Effect of Microenvironment pH on HeLa Cell Viability
Isabela Caicedo, Sara Galbraith, Blake McDonald, David Rojas
Introduction
This experiment observes the effects of pH
change on HeLa cell culture viability by adding
acetic acid and sodium hydroxide in varying
concentrations. This relationship is essential to
understanding ideal media conditions to sustain
cell culture. Based on literature regarding the
effects of pregnancy on pH, we believe that
acidic conditions will be optimal.
Methodology
Four 6-well plates were seeded with 300K
cells/well. All well plates were incubated for 24
hours to allow cell proliferation.
After the incubation period, confluencies were
recorded and 1 M acetic acid (AcOH) and 1 M
sodium hydroxide (NaOH) were added to wells
according to plate layouts (shown below). A pH
test was then performed utilizing litmus paper.
Following this test, the well plates underwent a
second 24 hour incubation period and
confluencies were again recorded.
Subsequently, an Alamar Blue Assay was
TM
performed. A BioTek Multi-Mode Microplate
Reader was utilized for absorbance analysis at
570 nm to assess cell viability.
Results
Cell confluencies observed pre- and post-acid/base treatment are reported
below. Non-statistical comparison between columns in each plate revealed
large discrepancies between confluencies despite uniform seeding density.
Fig. 1 Data are expressed as mean
absorbances and the differences
between them were analyzed
using a one-way ANOVA test
performed by MINITAB. p <
0.05 was considered statistically
significant. Cells without test
agent were used as a positive
control. The results show a
gradual decrease in absorbance as
Fig. 2
concentration of AcOH is
increased. Absorbance does not
follow any obvious trend as
concentration of NaOH is
increased. The error observed for
each mean absorbance in the base
treatment group is larger
compared to that seen in the acid
treatment group.
J. Crayton Pruitt Family Department of Biomedical Engineering
Conclusion
Based on the absorbance results from the
Alamar Blue Assay and the statistical analysis
of these results, a conclusion regarding the
most optimal condition cannot be reached due
to conflicting results in the basic experimental
group. We can conclude that increasing acidic
concentration had a negative effect on cell
viability compared to the control with pH of
approximately 7.4. The acidic groups were
able to sustain higher confluencies than the
basic group, suggesting more optimal
conditions.
Future Direction
Methods to consider changing on the next
iteration include
● Bubble CO2 into media
● Change CO2 concentration of incubator
● Use pH meter for quantitative
measurements
● Test comparably dissociative acid and
base
● Ensure similar confluencies in each
experimental group
References
[1] S. Riedewald, I. M. Kreutzmann, T. Heinze, and E.
Sailing, “Vaginal and cervical pH in normal pregnancy and
pregnancy complicated by preterm labor,” Pubmed.gov.