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Cells on Strike? 10 Reasons Your Cells Won't Attach to the Flask (Low Adhesion and How to Fix It!)
- CLYTE research team
- Oct 15
- 7 min read
Nothing is more frustrating in the world of cell culture than looking into the microscope and seeing your precious cells floating in the medium instead of happily attached to the flask. It's a common problem that can bring your research to a screeching halt. But don't worry, you're not alone, and the solution is often simpler than you think. In this comprehensive guide, we'll explore the top 10 reasons why your cells might be giving you the cold shoulder and, more importantly, what you can do about it.
You might also be interested in this article about tackling contamination! Ask Sophie why your cells aren't attaching to the flask?
1. Trouble in Paradise: Problems with Your Culture Vessel
Not all plastic is created equal, especially in the world of cell culture. Adherent cells require a specially treated surface to attach and grow. If your cells aren't sticking, the first thing to check is your flask.
Are you using TC-treated flasks? Most adherent cell lines require a tissue culture (TC) treated surface, which is modified to be more hydrophilic, allowing cell adhesion proteins to bind.
Is the flask new and clean? Scratches, residues from previous cultures, or manufacturing defects can all prevent cells from attaching.
Could there be static electricity? Especially in low humidity, static electricity can build up on plastic flasks and repel cells. You can wipe the outside of the flask with a damp, disinfected towel to dissipate the charge.
2. Unhappy Campers: Issues with Cell Health and Viability
Healthy, happy cells are much more likely to adhere and proliferate. If your cells are stressed or dying, they won't have the energy to adhere to the flask.
Check for viability: Use a trypan blue exclusion assay to determine the percentage of viable cells. A low viability count is a major red flag.
Don't over-passage: Cells that have been passaged too many times can undergo senescence and lose their ability to attach. Always use low-passage cells for your experiments.
Handle with care: Cells are delicate, especially after thawing or trypsinization. Avoid vigorous pipetting or centrifugation, which can cause mechanical damage.
3. The Secret Sauce: Media and Reagent Problems
Your cell culture medium is a complex cocktail of nutrients, growth factors, and other essential components. If something is wrong with the medium, your cells will be the first to know.
Is your FBS top-notch? Fetal Bovine Serum (FBS) is a critical component of many culture media, providing a source of adhesion factors like fibronectin and vitronectin. If you're having attachment problems, try increasing the FBS concentration (up to 20%) or using a different batch.
Glutamine degradation: Glutamine is an essential amino acid, but it's also unstable and degrades over time into toxic ammonia. Make sure your medium is fresh and properly stored.
Neutralize that trypsin! Trypsin is an enzyme used to detach cells from the flask. If it's not properly neutralized with medium containing FBS, it can continue to chew up cell surface proteins, preventing reattachment.
4. Location, Location, Location: Environmental Stress
Cells are creatures of habit and are very sensitive to their environment. Even small fluctuations in temperature, CO2, or humidity can cause them to detach.
Temperature troubles: Make sure your incubator is properly calibrated and maintaining a stable temperature.
CO2 chaos: The CO2 level in your incubator is critical for maintaining the pH of your medium. Check the CO2 tank and make sure the incubator's sensor is working correctly.
Humidity horrors: Low humidity can cause the medium to evaporate, leading to a toxic concentration of salts and other components.
5. Uninvited Guests: Contamination
Mycoplasma, bacteria, and fungi are the uninvited guests of the cell culture world. These contaminants can compete with your cells for nutrients and release toxic byproducts that can prevent adhesion.
Check for signs of contamination: Look for turbidity, color changes in the medium, or a "grainy" appearance under the microscope.
Get tested: If you suspect mycoplasma contamination, get your cells tested by a reputable service.
Practice good aseptic technique: The best way to deal with contamination is to prevent it in the first place.
6. Growing Pains: Scaling Up Issues
Moving your cells from a small flask to a larger, multi-layer flask can present new challenges.
Oxygen O-no: The oxygen tension in a multi-layer flask can be lower than in a single-layer flask, which can affect cell adhesion.
Cell density dilemmas: The optimal seeding density may be different in a multi-layer flask. You may need to adjust your cell numbers accordingly.
Coating concerns: The surface coating in a multi-layer flask may be different than in a single-layer flask. You may need to pre-coat the flask with an extracellular matrix (ECM) protein like collagen or fibronectin.
7. Too Close for Comfort (or Too Far Apart): Incorrect Cell Seeding Density
Seeding your cells at the correct density is crucial for their survival and attachment.
Too low: If you seed your cells at too low a density, they may not be able to produce enough of their own ECM to support adhesion.
Too high: If you seed your cells at too high a density, they may not have enough space to attach and spread out.
8. The Big Chill: Cryopreservation and Thawing
The process of freezing and thawing cells is a major stressor and a common cause of poor adhesion.
Thaw quickly: Thaw your cells rapidly in a 37°C water bath to minimize the formation of ice crystals.
Remove the cryoprotectant: DMSO is a common cryoprotectant, but it's toxic to cells at room temperature. Remove it as soon as possible after thawing.
Give them time to recover: It can take cells 24-48 hours to fully recover from the stress of thawing. Don't be too quick to discard floating cells.
9. A Little Too Much: Over-trypsinization
Leaving trypsin on your cells for too long can damage the cell surface proteins that are essential for adhesion.
Use the right concentration: Use the lowest concentration of trypsin that is effective for your cell line.
Don't over-incubate: Monitor your cells closely and neutralize the trypsin as soon as they have detached.
Be gentle: A gentle tap on the side of the flask is usually all that's needed to dislodge the cells.
10. A Helping Hand: Lack of Adhesion Factors
Some cell lines are just pickier than others and require a little extra help to attach.
Coat your flasks: Pre-coating your flasks with an ECM protein like collagen, fibronectin, or gelatin can provide a more favorable surface for adhesion. Depends on the cell type!
Use a specialty medium: Some commercially available media are specifically formulated to enhance cell adhesion.
Cell Adhesion Bottom Line
Poor cell adhesion is a common and frustrating problem, but it's usually solvable. By systematically working through these potential causes, you can identify the culprit and get your cells back on track. Remember to be patient, be observant, and don't be afraid to try something new. Happy culturing!
Frequently Asked Questions (FAQ)
Why are my cells not adhering to my flask?
This is a common issue with several potential causes. The most frequent culprits include:
Incorrect Flasks: You might be using non-treated plastic instead of tissue culture (TC) treated flasks, which have a modified surface to promote cell adhesion.
Poor Cell Health: The cells may have low viability due to improper handling, being over-passaged, or stress from the thawing process.
Media Issues: Your media could be the problem. This can be due to degraded glutamine, low-quality Fetal Bovine Serum (FBS) that lacks necessary adhesion factors, or an incorrect pH.
Contamination: A hidden contamination, especially from Mycoplasma, can stress cells and prevent them from attaching properly.
Technique Errors: Over-exposing cells to trypsin can damage the surface proteins they need to stick to the flask.
Why are my cells not attaching after thawing?
Thawing is one of the most stressful events a cell can endure. Poor adhesion after thawing is often due to:
Cryo-injury: If the cells were frozen too slowly or thawed too slowly, ice crystals can form and damage cell membranes, leading to low viability.
DMSO Toxicity: The cryoprotectant DMSO is toxic to cells at room temperature. It's crucial to dilute and remove it quickly by centrifuging the cells and resuspending them in fresh, warm medium.
Cellular Recovery Time: Cells need time to recover from the shock of being frozen. Their metabolic activity is low, and it can take them 24-48 hours to repair damage and begin producing the proteins needed for adhesion. Don't be too quick to discard floating cells; give them at least a day to recover and attach.
How to fix non-adherent cells?
To fix the problem, you need to identify the cause. Here is a troubleshooting checklist:
Confirm Your Flasks: Double-check that you are using new, unused TC-treated flasks.
Increase Serum: Try increasing the FBS concentration in your medium to 15% or even 20%. FBS contains vital adhesion factors like fibronectin.
Use Coated Flasks: For particularly tricky cell lines, coat the flask surface with an extracellular matrix (ECM) protein like collagen, fibronectin, or gelatin before seeding your cells.
Be Patient: Especially after thawing, give your cells 24 hours to attach before giving up on them.
Start Fresh: If you suspect low viability or contamination, it's best to discard the culture and thaw a new, low-passage vial of cells, following best practices for thawing.
How long does it take for cells to attach?
The time it takes for cells to attach depends heavily on the cell line and its health. However, here's a general timeline:
Initial Adhesion: Most healthy, fast-growing cell lines (like HEK293 or HeLa) will begin to attach within 2 to 4 hours after seeding.
Full Adhesion: You should see the majority of viable cells attached and spread out within 12 to 24 hours.
Post-Thaw Exception: Cells recovering from cryopreservation are slower. It may take them up to 48 hours to fully attach and begin dividing. If you see very few attached cells after 24 hours during a routine passage, it's time to start troubleshooting.
