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Cell Culture – Viability and Cell Attachment

45 min Read

Ensuring your cell cultures are happy and healthy can be tricky business! Recognizing healthy as well as dead/compromised cell cultures is an important foundational skill that every cell culture enthusiast should learn. In this blog post, we’ll cover:

  • Cell Culture Growth Behavior
  • Cell Morphology
  • Identifying Cell Death
  • Cell Counting
  • Components of Cell Culture Media/Vessel
  • Troubleshooting Guide

How do you know your cells are healthy and happy?

Cell Culture Growth Behavior

A few things to keep in mind before starting your cell culture are cell line growth characteristics such as proliferation rates and seeding density.

In culture, cell growth will follow the standard pattern of a lag phase, logarithmic growth phase, and a stationary phase (Figure 1). It is important to recognize which phase of growth your cell cultures are in to ensure you maintain healthy cells for your experiment.

Cell Culture Growth Curve

Figure 1 – The typical growth curve for cell culture takes on a sigmoidal (S) shaped curve with a lag phase, logarithmic phase, and a stationary phase.

Lag Phase

After thawing, cells begin acclimatizing to the new culture conditions and will be in lag phase. The length of the lag phase can depend on how stressed the cells are and the seeding density used. For adherent cells, attachment usually occurs within 24 hours.

Logarithmic Growth Phase

Once cells recover and begin actively dividing, they enter the logarithmic growth phase. Population doubling time and other cell characteristics should be assessed at this time. Cells are usually passaged late in the log growth phase before they reach 100% confluency.

Stationary Phase

If the cells are allowed to become overcrowded they become more susceptible to injury, senescence, and apoptosis. At this point, the cells are in stationary phase and less than one tenth of the cells are actively dividing.

Cell Morphology

Cell morphology describes a cell’s shape and overall appearance and is an important indicator of cell culture health. Improper aseptic technique can lead to contamination of your cell lines so it’s a good idea to check your cell’s morphology each time after use. Read our article about how to recognize signs of contamination in your cell culture.

Below are common cell types and their typical morphology:

Adherent cells

1. Forms monolayer of cells attached to surface

2. Two main morphological categories:

  • Epithelial-like: Polygonal shape, stationary, grows in patches
  • Fibroblast-like: Elongated shape, bipolar or multipolar, can migrate over substrate

Adherent Cells (epithelial-like)

Figure 2 – Adherent cells with epithelial-like morphology.

Adherent Cells (fibroblast-like)

Figure 3 – Adherent cells with fibroblast-like morphology.

Suspension cells

  • Free-floating in suspension (no monolayer formation)
  • Appear as single cells or small clumps
  • Lymphoblast-like spherical shape
  • Cells that appear detached and rounded but “refractile” are healthy cells

Suspension cell morphology

Figure 4 – Example of suspension cell morphology.

Identifying Cell Death

The death of your cell cultures can be disappointing and frustrating. That’s why it’s always a good idea to carefully observe signs of cell death early on in order to identify the cause and hopefully prevent further deterioration of your precious cultures. Here are some common signs of cell death:

  • The occurrence of cell clumping. After cell lysis, DNA and other cell debris is released into the medium and aggregates with cells, appearing as a small clump.
  • Detached, rounded adherent cells and clumped suspension cells that appear small and dark. (Cells that are detached and rounded but “refractile” are healthy cells.)
  • Cells that stain blue after the addition of trypan blue. When a cell has a compromised/damaged cell membrane, Trypan blue is able to enter the cell and stain intracellular proteins.
  • Cell crenation or blebbing.

Cell death

Figure 5 – Example of cell death.

Cell Counting

To get an accurate snapshot of your cell culture’s health you will need to count your cells regularly. Cell counting is an important part of daily cell culture routine and is essential for determining the cell concentration needed for passaging/seeding, to monitor growth rates of your cells, and to assess viability of cells after drug treatments. Two main pieces of equipment are commonly used for cell counting: a hemocytometer or an automated cell counter.


  • For manual cell counting
  • Contains glass slides with two counting chambers etched with grids of a specific dimension that allow counting of the total number of cells within an exact volume.

Automated Cell Counter

  • More precise way to determine cell counts but, in cases where electrical impedance method is used, you may also end up counting dead cells
  • Cells are counted as they pass within an aperture between two electrodes
  • Can be used in combination with viability assessment from a hemocytometer
Components of Cell Culture Media/Vessels

Cell culture media provides the essential nutrients, growth factors, hormones, and attachment factors necessary for healthy cell growth. Media lacking in the ideal nutrient profile may be a factor for poor cell attachment and cell culture viability.

Different cell lines have a variety of nutrient requirements. It’s important to check the composition of the media you are using when culturing your cells to ensure it is the ideal media to use. For example, different basal media have a range of glucose, amino acid, vitamins, salts, and other nutrient concentrations. Buffers also play an important role in maintaining the pH in media. Depending on the amount of CO2 in the growth environment, the amount of sodium bicarbonate in the medium can vary. Finally, be mindful that serum is heat sensitive and higher temperatures can inactivate the growth factors found in it.

Finally, it is a good idea to check the culture protocols for centrifuge speeds, and proper proteolytic enzyme concentrations to minimize the stress your cells experience during culturing.

If you’re handling adherent cells, it is important to be familiar with the cell culture vessel coatings that are commonly used:

  • Poly-Lysine (TM061): Positively charged synthetic polymer that helps cells attach to plate surfaces due to electrostatic interactions between the coated plate and the negatively charged cell membrane.
  • Poly-Ornithine (TM062): Positively charged synthetic chain of amino acids that is used to coat culture vessels, promoting cell attachment through electrostatic interactions between the cell membranes and the plate.
  • Collagen: Mammalian protein that is found in the extracellular matrix that allows for adhesion and growth of cells in connective tissue. It can be used to coat cell culture vessels to ensure attachment of certain cells.
  • Fibronectin: Glycoprotein that is found in the extracellular matrix. Used as a coating to promote the adhesion and growth of certain cells.
  • Laminin: Extracellular matrix protein that is found in the basal lamina and that can aid with cell adhesion.
  • Gelatin: Hydrolyzed collagen that is used as a culture dish coating for cell attachment.
Troubleshooting Guide

It’s a great idea to keep frequent and detailed records of each time you handle your cell culture, as you will find it easier to identify early signs of any cell culture growth problems and trace the cause to prevent future incidents.

Refer to the table below to help you quickly troubleshoot:

Problem Possible Cause Solution
Slow or no growth Incorrect or expired growth media, serum, etc. Use media as recommended by the supplier with supplements as required. Prepare a new lot of fresh medium and use pre-warmed.
Mycoplasma contamination Discard or treat cells with mycoplasma decontamination solutions. Disinfect working area thoroughly and use fresh media.
Cells reached 100% confluency Passage cells while they are still in the logarithmic growth phase.
Cells have been passaged too many times. Use cells with a lower passage number. When storage cells, it is always a good idea to freezer cells at lower passage numbers into multiple aliquots.
Seeded at too low a density Use recommended seeding density.
Incubator has wrong CO2 concentration or temperature Reduce the amount of time the incubator door is open to keep the incubator environment ideal. Check CO2 supply. Cells near the door may tend to grow slower - place cells at the back of the incubator.
Cell damage during thawing Follow exact thawing recommendations. Minimize the time cells are exposed to DMSO at room temperature.
Inaccurate cell counting Ensure cells are evenly mixed for accurate cell counts. Re-check your calculations.
Some media components (riboflavin, tryptophan and HEPES) are sensitive to fluorescent light and can convert to cytotoxic free radicals and H2O2. Store media and cell cultures in the dark.
Poor cell attachment Static electrical charges on plastic vessels. Occurs more often when relative humidity (RH) is low. Increase humidity, wipe the vessel on the outside with a clean damp cloth, or use an antistatic device. Rubbing the vessels against plastic packaging when opening them can increase the charge.
Over-trypsinization: trypsin concentration is too high, or did not add inactivation dissociating solution Check recommended subculture procedures. Inactivate trypsin by adding fresh medium or Trypsin Neutralizing solution and remove trypsin by centrifugation.
Medium does not contain sufficient serum or attachment factors Check medium composition and add attachment factors, if required.
Uneven cell attachment or clumping Roller Bottles are being rotated too quickly Recommended starting speed is 0.5 – 1.0 rpm. For difficult to attach cells, 0.1 – 0.4 rpm may be used until the cells attach (Corning Guide for Identifying and Correcting Common Cell Growth Problems).
Cell culture volume is too small Heavier growth will appear along the edges of the cell culture vessels due to more cells and medium in the meniscus. Use recommended medium volumes for culture vessels.
Inadequate mixing of cells Ensure cells are re-suspended thoroughly and mixed evenly in the fresh medium.
Cell inoculum or medium contains bubbles Carefully mix cell inoculum and dispense medium into culture vessel with pipette tip against inner side of the vessel.
Overdigestion with proteolytic enzymes such as trypsin This can cause cell lysis and clumping.Check subculture protocol and recommended trypsin concentration that is required.
Overgrowth of cells, causing cell debris and free DNA build-up Subculture your cells before they reach the stationary and death phases. Check recommended cell densities for passaging cells.
Vessels are directly sitting on a perforated metal shelf in the incubator Use an empty “dummy” vessel on the bottom of the stack to avoid direct contact with the shelf and create a more even temperature distribution.
Few viable cells Cells were frozen incorrectly Freeze cells at recommended density and in appropriate media and reagents. Cells should be frozen slowly to prevent formation of damaging ice crystals.
Cells were thawed incorrectly Follow recommended thawing procedures. DMSO is toxic at room temperature so cells should be thawed quickly. Use pre-warmed media. Do not vortex or centrifuge cells at high speeds.
Cells were diluted too much Seed at a higher density as recommended for better recovery.
Cell Death Overdigestion with proteolytic enzymes such as trypsin Follow recommended protocol for cell detachment. Do not expose cells to trypsin for more than 8-10 mins. Detachment can be sped up at 37°C.
Overgrowth Passage cells around 80% confluency, before they reach 100%.
Environmental stress Avoid repeated freeze/thaw cycles, vortexing, or centrifuging cells at too high of speeds.