Seeding, Subculturing, and Maintaining Cells

However, foundational processes such as establishing an appropriate seeding density, monitoring growth patterns, and adhering to the appropriate subculturing protocols are elements that all cells need to grow and proliferate successfully.The information and protocols on this page will help you develop a baseline understanding of the majority of hands-on culturing procedures.

Cell seeding is a fundamental process in the cell culture workflow that involves placing cells into a culture vessel or onto a surface to allow them to attach, grow, and proliferate. For adherent cells, seeding cells means plating the cells into a culture vessel with pre-warmed medium and incubating them under suitable conditions. In suspension cells, seeding involves transferring the calculated number of cells directly into the culture medium. This process starts with preparing a single-cell suspension from an active culture, a newly thawed vial, or a newly established cell line. This is followed by counting the cells to determine the appropriate seeding density.

In adherent cultures, seeding density is the number of cells plated per surface area unit, typically cm² for most common dishes, plates, and flasks. In suspension cultures, seeding density refers to the number of cells added per unit volume of the culture medium in a vessel. Seeding cells at the optimal density is crucial because it determines the initial cell concentration, which ultimately affects cell growth, nutrient availability, and space for proliferation. A proper cell seeding density provides adequate resources and room for cells to grow, leading to healthy and uniform cultures. Incorrect seeding density can result in overcrowding or sparse growth, negatively impacting experimental outcomes and cell viability. To achieve a consistent growth rate and yield appropriate for your cell type, always follow the instructions included with your product.

Seeding densities for common cell culture vessels

Successfully seeding cells for culture requires several essential materials to produce an environment conducive to cell proliferation. This involves choosing the tools and equipment you’ll employ during the experiment, but more importantly, the vessel and complete media formulation for the cell. Media composition directly influences proliferation and can be the root cause of experimental failures. Seeding density also influences confluence, so tracking cell reproduction with hemocytometers or automated cell counters is essential for checking baseline seeding densities and subsequent growth through the life of your culture.

You will need:

• Your cell line of interest (this may be acquired as an active culture, newly thawed vial, or a new cell line)
• Cell culture vessel of choice such as flasks, plates, and dishes
• Complete cell culture medium
• Hemocytometers or automated cell counter
• Sterile pipettes and pipette tips
• Incubator

The protocol below describes how to seed cells for the adherent or suspension culture of mammalian cells. Before you prepare your cells for the cell seeding protocol, it is recommended to ready your materials from the list above and complete the following calculations to limit the time cells may be exposed to contamination.

1. Choose the appropriate culture vessel based on the number of cells and the culture type required by your cell line.
2. Calculate the desired seeding density based on the experimental requirements and cell line specifications. Product information sheets for cell lines available from Thermo Fisher Scientific.
   Now, you may prepare your cells for seeding. The Gibco Cell Culture Basics Library features many protocols to help you harvest the cells based on where your cells are derived from. See Thawing Cells, Adherent Cell Culture Protocol, Suspension Cell Culture Protocol, and Cell Dissociation Protocol for information on preparing to seed your cells. Follow the protocols of the harvesting method. Once your cells are resuspended in fresh media, you are ready to begin seeding cells.
3. Measure the concentration of your fresh cell suspension, usually in cells/mL, using a hemocytometer or an automated cell counter. For additional support, view our protocols for trypan blue staining and counting cells with a hemocytometer or automated cell counter.
4. Use the cell concentration to determine the volume of cell suspension required to achieve the desired seeding density using the formula:
   Volume of cell suspension = desired number of cells / cell concentration
5. Transfer the calculated volume of cell suspension into the selected culture vessel.
6. Add the appropriate volume of complete growth medium to reach the final desired volume in the culture vessel.
7. Place the culture vessel in the incubator set to the recommended conditions for the cell type and medium (usually 37 °C with 5% CO₂ and the required humidity). Cell seeding is complete.
8. Following the cell seeding protocol, regularly check the cells under a microscope to ensure they are attaching and proliferating as expected. Change the medium as necessary to provide fresh nutrients and remove waste products, following a regular maintenance schedule. Cell viability should be at least 90%.

Subculturing, also referred to as splitting or passaging cells, is the removal of medium and transfer of cells from a previous culture into fresh growth medium, a procedure that enables the further propagation of the cell line or cell strain.

The difference between seeding and passaging cells is that seeding is the placement of cells in a vessel for the purpose of starting a new culture, while subculturing is meant to split up those cells to maintain optimal environments for each set of cells. Passaging gives the cells room to proliferate and presents nutrient stability for cells to remain in log phase growth. In short, seeding is diluting a concentrated cell suspension for new cultures, while subculturing, passaging, and splitting all refer to dividing up an active culture to encourage log phase growth.

Passaging cells requires diligent maintenance and adherence to aseptic techniques. While passaging, carefully record your results and monitor morphology and cell viability for signs of contamination or abnormal behavior.

You can find direction for subculturing adherent or suspension cells in the following protocols:

• Adherent Cell Culture Protocol
• Suspension Cell Culture Protocol

The criteria for subculturing is similar in adherent and suspension cultures. Both culture types should be passaged when they are in the log phase, before they reach confluence. Normal cells stop growing when they reach confluence (contact inhibition), and it takes them longer to recover when reseeded. Transformed cells can continue proliferating even after they reach confluence, but they usually deteriorate after about two doublings. If not passaged prior to confluency, suspension cells can clump together and the medium will appear turbid when the culture flask is swirled.

You should also subculture your cells if you observe a rapid drop in pH (>0.1–0.2 pH units) with an increase in cell concentration. A drop in the pH of the growth medium usually indicates a buildup of lactic acid, which is a by-product of cellular metabolism. Lactic acid can be toxic to the cells, and the decreased pH can be sub-optimal for cell growth. The rate of change of pH is generally dependent on the cell concentration in those cultures—a high cell concentration exhausts medium faster than cells at lower concentrations.

Learn more about the ideal cell culture environment

Monitoring and subculturing cells on a precise schedule allows you to closely monitor the health of your cells and helps ensure your results are reproducible. Vary the seeding density of your cultures until you achieve consistent growth rate and yield appropriate for your cell type from a given seeding density. Deviations from the growth patterns usually indicate that the culture is unhealthy (e.g., deterioration, contamination) or a component of your culture system is not functioning properly (e.g., temperature is not optimal, culture medium too old).

You can passage cells for as long as the cell line is maintaining the expected properties, though it is important to track the number of times the cells have been split. Repeated subculture can result in changes to the cell’s key characteristics and genomic stability and potentially affect your experimental outcome. Be cautious of continuous cell lines that have been passaged repeatedly, and keep thorough notes.

In addition, we strongly recommend that you keep a detailed cell culture log, listing the feeding and subculture schedules, types of media used, the dissociation procedure followed, split ratios, morphological observations, seeding concentrations, yields, and any antibiotic use.

It is best to perform experiments and other non-routine procedures (e.g., changing type of media) according to your pre-determined subculture schedule. If your experimental schedule does not fit the routine subculture schedule, make sure that you do not passage your cells while they are still in the lag period or when they have reached confluency and ceased growing.

Maintaining a regular culture care schedule is crucial to the viability of your cells throughout the duration of your experiment. This is because the growth of cells in culture follows a standard pattern. A lag after seeding is followed by a period of exponential growth, called the log phase. When cells in adherent cultures use all the nutrients in the growth media or cover the entire substrate with no space left to expand, they enter the stationary phase (also known as the plateau phase). Similarly, cells in suspension cultures will enter the stationary phase when they exceed the medium's capacity to support further growth. In this phase, cell proliferation significantly slows down or stops entirely.

Cells should be passaged, or subcultured, when they cover the plate, or the cell density exceeds the capacity of the suspension medium. This will keep cells at an optimal density for continued growth and stimulate further proliferation. Maintaining log phase growth will maximize the number of healthy cells for your experiment.

These useful numbers for cell culture can help you determine a starting seeding density and cells at confluency count for various sizes of cell culture dishes, plates, and flasks.

Learn more about cell confluency and how to calculate it

There are several common problems that you may encounter when culturing cells. Problems in primary cultures may have different causes than the same problem in established cell lines. The table below addresses some of the common problems, along with their possible causes and suggested solutions.

1. Freshney, R. (1987) Culture of Animal Cells: A Manual of Basic Technique, p. 220, Alan R. Liss, Inc., New York.
2. ATCC. "Passage number effects in cell lines | ATCC" ATCC. Accessed July 21, 2025. 
3. Marro BS, Zak J, Beheshti Zavareh R, et al. (2019) Discovery of small molecules for the reversal of T cell exhaustion. Cell Rep 29(10):3293–3302.e3. doi: 10.1016/j.celrep.2019.10.119.