: Cell harvesting

Step 3: Cell counting

The cell concentration can be calculated by the following formula:

Step 4: Plating