Guidelines for Cell Seeding and Maintenance

Cell Seeding and Maintenance

How to Get Set Up for Cell Culture Success: Seeding and Maintenance

Whether you're new to cell culture or an experienced researcher, setting up and maintaining your cells properly is crucial for obtaining accurate and reliable results.

From thawing frozen cells to counting and seeding them in appropriate culture vessels, to regular feeding and passaging to maintain cell health, we've got you covered.

So, let's dive in and learn how to set up and maintain your cell cultures.

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Where do I start with seeding and maintenance?


The setup and maintenance process involves several steps, including thawing frozen cells, counting cells, and seeding them into appropriate culture vessels. Once cells are established in culture, maintaining proper growth conditions is key to their health and viability. Let's take a closer look at each of these steps.


Thawing Frozen Cells

Do's and Don'ts of Thawing Frozen Cells

Freeze-thawing can be seriously detrimental to your cells, so it’s important to thaw cells quickly and carefully to prevent damage. Rapid thawing or exposure to too much heat or cold can damage cells, leading to reduced viability and altered behavior. The right equipment, such as a water bath or incubator, can help maintain a consistent temperature and prevent damage to cells during the thawing process.

Here are some key tips to ensure your cells survive the thaw:

  • Use a water bath set to 37 °C to thaw the cells quickly and evenly.
  • Use sterile techniques to prevent contamination during the thawing process. Always use a laminar flow hood to keep your cells safe.
  • After thawing, centrifuge the cells to remove the cryoprotectant and resuspend the cells in warm fresh growth media.


Cell Counting

Methods for Counting Cells

Accurately seeding your cells at the right density is crucial to their survival and growth rate. There are several methods for counting cells, including manual or automated methods. The right density will differ depending on your cell type.

Here are some general tips for counting cells:

  • Use fresh growth media to suspend cells for counting.
  • Dilute the cell suspension appropriately to ensure accurate counts.
  • Count cells in multiple fields to ensure accuracy.



Cell Seeding and Maintenance

Seeding Cells in Culture Vessels

Seeding cells at the correct density is important for proper growth. Seed at a low density and your cells will struggle to communicate and grow. Seed them at a high density and they will quickly become overcrowded, depleting critical nutrients. That means your cells may start to behave erratically.

Like all living things, your cells need a good home and the type of culture vessel you use can make all the difference. Here are some tips for making your cells feel more at home:

  • Use sterile techniques to prevent contamination during cell seeding.
  • Seed cells at the appropriate density for the specific cell line and culture vessel.
  • Gently swirl or rock the culture vessel to ensure cells are evenly distributed.
  • Choose a vessel that will encourage the growth and survival of your cell type. For example, start by seeding your cells into a smaller flask to give them a chance to initiate growth before passaging into a larger vessel.
Maintaining Cell Culture

How to Maintain Your Cell Culture

Once your cells are established, what comes next?
At this point, you are either maintaining cells for assays or expanding cell types to make larger stocks. Once cells are established in culture, it is important to maintain them properly to ensure their health and viability. This includes regular feeding and passaging of cells to prevent overgrowth and nutrient depletion.

Be sure to follow the tips below to maintain your cell culture:

  • Change growth media regularly to provide fresh nutrients and remove waste products.
  • Monitor cell growth and behavior regularly to detect any changes or problems.
  • Passage cells at 70 to 80% confluency to prevent overgrowth and nutrient depletion.

By investing in the right equipment and techniques, you can overcome the common challenges associated with the set-up and maintenance of cell cultures.



Frequently Asked Questions


How do I determine the appropriate cell density for seeding?


Cell density varies widely based on cell type, application, and vessel size. For example, some cell types require seeding at a lower cell density while expanding for experiments while others will need close contact to survive and grow. Generally, we would recommend consulting the literature and performing pilot experiments to determine the optimal seeding density for your specific experiment.

As a rule, use the following instructions to calculate seeding density:
● Determine how many cells/mL you require (X)
● Count cells using a hemocytometer.
● Multiply that number by the total volume to get the total number of cells.
● Centrifuge cells and resuspend in 1 mL of media or PBS (Y).
● Determine how much media to add (Z) to reach the final seeding density: X/Y = Z


How do I determine if my cells require passaging after seeding?


Visual cues like confluency (percentage of the culture surface covered by cells) and changes in cell morphology indicate when cells need passaging. Additionally, you may notice that the media is starting to change color as nutrients become depleted. Generally, cells should be passaged before they become over-confluent or exhibit signs of stress. Signs of stress include unusual morphology, particulate floating in the media, and the detachment of adherent cells.


How can I prevent contamination during cell seeding and maintenance?


To prevent contamination, maintain a sterile environment, work in a laminar flow hood, when possible, wear appropriate personal protective equipment, and follow aseptic techniques while handling cells and media. If you suspect contamination, it’s important to deal with it immediately to prevent it from spreading to other cultures. Add a disinfectant to the flask before discarding then disinfect the incubator and any other equipment that may have become contaminated.


Help. I still don’t know how to set up my culture. What do I need to do?

To summarize:

  • Thaw frozen cells according to instructions.
  • Count cells using a hemocytometer or automated cell counter.
  • Seed cells into appropriate culture vessels.
  • Provide cells with the proper growth conditions, including media, temperature, and CO2 levels.
  • Regularly feed and passage cells to prevent overgrowth and nutrient depletion.

If you are still unsure, seek guidance from an experienced researcher or consult resources such as scientific publications, online forums, or cell providers.


How do I know which vessel is right for my cell type?

Adherent cells require a surface for attachment, while suspension cells do not. Different vessels are available for different cell types, including flasks, cell culture plates, and bioreactors. It is important to choose a vessel suited for the specific cell type and culture conditions. Also, consider factors such as surface area, gas exchange, and nutrient supply.

The key is to do your research and choose the right vessel for your specific cell type. Don't just go with the first thing you find. Take time to find the best option to ensure the health and growth of your cells.

Check out our page on Growing Cell Cultures: Types of Cells to learn about the right labware for your cell type.


How often should you change cell culture media?

This depends on a few things. The rate of nutrient depletion, cell density, and the type of cells you are working with can all play a role. In general, media should be changed every 1 to 3 days for most cell types.

However, some cells may require more frequent changes or longer intervals. It is important to monitor the health and growth of your cells regularly and adjust your media change schedule accordingly. For example, if you notice your media is yellowing very quickly, you may well have a contamination issue. Then it’s time to start again.