Subculturing Cells: Techniques and Best Practices

Transferring cells from one culture vessel to another is referred to as subculturing. To maintain cell viability and health and to expand the number of cells available for a research study, this procedure is crucial in biological study and experimentation.

Key Takeaways

  1. During subculturing, cells are transferred from one culture vessel to another.
  2. Adherent or suspension cultures should be passaged in the log phase but before they reach confluence or are growing to densely.
  3. Trypsin is a commonly used enzyme for detaching cells from a culture dish, but other solutions such as Accutase can also be used.
  4. Optimal cell density for subculturing cells varies based on the type of cell and growth conditions.
  5. Different types of media can be used for culturing cells, such as DMEM and RPMI, which often have to be supplemented with FBS.
  6. Subculturing suspension cultures and mammalian versus insect cells have their own specific considerations and protocols.

Importance of subculturing in cell culture research and experimentation

Cell culture is essential to biological research and experimentation and cell subculturing is crucial to cell culture. Subculturing involves transferring cells to new culture vessels giving them enough room to grow. This article will discuss subculturing methods and how to keep cells healthy and viable.

Techniques for Subculturing Adherent Cells


Trypsinization is used to remove cells from a culture vessel. This method uses the enzyme trypsin, which is added to the culture vessel and left to work for a few minutes. Check the cells microscopically for detachment and transfer to a new culture medium and vessel once fully detached.

Other detachment solutions

Accutase or TrypLE Express are other widely utilized cell detachment solutions. Accutase and TrypLE Express are cell detachment solutions designed to remove cells from culture vessels reducing the risk of cell damage during dissociation. Accutase, unlike trypsin, uses a combination of proteolytic and collagenolytic enzymes to remove cells and serves as a straight alternative for trypsin and collagenase applications. With its ability to cleave peptide bonds at the C-terminal end of lysine and arginine, TrypLE Express is an ideal substitute for trypsin, offering the added benefits of being gentle to cells and free from substances of animal origin. Both solutions can be applied to a wide range of cell types, including primary cells, stem cells, and cancer cells such as HEK293, CHO or A549 and do not require the use of serum.


A cell scraper is used to remove cells from a culture vessel. This is typically done when the cells have reached confluency, meaning that they have completely covered the surface of the dish. Scraping cells is often used to harvest cells for downstream applications such as cell counting, flow cytometry, or cell culture.

Scraping can harvest cells that are hard to detach with trypsinization. This method works on cells that resist mechanical or enzyme-based dissociation. Since scraping activates signaling pathways and changes cell behavior, it can be used to study mechanical stress on cells.

Scraping cells can tear membranes and cause cell damage. Thus, proper cell scraping technique and minimal force are essential.

Techniques for Subculturing Suspension Cells

Subculturing suspension cultures involves transferring cells from liquid cultures to new vessels. This method is used to grow blood cells, stem cells, and some cancer cells in liquid media.

Consider these five steps when subculturing suspension cultures:

  1. Before subculturing, measure cell densities and seed the new culture at the right density.
  2. Subculturing can be done by transferring cells or diluting them with fresh media.
  3. To ensure that cells are uniformly distributed in a new culture, they should be gently mixed or shaken.
  4. Avoid overdiluting suspension cultures to prevent cell death and inhibited growth.
  5. Suspension cultures are subcultured 1:2 or 1:3 depending on cell growth. For every 1mL of culture, transfer 0.5 or 0.33mL to a new vessel and add fresh media. This provides cells with nutrients and room for growth.

Comparison of Subculturing Mammalian and Insect Cells

Mammalian cells

  • To avoid contact inhibition and deterioration of cells after two doublings, adherent cultures must be passed during log phase growth, prior to reaching confluence.
  • In addition, suspension cultures should be passed during the log phase of growth, prior to clumping and turbidity in the medium.
  • Rapid decrease in pH (>0.1-0.2 pH units) in conjunction with an increase in cell concentration indicates the need for subculture.

Insect Cells

  • In order to maintain optimal cell growth and viability, adherent and suspension cultures must be passed during log phase growth, prior to confluence.
  • Passage at densities beyond confluence reduces doubling times, viabilities, and attachment ability.
  • In general, insect cells are cultured in more acidic media, with a gradual increase in pH that does not exceed 6.4 but falls as cell density rises.

Note: When subculturing cells, culture medium and pH changes are crucial to cell growth and preservation.

Best Practices for Maintaining Cell Health and Viability

Sterility: Maintain sterility when subculturing cells. This includes sterile equipment, waste disposal, and a clean work environment.

Timing: Subculture cells before they reach confluence. This optimizes cell growth and proliferation.

Cell Number: Cell health depends on cell density. Overcrowding reduces cell health and viability, while undercrowding reduces cell availability for experimentation. Considerations include:

  1. Depending on the type of cells being grown, the optimal cell density for subculturing can vary.
  2. Cell density can be determined by microscopy. Confluence should be 80–90%.
  3. A hemocytometer or automated cell counter can also be used to determine the optimal cell density. Cell densities are measured in cells per unit area or volume. One milliliter of culture media contains 1x106 cells/ml.

Media and Conditions: Each cell culture requires specific nutrients, pH, and temperatures.

The media used for culturing cells will also depend on the type of cells being grown. Commonly used media include:

  1. Dulbecco's Modified Eagle Medium (DMEM)
  2. Minimum Essential Medium (MEM)
  3. RPMI-1640

These media supply cells with nutrients, vitamins, and minerals. pH buffers and other chemicals regulate pH and temperature for cellular growth. Some cells need FBS or other growth factors in their media. This depends on the cell type, so consult the literature. To find the best media, supplements, and methods for your cells, test a variety to find the optimal one for your particular type of cells.

To sum up

Subculturing cells is essential for cell culture research. It maintains cell health and viability. Trypsinization, scraping, and pipetting are different ways to subculture cells, but the right method depends on the cell line used. Sterile conditions, correct subculturing timing, cell density, and media and conditions have to be on point. Follow these guidelines for cell culture research and experimentation to succeed.