Who Can Benefit From pH Tracking? 

This month I would like to highlight some of the critical ways pH stability affects a wide scope in cell culture. If your lab falls under any of these categories, pH monitoring can benefit your lab to confirm, change, or improve your current practices.

The Andrology Lab can make or break an IVF program. Sperm cells function in a wide pH range (normal seminal fluid pH values ranging from 7.2-8.0) but there are some specifics I would like to highlight. Sperm become immotile in a low pH (<7) environment as seen in the epididymis[1], but remain tolerant of pH with optimal zona binding at a media pH of 7.5[2]. Alkalization of the sperm cytoplasm occurs during capacitation and triggers calcium influx, sperm activation and hyperactivated motility[3] and sperm internal pH has been found to increase linearly with external pH[4]. What this points to in a lab setting is that subtle physiological changes can occur when pH is not properly controlled. An Andrology lab that is using bicarbonate-based media should strive to maintain media pH as much as possible during normal cell manipulation workflow, including tracking pH when frequently opening incubator doors and in preparation for clinical protocols such at intrauterine inseminations (IUI) and in vitro fertilization (IVF).

Many types of cell culture require specific pH ranges: for example, embryo culture requires a pH range from 7.2-7.4[5,6]. Sperms cells are very tolerant of a wide pH range, but preparation for use in in vitro fertilization requires the culture media pH range be similar to embryo culture conditions (7.2-7.4).  Cell manipulations must be performed in non-ideal conditions outside of the incubator due to lab equipment restrictions and practicality. When cell cultures are manipulated outside of the incubator, differences in %CO₂ should be acknowledged, and work should be done as quickly as possible while maintaining accuracy. Improper protection of cell culture media from shifts in ambient CO₂ will be reflected as a rapid rise of the pH in the culture medium.

Monitoring culture media used in large batch cell culture can act as the first alert to contamination in your culture environment. Some forms of culture media contamination are undetectable without direct testing-  bacterial mycoplasma contamination shows no outer physical signs such as turbidity or pH changes.  Bacteria contamination, the most commonly found contaminate in cell culture, can be detected through qualitative observations such as a sudden pH drop in culture medium, medium turbidity, and analysis under a low-power microscope. Yeasts and molds colonies will have stable pH values at the start of contamination, but with time and propagation an increase in pH levels will be observed and create an adverse environment for cell survival [6].

A malfunction to the CO₂ system in your cell culture lab can lead unplanned increases in cell culture media pH. Direct measurements of the % CO₂ within an incubator is the most common quality control parameter recorded, with differences in the frequency of measurements depending on the lab’s personal preferences. Taking a singular %CO₂ measurement is misleading about the true environment of your cell culture, as %CO₂ values will change rapidly during cell culture workflow practices. Relying %CO₂ values portrayed on the exterior of an incubator can also be misleading if taken at face value. For example, we have previously discussed how differences in CO₂ detection equipment in the incubator can lead to inaccuracies from humidity and temperature inside incubators, as well as how weather or altitude can have unexpected effects on your expected pH values.

Continuous monitoring of pH values can give cell culture specialists critical information on the health of their culture systems and warn against critical problems.  The TrakStation pH monitoring system takes the guesswork out of your final media pH value and is currently being used in a wide variety of cell culture specialties, such as embryo culture, sperm preparation, stem cell research and animal reproductive science research.

1.        Nishigaki T. et al. (2014). Intracellular pH in sperm physiology. Biochem. Biophys. Res. Commun. 450, 1149–1158.
2.        Dale B, Menezo Y, Cohen J, DiMatteo L, Wilding M., (1998). Intracellular pH regulation in the human oocyte. Hum Reprod. 13:964–970.
3.        Ying Liu, Deng-Ke Wang, Li-Ming Chen.( 2012). The Physiology of Bicarbonate Transporters in Mammalian Reproduction, Biology of Reproduction, 86:4:1:99 1–13.
4.        Hamamah, S., Magnoux, E., Royere, D., Barthelemy, C., Dacheus, J.-L. and Gati, J.-L., (1996). Internal pH of human spermatozoa: effect of ions, human follicular fluid and progesterone. Mol Human Reprod. 2:219.
5.        Lane, M., Baltz, J.M., et al. (1998). Regulation of intracellular pH in hamster preimplantation embryos by the sodium hydrogen (Na+ / H+ ) antiporter. Biol. Reprod. 59, 1483–1490.
6.        John, D.P., Kiessling, A.A. (1988). Improved pronuclear mouse embryo development over an extended pH range in Ham’s F-10 medium without protein. Fertil. Steril. 49, 150–155.