Measure passage number-dependent cell proliferation

Researchers working with mammalian cells to develop gene therapy products do so from a frozen stock of cells that are thawed in aliquots and passaged several times. The passage number refers to the number of transfers from vessel to vessel.

Over time, this may impact cell characteristics including:

  • Morphology
  • Growth
  • Protein expression
  • Transfection efficiency

The Celigo Image Cytometer performs cell proliferation assays to identify issues and differences between culture passages that impact the consistency and predictability for downstream assays.

  1. Target cells at high and low passage numbers were seeded in a 96-well plate at day zero and allowed to adhere overnight
  2. The Celigo was used for whole-well imaging on the following two days to measure confluence percentages
  3. The cell proliferation results were compared between high and low passage number cell lines

Rapid label-free cell proliferation assay

The brightfield images demonstrate clear cell growth over time (Figure 1). The identified and measured cell confluence is overlaid with a pseudo-color green fill. The brightfield images show the same well containing low-passage cells imaged 24 hours apart. Within one day, the confluence increased from 51.3% to 63.6%. The graph shows data from two different plates containing low- and high-passage cells, and the low-passage cells showed slightly higher average confluence percentages.

cell confluence area for low-passage cells
low and high passage cell lines

Figure 1. (a) Zoomed-in brightfield images on day one and two with pseudo-color green fill indicating cell confluence area for low-passage cell cells. (b) Confluence for low and high passage cell lines determines cell proliferation.

According to the American Type Culture Collection, “Preventing passage-related effects from influencing experiments becomes a matter of determining the passage number range under which a set of experiments can be consistently performed for a given cell line.”

Celigo Image Cytometer can help researchers:

  • Avoid passage-related effects
  • Produce valid, reproducible results
  • Ensure good culture technique based on growth data