Determining cell viability using a LIVE/DEAD assay and fluorescence
microscopy
Emily Boggs
Biomedical Engineering Program, Lawrence Technological University, MI 48075
Abstract: Two different configurations of rat tail-based collagen gels were created for the
purpose of studying cell viability. One gel remained pure collagen, with fibroblasts subsequently
seeded onto it. The second gel was seeded with cells before it solidified, essentially creating a
cell-permeated gel. A LIVE/DEAD ® assay was performed on the gels one week later, and the
cells analyzed for viability via fluoroscopy. More cells were found on the seeded scaffolds than
the permeated ones, and there was a lower dead cell:live cell ratio in the former.
Keywords: collagen, scaffold, fibroblasts, LIVE/DEAD ® assay, fluorescence microscopy
1. Introduction
Collagen “sandwich” scaffolds have been used with the culturing of hepatocytes.
Hepatocytes grown between two layers of collagen show improved ability to maintain in vivo
qualities, like cytoskeleton structure and prolonged secretion of liver proteins versus cells
cultured on a single layer of collagen. In addition, the hepatocytes that failed to thrive on a
single layer of collagen showed more in vivo properties like those mentioned above after a
second layer above was applied [1]. Why is this? Dunn et al. [2] suggests that this sandwich
configuration more accurately mimics a hepatocyte’s in vivo environment.
Could the same be done for fibroblasts? The success for hepatocytes may have come from
the fact that the above and below polymerized collagen matrix provided by the sandwich
induced correct alignments of the cells [2]. Since fibroblasts’ functions as ligament cells are
highly dependent on their alignment in a tissue, the same principles may apply.
2. Materials and Methods
2.1 Materials
2.1.1 Making the collagen scaffolds
 2.5 mg/mL rat tail collagen in solution
 10X phosphate buffer saline (PBS)
 0.2 M sodium hydroxide (NaOH)
 100 mL distilled water
 8 g sodium chloride (NaCl)
 0.2 g KCl
 1.44 g disodium phosphate (Na2HPO4)
 0.24 g monopotassium phosphate (KH2PO4)
 35 mm culture plates
 0.75 mm membrane filter
 Human periodontal ligament fibroblasts (HPDLF)
 Fluorescent microscope with fluorescein and rhodamine optical filters
2.1.2 Assay preparation
 20 µL of 2 mM ethidium homodimer (EthD-1) solution
 5 µL of 4 mM calcein AM solution
2.2 Formation of collagen scaffolds
The 2.5 mg/mL collagen solution was first perfused through a filter with 0.75 mm diameter
pores. The solution was then mixed with PBS, NaOH, KCl, Na2HPO4, and KH2PO4. This
produced a viscous collagen mixture that would polymerize after being incubated at 37 °C for
approximately an hour. 1 mL of this mixture would be used for each well plate.
HPDLF cells that had been cultured previously were lifted from a culture plate, suspended in
media, and counted. The target seeding density for each plate was 1600 cells/cm2. Since each
plate enclosed an area of 10 cm2, around 16000 cells would be seeded onto each plate. In half
of the plates, cell suspension was mixed with the collagen mixture so that 1 mL of the mixture
contained 16000 cells. The other plates contained only collagen before polymerization.
Both sets of plates were incubated to induce polymerization; afterwards, cells were seeded
onto the collagen-only plates. Now, half of the plates contained gel with collagen solution and
cells mixed together, while the other half consisted of cells seeded onto collagen-only gels. The
cells were then allowed to grow for one week before characterization.
Characterization of cells via the LIVE/DEAD ® assay
The 2 mM EthD-1 solution is added to 10 mL of sterile PBS; EthD-1 binds to the nuclei of
dead cells and stains them a fluorescent read. This was then mixed with 4 mM calcein AM,
yielding a solution of approximately 2 µM EthD-1 and 4 µM calcein AM. The calcein AM stain is
picked up by living cells, turning them a fluorescent green color. This mixture was then applied
directly to the cells on the collagen scaffolds. The cell plates are then rinsed with warm PBS
and incubated in the dark at 37 °C for 15 to 20 minutes.
After incubation, the plates are ready for viewing under a fluorescent microscope. Green
fluorescent cells are alive and viable, whereas red are dead. By counting the number of dead
and alive cells in 2 different areas on the plate, an approximate percentage of cell viability can
be inferred.
3. Results and Discussion
Figure 1: Collagen/cell mixture.
Figure 2: Collagen/cell mixture.
Figure 3: Cells seeded onto collagen.
Figure 2: Cells seeded onto collagen.
Overall, the plates where cells were seeded onto pre-polymerized collagen exhibited a
higher number of cells. The number of cells counted in each image is the number of whole cells
visible. The first image for the collagen/cell mixture (Fig. 1) contains 6 cells, 5 of which are alive
(80% viability). The second collagen/cell mixture (Fig. 2) contains 11 cells, 10 of which are alive
(91% viability). The first image for the cell seeded collagen (Fig. 3) contains 16 cells, 14 of
which are alive (88%). The second image for the cell seeded collagen (Fig. 4) contains 18 cells,
15 of which are alive (83%). On average, the cell/collagen mixture exhibited 85.5% viability,
while the cell seeded collagen exhibited the same: 85.5% viability. This means that cells were
no more likely to die on either type of scaffold, but more cells were present (or maintained from
the initial 16000) on the cell seeded collagen scaffolds.
While the collagen-sandwich scaffold worked well for hepatocytes, better results were
achieved with standard seeding with respect to fibroblasts. One explanation for the diminished
cell numbers in the sandwich scaffold is that the polymerized matrix initially hindered the
development of the fibroblasts’ “spindled” ends (filopodia) [3]. By covering less area, fibroblasts
scattered in the days following seeding were less likely to come in contact with each other,
causing a large number of isolated cells to die off [4]. The collagen as a scaffold material would
seem to have little effect, given that both plates had the same viability.
However, these conclusions are hindered by the fact that the sample size from which these
images were taken was very small (only two plates). It is possible that given a larger sample
population, other trends delineating cell proliferation and viability may emerge. In, addition, the
cells were only grown for one week (as opposed to up to six weeks [1]) and neither of the plates
showed any significant fibroblast alignment, which should have been one of the main
advantages of using the collagen sandwich system [2].
4. Conclusion
When it comes to growing fibroblasts on collagen scaffolds, cell numbers are better
maintained using a traditional cell seeding approach. However, both the cell seeding and
collagen sandwich approaches showed the same overall viability.
References
[1] Dunn, J. C. Y., Tompkins, R. G., & Yarmush, M. L. (1991). Long-term in vitro function of adult
hepatocytes in a collagen sandwich configuration. Biotechnology Progress, 7, 237-245.
[2] Dunn, J. C. Y., Yarmush, M. L., Koebe, H. G., & Tompkins, R. G. (1989). Hepatocyte
function and extracellular matrix geometry: Long-term culture in a sandwich configuration.
FASEB, 3(2), 174-177.
[3] Lauffenburger, D. A., & Horwitz, A. F. (1996). Cell migration: A physically integrated
molecular process. Cell, 84, 359-369.
[4] Mattila, P. K., & Lappalainen, P. (2008). Filopodia: Molecular architecture and cellular
functions. Nature, 9, 446-454. doi: 10.1038/nrm2406
Appendix
“Collagen gel by Dunn and Yarmush” Protocol (see attachments)
“Live-Dead assay” Protocol (see attachments)
I have neither given nor received any unauthorized aid in completing this work, nor have I
presented someone else’s work as my own.
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